WorldWideScience

Sample records for chimerism

  1. Electroejaculation of chimeric rats

    OpenAIRE

    McCoy, Marina R.; Montonye, Daniel; Bryda, Elizabeth C.

    2013-01-01

    With the advent of genetic engineering of rodents came the need to assess fertility and germline competency, especially in chimeric rodents generated using embryonic stem cells. Traditional methods rely on natural mating and progeny testing, which is time- and cost-intensive. Electroejaculation is a faster method of collecting sperm for genetic analysis and offers the additional benefit of using fewer animals. This column describes a refined electroejaculation technique for chimeric rats usin...

  2. Electroejaculation of chimeric rats.

    Science.gov (United States)

    McCoy, Marina R; Montonye, Daniel; Bryda, Elizabeth C

    2013-06-01

    With the advent of genetic engineering of rodents came the need to assess fertility and germline competency, especially in chimeric rodents generated using embryonic stem cells. Traditional methods rely on natural mating and progeny testing, which is time- and cost-intensive. Electroejaculation is a faster method of collecting sperm for genetic analysis and offers the additional benefit of using fewer animals. This column describes a refined electroejaculation technique for chimeric rats using light gas anesthesia and a custom-made platform for sperm collection. PMID:23689457

  3. Chimeric Pestivirus Experimental Vaccines.

    Science.gov (United States)

    Reimann, Ilona; Blome, Sandra; Beer, Martin

    2016-01-01

    Chimeric pestiviruses have shown great potential as marker vaccine candidates against pestiviral infections. Exemplarily, we describe here the construction and testing of the most promising classical swine fever vaccine candidate "CP7_E2alf" in detail. The description is focused on classical cloning technologies in combination with reverse genetics. PMID:26458840

  4. Chimeric enzymes with improved cellulase activities

    Science.gov (United States)

    Xu, Qi; Baker, John O; Himmel, Michael E

    2015-03-31

    Nucleic acid molecules encoding chimeric cellulase polypeptides that exhibit improved cellulase activities are disclosed herein. The chimeric cellulase polypeptides encoded by these nucleic acids and methods to produce the cellulases are also described, along with methods of using chimeric cellulases for the conversion of cellulose to sugars such as glucose.

  5. Progress in Chimeric Vector and Chimeric Gene Based Cardiovascular Gene Therapy

    Institute of Scientific and Technical Information of China (English)

    HU Chun-Song; YOON Young-sup; ISNER Jeffrey M.; LOSORDO Douglas W.

    2003-01-01

    Gene therapy for cardiovascular diseases has developed from preliminary animal experiments to clinical trials. However, vectors and target genes used currently in gene therapy are mainly focused on viral, nonviral vector and single target gene or monogene. Each vector system has a series of advantages and limitations. Chimeric vectors which combine the advantages of viral and nonviral vector,chimeric target genes which combine two or more target genes and novel gene delivery modes are being developed. In this article, we summarized the progress in chimeric vectors and chimeric genes based cardiovascular gene therapy, which including proliferative or occlusive vascular diseases such as atheroslerosis and restenosis, hypertonic vascular disease such as hypertension and cardiac diseases such as myocardium ischemia, dilated cardiomyopathy and heart failure, even heart transplantation. The development of chimeric vector, chimeric gene and their cardiovascular gene therapy is promising.

  6. Blood chimerism in a dizygotic dichorionic pregnancy.

    Science.gov (United States)

    Jang, Ja-Hyun; Jung, Haiyoung; Kim, Jong-Hwa; Park, Won-Soon; Kim, Sun-Hee

    2010-10-01

    Blood chimerism in twins is known to occur through the transfer of hematopoietic stem cells between the fetuses via a common placenta. We present a case of blood chimerism in a dizygotic dichorionic twin pregnancy. The female twin was delivered at 34 weeks of gestation, and the male twin was stillborn. Pathologic examination confirmed dichorionic diamniotic placentas. The karyotype of the female child was obtained using peripheral blood sample, and it revealed a mixture of 46,XX and 46,XY cells (chi 46,XY[13]/46,XX[7]). FISH analysis performed on the buccal cells by using CEP X/Y probe (Abbott Molecular Inc., USA) revealed 100% XX signals (nuc ish Xcen(DXZ1x2)[500]). Gross examination of the external genitalia and abdominal ultrasonography revealed no definitive abnormal findings in relation to sex differentiation. When XX/XY chimerism is present in blood lymphocytes, careful examination of external genitalia and reproductive organs and further studies are required to detect chimerism in non-hematopoetic tissues. This is a rare case of blood chimerism in dichorionic placentas, in contrast to those in monochorionic placentas. PMID:20890086

  7. Chimerism in health, transplantation and autoimmunity

    NARCIS (Netherlands)

    Koopmans, Marije; Kremer Hovinga, Idske Cornelia Lydia

    2009-01-01

    The term “chimerism” originates from Greek mythology and refers to the creature Chimaera, whose body was in front a lion, the back a serpent and the midsection a goat. In medicine, the term chimerism refers to an individual, organ or part consisting of tissues of diverse genetic constitution. Pregna

  8. FACIAL EXPRESSION RECOGNITION WITH THE USE OF CHIMERIC FACE TECHNIQUE

    OpenAIRE

    Menshikova, Galina

    2010-01-01

    The aim of this study was to investigate holistic / feature processing for encoding face expressions employing the chimeric face technique. In the course of our experiment we tested the recognition accuracy of universal and chimeric countenance. As the study has revealed there was a considerable difference between distributions of subject responses depending on the localization of expression features (top / bottom parts of the face). For chimeric face identification accuracy substantially dec...

  9. An E2-Substituted Chimeric Pestivirus With DIVA Vaccine Properties

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Nielsen, Jens;

    An advantage of the use of chimeric pestiviruses as modified live vaccines against classical swine fever (CSF) resides in their capacity to be manipulated to achieve the characteristics desired for safe and efficacious DIVA vaccines. We have recently generated a new chimeric virus, Riems26_E2gif...

  10. Do chimeric sponges have improved chances of survival?

    OpenAIRE

    Maldonado, Manuel

    1998-01-01

    It has been suggested that the capacity of fusion with both kin and genetically unrelated conspecifics to form chimeras (i.e. individuals with a mixture of genetically different cells) is evolutionarily retained In several phyla because the resulting organism obtains some selective advantages over non-chimeric conspecifics. Many demosponges are known to have fusible larvae that form young chimeric sponges, but the ecological and evolutionary significance of this phenom...

  11. Chimeric autologous/allogeneic constructs for skin regeneration.

    Science.gov (United States)

    Rasmussen, Cathy Ann; Tam, Joshua; Steiglitz, Barry M; Bauer, Rebecca L; Peters, Noel R; Wang, Ying; Anderson, R Rox; Allen-Hoffmann, B Lynn

    2014-08-01

    The ideal treatment for severe cutaneous injuries would eliminate the need for autografts and promote fully functional, aesthetically pleasing autologous skin regeneration. NIKS progenitor cell-based skin tissues have been developed to promote healing by providing barrier function and delivering wound healing factors. Independently, a device has recently been created to "copy" skin by harvesting full-thickness microscopic tissue columns (MTCs) in lieu of autografts traditionally harvested as sheets. We evaluated the feasibility of combining these two technologies by embedding MTCs in NIKS-based skin tissues to generate chimeric autologous/allogeneic constructs. Chimeric constructs have the potential to provide immediate wound coverage, eliminate painful donor site wounds, and promote restoration of a pigmented skin tissue possessing hair follicles, sweat glands, and sebaceous glands. After MTC insertion, chimeric constructs and controls were reintroduced into air-interface culture and maintained in vitro for several weeks. Tissue viability, proliferative capacity, and morphology were evaluated after long-term culture. Our results confirmed successful MTC insertion and integration, and demonstrated the feasibility of generating chimeric autologous/allogeneic constructs that preserved the viability, proliferative capacity, and structure of autologous pigmented skin. These feasibility studies established the proof-of-principle necessary to further develop chimeric autologous/allogeneic constructs for the treatment of complex skin defects. PMID:25102552

  12. Developmental competence of porcine chimeric embryos produced by aggregation

    DEFF Research Database (Denmark)

    Li, Juan; Jakobsen, Jannik E.; Xiong, Qiang;

    2015-01-01

    either by parthenogenetic activation (PA) or handmade cloning (HMC). Results showed that the developmental competence of chimeric embryos, evaluated based on their blastocyst rate and total cell number per blastocyst, was increased when two whole 2-cell stage embryos (PA or HMC) were aggregated. In...... comparison, when two blastomeres were aggregated, the developmental competence of the chimeric embryos decreased if the blastomeres were either from PA or from HMC embryos, but not if they were from different sources, i.e. one PA and one HMC blastomere. To evaluate the cell contribution in embryo formation......The purpose of our study was to compare the developmental competence and blastomere allocation of porcine chimeric embryos formed by micro-well aggregation. Chimeras were created by aggregating either two blastomeres originating from 2-cell embryos or two whole embryos, where embryos were produced...

  13. Study of cancer-specific chimeric promoters induced by irradiation

    International Nuclear Information System (INIS)

    Objective: To combine the radio-inducible CArG element with cancer-specific human telomerase reverse transcriptase (hTERT) gene promoter, and to construct the novel chimeric promoters. Methods: The synthetic hTERT promoters containing different number of radio-inducible CArG elements were constructed, and the activities of the promoters in the cancer cells (HeLa, A549, and MHCC97 cells) and nomal cells (hEL cells) were detected by using luciferase-reporter assays after the treatment of irradiation (a single or fractionated irradiation dose). Results: Synthetic promoter containing 6 repeated CArG units was better in radio-inducibility than any other promoters containing different number of CArG units, and nearly maximum levels obtained at 4-6 Gy. The very low activities of the chimeric promoters could be detected in normal hEL cells. A similar level of reporter gene expression was observed after 3 fractionated doses of 2 Gy compared with a single dose of 6 Gy in cancer cells. Conclusions: The cancer-specific chimeric promoter containing 6 CArG elements showes the best radio-response, and the chimeric promoter system has the potential in cancer gene therapy. (authors)

  14. Virulence, immunogenicity and vaccine properties of a novel chimeric pestivirus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Reimann, Ilona;

    2007-01-01

    avirulent and neither chimeric virus nor viral RNA was detected in serum after vaccination. The safety of the vaccine was tested by horizontal transmission to sentinel pigs, which remained uninfected. The vaccine efficacy was examined by challenge infection with classical swine fever virus (CSFV) Eystrup...

  15. CONSTRUCTION AND EXPRESSION OF A HUMAN-MOUSE CHIMERIC ANTIBODY AGAINST HUMAN BLADDER CANCER

    Institute of Scientific and Technical Information of China (English)

    白银; 王琰; 周丽君; 俞莉章

    2001-01-01

    To construct and express a human-mouse chimeric antibody against human bladder cancer. Method: The variable region genes of anti-human bladder cancer monoclonal antibody BDI-1 were cloned by RT-PCR. A human-mouse chimeric antibody expression vector was constructed and transfected into CHO cells. The chimeric antibody against bladder cancer was expressed and characterized. Result: Eukaryotic expression vector of the chimeric antibody against human bladder carcinoma was successfully constructed, and was expressed in eukaryotic cells; the expressed chimeric antibody ch-BDI showed same specificity as its parent McAb against human bladder cancer cells. Conclusion: The constructed chimeric antibody was expressed successfully in eukaryotic cells, and the chimeric antibody had desired affinity against human bladder cancer cells.

  16. High affinity mouse-human chimeric Fab against Hepatitis B surface antigen

    Institute of Scientific and Technical Information of China (English)

    Biplab Bose; Navin Khanna; Subrat K Acharya; Subrata Sinha

    2005-01-01

    AIM: Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody.METHODS: We cloned the VH and VL genes of this mouse antibody; and fused them with CH1 domain of human IgG1 and CL domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. Coli.RESULTS: The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal.This chimeric antibody fragment was further expressed in different strains of E> coli to increase the yield.CONCLUSION: We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a fulllength chimeric antibody for therapeutic uses.

  17. Design of chimeric antigen receptors with integrated controllable transient functions

    OpenAIRE

    Alexandre Juillerat; Alan Marechal; Jean-Marie Filhol; Julien Valton; Aymeric Duclert; Laurent Poirot; Philippe Duchateau

    2016-01-01

    The ability to control T cells engineered to permanently express chimeric antigen receptors (CARs) is a key feature to improve safety. Here, we describe the development of a new CAR architecture with an integrated switch-on system that permits to control the CAR T-cell function. This system offers the advantage of a transient CAR T-cell for safety while letting open the possibility of multiple cytotoxicity cycles using a small molecule drug.

  18. Design of chimeric antigen receptors with integrated controllable transient functions.

    Science.gov (United States)

    Juillerat, Alexandre; Marechal, Alan; Filhol, Jean-Marie; Valton, Julien; Duclert, Aymeric; Poirot, Laurent; Duchateau, Philippe

    2016-01-01

    The ability to control T cells engineered to permanently express chimeric antigen receptors (CARs) is a key feature to improve safety. Here, we describe the development of a new CAR architecture with an integrated switch-on system that permits to control the CAR T-cell function. This system offers the advantage of a transient CAR T-cell for safety while letting open the possibility of multiple cytotoxicity cycles using a small molecule drug. PMID:26750734

  19. Chimeric Proteins to Detect DNA Damage and Mismatches

    Energy Technology Data Exchange (ETDEWEB)

    McCutchen-Maloney, S; Malfatti, M; Robbins, K M

    2002-01-14

    The goal of this project was to develop chimeric proteins composed of a DNA mismatch or damage binding protein and a nuclease, as well as methods to detect DNA mismatches and damage. We accomplished this through protein engineering based on using polymerase chain reactions (PCRs) to create chimeras with novel functions for damage and mismatch detection. This project addressed fundamental questions relating to disease susceptibility and radiation-induced damage in cells. It also supported and enhanced LLNL's competency in the emerging field of proteomics. In nature, DNA is constantly being subjected to damaging agents such as exposure to ultraviolet (UV) radiation and various environmental and dietary carcinogens. If DNA damage is not repaired however, mutations in DNA result that can eventually manifest in cancer and other diseases. In addition to damage-induced DNA mutations, single nucleotide polymorphisms (SNPs), which are variations in the genetic sequence between individuals, may predispose some to disease. As a result of the Human Genome Project, the integrity of a person's DNA can now be monitored. Therefore, methods to detect DNA damage, mutations, and SNPs are useful not only in basic research but also in the health and biotechnology industries. Current methods of detection often use radioactive labeling and rely on expensive instrumentation that is not readily available in many research settings. Our methods to detect DNA damage and mismatches employ simple gel electrophoresis and flow cytometry, thereby alleviating the need for radioactive labeling and expensive equipment. In FY2001, we explored SNP detection by developing methods based on the ability of the chimeric proteins to detect mismatches. Using multiplex assays with flow cytometry and fluorescent beads to which the DNA substrates where attached, we showed that several of the chimeras possess greater affinity for damaged and mismatched DNA than for native DNA. This affinity was

  20. Chimeras taking shape: potential functions of proteins encoded by chimeric RNA transcripts.

    Science.gov (United States)

    Frenkel-Morgenstern, Milana; Lacroix, Vincent; Ezkurdia, Iakes; Levin, Yishai; Gabashvili, Alexandra; Prilusky, Jaime; Del Pozo, Angela; Tress, Michael; Johnson, Rory; Guigo, Roderic; Valencia, Alfonso

    2012-07-01

    Chimeric RNAs comprise exons from two or more different genes and have the potential to encode novel proteins that alter cellular phenotypes. To date, numerous putative chimeric transcripts have been identified among the ESTs isolated from several organisms and using high throughput RNA sequencing. The few corresponding protein products that have been characterized mostly result from chromosomal translocations and are associated with cancer. Here, we systematically establish that some of the putative chimeric transcripts are genuinely expressed in human cells. Using high throughput RNA sequencing, mass spectrometry experimental data, and functional annotation, we studied 7424 putative human chimeric RNAs. We confirmed the expression of 175 chimeric RNAs in 16 human tissues, with an abundance varying from 0.06 to 17 RPKM (Reads Per Kilobase per Million mapped reads). We show that these chimeric RNAs are significantly more tissue-specific than non-chimeric transcripts. Moreover, we present evidence that chimeras tend to incorporate highly expressed genes. Despite the low expression level of most chimeric RNAs, we show that 12 novel chimeras are translated into proteins detectable in multiple shotgun mass spectrometry experiments. Furthermore, we confirm the expression of three novel chimeric proteins using targeted mass spectrometry. Finally, based on our functional annotation of exon organization and preserved domains, we discuss the potential features of chimeric proteins with illustrative examples and suggest that chimeras significantly exploit signal peptides and transmembrane domains, which can alter the cellular localization of cognate proteins. Taken together, these findings establish that some chimeric RNAs are translated into potentially functional proteins in humans. PMID:22588898

  1. Construction, purification, and characterization of a chimeric TH1 antagonist

    Directory of Open Access Journals (Sweden)

    Javier-González Luís

    2006-05-01

    Full Text Available Abstract Background TH1 immune response antagonism is a desirable approach to mitigate some autoimmune and inflammatory reactions during the course of several diseases where IL-2 and IFN-γ are two central players. Therefore, the neutralization of both cytokines could provide beneficial effects in patients suffering from autoimmune or inflammatory illnesses. Results A chimeric antagonist that can antagonize the action of TH1 immunity mediators, IFN-γ and IL-2, was designed, engineered, expressed in E. coli, purified and evaluated for its in vitro biological activities. The TH1 antagonist molecule consists of the extracellular region for the human IFNγ receptor chain 1 fused by a four-aminoacid linker peptide to human 60 N-terminal aminoacid residues of IL-2. The corresponding gene fragments were isolated by RT-PCR and cloned in the pTPV-1 vector. E. coli (W3110 strain was transformed with this vector. The chimeric protein was expressed at high level as inclusion bodies. The protein was partially purified by pelleting and washing. It was then solubilized with strong denaturant and finally refolded by gel filtration. In vitro biological activity of chimera was demonstrated by inhibition of IFN-γ-dependent HLA-DR expression in Colo 205 cells, inhibition of IFN-γ antiproliferative effect on HEp-2 cells, and by a bidirectional effect in assays for IL-2 T-cell dependent proliferation: agonism in the absence versus inhibition in the presence of IL-2. Conclusion TH1 antagonist is a chimeric protein that inhibits the in vitro biological activities of human IFN-γ, and is a partial agonist/antagonist of human IL-2. With these attributes, the chimera has the potential to offer a new opportunity for the treatment of autoimmune and inflammatory diseases.

  2. Chimeric creatures in Greek mythology and reflections in science.

    Science.gov (United States)

    Bazopoulou-Kyrkanidou, E

    2001-04-15

    "The Chimaera" in Homer's Iliad, "was of divine stock, not of men, in the forepart a lion, in the hinder a serpent, and in the midst a goat, ellipsis Bellerophon slew her, trusting in the signs of the gods." In Hesiod's Theogony it is emphasized that "Chimaera ellipsis had three heads, one of a grim-eyed lion, another of a goat, and another of a snakeellipsis". In addition to this interspecies animal chimera, human/animal chimeras are referred to in Greek mythology, preeminent among them the Centaurs and the Minotaur. The Centaurs, as horse/men, first appear in Geometric and early Archaic art, but in the literature not until early in the fifth century B.C. The bullheaded-man Minotaur, who is not certainly attested in the literary evidence until circa 500 B.C., first appears in art about 650 B.C. Attempts, in the fourth century B.C. and thereafter, to rationalize their mythical appearance were in vain; their chimeric nature retained its fascinating and archetypal form over the centuries. Early in the 1980s, experimental sheep/goat chimeras were produced removing the reproductive barrier between these two animal species. Late in the 1990s, legal, political, ethical, and moral fights loomed over a patent bid on human/animal chimeras. Chimeric technology is recently developed; however, the concept of chimerism has existed in literary and artistic form in ancient mythology. This is yet another example where art and literature precede scientific research and development. PMID:11337752

  3. Chimeric elk/mouse prion proteins in transgenic mice

    OpenAIRE

    Tamguney, G; Giles, K; Oehler, A.; Johnson, NL; DeArmond, SJ; Prusiner, SB

    2013-01-01

    Chronic wasting disease (CWD) of deer and elk is a highly communicable neurodegenerative disorder caused by prions. Investigations of CWD are hampered by slow bioassays in transgenic (Tg) mice. Towards the development of Tg mice that will be more susceptible to CWD prions, we created a series of chimeric elk/mouse transgenes that encode the N terminus of elk PrP (ElkPrP) up to residue Y168 and the C terminus of mouse PrP (MoPrP) beyond residue 169 (mouse numbering), designated Elk3M(SNIVVK). ...

  4. Construction and characterization of chimeric BHIV (BIV/HIV-1) viruses carrying the bovine immunodeficiency virus gag gene

    OpenAIRE

    Zhu, Yi-Xin; Liu, Chang; Liu, Xin-Lei; Qiao, Wen-Tao; Chen, Qi-Min; Zeng, Yi; Geng, Yun-Qi

    2005-01-01

    AIM: To explore the possibility of the replacement of the gag gene between human immunodeficiency virus and bovine immunodeficiency virus, to achieve chimeric virions, and thereby gain a new kind of AIDS vaccine based on BHIV chimeric viruses.

  5. Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae

    DEFF Research Database (Denmark)

    Stentebjerg-Olesen, Bodil; Pallesen, Lars; Jensen, Lars Bogø;

    1997-01-01

    inserted. Several of the chosen positions seemed amenable even for large foreign inserts; the chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Display of chimeric fimbriae was tested...

  6. Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae

    DEFF Research Database (Denmark)

    Stentebjerg-Olesen, Bodil; Pallesen, Lars; Jensen, Lars Bogø; Christiansen, Gunnar; Klemm, Per

    inserted. Several of the chosen positions seemed amenable even for large foreign inserts; the chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Display of chimeric fimbriae was tested...

  7. DIVA vaccine properties of the live chimeric pestivirus strain CP7_E2gif

    DEFF Research Database (Denmark)

    von Rosen, Tanya; Rangelova, Desislava Yordanova; Nielsen, Jens;

    2014-01-01

    Live modified vaccines to protect against classical swine fever virus (CSFV), based on chimeric pestiviruses, have been developed to enable serological Differentiation of Infected from Vaccinated Animals (DIVA). In this context, the chimeric virus CP7_E2gif vaccine candidate is unique as it does...

  8. 78 FR 16505 - Prospective Grant of Exclusive License: Chimeric West Nile/Dengue Viruses

    Science.gov (United States)

    2013-03-15

    ...: Chimeric West Nile/Dengue Viruses AGENCY: Centers for Disease Control and Prevention (CDC), Department of... license, in the field of use of in vitro diagnostics for dengue virus infection, to practice the... Application 61/049,342, filed 4/30/2008, entitled ``Engineered, Chimeric West Nile/Dengue Viruses;''...

  9. High-resolution air quality simulation over Europe with the chemistry transport model CHIMERE

    Directory of Open Access Journals (Sweden)

    E. Terrenoire

    2015-01-01

    The results suggest that future work should focus on the development of national bottom-up emission inventories including a better account for semi-volatile organic compounds and their conversion to SOA, the improvement of the CHIMERE urban parameterization, the introduction into CHIMERE of the coarse nitrate chemistry and an advanced parameterization accounting for windblown dust emissions.

  10. The Construction of Chimeric T-Cell Receptor with Spacer Base of Modeling Study of VHH and MUC1 Interaction

    OpenAIRE

    Nazanin Pirooznia; Sadegh Hasannia; Majid Taghdir; Fatemeh Rahbarizadeh; Morteza Eskandani

    2011-01-01

    Adaptive cell immunotherapy with the use of chimeric receptors leads to the best and most specific response against tumors. Chimeric receptors consist of a signaling fragment, extracellular spacer, costimulating domain, and an antibody. Antibodies cause immunogenicity; therefore, VHH is a good replacement for ScFv in chimeric receptors. Since peptide sequences have an influence on chimeric receptors, the effect of peptide domains on each other's conformation were investigated. CD3Zeta, CD28, ...

  11. MHC-mismatched mixed chimerism augments thymic regulatory T-cell production and prevents relapse of EAE in mice

    OpenAIRE

    Wu, Limin; Li, Nainong; Zhang, Mingfeng; XUE, SHENG-LI; Cassady, Kaniel; Lin, Qing; Riggs, Arthur D; Zeng, Defu

    2015-01-01

    Induction of MHC- or HLA-matched mixed chimerism does not cause graft-versus-host disease (GVHD) in animal models or humans, but matched mixed chimerism cannot reverse autoimmunity. MHC-mismatched mixed chimerism is required for reversal of autoimmunity. Here, we report that, using a clinically applicable conditioning regimen consisting of cyclophosphamide, pentostatin, and antithymocyte globulin, MHC-mismatched mixed chimerism is established in experimental autoimmune encephalomyelitis (EAE)...

  12. 78 FR 13691 - Prospective Grant of Exclusive License: The Development of m971 and m972 Chimeric Antigen...

    Science.gov (United States)

    2013-02-28

    ... m971 and m972 Chimeric Antigen Receptors (CARs) for the Treatment of B Cell Malignancies AGENCY... inventions embodied in (a) U.S. Patent Application 61/717,960 entitled ``M971 Chimeric Antigen Receptors... their cell surface using chimeric antigen receptors which contain the m971 or m972 antibody...

  13. Serotype Chimeric Human Adenoviruses for Cancer GeneTherapy

    Directory of Open Access Journals (Sweden)

    Akseli Hemminki

    2010-09-01

    Full Text Available Cancer gene therapy consists of numerous approaches where the common denominator is utilization of vectors for achieving therapeutic effect. A particularly potent embodiment of the approach is virotherapy, in which the replication potential of an oncolytic virus is directed towards tumor cells to cause lysis, while normal cells are spared. Importantly, the therapeutic effect of the initial viral load is amplified through viral replication cycles and production of progeny virions. All cancer gene therapy approaches rely on a sufficient level of delivery of the anticancer agent into target cells. Thus,enhancement of delivery to target cells, and reduction of delivery to non-target cells, in an approach called transductional targeting, is attractive. Both genetic and non-genetic retargeting strategies have been utilized. However, in the context of oncolytic viruses, it is beneficial to have the specific modification included in progeny virions and hence genetic modification may be preferable. Serotype chimerism utilizes serotype specific differences in receptor usage, liver tropism and seroprevalence in order to gain enhanced infection of target tissue. This review will focus on serotype chimeric adenoviruses for cancer gene therapy applications.

  14. Identification and analysis of pig chimeric mRNAs using RNA sequencing data

    Directory of Open Access Journals (Sweden)

    Ma Lei

    2012-08-01

    Full Text Available Abstract Background Gene fusion is ubiquitous over the course of evolution. It is expected to increase the diversity and complexity of transcriptomes and proteomes through chimeric sequence segments or altered regulation. However, chimeric mRNAs in pigs remain unclear. Here we identified some chimeric mRNAs in pigs and analyzed the expression of them across individuals and breeds using RNA-sequencing data. Results The present study identified 669 putative chimeric mRNAs in pigs, of which 251 chimeric candidates were detected in a set of RNA-sequencing data. The 618 candidates had clear trans-splicing sites, 537 of which obeyed the canonical GU-AG splice rule. Only two putative pig chimera variants whose fusion junction was overlapped with that of a known human chimeric mRNA were found. A set of unique chimeric events were considered middle variances in the expression across individuals and breeds, and revealed non-significant variance between sexes. Furthermore, the genomic region of the 5′ partner gene shares a similar DNA sequence with that of the 3′ partner gene for 458 putative chimeric mRNAs. The 81 of those shared DNA sequences significantly matched the known DNA-binding motifs in the JASPAR CORE database. Four DNA motifs shared in parental genomic regions had significant similarity with known human CTCF binding sites. Conclusions The present study provided detailed information on some pig chimeric mRNAs. We proposed a model that trans-acting factors, such as CTCF, induced the spatial organisation of parental genes to the same transcriptional factory so that parental genes were coordinatively transcribed to give birth to chimeric mRNAs.

  15. Construction and characterization of chimeric BHIV (BIV/HIV-1) viruses carrying the bovine immunodeficiency virus gag gene

    Institute of Scientific and Technical Information of China (English)

    Yi-Xin Zhu; Chang Liu; Xin-Lei Liu; Wen-Tao Qiao; Qi-Min Chen; Yi Zeng; Yun-Qi Geng

    2005-01-01

    AIM: To explore the possibility of the replacement of the gag gene between human immunodeficiency virus and bovine immunodeficiency virus, to achieve chimeric virions,and thereby gain a new kind of AIDS vaccine based on BHIV chimeric viruses.METHODS: A series of chimeric BHIV proviral DNAs differing in the replacement regions in gag gene were constructed, and then were transfected into 293T cells. The expression of chimeric viral genes was detected at the RNA and protein level. The supematant of 293T cell was ultra centrifuged to detect the probable chimeric virion. Once the chimeric virion was detected, its biological activities were also assayed by infecting HIV-sensitive MT4 cells.RESULTS: Four chimeric BHIV proviral DNAs were constructed. Genes in chimeric viruses expressed correctly in transfected 293T cells. All four constructs assembled chimeric virions with different degrees of efficiency. These virions had complete structures common to retroviruses and packaged genomic RNAs, but the cleavages of the precursor Gag proteins were abnormal to some extent. Three of these virions tested could attach and enter into MT4 cells, and one of them could complete the course of reverse transcription. Yet none of them could replicate in MT4 cells.CONCLUSION: The replacement of partial gag gene of HIV with BIV gaggene is feasible. Genes in chimeric BHIVs are accurately expressed, and virions are assembled. These chimeric BHIVs (proviral DNA together with virus particles) have the potential to become a new kind of HIV/AIDS vaccine.

  16. Modeling cognition and disease using human glial chimeric mice

    DEFF Research Database (Denmark)

    Goldman, Steven A.; Nedergaard, Maiken; Windrem, Martha S.

    2015-01-01

    cognition and information processing. In addition, the cellular humanization of these brains permits their use in studying glial infectious and inflammatory disorders unique to humans, and the effects of those disorders on the glial contributions to cognition. Perhaps most intriguingly, by pairing our...... ability to construct human glial chimeras with the production of patient-specific hGPCs derived from pluripotential stem cells, we may now establish mice in which a substantial proportion of resident glia are both human and disease-derived. These mice in particular may provide us new opportunities for...... studying the human-specific contributions of glia to psychopathology, as well as to higher cognition. As such, the assessment of human glial chimeric mice may provide us new insight into the species-specific contributions of glia to human cognitive evolution, as well as to the pathogenesis of human...

  17. Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy.

    Science.gov (United States)

    Dai, Hanren; Wang, Yao; Lu, Xuechun; Han, Weidong

    2016-07-01

    The genetic modification and characterization of T-cells with chimeric antigen receptors (CARs) allow functionally distinct T-cell subsets to recognize specific tumor cells. The incorporation of costimulatory molecules or cytokines can enable engineered T-cells to eliminate tumor cells. CARs are generated by fusing the antigen-binding region of a monoclonal antibody (mAb) or other ligand to membrane-spanning and intracellular-signaling domains. They have recently shown clinical benefit in patients treated with CD19-directed autologous T-cells. Recent successes suggest that the modification of T-cells with CARs could be a powerful approach for developing safe and effective cancer therapeutics. Here, we briefly review early studies, consider strategies to improve the therapeutic potential and safety, and discuss the challenges and future prospects for CAR T-cells in cancer therapy. PMID:26819347

  18. Confined Blood Chimerism in Monochorionic Dizygotic Twins Conceived Spontaneously

    Directory of Open Access Journals (Sweden)

    Takashi Kanda

    2013-05-01

    Full Text Available Traditionally, monochorionicity has been regarded as synonymous with monozygosity. However, several recent cases of monochorionic dizygotic twins have shown that monochorionic twins can be dizygous. We report a rare case of monochorionic diamnionic, gender-discordant twins who were conceived spontaneously. Initially, a monochorionic placenta was diagnosed by ultrasonography at 8 weeks of gestation and then confirmed by pathology after delivery. The twins had different genders. A comparison of cytogenetic analyses using peripheral blood lymphocytes and skin fibroblasts revealed that chimerism was confined to blood cells. We have experienced two cases of monochorionic dizygotic twins since 2003. These cases suggest that monochorionic dizygotic twins are not as rare as previously thought.

  19. Persistence of chicken herpesvirus and retroviral chimeric molecules upon in vivo passage.

    Science.gov (United States)

    Borenshtain, R; Witter, R L; Davidson, I

    2003-01-01

    Mareks disease virus (MDV), a herpesvirus, and avian leucosis virus subgroup J (ALV-J), a retrovirus, were used for experimental coinfection of chickens. Chimeric molecules having sequences of both viruses were detected by the hotspot-combined polymerase chain reaction (HS-cPCR) system. The detection of chimeric molecules provided evidence for avian retroviral inserts in the herpesvirus genome. The persistence of chimeric molecules on in vivo passage served to indicate the infectivity of the recombinant virus. The evaluation of formation and persistence of the chimeric molecules was performed in two trials involving three in vivo passages. The chimeric molecules were identified according to the primer sets, their product length, and pattern. The persistence of chimeric molecules on in vivo passages served as an indication of their ability to replicate in and infect chickens. In the first experimental passage, MDV and ALV-J prototype strains, MD11 and HC-1, were intraperitoneally (i.p.) injected into 1-day-old chicks. The second trial included two passages. Passage II chicks were injected i.p. and passage III chickens were in contact with the chickens of passage II. For passage II, enriched white blood cells from blood samples of chickens from the first trial that had chimeric molecules were injected i.p. into 1-day-old chicks. For passage III, uninfected chicks were included together with the infected chicks. Synthesis evidence for the various species of chimeric molecules was assessed in the tissues of birds of the second trial. DNA was extracted from blood and feathers and analyzed by the hotspot-combined PCR and by pulsed field gel electrophoresis. To overcome the limits of detection, three amplification assays followed by hybridization of the products to specific viral probes were conducted. A variety of chimeric molecules were detected in low concentrations. Five species of chimeric molecules were characterized in blood, tumors, and feathers. Chimeric

  20. Exploration of genetically encoded voltage indicators based on a chimeric voltage sensing domain

    Directory of Open Access Journals (Sweden)

    Yukiko Mishina

    2014-09-01

    We recently introduced a new VSFP design in which the voltage-sensing domain (VSD is sandwiched between a FRET pair of fluorescent proteins (termed VSFP-Butterflies and also demonstrated a series of chimeric VSD in which portions of the VSD of Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP are substituted by homologous portions of a voltage-gated potassium channel subunit. These chimeric VSD had faster sensing kinetics than that of the native Ci-VSD. Here, we describe a new set of VSFPs that combine chimeric VSD with the Butterfly structure. We show that these chimeric VSFP-Butterflies can report membrane voltage oscillations of up to 200 Hz in cultured cells and report sensory evoked cortical population responses in living mice. This class of GEVIs may be suitable for imaging of brain rhythms in behaving mammalians.

  1. A chimeric measles virus with a lentiviral envelope replicates exclusively in CD4+/CCR5+ cells

    International Nuclear Information System (INIS)

    We generated a replicating chimeric measles virus in which the hemagglutinin and fusion surface glycoproteins were replaced with the gp160 envelope glycoprotein of simian immunodeficiency virus (SIVmac239). Based on a previously cloned live-attenuated Schwarz vaccine strain of measles virus (MV), this chimera was rescued at high titers using reverse genetics in CD4+ target cells. Cytopathic effect consisted in the presence of large cell aggregates evolving to form syncytia, as observed during SIV infection. The morphology of the chimeric virus was identical to that of the parent MV particles. The presence of SIV gp160 as the only envelope protein on chimeric particles surface altered the cell tropism of the new virus from CD46+ to CD4+ cells. Used as an HIV candidate vaccine, this MV/SIVenv chimeric virus would mimic transient HIV-like infection, benefiting both from HIV-like tropism and the capacity of MV to replicate in dendritic cells, macrophages and lymphocytes.

  2. Quantitative chimerism kinetics in relapsed leukemia patients after allogeneic hematopoietic stem cell transplantation

    Institute of Scientific and Technical Information of China (English)

    QIN Xiao-ying; WANG Jing-zhi; ZHANG Xiao-hui; LI Jin-lan; LI Ling-di; LIU Kai-yan; HUANG Xiao-jun; LI Guo-xuan; QIN Ya-zhen; WANG Yu; WANG Feng-rong; LIU Dai-hong; XU Lan-ping; CHEN Huan; HAN Wei

    2012-01-01

    Background Chimerism analysis is an important tool for the surveillance of post-transplant engraftment.It offers the possibility of identifying impending graft rejection and recurrence of underlying malignant or non-malignant disease.Here we investigated the quantitative chimerism kinetics of 21 relapsed leukemia patients after allogeneic hematopoietic stem cell transplantation (HSCT).Methods A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time polymerase chain reaction (RT-PCR) to obtain the informative marker for every leukemia patient.Quantitative chimerism analysis of bone marrow (BM) samples of 21 relapsed patients and 20 patients in stable remission was performed longitudinally.The chimerisms of BM and peripheral blood (PB) samples of 14 patients at relapse were compared.Results Twenty-one patients experienced leukemia relapse at a median of 135 days (range,30-720 days) after transplantation.High recipient chimerism in BM was found in all patients at relapse,and increased recipient chimerism in BM samples was observed in 90% (19/21) of patients before relapse.With 0.5% recipient DNA as the cut-off,median time between the detection of increased recipient chimerism and relapse was 45 days (range,0-120 days),with 76% of patients showing increased recipient chimerism at least 1 month prior to relapse.Median percentage of recipient DNA in 20 stable remission patients was 0.28%,0.04%,0.05%,0.05%,0.08%,and 0.05% at 1,2,3,6,9,and 12 months,respectively,after transplantation.This was concordant with other specific fusion transcripts and fluorescent in situ hybridization examination.The recipient chimerisms in BM were significantly higher than those in PB at relapse (P=0.001).Conclusions This SP-based RT-PCR essay is a reliable method for chimerism analysis.Chimerism kinetics in BM can be used as a marker of impending leukemia relapse,especially when no other specific marker is available.Based on our findings

  3. Chimeric hepatitis B virus core particles as probes for studying peptide-integrin interactions.

    OpenAIRE

    Chambers, M A; Dougan, G; Newman, J.; Brown, F.; Crowther, J.; Mould, A P; Humphries, M J; Francis, M. J.; Clarke, B.; Brown, A L; Rowlands, D.

    1996-01-01

    An RGD-containing epitope from the foot-and-mouth disease virus (FMDV) VP1 protein was inserted into the e1 loop of the hepatitis B virus core (HBc) protein. This chimeric protein was expressed at high levels in Escherichia coli and spontaneously assembled into virus-like particles which could be readily purified. These fusion particles elicited high levels of both enzyme-linked immunosorbent assay- and FMDV-neutralizing antibodies in guinea pigs. The chimeric particles bound specifically to ...

  4. Assembling Single-Cell Genomes and Mini-Metagenomes From Chimeric MDA Products

    OpenAIRE

    Nurk, Sergey; Bankevich, Anton; Antipov, Dmitry; Gurevich, Alexey A.; Korobeynikov, Anton; Lapidus, Alla; Prjibelski, Andrey D.; Pyshkin, Alexey; Sirotkin, Alexander; Sirotkin, Yakov; Stepanauskas, Ramunas; Clingenpeel, Scott R.; Woyke, Tanja; Jeffrey S. McLean; Lasken, Roger

    2013-01-01

    Recent advances in single-cell genomics provide an alternative to largely gene-centric metagenomics studies, enabling whole-genome sequencing of uncultivated bacteria. However, single-cell assembly projects are challenging due to (i) the highly nonuniform read coverage and (ii) a greatly elevated number of chimeric reads and read pairs. While recently developed single-cell assemblers have addressed the former challenge, methods for assembling highly chimeric reads remain poorly explored. We p...

  5. Hematopoietic Chimerism Monitoring Based on STRs: Quantitative Platform Performance on Sequential Samples

    OpenAIRE

    Kristt, Don; Israeli, Moshe; Narinski, Ronit; Or, Hagit; Yaniv, I; Stein, Jerry; Klein, Tirza

    2005-01-01

    Hematopoietic stem cell transplantation (HSCT) creates a donor-recipient cellular chimerism in the patient, which is quantitatively assayed from peripheral blood based on STR-DNA. Since chimerism values often vary across a patient’s samples, it is important to determine to what extent this variability reflects technical aspects of platform performance. This issue is systematically assessed in the current study for the first time. Using the SGM Plus multiplex PCR kit and ABI platform, the long...

  6. Context Dependent Effects of Chimeric Peptide Morpholino Conjugates Contribute to Dystrophin Exon-skipping Efficiency

    OpenAIRE

    Yin, HaiFang; Boisguerin, Prisca; Moulton, Hong M.; Betts, Corinne; Seow, Yiqi; Boutilier, Jordan; Wang, Qingsong; Walsh, Anthony; Lebleu, Bernard; Wood, Matthew JA

    2013-01-01

    We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was ...

  7. Targeting duplex DNA with chimeric α,β-triplex-forming oligonucleotides

    OpenAIRE

    Kolganova, N. A.; Shchyolkina, A K; Chudinov, A. V.; Zasedatelev, A S; Florentiev, V L; Timofeev, E. N.

    2012-01-01

    Triplex-directed DNA recognition is strictly limited by polypurine sequences. In an attempt to address this problem with synthetic biology tools, we designed a panel of short chimeric α,β-triplex-forming oligonucleotides (TFOs) and studied their interaction with fluorescently labelled duplex hairpins using various techniques. The hybridization of hairpin with an array of chimeric probes suggests that recognition of double-stranded DNA follows complicated rules combining reversed Hoogsteen and...

  8. Mosaic origins of a complex chimeric mitochondrial gene in Silene vulgaris.

    Directory of Open Access Journals (Sweden)

    Helena Storchova

    Full Text Available Chimeric genes are significant sources of evolutionary innovation that are normally created when portions of two or more protein coding regions fuse to form a new open reading frame. In plant mitochondria astonishingly high numbers of different novel chimeric genes have been reported, where they are generated through processes of rearrangement and recombination. Nonetheless, because most studies do not find or report nucleotide variation within the same chimeric gene, evolution after the origination of these chimeric genes remains unstudied. Here we identify two alleles of a complex chimera in Silene vulgaris that are divergent in nucleotide sequence, genomic position relative to other mitochondrial genes, and expression patterns. Structural patterns suggest a history partially influenced by gene conversion between the chimeric gene and functional copies of subunit 1 of the mitochondrial ATP synthase gene (atp1. We identified small repeat structures within the chimeras that are likely recombination sites allowing generation of the chimera. These results establish the potential for chimeric gene divergence in different plant mitochondrial lineages within the same species. This result contrasts with the absence of diversity within mitochondrial chimeras found in crop species.

  9. Prognostic Utility of Routine Chimerism Testing at 2 – 6 Months after Allogeneic Hematopoietic Cell Transplantation

    Science.gov (United States)

    Mossallam, Ghada I.; Kamel, Azza M.; Storer, Barry; Martin, Paul J.

    2009-01-01

    The utility of routine chimerism analysis as a prognostic indicator of subsequent outcomes after allogeneic hematopoietic cell transplantation (HCT) with myeloablative conditioning regimens remains controversial. To address this controversy, routine chimerism test results at 2 – 6 months after HCT with myeloablative conditioning regimens were evaluated for association with subsequent risks of chronic graft versus host disease (GVHD), non-relapse mortality (NRM), relapse and overall mortality. Only 70 (5%) of 1304 patients had <95% donor-derived cells in the marrow. Low donor chimerism in the marrow occurred predominantly among patients with low risk disease as compared to higher risk diseases and was significantly associated with a reduced risk of chronic GVHD. Among 673 patients tested, 164 (24%) had <85% donor-derived T cells in the blood. Low donor T cell chimerism occurred predominantly among patients with low risk disease as compared to higher risk diseases, among those who had conditioning with busulfan as compared to TBI, and among those with lower grades of acute GVHD. Low donor T cell chimerism in the blood was significantly associated with a reduced risk of chronic GVHD, but not with the risks of relapse, NRM or overall mortality. Routine testing of chimerism in the marrow and blood at 2 – 6 months after HCT with myeloablative conditioning regimens may be helpful in documenting engraftment in clinical trials but provides only limited prognostic information in clinical practice. PMID:19203726

  10. Targeting duplex DNA with chimeric α,β-triplex-forming oligonucleotides.

    Science.gov (United States)

    Kolganova, N A; Shchyolkina, A K; Chudinov, A V; Zasedatelev, A S; Florentiev, V L; Timofeev, E N

    2012-09-01

    Triplex-directed DNA recognition is strictly limited by polypurine sequences. In an attempt to address this problem with synthetic biology tools, we designed a panel of short chimeric α,β-triplex-forming oligonucleotides (TFOs) and studied their interaction with fluorescently labelled duplex hairpins using various techniques. The hybridization of hairpin with an array of chimeric probes suggests that recognition of double-stranded DNA follows complicated rules combining reversed Hoogsteen and non-canonical homologous hydrogen bonding. In the presence of magnesium ions, chimeric TFOs are able to form highly stable α,β-triplexes, as indicated by native gel-electrophoresis, on-array thermal denaturation and fluorescence-quenching experiments. CD spectra of chimeric triplexes exhibited features typically observed for anti-parallel purine triplexes with a GA or GT third strand. The high potential of chimeric α,β-TFOs in targeting double-stranded DNA was demonstrated in the EcoRI endonuclease protection assay. In this paper, we report, for the first time, the recognition of base pair inversions in a duplex by chimeric TFOs containing α-thymidine and α-deoxyguanosine. PMID:22641847

  11. Protective and immunological behavior of chimeric yellow fever dengue vaccine.

    Science.gov (United States)

    Halstead, Scott B; Russell, Philip K

    2016-03-29

    Clinical observations from the third year of the Sanofi Pasteur chimeric yellow fever dengue tetravalent vaccine (CYD) trials document both protection and vaccination-enhanced dengue disease among vaccine recipients. Children who were 5 years-old or younger when vaccinated experienced a DENV disease resulting in hospitalization at 5 times the rate of controls. On closer inspection, hospitalized cases among vaccinated seropositives, those at highest risk to hospitalized disease accompanying a dengue virus (DENV) infection, were greatly reduced by vaccination. But, seronegative individuals of all ages after being vaccinated were only modestly protected from mild to moderate disease throughout the entire observation period despite developing neutralizing antibodies at high rates. Applying a simple epidemiological model to the data, vaccinated seronegative individuals of all ages were at increased risk of developing hospitalized disease during a subsequent wild type DENV infection. The etiology of disease in placebo and vaccinated children resulting in hospitalization during a DENV infection, while clinically similar are of different origin. The implications of the observed mixture of DENV protection and enhanced disease in CYD vaccinees are discussed. PMID:26873054

  12. Toxicities of chimeric antigen receptor T cells: recognition and management.

    Science.gov (United States)

    Brudno, Jennifer N; Kochenderfer, James N

    2016-06-30

    Chimeric antigen receptor (CAR) T cells can produce durable remissions in hematologic malignancies that are not responsive to standard therapies. Yet the use of CAR T cells is limited by potentially severe toxicities. Early case reports of unexpected organ damage and deaths following CAR T-cell therapy first highlighted the possible dangers of this new treatment. CAR T cells can potentially damage normal tissues by specifically targeting a tumor-associated antigen that is also expressed on those tissues. Cytokine release syndrome (CRS), a systemic inflammatory response caused by cytokines released by infused CAR T cells can lead to widespread reversible organ dysfunction. CRS is the most common type of toxicity caused by CAR T cells. Neurologic toxicity due to CAR T cells might in some cases have a different pathophysiology than CRS and requires different management. Aggressive supportive care is necessary for all patients experiencing CAR T-cell toxicities, with early intervention for hypotension and treatment of concurrent infections being essential. Interleukin-6 receptor blockade with tocilizumab remains the mainstay pharmacologic therapy for CRS, though indications for administration vary among centers. Corticosteroids should be reserved for neurologic toxicities and CRS not responsive to tocilizumab. Pharmacologic management is complicated by the risk of immunosuppressive therapy abrogating the antimalignancy activity of the CAR T cells. This review describes the toxicities caused by CAR T cells and reviews the published approaches used to manage toxicities. We present guidelines for treating patients experiencing CRS and other adverse events following CAR T-cell therapy. PMID:27207799

  13. Dosimetry of chimeric TNT in lung tumor patients

    Institute of Scientific and Technical Information of China (English)

    CHEN Yangchun; CHEN Shaoliang; JU Dianwen; SHI Hongcheng; YAO Zhifeng

    2007-01-01

    The purpose of this study was to assess the absorbed dose of tumor and main critical organs in 131I labeled chimeric tumor necrotic treatment (chTNT). In 9 patients, a single intravenous dose of (29.6±3.7) MBq/kg was administered. Blood samples were drawn at different time intervals, and urine was collected for up to one week. Tissue distribution of 131I -chTNT was followed for up to one week by gamma camera imaging. Absorbed doses to the whole body and to normal organs were computed according to the MIRD scheme using Mirdose-3 software. S-factors for lung tumors were estimated by comparison with lungs of similar mass and position in the body. It was found that mean serum disappearance half time values for 131I-chTNT were (4.93±9.36) h and (61.7±21.2) h for α, β respectively,while that for whole body was(99±10) h. Mean urine biological clearance half time value was (90±10) h. The absorbed dose to tumor was (8.28±2.65) Gy, and the tumor-to-nontumor dose ratio was 3.95±1.55. And the mean effective dose to patients was (1.02±0.29) mSv/MBq.

  14. Development of chimeric antigen receptors for multiple myeloma.

    Science.gov (United States)

    Martínez-Cingolani, Carolina; Bories, Jean Christophe

    2016-04-15

    Multiple myeloma (MM) is a haematologic malignancy characterized by the expansion of monoclonal plasma cells in the bone marrow. It is associated with serum or urine monoclonal protein and organ damage including renal failure, anaemia, hypercalcaemia and bone lesions. Despite recent improvements MM still remains an incurable disease. Previous studies have shown that the adoptive transfer of autologous T-cells modified to express chimeric antigen receptors (CAR) is effective in cases of acute and chronic lymphoid leukaemia. However, the adjustment of CAR-T-cell therapy to MM is hindered by the scarcity of antigens specific to the tumour plasma cells. Most candidate targets are shared by healthy tissues, and entail high risks of toxicity. Therefore several strategies have been proposed to regulate CAR-T-cell function as well as to enhance CAR-T-cell specificity against tumour cells. In this article we summarize the surface markers that have been investigated as targets to eliminate MM plasma cells and the MM-specific CARs that have been developed to date. Then we describe the different CAR-T-cell designs that could be applied in the case of MM to circumvent current problems of toxicity. PMID:27068946

  15. Dosimetry of chimeric TNT in lung tumor patients

    International Nuclear Information System (INIS)

    The purpose of this study was to assess the absorbed dose of tumor and main critical organs in 131I labeled chimeric tumor necrotic treatment (chTNT). In 9 patients, a single intravenous dose of (29.6±3.7) MBq/kg was administered. Blood samples were drawn at different time intervals, and urine was collected for up to one week. Tissue distribution of 131I -chTNT was followed for up to one week by gamma camera imaging. Absorbed doses to the whole body and to normal organs were computed according to the MIRD scheme using Mirdose-3 software. S-factors for lung tumors were estimated by comparison with lungs of similar mass and position in the body. It was found that mean serum disappearance half time values for 131I-chTNT were (4.934±9.36) h and (61.74±21.2) h for α, β respectively, while that for whole body was(99±10) h. Mean urine biological clearance half time value was (90±10) h. The ab- sorbed dose to tumor was (8.28±2.65) Gy, and the tumor-to-nontumor dose ratio was 3.95±1.55. And the mean effective dose to patients was (1.02±0.29) mSv/MBq. (authors)

  16. Immunological tolerance and tumor rejection in embryo-aggregated chimeric mice – Lessons for tumor immunity

    International Nuclear Information System (INIS)

    Rejection of transplanted tumors by the immune system is a rare event in syngeneic hosts, and is considered to be dependent on the local interaction of defensive immune reactions and tumor tolerance mechanisms. Here, we have enlisted the aid of a unique set of embryo-aggregated lineage chimeric mice derived from C57/BL6 and FVB donors to study the interplay between local and systemic tumor immunity and tolerance in rejection of mouse B16 melanoma cells, syngeneic to the C57/BL6 donor strain. Two variants of embryo-aggregated chimeric mice with either variable or no contribution of C57-derived cells to their skin were generated by the fusion of different ratios of morula stage blastomers. Chimeric mice were analyzed for s.c. growth of B16 tumors in comparison to their respective donor strains as well as normal F1 hybrids, and the relative frequencies of cellular components of the immune system by FACS analysis of peripheral blood or lymph node cells. B16 tumors grew significantly faster in mice with full chimerism in their skin as compared to syngeneic C57 or semi-syngeneic C57 × FVB F1 hosts. In contrast, s.c. tumor growth was either absent or significantly reduced in chimeric mice lacking C57-derived cells in their skin, but tolerant to C57 tissue in other organs. Comparison of the relative frequencies of various immune cells in the periphery via FACS-analysis did not reveal any significant differences between the two types of chimeric mice with respect to their donor strains. Our data suggest a complex interplay between mechanisms of local peripheral tolerance and innate antitumor mechanisms possibly involving NK cell allorecognition as a basis for the differential growth or rejection of B16 tumors in these unique chimeric mice, which we suggest to constitute a valuable new model system for the study of immune-mediated tumor rejection

  17. Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein

    International Nuclear Information System (INIS)

    The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV

  18. Immune Reconstitution Kinetics following Intentionally Induced Mixed Chimerism by Nonmyeloablative Transplantation.

    Directory of Open Access Journals (Sweden)

    Nayoun Kim

    Full Text Available Establishing mixed chimerism is a promising approach for inducing donor-specific transplant tolerance. The establishment and maintenance of mixed chimerism may enable long-term engraftment of organ transplants while minimizing the use of immunosuppressants. Several protocols for inducing mixed chimerism have been reported; however, the exact mechanism underlying the development of immune tolerance remains to be elucidated. Therefore, understanding the kinetics of engraftment during early post-transplant period may provide insight into establishing long-term mixed chimerism and permanent transplant tolerance. In this study, we intentionally induced allogeneic mixed chimerism using a nonmyeloablative regimen by host natural killer (NK cell depletion and T cell-depleted bone marrow (BM grafts in a major histocompatibility complex (MHC-mismatched murine model and analyzed the kinetics of donor (C57BL/6 and recipient (BALB/c engraftment in the weeks following transplantation. Donor BM cells were well engrafted and stabilized without graft-versus-host disease (GVHD as early as one week post-bone marrow transplantation (BMT. Donor-derived thymic T cells were reconstituted four weeks after BMT; however, the emergence of newly developed T cells was more obvious at the periphery as early as two weeks after BMT. Also, the emergence and changes in ratio of recipient- and donor-derived NKT cells and antigen presenting cells (APCs including dendritic cells (DCs and B cells were noted after BMT. Here, we report a longitudinal analysis of the development of donor- and recipient-originated hematopoietic cells in various lymphatic tissues of intentionally induced mixed chimerism mouse model during early post-transplant period. Through the understanding of immune reconstitution at early time points after nonmyeloablative BMT, we suggest guidelines on intentionally inducing durable mixed chimerism.

  19. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes.

    Science.gov (United States)

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F

    2015-08-18

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners--the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)--and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic--and plant and algal--lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller's ratchet--the origin of eukaryotic recombination, or sex--might have required surprisingly little evolutionary innovation. PMID:25733873

  20. Chimeric mouse-human IgG1 antibody that can mediate lysis of cancer cells

    International Nuclear Information System (INIS)

    A chimeric mouse-human antibody has been created that recognizes an antigen found on the surface of cells from many carcinomas. Immunoglobulin constant (C) domains of the mouse monoclonal antibody L6, C/sub γ2a/ and C/sub kappa/, were substituted by the human C/sub γ1/ and C/sub kappa/ by recombining cDNA modules encoding variable or C domains. The cDNA constructs were transfected into lymphoid cells for antibody production. The chimeric antibody and mouse L6 antibody bound to carcinoma cells with equal affinity and mediated complement-dependent cytolysis. In the presence of human effector cells, the chimeric antibody gave antibody-dependent cellular cytotoxicity at 100 times lower concentration than that needed for the mouse L6 antibody. The assay for lysis was carried out with 51Cr-labeled target calls. The chimeric antibody, but not the mouse L6 antibody, is effective against a melanoma line expressing small amounts of the L6 antigen. The findings point to the usefulness of the chimeric antibody approach for obtaining agents with strong antitumor activity for possible therapeutic use in man

  1. Recognition of base pair inversions in duplex by chimeric (alpha,beta) triplex-forming oligonucleotides.

    Science.gov (United States)

    Timofeev, Edward N; Goryaeva, Baira V; Florentiev, Vladimir L

    2006-10-01

    DNA recognition by triplex-forming oligonucleotides (TFOs) is usually limited by homopurine-homopyrimidine sequence in duplexes. Modifications of the third strand may overcome this limitation. Chimeric alpha-beta TFOs are expected to form triplex DNA upon binding to non-regular sequence duplexes. In the present study we describe binding properties of chimeric alpha-beta oligodeoxynucleotides in the respect to short DNA duplexes with one, three, and five base pair inversions. Non-natural chimeric TFO's contained alpha-thymidine residues inside (GT) or (GA) core sequences. Modified residues were addressed to AT/TA inversions in duplexes. It was found in the non-denaturing gel-electrophoresis experiments that single or five adjacent base pair inversions in duplexes may be recognized by chimeric alpha-beta TFO's at 10 degrees C and pH 7.8. Three dispersed base pair inversions in the double stranded DNA prevented triplex formation by either (GT) or (GA) chimeras. Estimation of thermal stability of chimeric alpha-beta triplexes showed decrease in T(m) values as compared with unmodified complexes. PMID:16928141

  2. Generation and evaluation of a chimeric classical swine fever virus expressing a visible marker gene.

    Science.gov (United States)

    Li, Yongfeng; Wang, Xiao; Sun, Yuan; Li, Lian-Feng; Zhang, Lingkai; Li, Su; Luo, Yuzi; Qiu, Hua-Ji

    2016-03-01

    Classical swine fever virus (CSFV) is a noncytopathogenic virus, and the incorporation of an enhanced green fluorescent protein (EGFP) tag into the viral genome provides a means of direct monitoring of viral infection without immunostaining. It is well established that the 3' untranslated region (3'-UTR) of the CSFV plays an important role in viral RNA replication. Although CSFV carrying a reporter gene and chimeric CSFV have been generated and evaluated, a chimeric CSFV with a visible marker has not yet been reported. Here, we generated and evaluated a chimeric virus containing the EGFP tag and the 3'-UTR from vaccine strain HCLV (C-strain) in the genetic background of the highly virulent CSFV Shimen strain. The chimeric marker CSFV was fluorescent and had an approximately 100-fold lower viral titer, lower replication level of viral genome, and weaker fluorescence intensity than the recombinant CSFV with only the EGFP tag or the parental virus. Furthermore, the marker chimera was avirulent and displayed no viremia in inoculated pigs, which were completely protected from lethal CSFV challenge as early as 15 days post-inoculation. The chimeric marker virus was visible in vitro and attenuated in vitro and in vivo, which suggests that CSFV can be engineered to produce attenuated variants with a visible marker to facilitate in vitro studies of CSFV infection and replication and to develop of novel vaccines against CSF. PMID:26614259

  3. Chimeric spider silk proteins mediated by intein result in artificial hybrid silks.

    Science.gov (United States)

    Lin, Senzhu; Chen, Gefei; Liu, Xiangqin; Meng, Qing

    2016-07-01

    Hybrid silks hold a great potential as specific biomaterials due to its controlled mechanical properties. To produce fibers with tunable properties, here we firstly made chimeric proteins in vitro, called W2C4CT and W2C8CT, with ligation of MaSp repetitive modules (C) with AcSp modules (W) by intein trans splicing technology from smaller precursors without final yield reduction. Intein mediated chimeric proteins form fibers at a low concentration of 0.4 mg/mL in 50 mM K3 PO4 pH 7.5 just drawn by hand. Hybrid fibers show smoother surface, and also have stronger chemical resistance as compared with fibers from W2CT (W fibers) and mixture of W2CT/C8CT (MHF8 fibers). Fibers from chimeric protein W2C4CT (HFH4) have improved mechanical properties than W fibers; however, with more C modules W2C8CT fibers (HFH8) properties decreased, indicates the length proportion of various modules is very important and should be optimized for fibers with specific properties. Generally, hybrid silks generated via chimeric proteins, which can be simplified by intein trans splicing, has greater potential to produce fibers with tunable properties. Our research shows that intein mediated directional protein ligation is a novel way to make large chimeric spider silk proteins and hybrid silks. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 385-392, 2016. PMID:26948769

  4. Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein

    KAUST Repository

    Mahfouz, Magdy M.

    2011-12-14

    Transcriptional activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria when they infect plants. TALEs contain a modular DNA binding domain that can be easily engineered to bind any sequence of interest, and have been used to provide user-selected DNA-binding modules to generate chimeric nucleases and transcriptional activators in mammalian cells and plants. Here we report the use of TALEs to generate chimeric sequence-specific transcriptional repressors. The dHax3 TALE was used as a scaffold to provide a DNA-binding module fused to the EAR-repression domain (SRDX) to generate a chimeric repressor that targets the RD29A promoter. The dHax3. SRDX protein efficiently repressed the transcription of the RD29A

  5. The determination of lymphoid cell chimerism using peripheral blood lymphocytes from murine bone marrow chimeras

    International Nuclear Information System (INIS)

    A simple, rapid and accurate method was devised for determining lymphoid cell chimerism in bone marrow-reconstituted mice. Chimeras were produced by reconstituting lethally irradiated mice with semi-allogeneic bone marrow cells. Lymphocytes from the peripheral blood of individual chimeric mice were purified by sedimentation in dextran solution and differential flotation in Ficoll-Hypaque gradients. From 250-500 μl of blood, 1-7 x 105 cells were routinely obtained. The extent of chimerism was determined serologically by using peripheral blood lymphocytes as target cells in a dye exclusion microcytotoxicity assay. Using this new technique, approximately 80% of the reconstituted mice were found to be repopulated with lymphocytes of the donor type. (Auth.)

  6. Chimerism a natural ability to tolerate kin, evolutionary traits connecting mammalian and protochordates

    Directory of Open Access Journals (Sweden)

    A Voskoboynik

    2009-03-01

    Full Text Available In the middle of the 20th century, Owen (1945, 1954 and Billingham et al. (1953 immunological studies suggested that fetal exposure to foreign antigens during pregnancy induce immunologic tolerance in the fetus. Recently, Mold et al. found that a substantial number of maternal cells crosses the placenta to reside in fetal lymph nodes and induces the development of regulatory T cells (Tregs that suppress fetal anti-maternal immunity. These Tregs cells persist till, at least, early adulthood. This result demonstrates how chimerism induces fetal tolerance to maternal antigens during mammalian pregnancy. Natural chimerism is the coexistence of two or more genomic lineages within the same individual. It is a common phenomenon which can be detected in a wide variety of multi-cellular organisms. In mammals, natural chimerism can be established during pregnancy between the mother and the fetus or between fetuses in a multiple embryos pregnancy. Restriction of natural chimerism mainly to kin is also observed in colonial marine protochordates. In protochordates, like Botryllus schlosseri, natural chimerism can be established through fusion of vasculature, between a parent colony and its progeny or between siblings (adult distinct colonies.The ability to tolerate a partial allogeneic individual and to create a chimeric entity between these colonies is determined by a single, highly polymorphic, fusion/histocompatibility locus (Fu/HC. Colonies that share at least one allele in their Fu/HC locus would fuse upon contact. A pair that does not share any Fu/HC allele would not. In the chimera, cells transmigrate between partners and in some cases, replace the germline and/or the somatic tissues of the host. This genotype replacement is mediated by stem cells (termed somatic/germ cell parasitism. Botryllus colonies propagate asexually through budding, therefore somatic stem cell parasitism in host colonies can induce the development of a partial allogeneic entity

  7. Interspecies chimeric complementation for the generation of functional human tissues and organs in large animal hosts.

    Science.gov (United States)

    Wu, Jun; Izpisua Belmonte, Juan Carlos

    2016-06-01

    The past decade's rapid progress in human pluripotent stem cell (hPSC) research has generated hope for meeting the rising demand of organ donation, which remains the only effective cure for end-stage organ failure, a major cause of death worldwide. Despite the potential, generation of transplantable organs from hPSCs using in vitro differentiation is far-fetched. An in vivo interspecies chimeric complementation strategy relying on chimeric-competent hPSCs and zygote genome editing provides an auspicious alternative for providing unlimited organ source for transplantation. PMID:26820411

  8. Molecular chimerization of Pasteurella haemolytica leukotoxin to interleukin-2: effects on cytokine and antigen function.

    OpenAIRE

    Hughes, H P; Campos, M.; Potter, A A; Babiuk, L. A.

    1992-01-01

    A chimeric recombinant protein composed of the lktA gene product from Pasteurella haemolytica fused to bovine interleukin-2 (IL-2) was made. The LKT-IL-2 chimera was compared with recombinant bovine IL-2 with regard to the ability to induce proliferative responses and LAK cell activity in bovine peripheral blood mononuclear cells in vitro. In both instances, chimerization had no effect on IL-2 activity. Similarly, the LKT component was unaffected in its ability to induce an effective immune r...

  9. Dosimetry of chimeric TNT in lung cancer patients

    International Nuclear Information System (INIS)

    Objective: To assess the irradiated absorptive dose of tumor and main critical organs chimeric tumor necrotic treatment (chTNT). Methods: In 9 lung cancer patients a single intravenous dose of 131I-chTNT (29.6±3.7) MBq/kg was administered. Blood samples were drawn at different time intervals and urine was collected for up to one week. Tissue distribution was followed for up to one week by gamma camera imaging. The geometric mean of the anterior and posterior counts was obtained from selected regions of interest (ROIs) to determine activity within the critical organs after being subtracted the background activity. Counts from thyroid were obtained from anterior images only. A background region was drawn below the thyroid gland to subtract underlying activity in the neck blood vessels. The geometric mean of the counts in the whole body scintigram at 0.5 h after injection was corrected for radioactive decay from the time of injection , this value being taken as 100%ID. The residence times for each critical organ and the remainder of the body were computed by dividing the area under their %ID/h curves by the 100%ID value. Absorbed doses to the whole body and to normal organ were computed from the residence time according to the MIRD scheme using Mirdose-3 software. Absorbed doses to tumor tissues were estimated using the same approach taken for normal organs. S-factors for tumors were estimated by comparison with normal organs of similar mass and position in the body. Results: Mean serum disappearance half time values for 131I-chTNT were α (4.9 ± 9.4) h, β (61.7 ± 21.2) h; and whole body, (99 ± 10) h. Mean urine biological clearance half time values was (90 ± 10) h. The absorbed dose of tumor was (8.28 ± 2.65) Gy, the tumor-to-nontumor ratio was 3.95 ± 1.55, while the absorbed dose of marrow was 0.44-0.73 Gy, thyroid was 0.47-23.09 Gy, ovaries was 0.50-0.77 Gy, testicles was 0.38-0.58 Gy, kidneys was 1.71- 4.55 Gy, liver was 1.18-2.63 Gy, and lungs was 1

  10. Thermostability of multidomain proteins: chimeric mesophilic/thermophilic elongation factors EF-Tu

    Czech Academy of Sciences Publication Activity Database

    Šanderová, Hana; Maloň, Petr; Hůlková, Marta; Jonák, Jiří

    Varšava : FEBS, 2004, s. 7. [FEBS Forum for Young Scientists. Varšava (PL), 24.06.2004-26.06.2004] R&D Projects: GA ČR GA303/02/0689 Institutional research plan: CEZ:AV0Z5052915 Keywords : thermostability * EF-Tu * chimeric protein Subject RIV: EB - Genetics ; Molecular Biology

  11. Thermostability of multidomain proteins: chimeric mesophilic/thermophilic elongation factors EF-Tu

    Czech Academy of Sciences Publication Activity Database

    Šanderová, Hana; Maloň, Petr; Hůlková, Marta; Jonák, Jiří

    Oxford : Blackwell Publishing, 2004, s. 219. [Meeting of the Federation of the European Biochemical Societies /29./. Varšava (PL), 26.06.2004-01.07.2004] R&D Projects: GA ČR GA303/02/0689 Institutional research plan: CEZ:AV0Z5052915 Keywords : thermostability * EF-Tu * chimeric protein Subject RIV: EB - Genetics ; Molecular Biology

  12. Preparation and Characterization of a Novel Chimeric Protein VEGI-CTT in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jiping Cai

    2008-08-01

    Full Text Available Vascular endothelial cell growth inhibitor (VEGI is a recently identified antiangiogenic cytokine that belongs to the TNF superfamily, and could effectively inhibit endothelial cell proliferation and angiogenesis. Synthetic peptide CTT (CTTHWGFTLC has been found to suppress invasion and migration of both tumor and endothelial cells by potent and selective inhibition of MMP-2 and MMP-9. To prepare chimeric protein VEGI-CTT for more potent antitumor therapy, the recombinant expression vector pET-VEGI-CTT was constructed. This fusion protein was expressed in inclusion bodies in E. coli BL21 (DE3, and was refolded and purified by immobilized metal affinity chromatography using His-tag. Purified VEGI-CTT protein was characterized by proliferation assays of the endothelial cells and casein degradation assay in vitro. The results demonstrated that chimeric protein VEGI-CTT had a potent activity of antiangiogenesis through inhibiting the proliferation of endothelial cells, and could effectively reduce the activity of MMP-2 and MMP-9. The preliminarily in vivo study demonstrated that chimeric protein VEGI-CTT had more potent antitumor activity than VEGI and/or CTT peptide against CA46 human lymphoma xenografts in nude mice. Thus, these facts that are derived from the present study suggest that the chimeric protein VEGI-CTT may be used for tumor therapy in the future.

  13. Trypanosoma cruzi Differentiates and Multiplies within Chimeric Parasitophorous Vacuoles in Macrophages Coinfected with Leishmania amazonensis.

    Science.gov (United States)

    Pessoa, Carina Carraro; Ferreira, Éden Ramalho; Bayer-Santos, Ethel; Rabinovitch, Michel; Mortara, Renato Arruda; Real, Fernando

    2016-05-01

    The trypanosomatids Leishmania amazonensis and Trypanosoma cruzi are excellent models for the study of the cell biology of intracellular protozoan infections. After their uptake by mammalian cells, the parasitic protozoan flagellates L. amazonensis and T. cruzi lodge within acidified parasitophorous vacuoles (PVs). However, whereas L. amazonensis develops in spacious, phagolysosome-like PVs that may enclose numerous parasites, T. cruzi is transiently hosted within smaller vacuoles from which it soon escapes to the host cell cytosol. To investigate if parasite-specific vacuoles are required for the survival and differentiation of T. cruzi, we constructed chimeric vacuoles by infection of L. amazonensis amastigote-infected macrophages with T. cruzi epimastigotes (EPIs) or metacyclic trypomastigotes (MTs). These chimeric vacuoles, easily observed by microscopy, allowed the entry and fate of T. cruzi in L. amazonensis PVs to be dynamically recorded by multidimensional imaging of coinfected cells. We found that although T. cruzi EPIs remained motile and conserved their morphology in chimeric vacuoles, T. cruzi MTs differentiated into amastigote-like forms capable of multiplying. These results demonstrate that the large adaptive vacuoles of L. amazonensis are permissive to T. cruzi survival and differentiation and that noninfective EPIs are spared from destruction within the chimeric PVs. We conclude that T. cruzi differentiation can take place in Leishmania-containing vacuoles, suggesting this occurs prior to their escape into the host cell cytosol. PMID:26975994

  14. Evidence for transcript networks composed of chimeric RNAs in human cells.

    Science.gov (United States)

    Djebali, Sarah; Lagarde, Julien; Kapranov, Philipp; Lacroix, Vincent; Borel, Christelle; Mudge, Jonathan M; Howald, Cédric; Foissac, Sylvain; Ucla, Catherine; Chrast, Jacqueline; Ribeca, Paolo; Martin, David; Murray, Ryan R; Yang, Xinping; Ghamsari, Lila; Lin, Chenwei; Bell, Ian; Dumais, Erica; Drenkow, Jorg; Tress, Michael L; Gelpí, Josep Lluís; Orozco, Modesto; Valencia, Alfonso; van Berkum, Nynke L; Lajoie, Bryan R; Vidal, Marc; Stamatoyannopoulos, John; Batut, Philippe; Dobin, Alex; Harrow, Jennifer; Hubbard, Tim; Dekker, Job; Frankish, Adam; Salehi-Ashtiani, Kourosh; Reymond, Alexandre; Antonarakis, Stylianos E; Guigó, Roderic; Gingeras, Thomas R

    2012-01-01

    The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5' and 3' transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network. PMID:22238572

  15. Viral Engineering of Chimeric Antigen Receptor Expression on Murine and Human T Lymphocytes.

    Science.gov (United States)

    Hammill, Joanne A; Afsahi, Arya; Bramson, Jonathan L; Helsen, Christopher W

    2016-01-01

    The adoptive transfer of a bolus of tumor-specific T lymphocytes into cancer patients is a promising therapeutic strategy. In one approach, tumor specificity is conferred upon T cells via engineering expression of exogenous receptors, such as chimeric antigen receptors (CARs). Here, we describe the generation and production of both murine and human CAR-engineered T lymphocytes using retroviruses. PMID:27581020

  16. Custom-engineered chimeric foot-and-mouth disease vaccine elicits protective immune responses in pigs

    Science.gov (United States)

    Chimeric foot-and-mouth disease viruses (FMDV) of which the antigenic properties can be readily manipulated is a potentially powerful approach in the control of foot-and-mouth disease (FMD) in sub-Saharan Africa. FMD vaccine application is complicated by the extensive variability of the South Africa...

  17. In Silico Design of a Chimeric Protein Containing Antigenic Fragments of Helicobacter pylori; A Bioinformatic Approach

    Science.gov (United States)

    Mohammad, Nazanin; Karsabet, Mehrnaz Taghipour; Amani, Jafar; Ardjmand, Abolfazl; Zadeh, Mohsen Razavi; Gholi, Mohammad Khalifeh; Saffari, Mahmood; Ghasemi, Amir

    2016-01-01

    Helicobacter pylori is a global health problem which has encouraged scientists to find new ways to diagnose, immunize and eradicate the H. pylori infection. In silico studies are a promising approach to design new chimeric antigen having the immunogenic potential of several antigens. In order to obtain such benefit in H. pylori vaccine study, a chimeric gene containing four fragments of FliD sequence (1-600 bp), UreB (327-334 bp),VacA (744-805 bp) and CagL(51-100 bp) which have a high density of B- and T-cell epitopes was designed. The secondary and tertiary structures of the chimeric protein and other properties such as stability, solubility and antigenicity were analyzed. The in silico results showed that after optimizing for the purpose of expression in Escherichia coli BL21, the solubility and antigenicity of the construct fragments were highly retained. Most regions of the chimeric protein were found to have a high antigenic propensity and surface accessibility. These results would be useful in animal model application and accounted for the development of an epitope-based vaccine against the H. pylori. PMID:27335622

  18. Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Desai, D M; Sap, J; Schlessinger, J;

    1993-01-01

    inactivate the EGFR-CD45 chimera in a manner that is dependent on dimerization of the chimeric protein. Inactivation of EGFR-CD45 chimera function results in the loss of TCR signaling, indicating that CD45 function is continuously required for TCR-mediated proximal signaling events. These results suggest...

  19. Alloreactive regulatory T cells allow the generation of mixed chimerism and transplant tolerance

    Directory of Open Access Journals (Sweden)

    Paulina eRuiz

    2015-11-01

    Full Text Available The induction of donor-specific transplant tolerance is one of the main goals of modern immunology. Establishment of a mixed chimerism state in the transplant recipient has proven to be a suitable strategy for the induction of long-term allograft tolerance; however, current experimental recipient preconditioning protocols have many side effects, and are not feasible for use in future therapies. In order to improve the current mixed chimerism induction protocols, we developed a non-myeloablative bone-marrow transplant protocol using retinoic acid induced alloantigen-specific Tregs, clinically available immunosuppressive drugs and lower doses of irradiation. We demonstrate that retinoic acid induced alloantigen-specific Tregs in addition to a non-myeloablative bone-marrow transplant protocol generates stable mixed chimerism and induce tolerance to allogeneic secondary skin allografts in mice. Therefore, the establishment of mixed chimerism through the use of donor-specific Tregs rather than non-specific immunosuppression could have a potential use in organ transplantation.

  20. SAT Type Foot-and-Mouth Disease (FMD) Chimeric Vaccine Elicits Protection in Pigs

    Science.gov (United States)

    The recent development of infectious cDNA clone technology for foot-and-mouth disease (FMD), Southern African Territories (SAT) viruses has provided a valuable tool for genetic and biological characterization of field and laboratory strains. Recombinant chimeric viruses, containing the capsid-coding...

  1. Origination of an X-linked testes chimeric gene by illegitimate recombination in Drosophila.

    Directory of Open Access Journals (Sweden)

    2006-05-01

    Full Text Available The formation of chimeric gene structures provides important routes by which novel proteins and functions are introduced into genomes. Signatures of these events have been identified in organisms from wide phylogenic distributions. However, the ability to characterize the early phases of these evolutionary processes has been difficult due to the ancient age of the genes or to the limitations of strictly computational approaches. While examples involving retrotransposition exist, our understanding of chimeric genes originating via illegitimate recombination is limited to speculations based on ancient genes or transfection experiments. Here we report a case of a young chimeric gene that has originated by illegitimate recombination in Drosophila. This gene was created within the last 2-3 million years, prior to the speciation of Drosophila simulans, Drosophila sechellia, and Drosophila mauritiana. The duplication, which involved the Bällchen gene on Chromosome 3R, was partial, removing substantial 3' coding sequence. Subsequent to the duplication onto the X chromosome, intergenic sequence was recruited into the protein-coding region creating a chimeric peptide with approximately 33 new amino acid residues. In addition, a novel intron-containing 5' UTR and novel 3' UTR evolved. We further found that this new X-linked gene has evolved testes-specific expression. Following speciation of the D. simulans complex, this novel gene evolved lineage-specifically with evidence for positive selection acting along the D. simulans branch.

  2. THE PERSISTENCE OF CHICKEN HERPES AND RETRO VIRAL CHIMERIC MOLECULES UPON IN VIVO PASSAGE

    Science.gov (United States)

    Marek's disease virus, a herpes virus, and avian leucosis virus subgroup J, a retrovirus were used for experimental co-infection of chicks. Two consecutive trials were performed in attempt to evaluate the formation and persistence of chimeric molecules that would indicate retro-viral integration int...

  3. The rapid generation of chimerical genes expanding protein diversity in zebrafish

    Directory of Open Access Journals (Sweden)

    Zou Ming

    2010-11-01

    Full Text Available Abstract Background Variation of gene number among species indicates that there is a general process of new gene origination. One of the major mechanism providing raw materials for the origin of new genes is gene duplication. Retroposition, as a special type of gene duplication- the RNA-based duplication, has been found to play an important role in new gene evolution in mammals and plants, but little is known about the process in the teleostei genome. Results Here we screened the zebrafish genome for identification of retrocopies and new chimerical retrogenes and investigated their origination and evolution. We identified 652 retrocopies, of which 440 are intact retrogenes and 212 are pseudogenes. Retrocopies have long been considered evolutionary dead ends without functional significance due to the presumption that retrocopies lack the regulatory element needed for expression. However, 437 transcribed retrocopies were identified from all of the retrocopies. This discovery combined with the substitution analysis suggested that the majority of all retrocopies are subject to negative selection, indicating that most of the retrocopies may be functional retrogenes. Moreover, we found that 95 chimerical retrogenes had recruited new sequences from neighboring genomic regions that formed de novo splice sites, thus generating new intron-containing chimeric genes. Based on our analysis of 38 pairs of orthologs between Cyprinus carpio and Danio rerio, we found that the synonymous substitution rate of zebrafish genes is 4.13×10-9 substitution per silent site per year. We also found 10 chimerical retrogenes that were created in the last 10 million years, which is 7.14 times the rate of 0.14 chimerical retrogenes per million years in the primate lineage toward human and 6.25 times the rate of 0.16 chimerical genes per million years in Drosophila. This is among the most rapid rates of generation of chimerical genes, just next to the rice. Conclusion There is

  4. Chimeric Peptides as Implant Functionalization Agents for Titanium Alloy Implants with Antimicrobial Properties

    Science.gov (United States)

    Yucesoy, Deniz T.; Hnilova, Marketa; Boone, Kyle; Arnold, Paul M.; Snead, Malcolm L.; Tamerler, Candan

    2015-04-01

    Implant-associated infections can have severe effects on the longevity of implant devices and they also represent a major cause of implant failures. Treating these infections associated with implants by antibiotics is not always an effective strategy due to poor penetration rates of antibiotics into biofilms. Additionally, emerging antibiotic resistance poses serious concerns. There is an urge to develop effective antibacterial surfaces that prevent bacterial adhesion and proliferation. A novel class of bacterial therapeutic agents, known as antimicrobial peptides (AMPs), are receiving increasing attention as an unconventional option to treat septic infection, partly due to their capacity to stimulate innate immune responses and for the difficulty of microorganisms to develop resistance towards them. While host and bacterial cells compete in determining the ultimate fate of the implant, functionalization of implant surfaces with AMPs can shift the balance and prevent implant infections. In the present study, we developed a novel chimeric peptide to functionalize the implant material surface. The chimeric peptide simultaneously presents two functionalities, with one domain binding to a titanium alloy implant surface through a titanium-binding domain while the other domain displays an antimicrobial property. This approach gains strength through control over the bio-material interfaces, a property built upon molecular recognition and self-assembly through a titanium alloy binding domain in the chimeric peptide. The efficiency of chimeric peptide both in-solution and absorbed onto titanium alloy surface was evaluated in vitro against three common human host infectious bacteria, Streptococcus mutans, Staphylococcus epidermidis, and Escherichia coli. In biological interactions such as occur on implants, it is the surface and the interface that dictate the ultimate outcome. Controlling the implant surface by creating an interface composed chimeric peptides may therefore

  5. Murine immune responses to a Plasmodium vivax-derived chimeric recombinant protein expressed in Brassica napus

    Directory of Open Access Journals (Sweden)

    Chung Nam-Jun

    2011-04-01

    Full Text Available Abstract Background To develop a plant-based vaccine against Plasmodium vivax, two P. vivax candidate proteins were chosen. First, the merozoite surface protein-1 (MSP-1, a major asexual blood stage antigen that is currently considered a strong vaccine candidate. Second, the circumsporozoite protein (CSP, a component of sporozoites that contains a B-cell epitope. Methods A synthetic chimeric recombinant 516 bp gene encoding containing PvMSP-1, a Pro-Gly linker motif, and PvCSP was synthesized; the gene, named MLC, encoded a total of 172 amino acids. The recombinant gene was modified with regard to codon usage to optimize gene expression in Brassica napus. The Ti plasmid inducible gene transfer system was used for MLC chimeric recombinant gene expression in B. napus. Gene expression was confirmed by polymerase chain reaction (PCR, beta-glucuronidase reporter gene (GUS assay, and Western blot. Results The MLC chimeric recombinant protein expressed in B. napus had a molecular weight of approximately 25 kDa. It exhibited a clinical sensitivity of 84.21% (n = 38 and a clinical specificity of 100% (n = 24 as assessed by enzyme-linked immunosorbent assay (ELISA. Oral immunization of BALB/c mice with MLC chimeric recombinant protein successfully induced antigen-specific IgG1 production. Additionally, the Th1-related cytokines IL-12 (p40, TNF, and IFN-γ were significantly increased in the spleens of the BALB/c mice. Conclusions The chimeric MLC recombinant protein produced in B. napus has potential as both as an antigen for diagnosis and as a valuable vaccine candidate for oral immunization against vivax malaria.

  6. Enhancement of mucosal immune responses by chimeric influenza HA/SHIV virus-like particles

    International Nuclear Information System (INIS)

    To enhance mucosal immune responses using simian/human immunodeficiency virus-like particles (SHIV VLPs), we have produced novel phenotypically mixed chimeric influenza HA/SHIV VLPs and used them to immunize C57BL/6J mice intranasally. Antibody and cytotoxic T-cell (CTL) responses as well as cytokine production in both systemic and mucosal sites were compared after immunization with SHIV VLPs or chimeric HA/SHIV VLPs. By using enzyme-linked immunosorbent assay (ELISA), the levels of serum IgG and mucosal IgA to the HIV envelope protein (Env) were found to be highest in the group immunized with chimeric HA/SHIV VLPs. Furthermore, the highest titer of serum neutralizing antibody against HIV Env was found with the group immunized with chimeric HA/SHIV VLPs. Analysis of the IgG1/IgG2a ratio indicated that a TH1-oriented immune response resulted from these VLP immunizations. HA/SHIV VLP-immunized mice also showed significantly higher CTL responses than those observed in SHIV VLP-immunized mice. Moreover, a MHC class I restricted T-cell activation ELISPOT assay showed a mixed type of TH1/TH2 cytokines in the HA/SHIV VLP-immunized mice, indicating that the chimeric VLPs can enhance both humoral and cellular immune responses to the HIV Env protein at multiple mucosal and systemic sites. The results indicate that incorporation of influenza HA into heterotypic VLPs may be highly effective for targeting vaccines to mucosal surfaces

  7. The expression and genetic immunization of chimeric fragment of Hantaan virus M and S segments

    International Nuclear Information System (INIS)

    Hemorrhagic fever with renal syndrome (HFRS), which is characterized by severe symptoms and high mortality, is caused by hantavirus. There are still no effective prophylactic vaccines directed to HFRS until now. In this research, we fused expressed G2 fragment of M segment and 0.7 kb fragment of S segment. We expect it could be a candidate vaccine. Chimeric gene G2S0.7 was first expressed in prokaryotic expression system pGEX-4T. After inducing expressed fusion proteins, GST-G2S0.7 was induced and its molecular weight was about 100 kDa. Meanwhile, the fusion protein kept the activity of its parental proteins. Further, BALB/c mice were vaccinated by the chimeric gene. ELISA, cell microculture neutralization test in vitro were used to detect the humoral immune response in immunized BALB/c mice. Lymphocyte proliferation assay was used to detect the cellular immune response. The results showed that the chimeric gene could simultaneously evoke specific antibody against nucleocapsid protein (NP) and glycoprotein (GP). And the immunized mice of every group elicited neutralizing antibodies with different titers. But the titers were low. Lymphocyte proliferation assay results showed that the stimulation indexes of splenocytes of chimeric gene to NP and GP were significantly higher than that of control. It suggested that the chimeric gene of Hantaan virus containing G2 fragment of M segment and 0.7 kb fragment of S segment could directly elicit specific anti-Hantaan virus humoral and cellular immune response in BALB/c mice

  8. Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants

    International Nuclear Information System (INIS)

    Virus-like particles (VLPs) are a promising vaccine platform due to the safety and efficiency. However, it is still unclear whether polyomavirus-based VLPs are useful for this purpose. Here, we attempted to evaluate the potential of polyomavirus VLPs for the antiviral vaccine using simian virus 40 (SV40). We constructed chimeric SV40-VLPs carrying an HLA-A⁎02:01-restricted, cytotoxic T lymphocyte (CTL) epitope derived from influenza A virus. HLA-A⁎02:01-transgenic mice were then immunized with the chimeric SV40-VLPs. The chimeric SV40-VLPs effectively induced influenza-specific CTLs and heterosubtypic protection against influenza A viruses without the need of adjuvants. Because DNase I treatment of the chimeric SV40-VLPs did not disrupt CTL induction, the intrinsic adjuvant property may not result from DNA contaminants in the VLP preparation. In addition, immunization with the chimeric SV40-VLPs generated long-lasting memory CTLs. We here propose that the chimeric SV40-VLPs harboring an epitope may be a promising CTL-based vaccine platform with self-adjuvant properties. - Highlights: • We constructed chimeric SV40-VLPs carrying an influenza virus-derived CTL epitope. • Chimeric SV40-VLPs induce influenza-specific CTLs in mice without adjuvants. • Chimeric SV40-VLPs induce heterosubtypic protection against influenza A viruses. • Chimeric SV40-VLPs induce long-lasting memory CTLs. • Chimeric SV40-VLPs is a promising vaccine platform with self-adjuvant properties

  9. Dosimetry of chimeric TNT in lung cancer patients

    International Nuclear Information System (INIS)

    Objective: A mouse-human chimeric Tumor-Necrosis-Therapy (chTNT) monoclonal antibody is directed against single-stranded DNA a universal nuclear antigen accessible in the necrotic cell within most solid tumors, which has potential for the radioimmunotherapy of many solid tumors. The radiation dosimetry of the 131I-chTNT was evaluated in 9 lung cancer patients (age 18-74 years, mean weight (49.6±6.5) kg). Methods: A single intravenous dose of (29.6±3.7) MBq/kg was administered. Blood samples were drawn at different time intervals and urine was collected for up to one week. Tissue distribution was followed for up to one week by gamma camera imaging. The geometric mean of the counts in the whole body scintigram at 0.5 h after infusion was corrected for radioactivity decay from the time of infusion, this value being taken as 100%ID. The geometric mean of the anterior and posterior counts was obtained from selected region of interested (ROI) to determine activity within the critical organs after being subtracted the background activity. Counts from thyroid were obtained from anterior images only. A background region was drawn below the thyroid gland to subtract underlying activity in the neck blood vessels. The residence times for brain, lungs, liver, spleen, kidneys, heart, thyroid and the whole body were computed by dividing the area under their %ID/h curves by the 100%ID value. Before integration, the activity-time curves were fitted to an algebraic function with an exponential tail (r>0.90). The absorbed doses were computed from the residence time according to the Medical Internal Radiation Dose (MIRD) scheme using MIRDOSE 3.0 software. The adult female and male phantoms were selected. The MIRDOSE 3.0 dynamic bladder model was used to calculate residence times for urinary bladder content assuming a urinary elimination fraction of 1.0 with a bladder-voiding interval of 4h. Absorbed doses to tumor tissues were estimated using the same approach taken for normal

  10. The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

    International Nuclear Information System (INIS)

    TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves' patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves' disease will be elucidated. (author). 25 refs

  11. The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ka Hee; Kim, Chang Min [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    1996-12-01

    TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves` patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves` disease will be elucidated. (author). 25 refs.

  12. Transfection of beta-casein chimeric gene and hormonal induction of its expression in primary murine mammary epithelial cells.

    OpenAIRE

    Yoshimura, M.; Oka, T

    1990-01-01

    To study the regulatory sequence elements responsible for casein gene expression, we constructed a chimeric gene containing 5.3 kilobases (kb) of the 5'-flanking sequence and 1.6 kb of the 3'-flanking sequence of the mouse beta-casein gene fused to the bacterial chloramphenicol acetyl-transferase (CAT) gene. The chimeric gene was transfected by the calcium phosphate-precipitation procedure into primary mouse mammary epithelial cells prepared from pregnant mice. The transfection procedure had ...

  13. Suicide Gene Therapy to Increase the Safety of Chimeric Antigen Receptor-Redirected T Lymphocytes

    Directory of Open Access Journals (Sweden)

    Monica Casucci, Attilio Bondanza

    2011-01-01

    Full Text Available Chimeric antigen receptors (CARs are generated by fusing the antigen-binding motif of a monoclonal antibody (mAb with the signal transduction machinery of the T-cell receptor (TCR. The genetic modification of T lymphocytes with chimeric receptors specific for tumor-associated antigens (TAAs allows for the redirection towards tumor cells. Clinical experience with CAR-redirected T cells suggests that antitumor efficacy associates with some degree of toxicity, especially when TAA expression is shared with healthy tissues. This situation closely resembles the case of allogeneic hematopoietic stem cell transplantation (HSCT, wherein allorecognition causes both the graft-versus-leukemia (GVL effect and graft-versus-host disease (GVHD. Suicide gene therapy, i.e. the genetic induction of a conditional suicide phenotype into donor T cells, enables dissociating the GVL effect from GVHD. Applying suicide gene modification to CAR-redirected T cells may therefore greatly increase their safety profile and facilitate their clinical development.

  14. Human glial chimeric mice reveal astrocytic dependence of JC virus infection

    DEFF Research Database (Denmark)

    Kondo, Yoichi; Windrem, Martha S; Zou, Lisa;

    2014-01-01

    with humanized white matter by engrafting human glial progenitor cells (GPCs) into neonatal immunodeficient and myelin-deficient mice. Intracerebral delivery of JCV resulted in infection and subsequent demyelination of these chimeric mice. Human GPCs and astrocytes were infected more readily than...... oligodendrocytes, and viral replication was noted primarily in human astrocytes and GPCs rather than oligodendrocytes, which instead expressed early viral T antigens and exhibited apoptotic death. Engraftment of human GPCs in normally myelinated and immunodeficient mice resulted in humanized white matter that was...... chimeric for human astrocytes and GPCs. JCV effectively propagated in these mice, which indicates that astroglial infection is sufficient for JCV spread. Sequencing revealed progressive mutation of the JCV capsid protein VP1 after infection, suggesting that PML may evolve with active infection. These...

  15. Very Long Term Stability of Mixed Chimerism after Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Hematologic Malignancies

    Directory of Open Access Journals (Sweden)

    Emmanuel Levrat

    2015-01-01

    Full Text Available The objective of this study is to analyze the evolution of chimerism of all patients transplanted for hematologic malignancies in our unit during a 20-year period, alive without relapse at 1 year after allogeneic hematopoietic stem cell transplantation (HSCT. Chimerism was tested using short tandem repeat polymorphisms after separation into mononuclear cells and granulocytes by Ficoll density gradient centrifugation. Of 155 patients studied, 89 had full chimerism (FC, 36 mononuclear cells mixed chimerism (MNC-MC, and 30 granulocytic MC with or without mononuclear cells MC (Gran-MC. Survival was significantly better in MNC-MC than in Gran-MC patients, with FC patients being intermediate. There was more disease relapse in the Gran-MC group but not in the MNC-MC group as compared to FC. MC was stable up to 21 years in the MNC-MC group and up to 19 years in the Gran-MC group. Of MC patients alive at 10 years, MC persisted in 83% in the MNC-MC and 57% in the Gran-MC groups. In conclusion, mixed chimerism may remain stable over a very long time period. In survivors without relapse at 1 year after HSCT, determining lineage specific chimerism may be useful as outcome differs, MNC-MC being associated with better outcome than Gran-MC.

  16. Skin Recurrence of Transformed Mycosis Fungoides Postumbilical Cord Blood Transplant despite Complete Donor Chimerism

    OpenAIRE

    Rahul Pawar; Anup Kasi Loknath Kumar; Janet Woodroof; Wei Cui; Joseph McGuirk; Sunil Abhyankar; Sid Ganguly; Anurag Singh; Tara Lin; Omar Aljitawi

    2014-01-01

    Background. Allogeneic stem cell transplant is the treatment of choice for systemic cutaneous T-cell lymphoma (CTCL) which provides graft-versus-lymphoma effect. Herein we discuss a case of recurrence of CTCL skin lesions after cord blood transplant in a patient who continued to have 100% donor chimerism in bone marrow. Case Presentation. A 48-year-old female with history of mycosis fungoides (MF) presented with biopsy proven large cell transformation of MF. PET scan revealed multiple adenop...

  17. Bone marrow chimeric mice reveal a dual role for CD36 in Plasmodium berghei ANKA infection

    Directory of Open Access Journals (Sweden)

    Febbraio Maria

    2007-03-01

    Full Text Available Abstract Background Adhesion of Plasmodium-infected red blood cells (iRBC to different host cells, ranging from endothelial to red blood cells, is associated to malaria pathology. In vitro studies have shown the relevance of CD36 for adhesion phenotypes of Plasmodium falciparum iRBC such as sequestration, platelet mediated clumping and non-opsonic uptake of iRBC. Different adhesion phenotypes involve different host cells and are associated with different pathological outcomes of disease. Studies with different human populations with CD36 polymorphisms failed to attribute a clear role to CD36 expression in human malaria. Up to the present, no in vivo model has been available to study the relevance of different CD36 adhesion phenotypes to the pathological course of Plasmodium infection. Methods Using CD36-deficient mice and their control littermates, CD36 bone marrow chimeric mice, expressing CD36 exclusively in haematopoietic cells or in non-haematopoietic cells, were generated. Irradiated CD36-/- and wild type mice were also reconstituted with syngeneic cells to control for the effects of irradiation. The reconstituted mice were infected with Plasmodium berghei ANKA and analysed for the development of blood parasitaemia and neurological symptoms. Results All mice reconstituted with syngeneic bone marrow cells as well as chimeric mice expressing CD36 exclusively in non-haematopoietic cells died from experimental cerebral malaria between day 6 and 12 after infection. A significant proportion of chimeric mice expressing CD36 only in haematopoietic cells did not die from cerebral malaria. Conclusion The analysis of bone marrow chimeric mice reveals a dual role of CD36 in P. berghei ANKA infection. Expression of CD36 in haematopoietic cells, most likely macrophages and dendritic cells, has a beneficial effect that is masked in normal mice by adverse effects of CD36 expression in non-haematopoietic cells, most likely endothelial cells.

  18. Chimeric viruses blur the borders between the major groups of eukaryotic single-stranded DNA viruses

    OpenAIRE

    Roux, Simon; Enault, Francois; Bronner, Gisèle; Vaulot, Daniel; Forterre, Patrick; Krupovic, Mart

    2013-01-01

    Metagenomic studies have uncovered an astonishing diversity of ssDNA viruses encoding replication proteins (Reps) related to those of eukaryotic Circoviridae, Geminiviridae or Nanoviridae; however, exact evolutionary relationships among these viruses remain obscure. Recently, a unique chimeric virus (CHIV) genome, which has apparently emerged via recombination between ssRNA and ssDNA viruses, has been discovered. Here we report on the assembly of 13 new CHIV genomes recovered f...

  19. Chimerism in M1 plants of Vicia faba, Capsicum annuum and Linum usitatissimum

    International Nuclear Information System (INIS)

    One important task of our group at IAEA is to develop procedures aiming to improve sampling of M2 seeds to facilitate the recovery of a maximum number of induced mutations in crop plants. Results from studies on three species are reported in this paper. Seeds have been mutagen treated and the chimeric M1 plants were progeny tested in M2. The position of the M2 seeds on the M1 plants has been recorded

  20. Oxidative stress and the mechanical properties of naturally occurring chimeric collagen-containing fibers.

    OpenAIRE

    Sun, C.; E. Vaccaro; Waite, J. H.

    2001-01-01

    The byssal threads of marine mussels are a fiber-reinforced composite material. Fibers are continuous, separated by matrix, and consist of chimeric collagens that encompass within the same primary protein structure domains corresponding to collagen, polyhistidine, and either elastin or dragline spider silk. The elastic modulus (stiffness) of the proximal portion of byssal threads was measured by cyclic stress-strain analysis at 50% extension. Before measurement, the threads were conditioned b...

  1. Chimeric nucleolin aptamer with survivin DNAzyme for cancer cell targeted delivery.

    Science.gov (United States)

    Subramanian, Nithya; Kanwar, Jagat R; Akilandeswari, Balachandran; Kanwar, Rupinder K; Khetan, Vikas; Krishnakumar, Subramanian

    2015-04-25

    A chimeric aptamer-DNAzyme conjugate was generated for the first time using a nucleolin aptamer (NCL-APT) and survivin Dz (Sur_Dz) and exhibited the targeted killing of cancer cells. This proof of concept of using an aptamer for the delivery of DNAzyme can be applied to other cancer types to target survivin in cancer cells in a specific manner. PMID:25797393

  2. Chimeric External Control to Quantify Cell Free DNA in Plasma Samples by Real Time PCR

    OpenAIRE

    Eini, Maryam; Behzad-Behbahani, Abbas; Takhshid, Mohammad Ali; Ramezani, Amin; Rafiei Dehbidi, Gholam Reza; Okhovat, Mohammad Ali; Farhadi, Ali; Alavi, Parniyan

    2016-01-01

    Background: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA (cfDNA) is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control (EDC) is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for ...

  3. T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors

    DEFF Research Database (Denmark)

    Jamnani, Fatemeh Rahimi; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad Ali;

    2014-01-01

    Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination of...... costimulatory endodomains for CAR construction to improve the effector functions of the engineered T cells. Camelid single-domain antibodies (VHHs), which are the smallest single domain antibodies, can endow great targeting ability to CAR-engineered T cells....

  4. Design and Development of Therapies using Chimeric Antigen Receptor-Expressing T cells

    OpenAIRE

    Dotti, Gianpietro; Gottschalk, Stephen; Savoldo, Barbara; Brenner, Malcolm K

    2014-01-01

    Investigators developed chimeric antigen receptors (CARs) for expression on T cells more than 25 years ago. When the CAR is derived from an antibody, the resultant cell should combine the desirable targeting features of an antibody (e.g. lack of requirement for major histocompatibility complex recognition, ability to recognize non-protein antigens) with the persistence, trafficking and effector functions of a T-cell. This article describes how the past two decades have seen a crescendo of res...

  5. Domain exchange: characterization of a chimeric lipase of hepatic lipase and lipoprotein lipase.

    OpenAIRE

    Wong, H; Davis, R. C.; Nikazy, J; Seebart, K E; Schotz, M C

    1991-01-01

    Hepatic lipase and lipoprotein lipase hydrolyze fatty acids from triacylglycerols and are critical in the metabolism of circulating lipoproteins. The two lipases are similar in size and amino acid sequence but are distinguished by functional differences in substrate preference and cofactor requirement. Presumably, these distinctions result from structural differences in functional domains. To begin localization of these domains, a chimeric lipase was constructed composed of the N-terminal 329...

  6. Remote control of therapeutic T cells through a small molecule-gated chimeric receptor

    OpenAIRE

    Wu, Chia-Yung; Kole T Roybal; Puchner, Elias M.; Onuffer, James; Lim, Wendell A.

    2015-01-01

    There is growing promise in using engineered cells as therapeutic agents. For example, synthetic Chimeric Antigen Receptors (CARs) can redirect T cells to recognize and eliminate tumor cells expressing specific antigens. Despite promising clinical results, excessive activity and poor control over such engineered T cells can cause severe toxicities. We present the design of “ON-switch” CARs that enable small molecule-control over T cell therapeutic functions, while still retaining antigen spec...

  7. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells

    OpenAIRE

    Frigault, Matthew J.; Lee, Jihyun; Basil, Maria Ciocca; Carpenito, Carmine; Motohashi, Shinichiro; Scholler, John; Kawalekar, Omkar U.; Guedan, Sonia; McGettigan, Shannon E; Posey, Avery D; Ang, Sonny; Cooper, Laurence J. N.; Platt, Jesse M.; Johnson, F. Brad; Paulos, Chrystal M.

    2015-01-01

    This study compared second generation chimeric antigen receptors encoding signaling domains composed of CD28, ICOS and 4-1BB. Here we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T-cell with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to three months following a single stimulation through the TCR. Sustained numeric expansion was independent of cognate antigen and...

  8. Topology of the RNA polymerase active center probed by chimeric rifampicin-nucleotide compounds.

    OpenAIRE

    Mustaev, A; Zaychikov, E; Severinov, K.; Kashlev, M; Polyakov, A.; Nikiforov, V.; Goldfarb, A

    1994-01-01

    Spatial organization of the binding sites for the priming substrate, the template DNA, and the transcription inhibitor rifampicin (Rif) in Escherichia coli RNA polymerase (EC 2.7.7.6) was probed with chimeric compounds in which Rif is covalently attached to a ribonucleotide. The compounds bind to RNA polymerase in bifunctional manner and serve as substrates for RNA chain extension, yielding chains up to 8 nucleotides in length, with Rif linked to their 5' termini. These products act as potent...

  9. Development of a high-throughput microfluidic integrated microarray for the detection of chimeric bioweapons.

    Energy Technology Data Exchange (ETDEWEB)

    Sheppod, Timothy; Satterfield, Brent; Hukari, Kyle W.; West, Jason A. A.; Hux, Gary A.

    2006-10-01

    The advancement of DNA cloning has significantly augmented the potential threat of a focused bioweapon assault, such as a terrorist attack. With current DNA cloning techniques, toxin genes from the most dangerous (but environmentally labile) bacterial or viral organism can now be selected and inserted into robust organism to produce an infinite number of deadly chimeric bioweapons. In order to neutralize such a threat, accurate detection of the expressed toxin genes, rather than classification on strain or genealogical decent of these organisms, is critical. The development of a high-throughput microarray approach will enable the detection of unknowns chimeric bioweapons. The development of a high-throughput microarray approach will enable the detection of unknown bioweapons. We have developed a unique microfluidic approach to capture and concentrate these threat genes (mRNA's) upto a 30 fold concentration. These captured oligonucleotides can then be used to synthesize in situ oligonucleotide copies (cDNA probes) of the captured genes. An integrated microfluidic architecture will enable us to control flows of reagents, perform clean-up steps and finally elute nanoliter volumes of synthesized oligonucleotides probes. The integrated approach has enabled a process where chimeric or conventional bioweapons can rapidly be identified based on their toxic function, rather than being restricted to information that may not identify the critical nature of the threat.

  10. Human-animal chimeras: ethical issues about farming chimeric animals bearing human organs.

    Science.gov (United States)

    Bourret, Rodolphe; Martinez, Eric; Vialla, François; Giquel, Chloé; Thonnat-Marin, Aurélie; De Vos, John

    2016-01-01

    Recent advances in stem cells and gene engineering have paved the way for the generation of interspecies chimeras, such as animals bearing an organ from another species. The production of a rat pancreas by a mouse has demonstrated the feasibility of this approach. The next step will be the generation of larger chimeric animals, such as pigs bearing human organs. Because of the dramatic organ shortage for transplantation, the medical needs for such a transgressive practice are indisputable. However, there are serious technical barriers and complex ethical issues that must be discussed and solved before producing human organs in animals. The main ethical issues are the risks of consciousness and of human features in the chimeric animal due to a too high contribution of human cells to the brain, in the first case, or for instance to limbs, in the second. Another critical point concerns the production of human gametes by such chimeric animals. These worst-case scenarios are obviously unacceptable and must be strictly monitored by careful risk assessment, and, if necessary, technically prevented. The public must be associated with this ethical debate. Scientists and physicians have a critical role in explaining the medical needs, the advantages and limits of this potential medical procedure, and the ethical boundaries that must not be trespassed. If these prerequisites are met, acceptance of such a new, borderline medical procedure may prevail, as happened before for in-vitro fertilization or preimplantation genetic diagnosis. PMID:27356872

  11. Skin Recurrence of Transformed Mycosis Fungoides Postumbilical Cord Blood Transplant despite Complete Donor Chimerism

    Directory of Open Access Journals (Sweden)

    Rahul Pawar

    2014-01-01

    Full Text Available Background. Allogeneic stem cell transplant is the treatment of choice for systemic cutaneous T-cell lymphoma (CTCL which provides graft-versus-lymphoma effect. Herein we discuss a case of recurrence of CTCL skin lesions after cord blood transplant in a patient who continued to have 100% donor chimerism in bone marrow. Case Presentation. A 48-year-old female with history of mycosis fungoides (MF presented with biopsy proven large cell transformation of MF. PET scan revealed multiple adenopathy in abdomen and chest suspicious for lymphoma and skin biopsy showed large cell transformation. She was treated with multiple cycles of chemotherapy. Posttherapy PET scan showed resolution of lymphadenopathy. Later she underwent ablative preparative regimen followed by single cord blood transplant. Bone marrow chimerism studies at day +60 after transplant showed 100% donor cells without presence of lymphoma. However 5 months after transplant she had recurrence of MF with the same genotype as prior skin lesion. Bone marrow chimerism study continued to show 100% donor cells. Conclusion. A differential graft-versus-lymphoma effect in our case prevented lymphoma recurrence systemically but failed to do so in skin. We hypothesize that this response may be due to presence of other factors in the bone marrow and lymph node microenvironments preventing recurrence in these sites.

  12. Venturing in coral larval chimerism: a compact functional domain with fostered genotypic diversity.

    Science.gov (United States)

    Rinkevich, Baruch; Shaish, Lee; Douek, Jacob; Ben-Shlomo, Rachel

    2016-01-01

    The globally distributed coral species Pocillopora damicornis is known to release either sexual or asexual derived planula-larvae in various reef locations. Using microsatellite loci as markers, we documented the release of asexually derived chimeric larvae (CL), originating from mosaicked maternal colonies that were also chimeras, at Thai and Philippines reefs. The CL, each presenting different combinations of maternal genotypic constituents, create genetically-complex sets of asexual propagules. This novel mode of inheritance in corals challenges classical postulations of sexual/asexual reproduction traits, as asexual derived CL represent an alliance between genotypes that significantly sways the recruits' absolute fitness. This type of inherited chimerism, while enhancing intra-entity genetic heterogeneity, is an evolutionary tactic used to increase genetic-heterogeneity, primarily in new areas colonized by a limited number of larvae. Chimerism may also facilitate combat global change impacts by exhibiting adjustable genomic combinations of within-chimera traits that could withstand alterable environmental pressures, helping Pocillopora become a successful cosmopolitan species. PMID:26758405

  13. Preparation and Evaluation of Human-Murine Chimeric Antibody against Protective Antigen of Bacillus anthracis

    Directory of Open Access Journals (Sweden)

    Lina Hao

    2014-10-01

    Full Text Available The aim of this research is to develop a human/murine chimeric Fab antibody which neutralizes the anthrax toxin, protective antigen (PA. The chimeric Fab was constructed using variable regions of murine anti-PA monoclonal antibody in combination with constant regions of human IgG. The chimeric PA6-Fab was expressed in E. coli. BL21 and evaluated by ELISA and co-immunoprecipitation- mass spectra. The potency of PA6-Fab to neutralize LeTx was examined in J774A.1 cell viability in vitro and in Fisher 344 rats in vivo. The PA6-Fab did not have domain similarity corresponding to the current anti PA mAbs, but specifically bound to anthrax PA at an affinity of 1.76 nM, and was able to neutralize LeTx in vitro and protected 56.9% cells at 20 μg/mL against anthrax LeTx. One hundred μg PA6-Fab could neutralize 300 μg LeTx in vivo. The PA6-Fab has potential as a therapeutic mAb for treatment of anthrax.

  14. Induced regulatory T cells in allograft tolerance via transient mixed chimerism

    Science.gov (United States)

    Hotta, Kiyohiko; Aoyama, Akihiro; Oura, Tetsu; Yamada, Yohei; Tonsho, Makoto; Huh, Kyu Ha; Kawai, Kento; Schoenfeld, David; Allan, James S.; Madsen, Joren C.; Benichou, Gilles; Smith, Rex-Neal; Colvin, Robert B.; Sachs, David H.; Cosimi, A. Benedict; Kawai, Tatsuo

    2016-01-01

    Successful induction of allograft tolerance has been achieved in nonhuman primates (NHPs) and humans via induction of transient hematopoietic chimerism. Since allograft tolerance was achieved in these recipients without durable chimerism, peripheral mechanisms are postulated to play a major role. Here, we report our studies of T cell immunity in NHP recipients that achieved long-term tolerance versus those that rejected the allograft (AR). All kidney, heart, and lung transplant recipients underwent simultaneous or delayed donor bone marrow transplantation (DBMT) following conditioning with a nonmyeloablative regimen. After DBMT, mixed lymphocyte culture with CFSE consistently revealed donor-specific loss of CD8+ T cell responses in tolerant (TOL) recipients, while marked CD4+ T cell proliferation in response to donor antigens was found to persist. Interestingly, a significant proportion of the proliferated CD4+ cells were FOXP3+ in TOL recipients, but not in AR or naive NHPs. In TOL recipients, CD4+FOXP3+ cell proliferation against donor antigens was greater than that observed against third-party antigens. Finally, the expanded Tregs appeared to be induced Tregs (iTregs) that were converted from non-Tregs. These data provide support for the hypothesis that specific induction of iTregs by donor antigens is key to long-term allograft tolerance induced by transient mixed chimerism. PMID:27446989

  15. Delineation of structural domains involved in the subtype specificity of tachykinin receptors through chimeric formation of substance P/substance K receptors.

    OpenAIRE

    Y. Yokota; Akazawa, C; Ohkubo, H; Nakanishi, S.

    1992-01-01

    The mammalian tachykinin receptors belong to the family of G protein-coupled receptors and consist of the substance P, substance K and neuromedin K receptors (SPR, SKR and NKR). We constructed 14 chimeric receptors in which seven transmembrane segments were sequentially exchanged between the rat SPR and SKR and examined the subtype specificity of the chimeric receptors by radioligand binding and inositol phosphate measurements after transfection into COS cells. All chimeric receptors showed m...

  16. Chimerism Analysis of Cell-Free DNA in Patients Treated with Hematopoietic Stem Cell Transplantation May Predict Early Relapse in Patients with Hematologic Malignancies

    OpenAIRE

    Mahmoud Aljurf; Hala Abalkhail; Amal Alseraihy; Said Y. Mohamed; Mouhab Ayas; Fahad Alsharif; Hazza Alzahrani; Abdullah Al-Jefri; Ghuzayel Aldawsari; Ali Al-Ahmari; Belgaumi, Asim F.; Claudia Ulrike Walter; Hassan El-Solh; Walid Rasheed; Maher Albitar

    2016-01-01

    Background. We studied DNA chimerism in cell-free DNA (cfDNA) in patients treated with HSCT. Methods. Chimerism analysis was performed on CD3+ cells, polymorphonuclear (PMN) cells, and cfDNA using 16 small tandem repeat loci. The resulting labeled PCR-products were size-fractionated and quantified. Results. Analyzing samples from 191 patients treated with HSCT for nonneoplastic hematologic disorders demonstrated that the cfDNA chimerism is comparable to that seen in PMN cells. Analyzing leuke...

  17. Co-transformation of canola by chimeric chitinase and tlp genes towards improving resistance to Sclerotinia sclerotiorum.

    Science.gov (United States)

    Aghazadeh, Rustam; Zamani, Mohammadreza; Motallebi, Mostafa; Moradyar, Mehdi; Moghadassi Jahromi, Zahra

    2016-09-01

    Canola (Brassica napus) plants were co-transformed with two pathogenesis-related protein genes expressing a Trichoderma atroviride chitinase with a chitin-binding domain (chimeric chitinase) and a thaumatin-like protein (tlp) from Oryza sativa conferring resistance to phytopatogenic fungi by Agrobacterium-mediated transformation. The putative transgenic plants were confirmed by PCR. After measuring the specific activity of the chimeric chitinase and glucanase activity for tlp genes, transgenic plants with high specific activity were selected for southern blot analysis to confirm the copy number of the genes. In vitro assays, the antifungal activity of crude extracted protein against Sclerotinia sclerotiorum showed that the inhibition percentage in double transgenic plants was between 55 and 62, whereas the inhibition percentage in single-gene transformants (chimeric chitinase) ranged from 35 to 45 percent. Importantly, in greenhouse conditions, the double transgenic plants showed significant resistance than the single-gene transformant and wild type plants. The results in T2 generation using the intact leaf inoculation method showed that the average lesion diameters were 10, 14.7 and 29 mm for the double transformant, single-gene transformant and non-transgenic plants, respectively. Combined expression of chimeric chitinase and tlp in transgenic plants showed significantly enhanced resistance against S. sclerotiorum than the one that express single-gene transformant plants. These results suggest that the co-expression of chimeric chitinase and tlp can confer enhanced disease resistance in canola plant. PMID:27430511

  18. Construction and evaluation of a chimeric protein made from Fasciola hepatica leucine aminopeptidase and cathepsin L1.

    Science.gov (United States)

    Hernández-Guzmán, K; Sahagún-Ruiz, A; Vallecillo, A J; Cruz-Mendoza, I; Quiroz-Romero, H

    2016-01-01

    Leucine aminopeptidase (LAP) and cathepsin L1 (CL1) are important enzymes for the pathogenesis and physiology of Fasciola hepatica. These enzymes were analysed in silico to design a chimeric protein containing the most antigenic sequences of LAP (GenBank; AAV59016.1; amino acids 192-281) and CL1 (GenBank CAC12806.1; amino acids 173-309). The cloned 681-bp chimeric fragment (rFhLAP-CL1) contains 270 bp from LAP and 411 bp from CL1, comprising three epitopes, DGRVVHLKY (amino acids 54-62) from LAP, VTGYYTVHSGSEVELKNLV (amino acids 119-137) and YQSQTCLPF (amino acids 161-169) from CL1. The ~25 kDa rFhLAP-CL1 chimeric protein was expressed from the pET15b plasmid in the Rosetta (DE3) Escherichia coli strain. The chimeric protein rFhLAP-CL1, which showed antigenic and immunogenic properties, was recognized in Western blot assays using F. hepatica-positive bovine sera, and induced strong, specific antibody responses following immunization in rabbits. The newly generated chimeric protein may be used as a diagnostic tool for detection of antibodies against F. hepatica in bovine sera and as an immunogen to induce protection against bovine fasciolosis. PMID:25274570

  19. Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants

    Energy Technology Data Exchange (ETDEWEB)

    Kawano, Masaaki [Department of Allergy and Immunology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Morikawa, Katsuma [Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501 (Japan); Suda, Tatsuya [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Laboratory for Immunopharmacology of Microbial Products, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Ohno, Naohito [Laboratory for Immunopharmacology of Microbial Products, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Matsushita, Sho [Department of Allergy and Immunology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Allergy Center, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Akatsuka, Toshitaka [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Handa, Hiroshi, E-mail: handa.h.aa@m.titech.ac.jp [Solutions Research Laboratory, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8503 (Japan); Matsui, Masanori, E-mail: mmatsui@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2014-01-05

    Virus-like particles (VLPs) are a promising vaccine platform due to the safety and efficiency. However, it is still unclear whether polyomavirus-based VLPs are useful for this purpose. Here, we attempted to evaluate the potential of polyomavirus VLPs for the antiviral vaccine using simian virus 40 (SV40). We constructed chimeric SV40-VLPs carrying an HLA-A{sup ⁎}02:01-restricted, cytotoxic T lymphocyte (CTL) epitope derived from influenza A virus. HLA-A{sup ⁎}02:01-transgenic mice were then immunized with the chimeric SV40-VLPs. The chimeric SV40-VLPs effectively induced influenza-specific CTLs and heterosubtypic protection against influenza A viruses without the need of adjuvants. Because DNase I treatment of the chimeric SV40-VLPs did not disrupt CTL induction, the intrinsic adjuvant property may not result from DNA contaminants in the VLP preparation. In addition, immunization with the chimeric SV40-VLPs generated long-lasting memory CTLs. We here propose that the chimeric SV40-VLPs harboring an epitope may be a promising CTL-based vaccine platform with self-adjuvant properties. - Highlights: • We constructed chimeric SV40-VLPs carrying an influenza virus-derived CTL epitope. • Chimeric SV40-VLPs induce influenza-specific CTLs in mice without adjuvants. • Chimeric SV40-VLPs induce heterosubtypic protection against influenza A viruses. • Chimeric SV40-VLPs induce long-lasting memory CTLs. • Chimeric SV40-VLPs is a promising vaccine platform with self-adjuvant properties.

  20. Prokaryotic expression and renaturation of engineering chimeric Fab antibody against human hepatoma

    Institute of Scientific and Technical Information of China (English)

    Jin-Liang Xing; Xiang-Min Yang; Xi-Ying Yao; Fei Song; Zhi-Nan Chen

    2004-01-01

    AIM: To express chimeric Fd (cFd) and chimeric light chain (cL) in E.coli respectively and refold them into chimeric Fab (cFab) antibody.METHODS: cFd and cL genes were respectively inserted into the prokaryotic expression vector pET32a to construct recombinant vectors pET32a/cFd and pET32a/cL. Then,the competent E. colicells were transformed by the recombinant vectors and induced by IPTG. Moreover, a large quantity of cFd and cL expression products were prepared and mixed with equal molar to refold into cFab by gradient dialysis. The refolded products were identified and analyzed by sodium SDS-PAGE, Western blotting,ELISA and HPLC.RESULTS: High efficient prokaryotic expressions of both cFd and cL in the form of non-fusion protein were obtained with the expression levels of 28.3% and 32.3% of total bacteria proteins, respectively. Their relative molecular masses were all 24 ku or so, and both of them mainly existed in the form of inclusion bodies. In addition, cFd and cL were successfully refolded into cFab by gradient dialysis, with about 59.45% of recovery when the starting total protein concentration was 100 μg/mL. The renatured cFab could specifically bind to related antigen with high affinity.CONCLUSION: The cFab antibody against human hepatoma was highly and efficiently expressed and refolded, which laid a solid foundation for studying its application in the treatment of hepatoma.

  1. Chimerism induction by nonmyeloablactive preconditioning and bone marrow infusion in rat small bowel transplantation

    Directory of Open Access Journals (Sweden)

    Bakonyi Neto Alexandre

    2003-01-01

    Full Text Available In our previous work we demonstrated that the use of donor specific bone marrow infusions ( DSBMI after small bowel transplantation did not improve the graft survival after a short course of immunossupression. PURPOSE: In the current study, we evaluated whether recipient preconditioning with different regimens of radiation combined with DSBMI may enhance small bowel allograft survival with minimum recipient morbidity. METHODS: Heterotopic small bowel transplantation (SBTx was performed with Lewis rats as recipients and DA rats as donors, which were immunossupressed with a short course of tacrolimus (FK 506 at 1mg/Kg/day for 5 days and distributed in 4 groups: group 1 (n= 4 without both irradiation and DSBMI; Groups 2 (n= 6, 3 (n= 9 and 4 (n= 6 received 100 x 10(6 DSBM cells at the time of the transplant. Groups 3 and 4 were irradiated with 250 and 400 rd respectively. Animals were examined daily for clinical signs of rejection or GVHD. Blood samples were taken weekly for chimeric studies by FC and intestinal biopsies were performed every 2 weeks. RESULTS: Animals in G1 and G2 had minimal rejection at day 15 after SBTx while GVHD was clinically and histologically characterized in G 3 and G 4. Total chimerism and T-cell chimerism was higher in irradiated groups when compared to non-irradiated groups. With exception of G1 and 2 where rejection was the cause of death, all animals in G3 and 4 died of GVHD. CONCLUSION:We concluded that low cytoreductive of irradiation can successfully decrease the graft rejection but not prevent the occurrence of GVHD.

  2. Design and Construction of Chimeric VP8-S2 Antigen for Bovine Rotavirus and Bovine Coronavirus

    Science.gov (United States)

    Nasiri, Khadijeh; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza; Zibaee, Saeed

    2016-01-01

    Purpose: Bovine Rotavirus and Bovine Coronavirus are the most important causes of diarrhea in newborn calves and in some other species such as pigs and sheep. Rotavirus VP8 subunit is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. Methods: In the present study, several prediction programs were used to predict B and T-cells epitopes, secondary and tertiary structures, antigenicity ability and enzymatic degradation sites. Finally, a chimeric antigen was designed using computational techniques. The chimeric VP8-S2 antigen was constructed. It was cloned and sub-cloned into pGH and pET32a(+) expression vector. The recombinant pET32a(+)-VP8-S2 vector was transferred into E.oli BL21CodonPlus (DE3) as expression host. The recombinant VP8-S2 protein was purified by Ni-NTA chromatography column. Results: The results of colony PCR, enzyme digestion and sequencing showed that the VP8-S2 chimeric antigen has been successfully cloned and sub-cloned into pGH and pET32a(+).The results showed that E.coli was able to express VP8-S2 protein appropriately. This protein was expressed by induction of IPTG at concentration of 1mM and it was confirmed by Ni–NTA column, dot-blotting analysis and SDS-PAGE electrophoresis. Conclusion: The results of this study showed that E.coli can be used as an appropriate host to produce the recombinant VP8-S2 protein. This recombinant protein may be suitable to investigate to produce immunoglobulin, recombinant vaccine and diagnostic kit in future studies after it passes biological activity tests in vivo in animal model and or other suitable procedure. PMID:27123423

  3. Chimeric plant virus particles administered nasally or orally induce systemic and mucosal immune responses in mice

    DEFF Research Database (Denmark)

    Brennan, F.R.; Bellaby, T.; Helliwell, S.M.;

    1999-01-01

    The humoral immune responses to the D2 peptide of fibronectin-binding protein B (FnBP) of Staphylococcus aureus, expressed on the plant virus cowpea mosaic virus (CPMV), were evaluated after mucosal delivery to mice. Intranasal immunization of these chimeric virus particles (CVPs), either alone or...... of CVPs to generate antibody at distant mucosal sites. IgG2a and TgG2b were the dominant IgG subclasses in sera to both CPMV and FnBP, demonstrating a bias in the response toward the T helper 1 type. The sera completely inhibited the binding of human fibronectin to the S. aureus FnBP. Oral...

  4. Rituximab chimeric anti-CD20 monoclonal antibody treatment for adult refractory idiopathic thrombocytopenic purpura

    DEFF Research Database (Denmark)

    Braendstrup, Peter; Bjerrum, Ole W; Nielsen, Ove J;

    2005-01-01

    . Recent studies have shown that rituximab, a chimeric anti-CD20 monoclonal antibody, is useful in the treatment of these patients, with overall response rates of about 50%. Most published reports have included a small number patients including case reports. The present study reports the results of a...... retrospective Danish multicenter study of rituximab in the treatment of adult patients with refractory ITP. Thirty-five patients (median age 52 years, range 17-82 years, 17 males) were included. One patient had immune thrombocytopenia and neutropenia. All patients had received prednisolone (Pred). Next to Pred...

  5. Chimeric antigen receptor T cell therapy: 25years in the making.

    Science.gov (United States)

    Gill, Saar; Maus, Marcela V; Porter, David L

    2016-05-01

    Chimeric antigen receptor (CAR) T cell therapy of cancer is generating enormous enthusiasm. Twenty-five years after the concept was first proposed, major advances in molecular biology, virology, and good manufacturing practices (GMP)-grade cell production have transformed antibody-T cell chimeras from a scientific curiosity to a fact of life for academic cellular immunotherapy researchers and, increasingly, for patients. In this review, we explain the preclinical concept, outline how it has been translated to the clinic, and draw lessons from the first years of CAR T cell therapy for the practicing clinician. PMID:26574053

  6. High affinity mouse-human chimeric Fab against Hepatitis B surface antigen

    OpenAIRE

    Bose, Biplab; Khanna, Navin; Acharya, Subrat K; Sinha, Subrata

    2005-01-01

    AIM: Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this a...

  7. T cells expressing chimeric antigen receptors can cause anaphylaxis in humans

    OpenAIRE

    Maus, Marcela V.; Haas, Andrew R; Beatty, Gregory L.; Albelda, Steven M.; Levine, Bruce L.; Liu, Xiaojun; Zhao, Yangbing; Kalos, Michael; June, Carl H.

    2013-01-01

    T cells can be redirected to overcome tolerance to cancer by engineering with integrating vectors to express a chimeric antigen receptor (CAR). In preclinical models, we have previously demonstrated that transfection of T cells with messenger RNA (mRNA) coding for a CAR is an alternative strategy that has antitumor efficacy and the potential to evaluate the on-target off-tumor toxicity of new CAR targets safely due to transient mRNA CAR expression. Here, we report the safety observed in four ...

  8. The chimeric VirA-tar receptor protein is locked into a highly responsive state.

    OpenAIRE

    Turk, S C; van Lange, R P; Sonneveld, E; Hooykaas, P J

    1993-01-01

    The wild-type VirA protein is known to be responsive not only to phenolic compounds but also to sugars via the ChvE protein (G. A. Cangelosi, R. G. Ankenbauer, and E. W. Nester, Proc. Natl. Acad. Sci. USA 87:6708-6712, 1990, and N. Shimoda, A. Toyoda-Yamamoto, J. Nagamine, S. Usami, M. Katayama, Y. Sakagami, and Y. Machida, Proc. Natl. Acad. Sci. USA 87:6684-6688, 1990). It is shown here that the mutant VirA(Ser-44, Arg-45) protein and the chimeric VirA-Tar protein are no longer responsive to...

  9. Multi-petal cyclamen flowers produced by AGAMOUS chimeric repressor expression

    OpenAIRE

    Yuri Tanaka; Yoshimi Oshima; Tomomichi Yamamura; Masao Sugiyama; Nobutaka Mitsuda; Norihiro Ohtsubo; Masaru Ohme-Takagi; Teruhiko Terakawa

    2013-01-01

    Cyclamen persicum (cyclamen) is a commercially valuable, winter-blooming perennial plant. We cloned two cyclamen orthologues of AGAMOUS (AG), CpAG1 and CpAG2, which are mainly expressed in the stamen and carpel, respectively. Cyclamen flowers have 5 petals, but expression of a chimeric repressor of CpAG1 (CpAG1-SRDX) caused stamens to convert into petals, resulting in a flower with 10 petals. By contrast, CpAG2-SRDX only caused incomplete formation of stamens and carpels. Expression in Arabid...

  10. Dizygotic monochorionic twin pregnancy conceived following intracytoplasmic sperm injection treatment and complicated by twin-twin transfusion syndrome and blood chimerism

    DEFF Research Database (Denmark)

    Ekelund, Charlotte Kvist; Skibsted, L.; Søgaard, Kirsten; Main, Katharina Maria; Dziegiel, M.H.; Schwartz, M.; Moeller, N.; Roos, L.; Tabor, A.

    2008-01-01

    were phenotypically a normal male and a normal female. Histology of the placenta showed it to be monochorionic diamniotic. Blood chimerism was found postnatally as both infants had the karyotypes 46,XX[13]/46,XY[17]. Chimerism was not found in cells from a buccal swab at 6 months of age. This is one of...

  11. T-cell chimerism is valuable in predicting early mortality in steroid-resistant acute graft-versus-host disease after myeloablative allogeneic cell transplantation

    DEFF Research Database (Denmark)

    Minculescu, Lia; Madsen, Hans O.; Sengeløv, Henrik

    2014-01-01

    The main aim of this study was to evaluate the impact of early T-cell chimerism status on the incidence and clinical course of acute graft-versus-host disease (aGVHD) in allogeneic transplant recipients after myeloablative conditioning. Of 62 patients, 38 (61%) had complete T-cell donor chimerism...

  12. Pre-clinical evaluation of CD38 chimeric antigen receptor engineered T cells for the treatment of multiple myeloma.

    Science.gov (United States)

    Drent, Esther; Groen, Richard W J; Noort, Willy A; Themeli, Maria; Lammerts van Bueren, Jeroen J; Parren, Paul W H I; Kuball, Jürgen; Sebestyen, Zsolt; Yuan, Huipin; de Bruijn, Joost; van de Donk, Niels W C J; Martens, Anton C M; Lokhorst, Henk M; Mutis, Tuna

    2016-05-01

    Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody sequences to generate second-generation retroviral CD38-chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite becoming CD38-negative during culture. CD38-chimeric antigen receptor-transduced T cells also displayed significant anti-tumor effects in a xenotransplant model, in which multiple myeloma tumors were grown in a human bone marrow-like microenvironment. CD38-chimeric antigen receptor-transduced T cells also appeared to lyse the CD38(+) fractions of CD34(+) hematopoietic progenitor cells, monocytes, natural killer cells, and to a lesser extent T and B cells but did not inhibit the outgrowth of progenitor cells into various myeloid lineages and, furthermore, were effectively controllable with a caspase-9-based suicide gene. These results signify the potential importance of CD38-chimeric antigen receptor-transduced T cells as therapeutic tools for CD38(+) malignancies and warrant further efforts to diminish the undesired effects of this immunotherapy using appropriate strategies. PMID:26858358

  13. In vivo tumor localization and biodistribution in the human tumor xenografts models of an anti-CD71 mouse/human chimeric antibody

    International Nuclear Information System (INIS)

    Objective: In order to investigate the tumor localization and biodistribution of the anti-CD71 mouse/human chimeric antibody (D2C). Methods: The tumor localization and biodistribution of the chimeric antibody (D2C) were observed by labeling the chimeric Ab with radioiodine (131I) and injecting it into nude mice (Balb/c nu/nu) transplanted with human hepatocellular carcinoma cells (SMMC-7721). Results: The labeled chimeric Ab (D2C), with intraperitoneal as well as tumor regional administration, was significantly localized in the tumor and the location of the tumor was successfully visualized by SPECT. The in vivo D2C Ab's biodistribution of organs and tissues showed that non-specific binding in the tumor regional administration was lower than those in the intraperitoneal. Conclusion: The human/mouse chimeric antibody (D2C) can exert in specific tumor localization in vivo and can be utilized for radio-immunoimaging

  14. Cross-presentation of HCMV chimeric protein enables generation and measurement of polyclonal T cells.

    Science.gov (United States)

    Nguyen, Thi H O; Sullivan, Lucy C; Kotsimbos, Tom C; Schwarer, Anthony P; Mifsud, Nicole A

    2010-08-01

    CD8(+) T cell immunity has a critical function in controlling human cytomegalovirus (HCMV) infection. In immunocompromized individuals, HCMV reactivation or disease can lead to increased morbidity and mortality, particularly in transplant recipients. In this setting, adoptive transfer of HCMV-specific CD8(+) T cells is a promising vaccine strategy to restore viral immunity, with most clinical approaches focussing on the use of peptides for the generation of single epitope-specific CD8(+) T cells. We show that using an IE1-pp65 chimeric protein as the antigen source promotes effective cross-presentation, by monocyte-derived dendritic cells (MoDCs), to generate polyclonal CD8(+) T cell epitopes. By exploring human leukocyte antigen (HLA)-restricted immunodominance hierarchies both within and across two immunodominant proteins, we show that HLA-B7 epitopes elicit higher CD8(+) T cell responses compared with HLA-A1, -A2 or -B8. This study provides important evidence highlighting both the efficacy of the IE1-pp65 chimeric protein and the importance of immunodominance in designing future therapeutic vaccines. PMID:20195281

  15. Incorporation of chimeric HIV-SIV-Env and modified HIV-Env proteins into HIV pseudovirions

    International Nuclear Information System (INIS)

    Low level incorporation of the viral glycoprotein (Env) into human immunodeficiency virus (HIV) particles is a major drawback for vaccine strategies against HIV/AIDS in which HIV particles are used as immunogen. Within this study, we have examined two strategies aimed at achieving higher levels of Env incorporation into non-infectious pseudovirions (PVs). First, we have generated chimeric HIV/SIV Env proteins containing the truncated C-terminal tail region of simian immunodeficiency virus (SIV)mac239-Env767stop, which mediates strongly increased incorporation of SIV-Env into SIV particles. In a second strategy, we have employed a truncated HIV-Env protein (Env-Tr752N750K) which we have previously demonstrated to be incorporated into HIV virions, generated in infected T-cells, to a higher level than that of Wt-HIV-Env. Although the chimeric HIV/SIV Env proteins were expressed at the cell surface and induced increased levels of cell-cell fusion in comparison to Wt-HIV-Env, they did not exhibit increased incorporation into either HIV-PVs or SIV-PVs. Only Env-Tr752N750K exhibited significantly higher (threefold) levels of incorporation into HIV-PVs, an improvement, which, although not dramatic, is worthwhile for the large-scale preparation of non-infectious PVs for vaccine studies aimed at inducing Env humoral responses

  16. A Simple Methodology for Conversion of Mouse Monoclonal Antibody to Human-Mouse Chimeric Form

    Directory of Open Access Journals (Sweden)

    Vinh T. Dang

    2013-01-01

    Full Text Available Passive immunotherapy has mainly been used as a therapy against cancer and inflammatory conditions. Recent studies have shown that monoclonal antibody-(mAb- based passive immunotherapy is a promising approach to combat virus infection. Specific mouse mAbs can be routinely generated in large amounts with the use of hybridoma technology but these cannot be used for therapy in human beings due to their immunogenicity. Therefore, the development of chimeric and humanized mAbs is important for therapeutic purpose. This is facilitated by a variety of molecular techniques like recombinant DNA technology and the better understanding of the structure and function of antibody. The human-mouse chimeric forms allow detailed analysis of the mechanism of inhibition and the potential for therapeutic applications. Here, a step-by-step description of the conversion process will be described. The commercial availability of the reagents required in each step means that this experimentation can be easily set up in research laboratories.

  17. Use of recombinant chimeric antigens for the serodiagnosis of Mycoplasma pneumoniae infection.

    Science.gov (United States)

    Montagnani, F; De Paolis, F; Beghetto, E; Gargano, N

    2010-11-01

    In this paper, we have evaluated the diagnostic utility of three antigenic regions of the Mycoplasma pneumoniae P1, P30, and MPN456 gene products in order to replace the soluble, whole-cell bacterial extract in serological assays. Antigenic regions, being previously identified as B-cell epitopes, were used individually or assembled in a recombinant chimeric antigen by genetic engineering. Paired serum samples from 47 patients with M. pneumoniae infection and from 39 subjects with a clinical picture of atypical pneumonia but without a defined diagnosis of M. pneumoniae infection were included. Immunoglobulin G (IgG) antibodies against epitopes carried by recombinant antigens were measured by performing recombinant enzyme-linked immunosorbent assays (Rec-ELISAs). Rec-ELISA results were compared to those obtained by a commercial assay using the whole-cell Mycoplasma antigen. Our study demonstrates that all IgG Rec-ELISAs using recombinant antigens have better sensitivity with respect to the commercial assay. Furthermore, we show that the use of chimeric antigens improve the performance of the assays. The use of recombinant antigens is effective in distinguishing M. pneumoniae-infected patients from uninfected individuals and shows that immunoassays based on recombinant antigens could provide the basis for standardized commercial tests for the serodiagnosis of M. pneumoniae diseases. PMID:20632053

  18. Chimeric piggyBac transposases for genomic targeting in human cells.

    Science.gov (United States)

    Owens, Jesse B; Urschitz, Johann; Stoytchev, Ilko; Dang, Nong C; Stoytcheva, Zoia; Belcaid, Mahdi; Maragathavally, Kommineni J; Coates, Craig J; Segal, David J; Moisyadi, Stefan

    2012-08-01

    Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4-PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24% of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy. PMID:22492708

  19. Establishment of permanent chimerism in a lactate dehydrogenase-deficient mouse mutant with hemolytic anemia

    International Nuclear Information System (INIS)

    Pluripotent hemopoietic stem cell function was investigated in the homozygous muscle type lactate dehydrogenase (LDH-A) mutant mouse using bone marrow transplantation experiments. Hemopoietic tissues of LDH-A mutants showed a marked decreased in enzyme activity that was associated with severe hemolytic anemia. This condition proved to be transplantable into wild type mice (+/+) through total body irradiation (TBI) at a lethal dose of 8.0 Gy followed by engraftment of mutant bone marrow cells. Since the mutants are extremely radiosensitive (lethal dose50/30 4.4 Gy vs 7.3 Gy in +/+ mice), 8.0-Gy TBI followed by injection of even high numbers of normal bone marrow cells did not prevent death within 5-6 days. After a nonlethal dose of 4.0 Gy and grafting of normal bone marrow cells, a transient chimerism showing peripheral blood characteristics of the wild type was produced that returned to the mutant condition within 12 weeks. The transfusion of wild type red blood cells prior to and following 8.0-Gy TBI and reconstitution with wild type bone marrow cells prevented the early death of the mutants and permanent chimerism was achieved. The chimeras showed all hematological parameters of wild type mice, and radiosensitivity returned to normal. It is concluded that the mutant pluripotent stem cells are functionally comparable to normal stem cells, emphasizing the significance of this mouse model for studies of stem cell regulation

  20. Co-receptor and co-stimulation blockade for mixed chimerism and tolerance without myelosuppressive conditioning

    Directory of Open Access Journals (Sweden)

    Fairchild Paul J

    2006-04-01

    Full Text Available Abstract Background A major challenge in the application of marrow transplantation as a route to immunological tolerance of a transplanted organ is to achieve hematopoietic stem cell (HSC engraftment with minimal myelosuppressive treatments. Results We here describe a combined antibody protocol which can achieve long-term engraftment with clinically relevant doses of MHC-mismatched bone marrow, without the need for myelosuppressive drugs. Although not universally applicable in all strains, we achieved reliable engraftment in permissive strains with a two-stage strategy: involving first, treatment with anti-CD8 and anti-CD4 in advance of transplantation; and second, treatment with antibodies targeting CD4, CD8 and CD40L (CD154 at the time of marrow transplantation. Long-term mixed chimerism through co-receptor and co-stimulation blockade facilitated tolerance to donor-type skin grafts, without any evidence of donor-antigen driven regulatory T cells. Conclusion We conclude that antibodies targeting co-receptor and co-stimulatory molecules synergise to enable mixed hematopoietic chimerism and central tolerance, showing that neither cytoreductive conditioning nor 'megadoses' of donor bone marrow are required for donor HSC to engraft in permissive strains.

  1. The identification of a spontaneous 47, XX, +21/46, XY chimeric fetus with male genitalia

    Directory of Open Access Journals (Sweden)

    Lee Kuei-Fang

    2012-09-01

    Full Text Available Abstract Background Approximately 30 sex-chromosome discordant chimera cases have been reported to date, of which only four cases carried trisomy 21. Here, we present an additional case, an aborted fetus with a karyotype of 47,XX, +21/46,XY. Case presentation Autopsy demonstrated that this fetus was normally developed and had male genitalia. Major characteristics of Down syndrome were not observed except an enlarged gap between the first and second toes. Karyotyping of tissues cultured from the fetus revealed the same chimeric chromosomal composition detected in the amniotic fluid but with a different ratio of [47,XX,+21] to [46,XY]. Further short tandem repeat analysis indicated a double paternal contribution and single maternal contribution to the fetus, with the additional chromosome 21 in the [47,XX,+21] cell lineage originating from the paternal side. Conclusion We thus propose that this chimeric fetus was formed via the dispermic fertilization of a parthenogenetic ovum with one (Y sperm and one (X,+21 sperm.

  2. Establishment and characterization of a chimeric infectious cDNA clone of classical swine fever virus.

    Science.gov (United States)

    Zhao, T S; Xia, Y H

    2016-01-01

    Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. There are two important CSFV strains in China, Shimen and hog cholera lapinized virus (HCLV). Shimen strain is highly virulent while HCLV, also referred to as C-strain, is a live attenuated vaccine strain considered to be one of the most effective and safest live vaccines. In this study, a chimeric infectious cDNA clone of CSFV named pT7SM-c was engineered by replacing the Erns genomic region of an infectious clone of CSFV Shimen strain, pT7SM, with the same region obtained from HCLV. RNA transcripts of pT7SM-c containing an engineered EcoRI site that served as a genetic marker were directly infectious in PK15 cells. The rescued virus vT7SM-c showed similar growth kinetics and cytopathic effect with the parental virus vT7SM in the cells. The chimeric infectious cDNA clone can be used as a practical tool for further studying of the virulence, protein function and pathogenesis of CSFV through genetic manipulation. PMID:27265471

  3. Enhanced antigen detection in immunohistochemical staining using a 'digitized' chimeric antibody.

    Science.gov (United States)

    Eng, Hui-Yan; Wang, Cheng-I; Xue, Yuezhen; Lee, Chia-Yin; Zulkifli, Sarah Binte; Chiam, Poh-Cheang; Ghadessy, Farid J; Lane, David P

    2016-01-01

    The immunohistochemical (IHC) staining of mouse tissue sections using antibodies of mouse origin can result in high nonspecific background due to the staining of endogenous immunoglobulins (Igs) by enzyme-conjugated secondary antibodies. In order to obviate this issue, we developed a chimeric mouse-human anti-p53 monoclonal antibody (MH242) by grafting the variable regions of a known mouse antibody into a human Ig scaffold. This facilitated use of an anti-human secondary antibody, and resulted in near-zero background when compared with its parental mouse monoclonal antibody (PAb242). Furthermore, the chimeric antibody enabled reproducible detection of mutant p53 (homozygous R172H) expression in mouse tissue, an observation hitherto largely equivocal based on the use of existing antibodies. The approach we describe leads to the generation of tractable antibody reagents, whose integrity can be readily verified through DNA sequencing of expressor plasmids. The wide-spread adoption of such 'digitized' antibodies should reduce experimental disparities that can commonly arise through variations in antibody quality. PMID:26508747

  4. EspA-Intimin chimeric protein, a candidate vaccine against Escherichia coli O157:H7.

    Directory of Open Access Journals (Sweden)

    Hamid Sedighian Rad

    2013-09-01

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC O157:H7 is an important enteric pathogen in human causing bloody or nonbloody diarrhea, which may be complicated by hemolytic uremic syndrome (HUS. Cattle are an important reservoir of EHEC. This research aims at vaccination with a divalent chimer protein composed of EspA120 and Intimin 282 and its preventive effect of EHEC O157 colonization in mice rectal epithelium.A divalent recombinant EspA-Intimin (EI protein containing EspA120 and Intimin280 attached with a linker was amplified from a trivalent construct and cloned in pET-28a (+ vector. The immunization was conducted in mice after expression and purification of the recombinant EI (rEI.Mice subcutaneously immunized with rEI, elicited significant rEI specific serum IgG antibodies and showed significantly decreased E.coli O157:H7 shedding compared to the control group.The chimeric recombinant protein induced strong humoral response as well as protection against oral challenges with live E.coli O157:H7.

  5. Protection of Mice from Lethal Endotoxemia by Chimeric Human BPI-Fcγ1 Gene Delivery

    Institute of Scientific and Technical Information of China (English)

    Chen Li; Jing Li; Zhe Lv; Xinghua Guo; Qinghua Chen; Qingli Kong; Yunqing An

    2006-01-01

    To evaluate the potentiality of applying gene therapy to endotoxemia in high-risk patients, we investigated the effects of transferring an adeno-associated virus serotype 2 (AAV2)-mediated BPI-Fcγ1 gene on protecting mice from challenge of lethal endotoxin. The chimeric BPI-Fcγ1 gene consists of two parts, one encods functional N-terminus (1 to 199 amino acidic residues) of human BPI, which is a bactericidal/permeability-increasing protein,and the other encodes Fc segment of human immunoglobulin G1 (Fcγ1). Our results indicated that the target protein could be expressed and secreted into the serum of the gene-transferred mice. After lethal endotoxin challenge, the levels of endotoxin and TNF-α in the gene-transferred mice were decreased. The survival rate of the BPI-Fcγ1 gene-transferred mice was markedly increased. Our data suggest that AAV2-mediated chimeric BPI-Fcγ1 gene delivery can potentially be used clinically for the protection and treatment of endotoxemia and endotoxic shock in high-risk individuals.

  6. Development of GR/MR Chimeric Receptors and Their Response to Steroid Hormones

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    We have established an effective and reliable technique of developing GR/MR chimeric receptors by DNA homologous recombination. To develop the method we transformed several different E. coli strains with a linearized plasmid containing full length of mGR(mouse GR) and hormone binding domain(HBD) of rMR(rat MR), the linear DNA undergoes recombination due to the homology of the mGR and the rMR and recircularize , and propagation in E. coli. PCR was performed to screen correct construction in which fusion between GR and MR took place. The constructs were digested with appropriate restriction endonucleases to test probable fusion sites of GR and HBD of MR. Precise fusion sites of GR and MR for constructs AB1157 # 2 , AB1157 # 18, AB 1157 # 22, AB1157 # 32, CMK603 # 6 were verified by DNA sequencing. Trans fection of COS- 7 cells with the constructs and subsequent treatment of transfected COS-7 cells with steroid hormones were carried out, the results showed that the constructs gave response to tested hormones. The study suggested that the GR/MR chimeric receptors can give rise to fusion proteins and their interactive function between hormone and receptor.

  7. Simulating aerosols over Arabian Peninsula with CHIMERE: Sensitivity to soil, surface parameters and anthropogenic emission inventories

    Science.gov (United States)

    Beegum, S. Naseema; Gherboudj, Imen; Chaouch, Naira; Couvidat, Florian; Menut, Laurent; Ghedira, Hosni

    2016-03-01

    A three dimensional chemistry transport model, CHIMERE, was used to simulate the aerosol optical depths (AOD) over the Arabian Peninsula desert with an offline coupling of Weather Research and Forecasting (WRF) model. The simulations were undertaken with: (i) different horizontal and vertical configurations, (ii) new datasets derived for soil/surface properties, and (iii) EDGAR-HTAP anthropogenic emissions inventories. The model performance evaluations were assessed: (i) qualitatively using MODIS (Moderate-Resolution Imaging Spectroradiometer) deep blue (DB) AOD data for the two local dust events of August 6th and 23rd (2013), and (ii) quantitatively using AERONET (Aerosol Robotic Network) AOD observations, CALIPSO (Cloud-Aerosol Lidar and Infrared Pathfinder Satellite Observation) aerosol extinction profiles, and AOD simulations from various forecast models. The model results were observed to be highly sensitive to erodibility and aerodynamic surface roughness length. The use of new datasets on soil erodibility, derived from the MODIS reflectance, and aerodynamic surface roughness length (z0), derived from the ERA-Interim datasets, significantly improved the simulation results. Simulations with the global EDGAR-HTAP anthropogenic emission inventories brought the simulated AOD values closer to the observations. Performance testing of the adapted model for the Arabian Peninsula domain with improved datasets showed good agreement between AERONET AOD measurements and CHIMERE simulations, where the correlation coefficient (R) is 0.6. Higher values of the correlation coefficients and slopes were observed for the dusty periods compared to the non-dusty periods.

  8. Chimeric Mouse model to track the migration of bone marrow derived cells in glioblastoma following anti-angiogenic treatments.

    Science.gov (United States)

    Achyut, B R; Shankar, Adarsh; Iskander, A S M; Ara, Roxan; Knight, Robert A; Scicli, Alfonso G; Arbab, Ali S

    2016-03-01

    Bone marrow derived cells (BMDCs) have been shown to contribute in the tumor development. In vivo animal models to investigate the role of BMDCs in tumor development are poorly explored. We established a novel chimeric mouse model using as low as 5 × 10(6) GFP+ BM cells in athymic nude mice, which resulted in >70% engraftment within 14 d. In addition, chimera was established in NOD-SCID mice, which displayed >70% with in 28 d. Since anti-angiogenic therapies (AAT) were used as an adjuvant against VEGF-VEGFR pathway to normalize blood vessels in glioblastoma (GBM), which resulted into marked hypoxia and recruited BMDCs to the tumor microenvironment (TME). We exploited chimeric mice in athymic nude background to develop orthotopic U251 tumor and tested receptor tyrosine kinase inhibitors and CXCR4 antagonist against GBM. We were able to track GFP+ BMDCs in the tumor brain using highly sensitive multispectral optical imaging instrument. Increased tumor growth associated with the infiltration of GFP+ BMDCs acquiring suppressive myeloid and endothelial phenotypes was seen in TME following treatments. Immunofluorescence study showed GFP+ cells accumulated at the site of VEGF, SDF1 and PDGF expression, and at the periphery of the tumors following treatments. In conclusion, we developed a preclinical chimeric model of GBM and phenotypes of tumor infiltrated BMDCs were investigated in context of AATs. Chimeric mouse model could be used to study detailed cellular and molecular mechanisms of interaction of BMDCs and TME in cancer. PMID:26797476

  9. Fluctuations between multiple EF-G-induced chimeric tRNA states during translocation on the ribosome

    Science.gov (United States)

    Adio, Sarah; Senyushkina, Tamara; Peske, Frank; Fischer, Niels; Wintermeyer, Wolfgang; Rodnina, Marina V.

    2015-06-01

    The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement.

  10. The construction of chimeric T-Cell receptor with spacer base of modeling study of VHH and MUC1 interaction.

    Science.gov (United States)

    Pirooznia, Nazanin; Hasannia, Sadegh; Taghdir, Majid; Rahbarizadeh, Fatemeh; Eskandani, Morteza

    2011-01-01

    Adaptive cell immunotherapy with the use of chimeric receptors leads to the best and most specific response against tumors. Chimeric receptors consist of a signaling fragment, extracellular spacer, costimulating domain, and an antibody. Antibodies cause immunogenicity; therefore, VHH is a good replacement for ScFv in chimeric receptors. Since peptide sequences have an influence on chimeric receptors, the effect of peptide domains on each other's conformation were investigated. CD3Zeta, CD28, VHH and CD8α, and FcgIIα are used as signaling moieties, costimulating domain, antibody, and spacers, respectively. To investigate the influence of the ligation of spacers on the conformational structure of VHH, models of VHH were constructed. Molecular dynamics simulation was run to study the influence of the presence of spacers on the conformational changes in the binding sites of VHH. Root mean square deviation and root mean square fluctuation of critical segments in the binding site showed no noticeable differences with those in the native VHH. Results from molecular docking revealed that the presence of spacer FcgIIα causes an increasing effect on VHH with MUC1 interaction. Each of the constructs was transformed into the Jurkat E6.1. Expression analysis and evaluation of their functions were examined. The results showed good expression and function. PMID:21869862

  11. The Construction of Chimeric T-Cell Receptor with Spacer Base of Modeling Study of VHH and MUC1 Interaction

    Directory of Open Access Journals (Sweden)

    Nazanin Pirooznia

    2011-01-01

    Full Text Available Adaptive cell immunotherapy with the use of chimeric receptors leads to the best and most specific response against tumors. Chimeric receptors consist of a signaling fragment, extracellular spacer, costimulating domain, and an antibody. Antibodies cause immunogenicity; therefore, VHH is a good replacement for ScFv in chimeric receptors. Since peptide sequences have an influence on chimeric receptors, the effect of peptide domains on each other's conformation were investigated. CD3Zeta, CD28, VHH and CD8α, and FcgIIα are used as signaling moieties, costimulating domain, antibody, and spacers, respectively. To investigate the influence of the ligation of spacers on the conformational structure of VHH, models of VHH were constructed. Molecular dynamics simulation was run to study the influence of the presence of spacers on the conformational changes in the binding sites of VHH. Root mean square deviation and root mean square fluctuation of critical segments in the binding site showed no noticeable differences with those in the native VHH. Results from molecular docking revealed that the presence of spacer FcgIIα causes an increasing effect on VHH with MUC1 interaction. Each of the constructs was transformed into the Jurkat E6.1. Expression analysis and evaluation of their functions were examined. The results showed good expression and function.

  12. Chimerism 47,XY,+21/46,XX in a female infant with anencephaly and other congenital defects

    OpenAIRE

    Danielle R. Lucon; Luciene M. Zanchetta; Cavalcanti, Denise P.

    2006-01-01

    Chimerism is rare in humans and is usually discovered accidentally when a 46,XX and 46,XY karyotype is found in a same individual. We describe a malformed female infant with neural tube defect (NTD) and a 47,XY,+21[5]/46,XX[30] karyotype.

  13. Chimerism 47,XY,+21/46,XX in a female infant with anencephaly and other congenital defects

    Directory of Open Access Journals (Sweden)

    Danielle R. Lucon

    2006-01-01

    Full Text Available Chimerism is rare in humans and is usually discovered accidentally when a 46,XX and 46,XY karyotype is found in a same individual. We describe a malformed female infant with neural tube defect (NTD and a 47,XY,+21[5]/46,XX[30] karyotype.

  14. Targeting apoptosis to induce stable mixed hematopoietic chimerism and long-term allograft survival without myelosuppressive conditioning in mice.

    Science.gov (United States)

    Cippà, Pietro E; Gabriel, Sarah S; Chen, Jin; Bardwell, Philip D; Bushell, Andrew; Guimezanes, Annick; Kraus, Anna K; Wekerle, Thomas; Wüthrich, Rudolf P; Fehr, Thomas

    2013-08-29

    Induction of mixed hematopoietic chimerism results in donor-specific immunological tolerance by apoptosis-mediated deletion of donor-reactive lymphocytes. A broad clinical application of this approach is currently hampered by limited predictability and toxicity of the available conditioning protocols. We developed a new therapeutic approach to induce mixed chimerism and tolerance by a direct pharmacological modulation of the intrinsic apoptosis pathway in peripheral T cells. The proapoptotic small-molecule Bcl-2 inhibitor ABT-737 promoted mixed chimerism induction and reversed the antitolerogenic effect of calcineurin inhibitors by boosting the critical role of the proapoptotic Bcl-2 factor Bim. A short conditioning protocol with ABT-737 in combination with costimulation blockade and low-dose cyclosporine A resulted in a complete deletion of peripheral donor-reactive lymphocytes and was sufficient to induce mixed chimerism and robust systemic tolerance across full major histocompatibility complex barriers, without myelosuppression and by using moderate doses of bone marrow cells. Thus, immunological tolerance can be achieved by direct modulation of the intrinsic apoptosis pathway in peripheral lymphocytes-a new approach to translate immunological tolerance into clinically applicable protocols. PMID:23869083

  15. Performance Assessment of Four Chimeric Trypanosoma cruzi Antigens Based on Antigen-Antibody Detection for Diagnosis of Chronic Chagas Disease.

    Science.gov (United States)

    Santos, Fred Luciano Neves; Celedon, Paola Alejandra Fiorani; Zanchin, Nilson Ivo Tonin; Brasil, Tatiana de Arruda Campos; Foti, Leonardo; Souza, Wayner Vieira de; Silva, Edmilson Domingos; Gomes, Yara de Miranda; Krieger, Marco Aurélio

    2016-01-01

    The performance of serologic tests in chronic Chagas disease diagnosis largely depends on the type and quality of the antigen preparations that are used for detection of anti-Trypanosoma cruzi antibodies. Whole-cell T. cruzi extracts or recombinant proteins have shown variation in the performance and cross-reactivity. Synthetic chimeric proteins comprising fragments of repetitive amino acids of several different proteins have been shown to improve assay performances to detect Chagasic infections. Here, we describe the production of four chimeric T. cruzi proteins and the assessment of their performance for diagnostic purposes. Circular Dichroism spectra indicated the absence of well-defined secondary structures, while polydispersity evaluated by Dynamic Light Scattering revealed only minor aggregates in 50 mM carbonate-bicarbonate (pH 9.6), demonstrating that it is an appropriate buffering system for sensitizing microplates. Serum samples from T. cruzi-infected and non-infected individuals were used to assess the performance of these antigens for detecting antibodies against T. cruzi, using both enzyme-linked immunosorbent assay and a liquid bead array platform. Performance parameters (AUC, sensitivity, specificity, accuracy and J index) showed high diagnostic accuracy for all chimeric proteins for detection of specific anti-T. cruzi antibodies and differentiated seropositive individuals from those who were seronegative. Our data suggest that these four chimeric proteins are eligible for phase II studies. PMID:27517281

  16. CHIMERIC WEST NILE/DENGUE VIRUS VACCINE CANDIDATE: PRECLINICAL EVALUATION IN MICE, GEESE, AND MONKEYS FOR SAFETY AND IMMUNOGENICITY

    Science.gov (United States)

    A live attenuated virus vaccine is being developed to protect against West Nile virus (WN) disease in humans. Previously, it was found that chimeric West Nile/dengue viruses (WN/DEN4 and WN/DEN4-delta-30) bearing the membrane precursor and envelope protein genes of WN on a backbone of dengue type 4 ...

  17. Chimeric Foot-and-Mouth Disease Viruses: Evaluation of Their Efficacy as Potential Marker Vaccines in Cattle

    Science.gov (United States)

    Previous work in swine has demonstrated that full protection against Foot-and-Mouth Disease (FMD) can be achieved following vaccination with chimeric Foot-and-Mouth Disease Virus (FMDV) vaccines, whereby the VP1 G-H loop has been substituted with a non-homologous alternative. If proven to be effect...

  18. Cloning, expression, and purification of a highly immunogenic recombinant gonadotropin-releasing hormone (GnRH) chimeric peptide.

    Science.gov (United States)

    Xu, Jinshu; Zhu, Zheng; Duan, Peng; Li, Wenjia; Zhang, Yin; Wu, Jie; Hu, Zhuoyi; Roque, Rouel S; Liu, Jingjing

    2006-12-01

    To design an anti-gonadotropin-releasing hormone (GnRH) vaccine capable of eliciting strong immunogenicity, a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The chimeric peptide called GnRH3-hinge-MVP contained three linear repeats of GnRH (GnRH3), a fragment of the human IgG1 hinge region, and a T-cell epitope of measles virus protein (MVP). The expression plasmid contained the GnRH3-hinge-MVP construct ligated to its fusion partner (AnsB-C) via an unique acid labile Asp-Pro linker. The recombinant fusion protein was expressed in an inclusion body in Escherichia coli under IPTG or lactose induction and the target peptide was easily purified using washing of urea and ethanol precipitation. The target chimeric peptide was isolated from the fusion partner following acid hydrolysis and purified using DEAE-Sephacel chromatography. The purified GnRH3-hinge-MVP was determined to be highly homogeneous by IEF analysis and the N-terminal sequencing. Further, immunization of female mice with the recombinant chimeric peptide resulted in generation of high-titer antibodies specific for GnRH. The results showed that GnRH3-hinge-MVP could be considered as a candidate anti-GnRH vaccine. PMID:17064933

  19. Chimeric HIV-1 envelope glycoproteins with potent intrinsic granulocyte-macrophage colony-stimulating factor (GM-CSF activity.

    Directory of Open Access Journals (Sweden)

    Gözde Isik

    Full Text Available HIV-1 acquisition can be prevented by broadly neutralizing antibodies (BrNAbs that target the envelope glycoprotein complex (Env. An ideal vaccine should therefore be able to induce BrNAbs that can provide immunity over a prolonged period of time, but the low intrinsic immunogenicity of HIV-1 Env makes the elicitation of such BrNAbs challenging. Co-stimulatory molecules can increase the immunogenicity of Env and we have engineered a soluble chimeric Env trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF domain. This chimeric molecule induced enhanced B and helper T cell responses in mice compared to Env without GM-CSF. We studied whether we could optimize the activity of the embedded GM-CSF as well as the antigenic structure of the Env component of the chimeric molecule. We assessed the effect of truncating GM-CSF, removing glycosylation-sites in GM-CSF, and adjusting the linker length between GM-CSF and Env. One of our designed Env(GM-CSF chimeras improved GM-CSF-dependent cell proliferation by 6-fold, reaching the same activity as soluble recombinant GM-CSF. In addition, we incorporated GM-CSF into a cleavable Env trimer and found that insertion of GM-CSF did not compromise Env cleavage, while Env cleavage did not compromise GM-CSF activity. Importantly, these optimized Env(GM-CSF proteins were able to differentiate human monocytes into cells with a macrophage-like phenotype. Chimeric Env(GM-CSF should be useful for improving humoral immunity against HIV-1 and these studies should inform the design of other chimeric proteins.

  20. Application of functional genomics to the chimeric mouse model of HCV infection: optimization of microarray protocols and genomics analysis

    Directory of Open Access Journals (Sweden)

    Smith Maria W

    2006-05-01

    Full Text Available Abstract Background Many model systems of human viral disease involve human-mouse chimeric tissue. One such system is the recently developed SCID-beige/Alb-uPA mouse model of hepatitis C virus (HCV infection which involves a human-mouse chimeric liver. The use of functional genomics to study HCV infection in these chimeric tissues is complicated by the potential cross-hybridization of mouse mRNA on human oligonucleotide microarrays. To identify genes affected by mouse liver mRNA hybridization, mRNA from identical human liver samples labeled with either Cy3 or Cy5 was compared in the presence and absence of known amounts of mouse liver mRNA labeled in only one dye. Results The results indicate that hybridization of mouse mRNA to the corresponding human gene probe on Agilent Human 22 K oligonucleotide microarray does occur. The number of genes affected by such cross-hybridization was subsequently reduced to approximately 300 genes both by increasing the hybridization temperature and using liver samples which contain at least 80% human tissue. In addition, Real Time quantitative RT-PCR using human specific probes was shown to be a valid method to verify the expression level in human cells of known cross-hybridizing genes. Conclusion The identification of genes affected by cross-hybridization of mouse liver RNA on human oligonucleotide microarrays makes it feasible to use functional genomics approaches to study the chimeric SCID-beige/Alb-uPA mouse model of HCV infection. This approach used to study cross-species hybridization on oligonucleotide microarrays can be adapted to other chimeric systems of viral disease to facilitate selective analysis of human gene expression.

  1. An avirulent chimeric Pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus

    International Nuclear Information System (INIS)

    A chimeric Pestivirus was constructed using an infectious cDNA clone of bovine viral diarrhea virus (BVDV) [J. Virol. 70 (1996) 8606]. After deletion of the envelope protein E2-encoding region, the respective sequence of classical swine fever virus (CSFV) strain Alfort 187 was inserted in-frame resulting in plasmid pA/CP7E2alf. After transfection of in vitro-transcribed CP7E2alf RNA, autonomous replication of chimeric RNA in bovine and porcine cell cultures was observed. Efficient growth of chimeric CP7E2alf virus, however, could only be demonstrated on porcine cells, and in contrast to the parental BVDV strain CP7, CP7E2alf only inefficiently infected and propagated in bovine cells. The virulence, immunogenicity, and 'marker vaccine' properties of the generated chimeric CP7E2alf virus were determined in an animal experiment using 27 pigs. After intramuscular inoculation of 1 x 107 TCID50, CP7E2alf proved to be completely avirulent, and neither viremia nor virus transmission to contact animals was observed; however, CSFV-specific neutralizing antibodies were detected from day 11 after inoculation. In addition, sera from all animals reacted positive in an E2-specific CSFV-antibody ELISA, but were negative for CSFV-ERNS-specific antibodies as determined with a CSFV marker ELISA. After challenge infection with highly virulent CSFV strain Eystrup, pigs immunized with CP7E2alf were fully protected against clinical signs of CSFV infection, viremia, and shedding of challenge virus, and almost all animals scored positive in a CSFV marker ELISA. From our results, we conclude that chimeric CP7E2alf may not only serve as a tool for a better understanding of Pestivirus attachment, entry, and assembly, but also represents an innocuous and efficacious modified live CSFV 'marker vaccine'

  2. Expression and immunoactivity of chimeric particulate antigens of receptor binding site-core antigen of hepatitis B virus

    Institute of Scientific and Technical Information of China (English)

    Hai-Jie Yang; Ning-Shao Xia; Min Chen; Tong Cheng; Shui-Zhen He; Shao-Wei Li; Bao-Quan Guan; Zi-Heng Zhu; Ying Gu; Jun Zhang

    2005-01-01

    AIM: To improve the immunogenicity of receptor binding site of hepatitis B virus (HBV) on preS1 antigen using HBV core antigen as an immuno-carrier.METHODS: One to 6 tandem copies of HBV preS1 (21-47)fragment were inserted into HBcAg at the sites of aa 78 and 82, and expressed in E. coli. ELISA, Western blot and animal immunization were used to analyze the antigenicity and immmunogenicity of purified particulate antigens. The ability to capture HBV by antibodies elicited by chimeric partides was detected with immuno-capture PCR.RESULTS: Recombinant antigens CⅠ, CⅡ, CⅢ carrying 1-3 copies of HBV preS1 (21-47) individually could form viruslike particles (VLPs), similar to HBcAg in morphology. But recombinant antigens carrying 4-6 copies of HBV preS1 (21-47) were poorly expressed in E.coli. Chimeric antigens were lacking of immunoreactivity with anti-HBc monoclonal antibodies (McAbs), but still reserved good immunoreactivity with anti-HBe McAbs. CⅠ, CⅡ, CⅢ could strongly react with anti-preS1 McAb, suggesting that preS1 (21-47) fragment was well exposed on the surface of chimeric VLPs. Three chimeric VLP antigens (CⅠ, CⅡ and CⅢ) could stimulate mice to produce high-level antibody responses, and their immunogenicity was stronger than non-particulate antigen 21-47*6, containing 6 copies of preS1 (21-47). Mouse antibodies to CⅠ, CⅡ and CⅢ were able to capture HBV virions in immuno-capture PCR assay in vitro.CONCLUSION: Chimeric particulate antigens of receptor binding site-core antigen of HBV can elicit strong antibody responses to preS1. They have a potential to be developed into prophylactic or therapeutic vaccines against HBV infection.

  3. PRODUCTION IN PICHIA PASTORIS AND CHARACTERIZATION OF GENETIC ENGINEERED CHIMERIC HBV/HEV VIRUS-LIKE PARTICLES

    Institute of Scientific and Technical Information of China (English)

    Hong-zhao Li; Hong-ying Gang; Qiang-ming Sun; Xiao Liu; Yan-bing Ma; Mao-sheng Sun; Chang-bai Dai

    2004-01-01

    Objective To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV)on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg).Methods The gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg.The resulted fusion gene was then integrated through transformation into the genome of Pichiapastoris under the control of a methanol-induced alcohol oxidase 1 (A OX 1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualization.Results The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichiapastoris with an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological, physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg.Conclusion The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.

  4. MHC-mismatched mixed chimerism augments thymic regulatory T-cell production and prevents relapse of EAE in mice.

    Science.gov (United States)

    Wu, Limin; Li, Nainong; Zhang, Mingfeng; Xue, Sheng-Li; Cassady, Kaniel; Lin, Qing; Riggs, Arthur D; Zeng, Defu

    2015-12-29

    Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system with demyelination, axon damage, and paralysis. Induction of mixed chimerism with allogeneic donors has been shown to not cause graft-versus-host disease (GVHD) in animal models and humans. We have reported that induction of MHC-mismatched mixed chimerism can cure autoimmunity in autoimmune NOD mice, but this approach has not yet been tested in animal models of MS, such as experimental autoimmune encephalomyelitis (EAE). Here, we report that MHC-mismatched mixed chimerism with C57BL/6 (H-2(b)) donor in SJL/J (H-2(s)) EAE recipients eliminates clinical symptoms and prevents relapse. This cure is demonstrated by not only disappearance of clinical signs but also reversal of autoimmunity; elimination of infiltrating T, B, and macrophage cells in the spinal cord; and regeneration of myelin sheath. The reversal of autoimmunity is associated with a marked reduction of autoreactivity of CD4(+) T cells and significant increase in the percentage of Foxp3(+) Treg among host-type CD4(+) T cells in the spleen and lymph nodes. The latter is associated with a marked reduction of the percentage of host-type CD4(+)CD8(+) thymocytes and an increase of Treg percentage among the CD4(+)CD8(+) and CD4(+)CD8(-) thymocytes. Thymectomy leads to loss of prevention of EAE relapse by induction of mixed chimerism, although there is a dramatic expansion of host-type Treg cells in the lymph nodes. These results indicate that induction of MHC-mismatched mixed chimerism can restore thymic negative selection of autoreactive CD4(+) T cells, augment production of Foxp3(+) Treg, and cure EAE. PMID:26647186

  5. Efficient, trans-complementing packaging systems for chimeric, pseudoinfectious dengue 2/yellow fever viruses

    International Nuclear Information System (INIS)

    In our previous studies, we have stated to build a new strategy for developing defective, pseudoinfectious flaviviruses (PIVs) and applying them as a new type of vaccine candidates. PIVs combined the efficiency of live vaccines with the safety of inactivated or subunit vaccines. The results of the present work demonstrate further development of chimeric PIVs encoding dengue virus 2 (DEN2V) glycoproteins and yellow fever virus (YFV)-derived replicative machinery as potential vaccine candidates. The newly designed PIVs have synergistically functioning mutations in the prM and NS2A proteins, which abolish processing of the latter proteins and make the defective viruses capable of producing either only noninfectious, immature and/or subviral DEN2V particles. The PIV genomes can be packaged to high titers into infectious virions in vitro using the NS1-deficient YFV helper RNAs, and both PIVs and helpers can then be passaged as two-component genome viruses at an escalating scale.

  6. Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer

    Directory of Open Access Journals (Sweden)

    Marc Cartellieri

    2010-01-01

    Full Text Available CD4+ and CD8+ T lymphocytes are powerful components of adaptive immunity, which essentially contribute to the elimination of tumors. Due to their cytotoxic capacity, T cells emerged as attractive candidates for specific immunotherapy of cancer. A promising approach is the genetic modification of T cells with chimeric antigen receptors (CARs. First generation CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen and a lymphocyte activating signaling chain. The CAR-mediated recognition induces cytokine production and tumor-directed cytotoxicity of T cells. Second and third generation CARs include signal sequences from various costimulatory molecules resulting in enhanced T-cell persistence and sustained antitumor reaction. Clinical trials revealed that the adoptive transfer of T cells engineered with first generation CARs represents a feasible concept for the induction of clinical responses in some tumor patients. However, further improvement is required, which may be achieved by second or third generation CAR-engrafted T cells.

  7. Detecting chimeric 5′/3′UTRs with cross-chromosomal splicing by bioinformatics

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhihua; ZHANG Yong; SHI Baochen; DENG Wei; ZHAO Yi; CHEN Runsheng

    2004-01-01

    The 5′/3′ UTRs of mRNA are crucial in translational regulation, and several serious diseases are believed to be associated with abnormal splicing of these parts of the mRNA sequence. In this work a novel method which uses sequence alignment database searching for detecting chimeric 5′3′ UTRs with cross-chromosomal splicing is reported. Eight highly credible instances of cross-chromosomal splicing have been found using this method, representing additional confirmation of the existence of cross-chromosomal splicing events provided by bioinformatics tools. Since no conserved motif has been found in any of the eight instances, and at the same time current prediction algorithms produce only trivial secondary structures at the "splicing sites", it is not possible to identify any specific signal leading to the splicing.

  8. A recombinant, chimeric tetravalent dengue vaccine candidate based on a dengue virus serotype 2 backbone.

    Science.gov (United States)

    Osorio, Jorge E; Wallace, Derek; Stinchcomb, Dan T

    2016-04-01

    Dengue fever is caused by infection with one of four dengue virus (DENV) serotypes (DENV-1-4), necessitating tetravalent dengue vaccines that can induce protection against all four DENV. Takeda's live attenuated tetravalent dengue vaccine candidate (TDV) comprises an attenuated DENV-2 strain plus chimeric viruses containing the prM and E genes of DENV-1, -3 and -4 cloned into the attenuated DENV-2 'backbone'. In Phase 1 and 2 studies, TDV was well tolerated by children and adults aged 1.5-45 years, irrespective of prior dengue exposure; mild injection-site symptoms were the most common adverse events. TDV induced neutralizing antibody responses and seroconversion to all four DENV as well as cross-reactive T cell-mediated responses that may be necessary for broad protection against dengue fever. PMID:26635182

  9. Chimeric viruses blur the borders between the major groups of eukaryotic single-stranded DNA viruses.

    Science.gov (United States)

    Roux, Simon; Enault, François; Bronner, Gisèle; Vaulot, Daniel; Forterre, Patrick; Krupovic, Mart

    2013-01-01

    Metagenomic studies have uncovered an astonishing diversity of ssDNA viruses encoding replication proteins (Reps) related to those of eukaryotic Circoviridae, Geminiviridae or Nanoviridae; however, exact evolutionary relationships among these viruses remain obscure. Recently, a unique chimeric virus (CHIV) genome, which has apparently emerged via recombination between ssRNA and ssDNA viruses, has been discovered. Here we report on the assembly of 13 new CHIV genomes recovered from various environments. Our results indicate a single event of capsid protein (CP) gene capture from an RNA virus in the history of this virus group. The domestication of the CP gene was followed by an unprecedented recurrent replacement of the Rep genes in CHIVs with distant counterparts from diverse ssDNA viruses. We suggest that parasitic and symbiotic interactions between unicellular eukaryotes were central for the emergence of CHIVs and that such turbulent evolution was primarily dictated by incongruence between the CP and Rep proteins. PMID:24193254

  10. Chimeric plant virus particles administered nasally or orally induce systemic and mucosal immune responses in mice

    DEFF Research Database (Denmark)

    Brennan, F.R.; Bellaby, T.; Helliwell, S.M.; Jones, T.D.; Kamstrup, Søren; Dalsgaard, Kristian; Flock, J.I.; Hamilton, W.D.O.

    1999-01-01

    of CVPs to generate antibody at distant mucosal sites. IgG2a and TgG2b were the dominant IgG subclasses in sera to both CPMV and FnBP, demonstrating a bias in the response toward the T helper 1 type. The sera completely inhibited the binding of human fibronectin to the S. aureus FnBP. Oral......The humoral immune responses to the D2 peptide of fibronectin-binding protein B (FnBP) of Staphylococcus aureus, expressed on the plant virus cowpea mosaic virus (CPMV), were evaluated after mucosal delivery to mice. Intranasal immunization of these chimeric virus particles (CVPs), either alone or....... These studies demonstrate for the first time that recombinant plant viruses have potential as mucosal vaccines without the requirement for adjuvant and that the nasal route is most effective for the delivery of these nonreplicating particles....

  11. Case of 46,XX/47,XY, +21 chimerism in a newborn infant with ambiguous genitalia

    Energy Technology Data Exchange (ETDEWEB)

    Sawai, Tomoko; Yoshimoto, Masaaki; Kinoshita, Ei-ichi; Baba, Tsuneyoshi; Matsumoto, Tadashi; Tsuji, Yoshiro, Niikawa, Norio [Nagasaki Univ. School of Medicine, Nagasaki (Japan); Fukuda, Shinpei [Ohmura Municipal Hospital, Ohmura (Japan); Harada, Naoki [Kyushu Medical Science, Nagasaki (Japan)

    1994-02-15

    The authors describe the whole-body chimerism in a newborn infant with small phallus, pseudo-vaginal perineal hypospadias, and a bifid scrotum containing gonads. The human testis determining factor gene (SRY) was detected by PCR amplification. GTG-banding chromosome analysis in peripheral blood lymphocytes and cultured fibroblasts derived from right cubital skin showed a 46,XX/47,XY, +21 karyotype. Their ratios in each cell line were 294:5 and 178:7, respectively. QFQ-banding chromosome analysis documented 3 heteromorphic satellites on trisomic chromsomes 21 in the 47,XY,+21 cell line and a homozygous satellite pattern in the 46,XX cell line. Heteromorphic patterns of chromsomes 4, 13, 14, and 22 were also different between the two cell lines. To our knowledge, such disomy/trisomy chimeras have not been described previously. 10 refs., 3 figs.

  12. Giant trochanteric pressure sore: Use of a pedicled chimeric perforator flap for cover

    Directory of Open Access Journals (Sweden)

    Mehrotra Sandeep

    2009-01-01

    Full Text Available Pressure sores are increasing in frequency commensurate with an ageing population with multi-system disorders and trauma. Numerous classic options are described for providing stable wound cover. With the burgeoning knowledge on perforator anatomy, recent approaches focus on the use of perforator-based flaps in bedsore surgery. A giant neglected trochanteric pressure sore in a paraplegic is presented. Since conventional options of reconstruction appeared remote, the massive ulcer was successfully managed by a chimeric perforator-based flap. The combined muscle and fasciocutaneous flaps were raised as separate paddles based on the anterolateral thigh perforator branches and provided stable cover without complications. Perforators allow versatility in managing complex wounds without compromising on established principles.

  13. Improving therapy of chronic lymphocytic leukemia with chimeric antigen receptor T cells.

    Science.gov (United States)

    Fraietta, Joseph A; Schwab, Robert D; Maus, Marcela V

    2016-04-01

    Adoptive cell immunotherapy for the treatment of chronic lymphocytic leukemia (CLL) has heralded a new era of synthetic biology. The infusion of genetically engineered, autologous chimeric antigen receptor (CAR) T cells directed against CD19 expressed by normal and malignant B cells represents a novel approach to cancer therapy. The results of recent clinical trials of CAR T cells in relapsed and refractory CLL have demonstrated long-term disease-free remissions, underscoring the power of harnessing and redirecting the immune system against cancer. This review will briefly summarize T-cell therapies in development for CLL disease. We discuss the role of T-cell function and phenotype, T-cell culture optimization, CAR design, and approaches to potentiate the survival and anti-tumor effects of infused lymphocytes. Future efforts will focus on improving the efficacy of CAR T cells for the treatment of CLL and incorporating adoptive cell immunotherapy into standard medical management of CLL. PMID:27040708

  14. Discovery of mitochondrial chimeric-gene associated with cytoplasmic male sterility of HL-rice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The mitochondrial genome libraries of HL-type sterile line(A) and maintainer line(B) have been constructed.Mitochondrial gene, atp6, was used to screen libraries, due to the different Southern and Northern blot results between sterile and maintainer line. Sequencing analysis of positive clones proved that there were two copies of atp6 gene in sterile line and only one in maintainer line. One copy of atpt6 in sterile line was same to that in maintainer line; the other showed different flanking sequence from the 49th nucleotide downstream of the termination codon of atp6 gene. A new chimeric gene, orfH79, was found in the region. OrfH79 had homology to mitochondrial gene coxⅡ and orfl07, and was special to HL-sterile cytoplasm.``

  15. Chimeric External Control to Quantify Cell Free DNA in Plasma Samples by Real Time PCR

    Science.gov (United States)

    Eini, Maryam; Behzad-Behbahani, Abbas; Takhshid, Mohammad Ali; Ramezani, Amin; Rafiei Dehbidi, Gholam Reza; Okhovat, Mohammad Ali; Farhadi, Ali; Alavi, Parniyan

    2016-01-01

    Background: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA (cfDNA) is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control (EDC) is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for monitoring stability of cfDNA concentration in blood samples over time. Methods: A0 DNA fragment of 167 bp chimeric sequence of parvovirus B19 and pBHA designated as EDC fragment was designed. To determine the impact of different factors during DNA extraction processing on quantification of cfDNA, blood samples were collected from normal subjects and divided into aliquots with and without specific treatment. In time intervals, the plasma samples were isolated. The amplicon of 167 bp EDC fragment in final concentration of 1.1 pg/500 μl was added to each plasma sample and total DNA was extracted by an in house method. Relative and absolute quantification real time PCR was performed to quantify both EDC fragment and cfDNA in extracted samples. Results: Comparison of real time PCR threshold cycle (Ct) for cfDNA fragment in tubes with and without specific treatment indicated a decrease in untreated tubes. In contrast, the threshold cycle was constant for EDC fragment in treated and untreated tubes, indicating the difference in Ct values of the cfDNA is because of specific treatments that were made on them. Conclusions: Spiking of DNA fragment size relevant to cfDNA into the plasma sample can be useful to minimize the bias due to sample preparation and extraction processing. Therefore, it is highly recommended that standard external DNA control be employed for the extraction and quantification of cfDNA for accurate data analysis.

  16. Synthetic metabolic engineering-a novel, simple technology for designing a chimeric metabolic pathway

    Directory of Open Access Journals (Sweden)

    Ye Xiaoting

    2012-09-01

    Full Text Available Abstract Background The integration of biotechnology into chemical manufacturing has been recognized as a key technology to build a sustainable society. However, the practical applications of biocatalytic chemical conversions are often restricted due to their complexities involving the unpredictability of product yield and the troublesome controls in fermentation processes. One of the possible strategies to overcome these limitations is to eliminate the use of living microorganisms and to use only enzymes involved in the metabolic pathway. Use of recombinant mesophiles producing thermophilic enzymes at high temperature results in denaturation of indigenous proteins and elimination of undesired side reactions; consequently, highly selective and stable biocatalytic modules can be readily prepared. By rationally combining those modules together, artificial synthetic pathways specialized for chemical manufacturing could be designed and constructed. Results A chimeric Embden-Meyerhof (EM pathway with balanced consumption and regeneration of ATP and ADP was constructed by using nine recombinant E. coli strains overproducing either one of the seven glycolytic enzymes of Thermus thermophilus, the cofactor-independent phosphoglycerate mutase of Pyrococcus horikoshii, or the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase of Thermococcus kodakarensis. By coupling this pathway with the Thermus malate/lactate dehydrogenase, a stoichiometric amount of lactate was produced from glucose with an overall ATP turnover number of 31. Conclusions In this study, a novel and simple technology for flexible design of a bespoke metabolic pathway was developed. The concept has been testified via a non-ATP-forming chimeric EM pathway. We designated this technology as “synthetic metabolic engineering”. Our technology is, in principle, applicable to all thermophilic enzymes as long as they can be functionally expressed in the host, and thus would be

  17. Fibrinogen interaction of CHO cells expressing chimeric αIIb/αvβ3 integrin

    Institute of Scientific and Technical Information of China (English)

    Juan-juan CHEN; Xiao-yu SU; Xiao-dong XI; Li-ping LIN; Jian DING; He LU

    2008-01-01

    Aim: The molecular mechanisms of the affinity regulation of αvβ3 integrin are important in tumor development, wound repairing, and angiogenesis. It has been established that the cytoplasmic domains of αvβ3 integrin play an important role in integrin-ligand affinity regulation. However, the relationship of structure-func-tion within these domains remains unclear. Methods: The extracellular and trans-membrane domain of αⅡb was fused to the αv integrin cytoplasmic domain, and the chimeric α subunit was coexpressed in Chinese hamster ovary (CHO) cells with the wild-type β3 subunit or with 3 mutant 133 sequences bearing truncations at the positions of T741, Y747, and F754, respectively. The CHO cells expressing these recombinant integrins were tested for soluble fibrinogen binding and the cell adhesion and spreading on immobilized fibrinogen. Results: All 4 types of integrins bound soluble fibrinogen in the absence of agonist stimulation, and only the cells expressing the chimeric α subunit with the wild-type β3 subunit, but not those with truncated β3, could adhere to and spread on immobilized fibrinogen. Conclusion: The substitution αⅡb at the cytoplasmic domain with the ctv cyto-plasmic sequence rendered the extracellular αⅡbβ3 a constitutively activated con-formation for ligands without the need of "inside-out" signals. Our results also indicated that the COOH-terminal sequence of β3 might play a key role in integrin αⅡb/αvβ3-mediated cell adhesion and spreading on immobilized fibrinogen. The cells expressing αⅡb/αvβ3 have enormous potential for facilitating drug screen-ing for antagonists either to αvβ3 intracellular interactions or to αⅡbβ3 receptor functions.

  18. Enhancement by dimethyl myleran of donor type chimerism in murine recipients of bone marrow allografts

    International Nuclear Information System (INIS)

    A major problem in using murine models for studies of bone marrow allograft rejection in leukemia patients is the narrow margin in which graft rejection can be analyzed. In mice irradiated with greater than 9 Gy total body irradiation (TBI) rejection is minimal, whereas after administration of 8 Gy TBI, which spares a significant number of clonable T cells, a substantial frequency of host stem cells can also be detected. In current murine models, unlike in humans, bone marrow allograft rejection is generally associated with full autologous hematopoietic reconstitution. In the present study, we investigated the effect of the myeloablative drug dimethyl myleran (DMM) on chimerism status following transplantation of T cell-depleted allogenic bone marrow (using C57BL/6 donors and C3H/HeJ recipients, conditioned with 8 Gy TBI). Donor type chimerism 1 to 2 months post-transplant of 1 to 3 x 10(6) bone marrow cells was markedly enhanced by using DMM one day after TBI and prior to transplantation. Conditioning with cyclophosphamide instead of DMM, in combination with 8 Gy TBI, did not enhance engraftment of donor type cells. Artificial reconstitution of T cells, after conditioning with TBI plus DMM, by adding mature thymocytes, or presensitization with irradiated donor type spleen cells 1 week before TBI and DMM, led to strong graft rejection and consequently to severe anemia. The anti-donor responses in these models were proportional to the number of added T cells and to the number of cells used for presensitization, and they could be neutralized by increasing the bone marrow inoculum

  19. Enhanced protective efficacy of a chimeric form of the schistosomiasis vaccine antigen Sm-TSP-2.

    Directory of Open Access Journals (Sweden)

    Mark S Pearson

    Full Text Available The large extracellular loop of the Schistosoma mansoni tetraspanin, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. Moreover, Sm-TSP-2 is uniquely recognised by IgG(1 and IgG(3 from putatively resistant individuals resident in S. mansoni endemic areas in Brazil. In the present study, we expressed Sm-TSP-2 at high yield and in soluble form in E. coli without the need for a solubility enhancing fusion partner. We also expressed in E. coli a chimera called Sm-TSP-2/5B, which consisted of Sm-TSP-2 fused to the immunogenic 5B region of the hookworm aspartic protease and vaccine antigen, Na-APR-1. Sm-TSP-2 formulated with alum/CpG showed significant reductions in adult worm and liver egg burdens in two separate murine schistosomiasis challenge studies. Sm-TSP-2/5B afforded significantly greater protection than Sm-TSP-2 alone when both antigens were formulated with alum/CpG. The enhanced protection obtained with the chimeric fusion protein was associated with increased production of anti-Sm-TSP-2 antibodies and IL-4, IL-10 and IFN-γ from spleen cells of vaccinated animals. Sera from 666 individuals from Brazil who were infected with S. mansoni were screened for potentially deleterious IgE responses to Sm-TSP-2. Anti-Sm-TSP-2 IgE to this protein was not detected (also shown previously for Na-APR-1, suggesting that the chimeric antigen Sm-TSP-2/5B could be used to safely and effectively vaccinate people in areas where schistosomes and hookworms are endemic.

  20. Enhanced protective efficacy of a chimeric form of the schistosomiasis vaccine antigen Sm-TSP-2.

    Science.gov (United States)

    Pearson, Mark S; Pickering, Darren A; McSorley, Henry J; Bethony, Jeffrey M; Tribolet, Leon; Dougall, Annette M; Hotez, Peter J; Loukas, Alex

    2012-01-01

    The large extracellular loop of the Schistosoma mansoni tetraspanin, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. Moreover, Sm-TSP-2 is uniquely recognised by IgG(1) and IgG(3) from putatively resistant individuals resident in S. mansoni endemic areas in Brazil. In the present study, we expressed Sm-TSP-2 at high yield and in soluble form in E. coli without the need for a solubility enhancing fusion partner. We also expressed in E. coli a chimera called Sm-TSP-2/5B, which consisted of Sm-TSP-2 fused to the immunogenic 5B region of the hookworm aspartic protease and vaccine antigen, Na-APR-1. Sm-TSP-2 formulated with alum/CpG showed significant reductions in adult worm and liver egg burdens in two separate murine schistosomiasis challenge studies. Sm-TSP-2/5B afforded significantly greater protection than Sm-TSP-2 alone when both antigens were formulated with alum/CpG. The enhanced protection obtained with the chimeric fusion protein was associated with increased production of anti-Sm-TSP-2 antibodies and IL-4, IL-10 and IFN-γ from spleen cells of vaccinated animals. Sera from 666 individuals from Brazil who were infected with S. mansoni were screened for potentially deleterious IgE responses to Sm-TSP-2. Anti-Sm-TSP-2 IgE to this protein was not detected (also shown previously for Na-APR-1), suggesting that the chimeric antigen Sm-TSP-2/5B could be used to safely and effectively vaccinate people in areas where schistosomes and hookworms are endemic. PMID:22428079

  1. Comparing regional modeling (CHIMERE) and satellite observations of aerosols (PARASOL): Methodology and case study over Mexico

    Science.gov (United States)

    Stromatas, Stavros

    2010-05-01

    S. Stromatas (1), S. Turquety (1), H. Chepfer (1), L. Menut (1), B. Bessagnet (2), JC Pere (2), D. Tanré (3) . (1) Laboratoire de Météorologie Dynamique, CNRS/IPSL, École Polytechnique, 91128 Palaiseau Cedex, France, (2) INERIS, Institut National de l'Environnement Industriel et des Risques, Parc technologique ALATA, 60550 Verneuil en Halatte, FRANCE, (3) Laboratoire d'Optique Atmosphérique/CNRS Univ. des Sciences et Tech. de Lille, 59650 - Villeneuve d'Ascq, France. Atmospheric suspended particles (aerosols) have significant radiative and environmental impacts, affecting human health, visibility and climate. Therefore, they are regulated by air quality standards worldwide, and monitored by regional observation networks. Satellite observations vastly improve the horizontal and temporal coverage, providing daily distributions. Aerosols are currently estimated using aerosol optical depth (AOD) retrievals, a quantitative measure of the extinction of solar radiation by aerosol scattering and absorption between the point of observation and the top of the atmosphere. Even though remarkable progresses in aerosol modeling by chemistry-transport models (CTM) and measurement experiments have been made in recent years, there is still a significant divergence between the modeled and observed results. However, AOD retrievals from satellites remains a highly challenging task mostly because it depends on a variety of different parameters such as cloud contamination, surface reflectance contributions and a priori assumptions on aerosol types, each one of them incorporating its own difficulties. Therefore, comparisons between CTM and observations are often difficult to interpret. In this presentation, we will discuss comparisons between regional modeling (CHIMERE CTM) over Mexico and satellite observations obtained by the POLDER instrument embarked on PARASOL micro-satellite. After a comparison of the model AOD with the retrieved L2 AOD, we will present an alternative

  2. Local and systemic immune responses induced by a recombinant chimeric protein containing Mycoplasma hyopneumoniae antigens fused to the B subunit of Escherichia coli heat-labile enterotoxin LTB.

    Science.gov (United States)

    Marchioro, Silvana Beutinger; Fisch, Andressa; Gomes, Charles K; Jorge, Sérgio; Galli, Vanessa; Haesebrouck, Freddy; Maes, Dominiek; Dellagostin, Odir; Conceição, Fabricio R

    2014-09-17

    A multi-antigen chimera composed of three antigens of Mycoplasma hyopneumoniae (R1, P42, and NrdF) and the mucosal adjuvant Escherichia coli heat-labile enterotoxin B subunit (LTB) was constructed, and its antigenic and immunogenic properties were evaluated in mice and pigs. In addition, we compared the effect of the fusion and co-administration of these proteins in mice. Antibodies against each subunit recognized the chimeric protein. Intranasal and intramuscular immunization of mice with the chimeric protein significantly increased IgG and IgA levels in the serum and tracheobronchial lavages, respectively, against some of the antigens present in the chimeric. Swine immunized with the chimeric protein developed an immune response against all M. hyopneumoniae antigens present in the fusion with a statistically significant difference (Phyopneumoniae infection. PMID:25091529

  3. An Unusual Chimeric Diterpene Synthase from Emericella variecolor and Its Functional Conversion into a Sesterterpene Synthase by Domain Swapping.

    Science.gov (United States)

    Qin, Bin; Matsuda, Yudai; Mori, Takahiro; Okada, Masahiro; Quan, Zhiyang; Mitsuhashi, Takaaki; Wakimoto, Toshiyuki; Abe, Ikuro

    2016-01-01

    Di- and sesterterpene synthases produce C20 and C25 isoprenoid scaffolds from geranylgeranyl pyrophosphate (GGPP) and geranylfarnesyl pyrophosphate (GFPP), respectively. By genome mining of the fungus Emericella variecolor, we identified a multitasking chimeric terpene synthase, EvVS, which has terpene cyclase (TC) and prenyltransferase (PT) domains. Heterologous gene expression in Aspergillus oryzae led to the isolation of variediene (1), a novel tricyclic diterpene hydrocarbon. Intriguingly, in vitro reaction with the enzyme afforded the new macrocyclic sesterterpene 2 as a minor product from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The TC domain thus produces the diterpene 1 and the sesterterpene 2 from GGPP and GFPP, respectively. Notably, a domain swap of the PT domain of EvVS with that of another chimeric sesterterpene synthase, EvSS, successfully resulted in the production of 2 in vivo as well. Cyclization mechanisms for the production of these two compounds are proposed. PMID:26546087

  4. ReX: A suite of computational tools for the design, visualization, and analysis of chimeric protein libraries.

    Science.gov (United States)

    Huang, Weiliang; Johnston, Wayne A; Boden, Mikael; Gillam, Elizabeth M J

    2016-02-01

    Directed evolution has greatly facilitated protein engineering and provided new insights into protein structure-function relationships. DNA shuffling using restriction enzymes is a particularly simple and cost-effective means of recombinatorial evolution that is well within the capability of most molecular biologists, but tools for the design and analysis of such experiments are limited. Here we introduce a suite of freely available online tools to make the construction and analysis of chimeric libraries readily accessible to the novice. REcut (http://qpmf.rx.umaryland.edu/REcut.html) facilitates the choice of DNA fragmentation strategy, while Xover (http://qpmf.rx.umaryland.edu/Xover.html) analyzes chimeric mutants to reveal recombination patterns and extract quantitative data. PMID:26842355

  5. A Chimeric Protein That Functions as both an Anthrax Dual-Target Antitoxin and a Trivalent Vaccine▿

    OpenAIRE

    Wu, Gaobing; Hong, Yuzhi; Guo, Aizhen; Feng, Chunfang; Cao, Sha; Zhang, Cheng-Cai; Shi, Ruiping; Tan, Yadi; Liu, Ziduo

    2010-01-01

    Effective measures for the prophylaxis and treatment of anthrax are still required for counteracting the threat posed by inhalation anthrax. In this study, we first demonstrated that the chimeric protein LFn-PA, created by fusing the protective antigen (PA)-binding domain of lethal factor (LFn) to PA, retained the functions of the respective molecules. On the basis of this observation, we attempted to develop an antitoxin that targets the binding of lethal factor (LF) and/or edema factor (EF)...

  6. MURINE MOBILIZED PERIPHERAL BLOOD STEM CELLS HAVE A LOWER CAPACITY THAN BONE MARROW TO INDUCE MIXED CHIMERISM AND TOLERANCE

    OpenAIRE

    Koporc, Zvonimir; Pilat, Nina; Nierlich, Patrick; Blaha, Peter; Bigenzahn, Sinda; Pree, Ines; Selzer, Edgar; Sykes, Megan; Muehlbacher, Ferdinand; Wekerle, Thomas

    2008-01-01

    Allogeneic bone marrow transplantation (BMT) under costimulation blockade allows induction of mixed chimerism and tolerance without global T cell depletion. The mildest such protocols without recipient cytoreduction, however, require clinically impracticable bone marrow (BM) doses. The successful use of mobilized peripheral blood stem cells (PBSC) instead of BM in such regimens would provide a substantial advance, allowing transplantation of higher doses of hematopoietic donor cells. We thus ...

  7. Chimeric β-lactamases: global conservation of parental function and fast time-scale dynamics with increased slow motions.

    Directory of Open Access Journals (Sweden)

    Christopher M Clouthier

    Full Text Available Enzyme engineering has been facilitated by recombination of close homologues, followed by functional screening. In one such effort, chimeras of two class-A β-lactamases - TEM-1 and PSE-4 - were created according to structure-guided protein recombination and selected for their capacity to promote bacterial proliferation in the presence of ampicillin (Voigt et al., Nat. Struct. Biol. 2002 9:553. To provide a more detailed assessment of the effects of protein recombination on the structure and function of the resulting chimeric enzymes, we characterized a series of functional TEM-1/PSE-4 chimeras possessing between 17 and 92 substitutions relative to TEM-1 β-lactamase. Circular dichroism and thermal scanning fluorimetry revealed that the chimeras were generally well folded. Despite harbouring important sequence variation relative to either of the two 'parental' β-lactamases, the chimeric β-lactamases displayed substrate recognition spectra and reactivity similar to their most closely-related parent. To gain further insight into the changes induced by chimerization, the chimera with 17 substitutions was investigated by NMR spin relaxation. While high order was conserved on the ps-ns timescale, a hallmark of class A β-lactamases, evidence of additional slow motions on the µs-ms timescale was extracted from model-free calculations. This is consistent with the greater number of resonances that could not be assigned in this chimera relative to the parental β-lactamases, and is consistent with this well-folded and functional chimeric β-lactamase displaying increased slow time-scale motions.

  8. Broad neutralization of calcium-permeable amyloid pore channels with a chimeric Alzheimer/Parkinson peptide targeting brain gangliosides.

    Science.gov (United States)

    Di Scala, Coralie; Yahi, Nouara; Flores, Alessandra; Boutemeur, Sonia; Kourdougli, Nazim; Chahinian, Henri; Fantini, Jacques

    2016-02-01

    Growing evidence supports a role for brain gangliosides in the pathogenesis of neurodegenerative diseases including Alzheimer's and Parkinson's. Recently we deciphered the ganglioside-recognition code controlling specific ganglioside binding to Alzheimer's β-amyloid (Aβ1-42) peptide and Parkinson's disease-associated protein α-synuclein. Cracking this code allowed us to engineer a short chimeric Aβ/α-synuclein peptide that recognizes all brain gangliosides. Here we show that ganglioside-deprived neural cells do no longer sustain the formation of zinc-sensitive amyloid pore channels induced by either Aβ1-42 or α-synuclein, as assessed by single-cell Ca(2+) fluorescence microscopy. Thus, amyloid channel formation, now considered a key step in neurodegeneration, is a ganglioside-dependent process. Nanomolar concentrations of chimeric peptide competitively inhibited amyloid pore formation induced by Aβ1-42 or α-synuclein in cultured neural cells. Moreover, this peptide abrogated the intracellular calcium increases induced by Parkinson's-associated mutant forms of α-synuclein (A30P, E46K and A53T). The chimeric peptide also prevented the deleterious effects of Aβ1-42 on synaptic vesicle trafficking and decreased the Aβ1-42-induced impairment of spontaneous activity in rat hippocampal slices. Taken together, these data show that the chimeric peptide has broad anti-amyloid pore activity, suggesting that a common therapeutic strategy based on the prevention of amyloid-ganglioside interactions is a reachable goal for both Alzheimer's and Parkinson's diseases. PMID:26655601

  9. Inducible Expression of Chimeric EWS/ETS Proteins Confers Ewing's Family Tumor-Like Phenotypes to Human Mesenchymal Progenitor Cells

    OpenAIRE

    Miyagawa, Yoshitaka; Okita, Hajime; Nakaijima, Hideki; Horiuchi, Yasuomi; Sato, Ban; TAGUCHI, Tomoko; Toyoda, Masashi; Katagiri, Yohko U; Fujimoto, Junichiro; Hata, Jun-Ichi; Umezawa, Akihiro; Kiyokawa, Nobutaka

    2008-01-01

    Ewing's family tumor (EFT) is a rare pediatric tumor of unclear origin that occurs in bone and soft tissue. Specific chromosomal translocations found in EFT cause EWS to fuse to a subset of ets transcription factor genes (ETS), generating chimeric EWS/ETS proteins. These proteins are believed to play a crucial role in the onset and progression of EFT. However, the mechanisms responsible for the EWS/ETS-mediated onset remain unclear. Here we report the establishment of a tetracycline-controlle...

  10. Using Chimeric Hypoviruses To Fine-Tune the Interaction between a Pathogenic Fungus and Its Plant Host

    OpenAIRE

    CHEN, BAOSHAN; Geletka, Lynn M.; Nuss, Donald L.

    2000-01-01

    Infectious cDNA clones of mild (CHV1-Euro7) and severe (CHV1-EP713) hypovirus strains responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica were used to construct viable chimeric viruses. Differences in virus-mediated alterations of fungal colony morphology, growth rate, and canker morphology were mapped to a region of open reading frame B extending from nucleotides 2,363 to 9,904. By swapping domains within this region, it was possible t...

  11. Cellular and humoral immune responses to chimeric EGFP-pseudocapsids derived from the mouse polyomavirus after their intranasal administration

    Czech Academy of Sciences Publication Activity Database

    Frič, Jan; Marek, M.; Hrušková, V.; Holáň, Vladimír; Forstová, J.

    2008-01-01

    Roč. 26, č. 26 (2008), s. 3242-3251. ISSN 0264-410X R&D Projects: GA MŠk 1M0506; GA MŠk LC545 Grant ostatní: GA Mšk(CZ) 1M0508 Institutional research plan: CEZ:AV0Z50520514 Keywords : mouse polyomavirus pseudocapsids * chimeric VLPs * antigen carrier and adjuvant Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.298, year: 2008

  12. Chimeric Rabies Virus-Like Particles Containing Membrane-Anchored GM-CSF Enhances the Immune Response against Rabies Virus

    OpenAIRE

    Hongtao Kang; Yinglin Qi; Hualei Wang; Xuexing Zheng; Yuwei Gao; Nan Li; Songtao Yang; Xianzhu Xia

    2015-01-01

    Rabies remains an important public health threat in most developing countries. To develop a more effective and safe vaccine against rabies, we have constructed a chimeric rabies virus-like particle (VLP), which containing glycoprotein (G) and matrix protein (M) of rabies virus (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain, and membrane-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF), and it was named of EVLP-G. The immunogenicity and protective efficacy of EVLP-G against R...

  13. Mutation of the Fiber Shaft Heparan Sulphate Binding Site of a 5/3 Chimeric Adenovirus Reduces Liver Tropism

    OpenAIRE

    Koski, Anniina; Karli, Eerika; Kipar, Anja; Escutenaire, Sophie; Kanerva, Anna; Hemminki, Akseli

    2013-01-01

    Natural tropism to the liver is a major obstacle in systemic delivery of adenoviruses in cancer gene therapy. Adenovirus binding to soluble coagulation factors and to cellular heparan sulphate proteoglycans via the fiber shaft KKTK domain are suggested to cause liver tropism. Serotype 5 adenovirus constructs with mutated KKTK regions exhibit liver detargeting, but they also transduce tumors less efficiently, possibly due to altered fiber conformation. We constructed Ad5/3lucS*, a 5/3 chimeric...

  14. Hematopoietic microenvironment. Origin, lineage, and transplantability of the stromal cells in long-term bone marrow cultures from chimeric mice.

    OpenAIRE

    Perkins, S; Fleischman, R A

    1988-01-01

    Studies of bone marrow transplant patients have suggested that the stromal cells of the in vitro hematopoietic microenvironment are transplantable into conditioned recipients. Moreover, in patients with myeloproliferative disorders, all of the stromal cells, which include presumptive endothelial cells, appear to be derived from hematopoietic precursors. To confirm these findings, we have constructed two chimeric mouse models: (a) traditional radiation chimeras, and (b) fetal chimeras, produce...

  15. Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR) expression by flow cytometry

    OpenAIRE

    Zheng Zhili; Chinnasamy Nachimuthu; Morgan Richard A

    2012-01-01

    Abstract Background There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR) for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymp...

  16. Regulation of expression of two LY-6 family genes by intron retention and transcription induced chimerism

    Directory of Open Access Journals (Sweden)

    Mallya Meera

    2008-09-01

    Full Text Available Abstract Background Regulation of the expression of particular genes can rely on mechanisms that are different from classical transcriptional and translational control. The LY6G5B and LY6G6D genes encode LY-6 domain proteins, whose expression seems to be regulated in an original fashion, consisting of an intron retention event which generates, through an early premature stop codon, a non-coding transcript, preventing expression in most cell lines and tissues. Results The MHC LY-6 non-coding transcripts have shown to be stable and very abundant in the cell, and not subject to Nonsense Mediated Decay (NMD. This retention event appears not to be solely dependent on intron features, because in the case of LY6G5B, when the intron is inserted in the artificial context of a luciferase expression plasmid, it is fully spliced but strongly stabilises the resulting luciferase transcript. In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript. EST database analysis revealed that these genes have an alternative expression pathway with the formation of Transcription Induced Chimeras (TIC. This data was confirmed by RT-PCR, revealing the presence of different transcripts that would encode the chimeric proteins CSNKβ-LY6G5B and G6F-LY6G6D, in which the LY-6 domain would join to a kinase domain and an Ig-like domain, respectively. Conclusion In conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms. In addition, these genes form chimeric transcripts with their neighbouring same orientation 5' genes. Of interest is the fact that the 5' genes (CSNKβ or G6F undergo differential splicing only in the context of the chimera (CSNKβ-LY6G5B or G6F-LY6G6C and not on their own.

  17. Assessing the ammonium nitrate formation regime in the Paris megacity and its representation in the CHIMERE model

    Science.gov (United States)

    Petetin, Hervé; Sciare, Jean; Beekmann, Matthias; Sanchez, Olivier; Rosso, Amandine; Denier van der Gon, Hugo

    2014-05-01

    Ammonium nitrates significantly contribute to the fine particulate matter load, in particular in the Paris agglomeration where two measurement campaigns, PARTICULES and FRANCIPOL, have recently made available a large database on this compound and its gaseous precursors, nitric acid and ammonia. These new observations give the opportunity (for the first time in France) to assess the ammonium nitrate formation regime (in terms of limited species) as well as the ability of the CHIMERE chemistry-transport model to simulate each species and to reproduce in fine the observed regime. Quite satisfactory results are obtained on nitrates, mainly due to a significant contribution of imports from outside the agglomeration. However, significant biases affect both gaseous precursors. Various uncertainty sources are discussed, including those relative to ammonia trafic and agricultural emissions, thermodynamic equilibria or oxidative capacity of the atmosphere. Despite these errors, CHIMERE manages to simulate a HNO3-limited regime, in agreement with observations, at least at the daily scale. This study especially confirms that further work on the OH radical characterization in the CHIMERE model and agricultural ammonia emissions are required to improve the simulation of the ammonium nitrate formation regime.

  18. Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

    Energy Technology Data Exchange (ETDEWEB)

    Iri-Sofla, Farnoush Jafari [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Rahbarizadeh, Fatemeh, E-mail: rahbarif@modares.ac.ir [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Ahmadvand, Davoud [Center of Pharmaceutical Nanotechnology and Nanotoxicology, Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen O (Denmark); Rasaee, Mohammad J. [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of)

    2011-11-01

    The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3{zeta}/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of Fc{gamma}RII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.

  19. Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

    International Nuclear Information System (INIS)

    The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3ζ/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of FcγRII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.

  20. Functional participation of a nifH-arsA2 chimeric fusion gene in arsenic reduction by Escherichia coli

    International Nuclear Information System (INIS)

    The NifH (dimer) and ArsA proteins are structural homologs and share common motifs like nucleotide-binding domains, signal transduction domains and also possible similar metal center ligands. Given the similarity between two proteins, we investigated if the NifH protein from Azotobacter vinelandii could functionally substitute for the ArsA1 half of the ArsA protein of Escherichia coli. The chimeric NifH-ArsA2 protein was expressed and detected in the E. coli strain by Western blotting. Growth comparisons of E. coli strains containing plasmids encoding for complete ArsA, partial ArsA (ArsA2) or chimeric ArsA (NifH-ArsA2) in media with increasing sodium arsenite concentrations (0-5 mM) showed that the chimeric NifH-ArsA2 could substitute for the ArsA. This functional complementation demonstrated the strong conservation of essential domains that have been maintained in NifH and ArsA even after their divergence to perform varied functions

  1. Construction of an allogenic chimeric mouse model for the study of the behaviors of donor stem cells in vivo

    Institute of Scientific and Technical Information of China (English)

    WANG Mo-lin; YAN Jing-bin; XIAO Yan-ping; HUANG Shu-zhen

    2005-01-01

    Background It is essential to establish an animal model for the elucidation of the biological behaviors of stem cells in vivo. We constructed a chimeric animal model by in utero transplantation for investigation of stem cell transplantation.Methods This chimerism was achieved by injecting the stem cells derived from the bone marrow of green fluorescence protein (GFP)-transgenic mice into fetal mice at 13.5 days of gestation. Several methods such as polymerase chain reaction (PCR), real-time PCR, fluorescence-assisted cell sorting (FACS) and fluorescence in situ hybridization (FISH) were used for the observation of donor cells.Results Under a fluorescence microscope, we observed the GFP cells of donor-origin in a recipient. PCR, FACS analysis and FISH indicated chimerism at various intervals. Real-time PCR indicated that some donor cells existed in chimera for more than 6 months.Conclusions Allogenic stem cells may exist in recipients for a long time and this allogenic animal model provides a useful tool for studying the behavior of hematopoietic stem cells and also offers an effective model system for the study of stem cells.

  2. Replication of a chimeric origin containing elements from Epstein-Barr virus ori P and bovine papillomavirus minimal origin.

    Science.gov (United States)

    Kivimäe, S; Allikas, A; Kurg, R; Ustav, M

    2001-05-01

    The bovine papillomavirus E2 protein is a multifunctional protein that activates viral transcription, co-operates in initiation of viral DNA replication, and is required for long-term episomal maintenance of viral genomes. The EBNA1 protein of Epstein-Barr virus is required for synthesis and maintenance of Epstein-Barr virus genomes. Both viral proteins act through direct interactions with their respective DNA sequences in their origins of replication. The chimeric protein E2:EBNA1, which consists of an transactivation domain of E2 and DNA binding domain of EBNA1 supported the replication of the chimeric origin that contained EBNA1 binding sites in place of the E2 binding sites principally as full-length E2 did in the case of papillomavirus minimal origin. This indicates that the chimeric protein E2:EBNA1 is competent to assemble a replication complex similar to the E2 protein. These data confirm the earlier observations that the only part of E2 specifically required for its activity in replication is the N-terminal activation domain and the function of the DNA binding domain of E2 in the initiation of replication is to tether the transactivation domain of E2 to the origin of replication. PMID:11311423

  3. Ag85A/ESAT-6 chimeric DNA vaccine induces an adverse response in tuberculosis-infected mice

    Science.gov (United States)

    Liang, Yan; Bai, Xuejuang; Zhang, Junxian; Song, Jingying; Yang, Yourong; Yu, Qi; Li, Ning; Wu, Xueqiong

    2016-01-01

    The Mycobacterium tuberculosis (M. tb) antigens encoded by the 6 kDa early secretory antigenic target (esat-6) and antigen 85A (ag85a) genes are known to exert protective effects against tuberculosis in animal models. In addition, these antigens represent vaccine components that were tested in early human clinical trials. In the present study, a chimeric DNA vaccine was constructed that contained two copies of the esat-6 gene inserted into the ag85a gene from M. tb. BALB/c mice were treated with this chimeric vaccine following infection with either M. tb H37Rv or a clinical multi drug resistant tuberculosis isolate. Treatment of both groups of mice with the chimeric vaccine resulted in accelerated mortality. These findings are in contrast with previous results, which indicated that DNA vaccines expressing the individual antigens were either beneficial or at least not harmful. The results of the present study suggested that the ESAT-6 antigen is not suitable for inclusion in therapeutic vaccines. PMID:27279275

  4. Identification of two amino acids within E2 important for the pathogenicity of chimeric classical swine fever virus.

    Science.gov (United States)

    Wu, Rui; Li, Ling; Zhao, Yu; Tu, Jun; Pan, Zishu

    2016-01-01

    Our previous study demonstrated that a chimeric classical swine fever virus (CSFV) vSM/CE2 containing the E2 gene of the vaccine C-strain on the genetic background of the virulent CSFV strain Shimen (vSM) was attenuated in swine but reversed to virulence after serial passages in PK15 cells. To investigate the molecular basis of the pathogenicity, the genome of the 11th passage vSM/CE2 variant (vSM/CE2-p11) was sequenced, and two amino acid mutations, T745I and M979K, within E2 of vSM/CE2-p11 were observed. Based on reverse genetic manipulation of the chimeric cDNA clone pSM/CE2, the mutated viruses vSM/CE2/T745I, vSMCE2/M979K and vSM/CE2/T745I;M979K were rescued. The data from infection of pigs demonstrated that the M979K amino acid substitution was responsible for pathogenicity. Studies in vitro indicated that T745I and M979K increased infectious virus production and replication. Our results indicated that two residues located at sites 745 and 979 within E2 play a key role in determining the replication in vitro and pathogenicity in vivo of chimeric CSFV vSM/CE2. PMID:26454191

  5. A chimeric protein of aluminum-activated malate transporter generated from wheat and Arabidopsis shows enhanced response to trivalent cations.

    Science.gov (United States)

    Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Ryan, Peter R; Yamamoto, Yoko

    2016-07-01

    TaALMT1 from wheat (Triticum aestivum) and AtALMT1 from Arabidopsis thaliana encode aluminum (Al)-activated malate transporters, which confer acid-soil tolerance by releasing malate from roots. Chimeric proteins from TaALMT1 and AtALMT1 (Ta::At, At::Ta) were previously analyzed in Xenopus laevis oocytes. Those studies showed that Al could activate malate efflux from the Ta::At chimera but not from At::Ta. Here, functions of TaALMT1, AtALMT1 and the chimeric protein Ta::At were compared in cultured tobacco BY-2 cells. We focused on the sensitivity and specificity of their activation by trivalent cations. The activation of malate efflux by Al was at least two-fold greater in the chimera than the native proteins. All proteins were also activated by lanthanides (erbium, ytterbium, gadolinium, and lanthanum), but the chimera again released more malate than TaALMT1 or AtALMT1. In Xenopus oocytes, Al, ytterbium, and erbium activated inward currents from the native TaALMT1 and the chimeric protein, but gadolinium only activated currents from the chimera. Lanthanum inhibited currents from both proteins. These results demonstrated that function of the chimera protein was altered compared to the native proteins and was more responsive to a range of trivalent cations when expressed in plant cells. PMID:27039280

  6. DPPC/poly(2-methyl-2-oxazoline)-grad-poly(2-phenyl-2-oxazoline) chimeric nanostructures as potential drug nanocarriers

    Energy Technology Data Exchange (ETDEWEB)

    Pippa, Natassa [Faculty of Pharmacy, National and Kapodistrian University of Athens, Department of Pharmaceutical Technology (Greece); Kaditi, Eleni; Pispas, Stergios [Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation (Greece); Demetzos, Costas, E-mail: demetzos@pharm.uoa.gr [Faculty of Pharmacy, National and Kapodistrian University of Athens, Department of Pharmaceutical Technology (Greece)

    2013-06-15

    In this study, we report on the self assembly behavior and on stability studies of mixed (chimeric) nanosystems consisting of dipalmitoylphosphatidylcholine (DPPC) and poly(2-methyl-2-oxazoline)-grad-poly(2-phenyl-2-oxazoline) (MPOx) gradient copolymer in aqueous media and in fetal bovine serum (FBS). A gamut of light scattering techniques and fluorescence spectroscopy were used in order to extract information on the size and morphological characteristics of the nanoassemblies formed, as a function of gradient block copolymer content, as well as temperature. The hydrodynamic radii (R{sub h}) of nanoassemblies decreased in the process of heating up to 50 Degree-Sign C, while the fractal dimension (d{sub f}) values, also increased. Indomethacin was successfully incorporated into these chimeric nanocarriers. Drug release was depended on the components ratio. The present studies show that there are a number of parameters that can be used in order to alter the properties of chimeric nanosystems, and this is advantageous to the development of 'smart' nanocarriers for drug delivery.

  7. Cloning, purification, crystallization and preliminary X-ray analysis of a chimeric NADPH-cytochrome P450 reductase

    International Nuclear Information System (INIS)

    A 2.5 Å resolution data set was collected from a crystal of a soluble chimeric form of NADPH-cytochrome P450 reductase (CPR) produced using a fusion gene composed of the yeast FMN and the human FAD domains. The chimeric protein was crystallized in a modified conformation compared with the previously solved structures. NADPH-cytochrome P450 reductase (CPR) is the favoured redox partner of microsomal cytochromes P450. This protein is composed of two flavin-containing domains (FMN and FAD) connected by a structured linker. An active CPR chimera consisting of the yeast FMN and human FAD domains has been produced, purified and crystallized. The crystals belonged to the monoclinic space group C2 and contained one molecule per asymmetric unit. Molecular replacement was performed using the published rat and yeast structures as search models. The initial electron-density maps revealed that the chimeric enzyme had crystallized in a conformation that differed from those of previously solved structures

  8. Analysis of beta-globin mutations shows stable mixed chimerism in patients with thalassemia after bone marrow transplantation.

    Science.gov (United States)

    Kapelushnik, J; Or, R; Filon, D; Nagler, A; Cividalli, G; Aker, M; Naparstek, E; Slavin, S; Oppenheim, A

    1995-10-15

    Beta-thalassemia major (TM) is caused by any of approximately 150 mutations within the beta-globin gene. To establish the degree of chimerism after bone marrow transplantation (BMT), we have performed molecular analysis of beta-globin mutations in 14 patients with TM over a period of 10 years. All patients underwent T cell-depleted allogeneic BMT from HLA-identical related donors, using either in vitro T-cell depletion with CAMPATH 1M and complement or in vivo depletion using CAMPATH 1G in the bone marrow collection bag. To date, at different time periods after BMT, seven patients have some degree of chimerism; six of these patients, all blood transfusion-independent, have donor cells in the range of 70% to 95%, with stable mixed chimerism (MC). The seventh patient has less than 10% donor cells with, surprisingly, only minimal transfusion requirements. The detection of beta-globin gene point mutation, as used here, is a highly specific and sensitive marker for engraftment and MC in patients with thalassemia. In light of its specificity, the method is applicable in all cases of TM, as it is independent of sex and other non-globin-related DNA markers. The high incidence of MC found in our patients may be a consequence of the pre-BMT T-cell depletion. Because MC was associated with transfusion independence, complete eradication of residual host cells for effective treatment of TM and possibly other genetic diseases may prove not to be essential. PMID:7579421

  9. Construction, Expression and Characterization of a Chimeric Protein Targeting Carcinoembryonic Antigen in Lung Cancer

    Institute of Scientific and Technical Information of China (English)

    LI Yang; HUA Shu-cheng; MA Cheng-yuan; YU Zhen-xiang; XU Li-jun; LI Dan; SUN Li-li; LI Xiao; PENG Li-ping

    2011-01-01

    The carcinoembryonic antigen(CEA) is an oncofetal glycoprotein known as an important clinical tumor marker and is overexpressed in several types of tumors, including colorectal and lung carcinomas. We constructed a chimeric protein that exhibits both specific binding and immune stimulating activities, by fusing staphylococcal enterotoxin A(SEA) to the C-terminus of an anti-CEA single-chain disulfide-stabilized Fv(scdsFv) antibody (single-chain-C-terminus/SEA, SC-C/SEA). The SC-C/SEA protein was expressed in Escherichia coli(E. coli), refolded, and purified on an immobilized Ni2+ affinity chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot analysis reveal that the target protein was expressed sufficiently. We used immunofluorescence assays to demonstrate that SC-C/SEA could bind specifically to human lung carcinoma cells(A549), but almost human uterine cervix cells(HeLa). We also used the L-lactate dehydrogenase(LDH) release assay to show that SC-C/SEA elicits a strong A549 tumor-specific cytotoxic T lymphocyte(CTL) response in vitro. The results suggest that SC-C/SEA shows specific activity against CEA-positive cells and has potential application in CEA-targeted cancer immunotherapy.

  10. Chimeric DNA Vaccines against ErbB2+ Carcinomas: From Mice to Humans

    International Nuclear Information System (INIS)

    DNA vaccination exploits a relatively simple and flexible technique to generate an immune response against microbial and tumor-associated antigens (TAAs). Its effectiveness is enhanced by the application of an electrical shock in the area of plasmid injection (electroporation). In our studies we exploited a sophisticated electroporation device approved for clinical use (Cliniporator, IGEA, Carpi, Italy). As the target antigen is an additional factor that dramatically modulates the efficacy of a vaccine, we selected ErbB2 receptor as a target since it is an ideal oncoantigen. It is overexpressed on the cell membrane by several carcinomas for which it plays an essential role in driving their progression. Most oncoantigens are self-tolerated molecules. To circumvent immune tolerance we generated two plasmids (RHuT and HuRT) coding for chimeric rat/human ErbB2 proteins. Their immunogenicity was compared in wild type mice naturally tolerant for mouse ErbB2, and in transgenic mice that are also tolerant for rat or human ErbB2. In several of these mice, RHuT and HuRT elicited a stronger anti-tumor response than plasmids coding for fully human or fully rat ErbB2. The ability of heterologous moiety to blunt immune tolerance could be exploited to elicit a significant immune response in patients. A clinical trial to delay the recurrence of ErbB2+ carcinomas of the oral cavity, oropharynx and hypopharynx is awaiting the approval of the Italian authorities

  11. Assessment of fetal cell chimerism in transgenic pig lines generated by Sleeping beauty transposition.

    Science.gov (United States)

    Garrels, Wiebke; Holler, Stephanie; Taylor, Ulrike; Herrmann, Doris; Niemann, Heiner; Ivics, Zoltan; Kues, Wilfried A

    2014-01-01

    Human cells migrate between mother and fetus during pregnancy and persist in the respective host for long-term after birth. Fetal microchimerism occurs also in twins sharing a common placenta or chorion. Whether microchimerism occurs in multiparous mammals such as the domestic pig, where fetuses have separate placentas and chorions, is not well understood. Here, we assessed cell chimerism in litters of wild-type sows inseminated with semen of transposon transgenic boars. Segregation of three independent monomeric transposons ensured an excess of transgenic over non-transgenic offspring in every litter. Transgenic siblings (n = 35) showed robust ubiquitous expression of the reporter transposon encoding a fluorescent protein, and provided an unique resource to assess a potential cell trafficking to non-transgenic littermates (n = 7) or mothers (n = 4). Sensitive flow cytometry, fluorescence microscopy, and real-time PCR provided no evidence for microchimerism in porcine littermates, or piglets and their mothers in both blood and solid organs. These data indicate that the epitheliochorial structure of the porcine placenta effectively prevents cellular exchange during gestation. PMID:24811124

  12. Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

    Science.gov (United States)

    Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

    2000-04-01

    Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy. PMID:10811469

  13. Design of Switchable Chimeric Antigen Receptor T Cells Targeting Breast Cancer.

    Science.gov (United States)

    Cao, Yu; Rodgers, David T; Du, Juanjuan; Ahmad, Insha; Hampton, Eric N; Ma, Jennifer S Y; Mazagova, Magdalena; Choi, Sei-Hyun; Yun, Hwa Young; Xiao, Han; Yang, Pengyu; Luo, Xiaozhou; Lim, Reyna K V; Pugh, Holly M; Wang, Feng; Kazane, Stephanie A; Wright, Timothy M; Kim, Chan Hyuk; Schultz, Peter G; Young, Travis S

    2016-06-20

    Chimeric antigen receptor T (CAR-T) cells have demonstrated promising results against hematological malignancies, but have encountered significant challenges in translation to solid tumors. To overcome these hurdles, we have developed a switchable CAR-T cell platform in which the activity of the engineered cell is controlled by dosage of an antibody-based switch. Herein, we apply this approach to Her2-expressing breast cancers by engineering switch molecules through site-specific incorporation of FITC or grafting of a peptide neo-epitope (PNE) into the anti-Her2 antibody trastuzumab (clone 4D5). We demonstrate that both switch formats can be readily optimized to redirect CAR-T cells (specific for the corresponding FITC or PNE) to Her2-expressing tumor cells, and afford dose-titratable activation of CAR-T cells ex vivo and complete clearance of the tumor in rodent xenograft models. This strategy may facilitate the application of immunotherapy to solid tumors by affording comparable efficacy with improved safety owing to switch-based control of the CAR-T response. PMID:27145250

  14. Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

    Science.gov (United States)

    Liu, Z.; Xia, M.; Poovaiah, B. W.

    1998-01-01

    cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

  15. Chimeric DNA Vaccines against ErbB2{sup +} Carcinomas: From Mice to Humans

    Energy Technology Data Exchange (ETDEWEB)

    Quaglino, Elena; Riccardo, Federica; Macagno, Marco; Bandini, Silvio; Cojoca, Rodica; Ercole, Elisabetta [Molecular Biotechnology Center, Department of Clinical and Biological Sciences, University of Turin, 10126 Turin (Italy); Amici, Augusto [Department of Molecular Cellular and Animal Biology, University of Camerino, 62032 Camerino (Italy); Cavallo, Federica, E-mail: federica.cavallo@unito.it [2 Department of Molecular Cellular and Animal Biology, University of Camerino, 62032 Camerino (Italy)

    2011-08-10

    DNA vaccination exploits a relatively simple and flexible technique to generate an immune response against microbial and tumor-associated antigens (TAAs). Its effectiveness is enhanced by the application of an electrical shock in the area of plasmid injection (electroporation). In our studies we exploited a sophisticated electroporation device approved for clinical use (Cliniporator, IGEA, Carpi, Italy). As the target antigen is an additional factor that dramatically modulates the efficacy of a vaccine, we selected ErbB2 receptor as a target since it is an ideal oncoantigen. It is overexpressed on the cell membrane by several carcinomas for which it plays an essential role in driving their progression. Most oncoantigens are self-tolerated molecules. To circumvent immune tolerance we generated two plasmids (RHuT and HuRT) coding for chimeric rat/human ErbB2 proteins. Their immunogenicity was compared in wild type mice naturally tolerant for mouse ErbB2, and in transgenic mice that are also tolerant for rat or human ErbB2. In several of these mice, RHuT and HuRT elicited a stronger anti-tumor response than plasmids coding for fully human or fully rat ErbB2. The ability of heterologous moiety to blunt immune tolerance could be exploited to elicit a significant immune response in patients. A clinical trial to delay the recurrence of ErbB2{sup +} carcinomas of the oral cavity, oropharynx and hypopharynx is awaiting the approval of the Italian authorities.

  16. Plant-derived chimeric virus particles for the diagnosis of primary Sjögren syndrome

    Directory of Open Access Journals (Sweden)

    Elisa eTinazzi

    2015-12-01

    Full Text Available Plants are ideal for the production of protein-based nanomaterials because they synthesize and assemble complex multimeric proteins that cannot be expressed efficiently using other platforms. Plant viruses can be thought of as self-replicating proteinaceous nanomaterials generally stable and easily produced in high titers. We used Potato virus X (PVX chimeric virus particles (CVPs and Cowpea mosaic virus (CPMV empty virus-like particles (eVLPs to display a linear peptide (lipo derived from human lipocalin , which is immunodominant in Sjögren’s syndrome (SjS and is thus recognized by autoantibodies in SjS patient serum. These virus-derived nanoparticles (VNPs were thus used to develop a diagnostic assay for SjS based on a direct enzyme linked immunosorbent assay (ELISA format. We found that PVX-lipo formulations were more sensitive than the chemically synthesized immunodominant peptide and equally specific when used to distinguish between healthy individuals and SjS patients. Our novel assay therefore allows the diagnosis of SjS using a simple, low-invasive serum test, contrasting with the invasive labial biopsy required for current tests. Our results demonstrate that nanomaterials based on plant viruses can be used as diagnostic reagents for SjS, and could also be developed for the diagnosis of other diseases.

  17. Plant-Derived Chimeric Virus Particles for the Diagnosis of Primary Sjögren Syndrome.

    Science.gov (United States)

    Tinazzi, Elisa; Merlin, Matilde; Bason, Caterina; Beri, Ruggero; Zampieri, Roberta; Lico, Chiara; Bartoloni, Elena; Puccetti, Antonio; Lunardi, Claudio; Pezzotti, Mario; Avesani, Linda

    2015-01-01

    Plants are ideal for the production of protein-based nanomaterials because they synthesize and assemble complex multimeric proteins that cannot be expressed efficiently using other platforms. Plant viruses can be thought of as self-replicating proteinaceous nanomaterials generally stable and easily produced in high titers. We used Potato virus X (PVX), chimeric virus particles, and Cowpea mosaic virus, empty virus-like particles to display a linear peptide (lipo) derived from human lipocalin, which is immunodominant in Sjögren's syndrome (SjS) and is thus recognized by autoantibodies in SjS patient serum. These virus-derived nanoparticles were thus used to develop a diagnostic assay for SjS based on a direct enzyme linked immunosorbent assay format. We found that PVX-lipo formulations were more sensitive than the chemically synthesized immunodominant peptide and equally specific when used to distinguish between healthy individuals and SjS patients. Our novel assay therefore allows the diagnosis of SjS using a simple, low-invasive serum test, contrasting with the invasive labial biopsy required for current tests. Our results demonstrate that nanomaterials based on plant viruses can be used as diagnostic reagents for SjS, and could also be developed for the diagnosis of other diseases. PMID:26648961

  18. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    Science.gov (United States)

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

  19. Homogeneized modeling of mineral dust emissions over Europe and Africa using the CHIMERE model

    Directory of Open Access Journals (Sweden)

    R. Briant

    2014-05-01

    Full Text Available In the region including Africa and Europe, the main part of mineral dust emissions is observed in Africa. The particles are thus transported towards Europe and constitute a non-negligible part of the surface aerosols measured and controlled in the framework of the European air quality legislation. The modelling of these African dust emissions fluxes and transport is widely studied and complex parameterizations are already used in regional to global model for this Sahara-Sahel region. In a lesser extent, mineral dust emissions occur locally in Europe, mainly over agricultural areas. Their modelling is generally poorly done or just ignored. But in some cases, this contribution may be important and may impact the European air quality budget. In this study, we propose an homogeneized calculations of mineral dust fluxes for Europe and Africa. For that, we extended the CHIMERE dust production model (DPM by using new soil and surface datasets, and the global aeolian roughness length dataset provided by GARLAP from microwave and visible satellite observations. This DPM is detailed along with academic tests case results and simulation on a real case results.

  20. Multiscale characterization of a chimeric biomimetic polypeptide for stem cell culture

    International Nuclear Information System (INIS)

    Mesenchymal stem cells have attracted great interest in the field of tissue engineering and regenerative medicine because of their multipotentiality and relative ease of isolation from adult tissues. The medical application of this cellular system requires the inclusion in a growth and delivery scaffold that is crucial for the clinical effectiveness of the therapy. In particular, the ideal scaffolding material should have the needed porosity and mechanical strength to allow a good integration with the surrounding tissues, but it should also assure high biocompatibility and full resorbability. For such a purpose, protein-inspired biomaterials and, in particular, elastomeric-derived polypeptides are playing a major role, in which they are expected to fulfil many of the biological and mechanical requirements. A specific chimeric protein, designed starting from elastin, resilin and collagen sequences, was characterized over different length scales. Single-molecule mechanics, aggregation properties and compatibility with human mesenchymal stem cells were tested, showing that the engineered compound is a good candidate as a stem cell scaffold to be used in tissue engineering applications. (paper)

  1. The developmental fate of green fluorescent mouse embryonic germ cells in chimeric embryos

    Institute of Scientific and Technical Information of China (English)

    XUXIN; SUMIOSUGANO; 等

    1999-01-01

    Primordial germ cells (PGCs),as precursors of mammalian germ lineage,have been gaining more attention as a new resource of pluripotent stem cells,which bring a great possibility to study developmental events of germ cell in vitro and at animal level.EG4 cells derived from 10.5 days post coitum (dpc) PGCs of 129/svJ strain mouse were established and maintained in an undifferentiated state.With an attempt to study the differentiation capability of EG4 cells with a reporter protein:green fluorescence protein,and the possible application of EG4 cells in the research of germ cell development,we have generated several EG4-GFP cell lines expressing enhanced green fluorescence protein (EGFP) and still maintaining typical characteristics of pluripotent stem cells.Then,the differentiation of EG4-GFP cells in vitro as well as their developmental fate in chimeric embryos which were produced by aggregating EG4-GFP cells to 8-cell stage embryos were studied.The results showed that EG4 cells carrying green fluorescence have a potential use in the research of germ cell development and other related studies.

  2. Chimeric Mice with Competent Hematopoietic Immunity Reproduce Key Features of Severe Lassa Fever.

    Science.gov (United States)

    Oestereich, Lisa; Lüdtke, Anja; Ruibal, Paula; Pallasch, Elisa; Kerber, Romy; Rieger, Toni; Wurr, Stephanie; Bockholt, Sabrina; Pérez-Girón, José V; Krasemann, Susanne; Günther, Stephan; Muñoz-Fontela, César

    2016-05-01

    Lassa fever (LASF) is a highly severe viral syndrome endemic to West African countries. Despite the annual high morbidity and mortality caused by LASF, very little is known about the pathophysiology of the disease. Basic research on LASF has been precluded due to the lack of relevant small animal models that reproduce the human disease. Immunocompetent laboratory mice are resistant to infection with Lassa virus (LASV) and, to date, only immunodeficient mice, or mice expressing human HLA, have shown some degree of susceptibility to experimental infection. Here, transplantation of wild-type bone marrow cells into irradiated type I interferon receptor knockout mice (IFNAR-/-) was used to generate chimeric mice that reproduced important features of severe LASF in humans. This included high lethality, liver damage, vascular leakage and systemic virus dissemination. In addition, this model indicated that T cell-mediated immunopathology was an important component of LASF pathogenesis that was directly correlated with vascular leakage. Our strategy allows easy generation of a suitable small animal model to test new vaccines and antivirals and to dissect the basic components of LASF pathophysiology. PMID:27191716

  3. Chimeric Mice with Competent Hematopoietic Immunity Reproduce Key Features of Severe Lassa Fever.

    Directory of Open Access Journals (Sweden)

    Lisa Oestereich

    2016-05-01

    Full Text Available Lassa fever (LASF is a highly severe viral syndrome endemic to West African countries. Despite the annual high morbidity and mortality caused by LASF, very little is known about the pathophysiology of the disease. Basic research on LASF has been precluded due to the lack of relevant small animal models that reproduce the human disease. Immunocompetent laboratory mice are resistant to infection with Lassa virus (LASV and, to date, only immunodeficient mice, or mice expressing human HLA, have shown some degree of susceptibility to experimental infection. Here, transplantation of wild-type bone marrow cells into irradiated type I interferon receptor knockout mice (IFNAR-/- was used to generate chimeric mice that reproduced important features of severe LASF in humans. This included high lethality, liver damage, vascular leakage and systemic virus dissemination. In addition, this model indicated that T cell-mediated immunopathology was an important component of LASF pathogenesis that was directly correlated with vascular leakage. Our strategy allows easy generation of a suitable small animal model to test new vaccines and antivirals and to dissect the basic components of LASF pathophysiology.

  4. Chimeric Mice with Competent Hematopoietic Immunity Reproduce Key Features of Severe Lassa Fever

    Science.gov (United States)

    Oestereich, Lisa; Lüdtke, Anja; Ruibal, Paula; Pallasch, Elisa; Kerber, Romy; Rieger, Toni; Wurr, Stephanie; Bockholt, Sabrina; Krasemann, Susanne

    2016-01-01

    Lassa fever (LASF) is a highly severe viral syndrome endemic to West African countries. Despite the annual high morbidity and mortality caused by LASF, very little is known about the pathophysiology of the disease. Basic research on LASF has been precluded due to the lack of relevant small animal models that reproduce the human disease. Immunocompetent laboratory mice are resistant to infection with Lassa virus (LASV) and, to date, only immunodeficient mice, or mice expressing human HLA, have shown some degree of susceptibility to experimental infection. Here, transplantation of wild-type bone marrow cells into irradiated type I interferon receptor knockout mice (IFNAR-/-) was used to generate chimeric mice that reproduced important features of severe LASF in humans. This included high lethality, liver damage, vascular leakage and systemic virus dissemination. In addition, this model indicated that T cell-mediated immunopathology was an important component of LASF pathogenesis that was directly correlated with vascular leakage. Our strategy allows easy generation of a suitable small animal model to test new vaccines and antivirals and to dissect the basic components of LASF pathophysiology. PMID:27191716

  5. Current status and regulatory perspective of chimeric antigen receptor-modified T cell therapeutics.

    Science.gov (United States)

    Kim, Mi-Gyeong; Kim, Dongyoon; Suh, Soo-Kyung; Park, Zewon; Choi, Min Joung; Oh, Yu-Kyoung

    2016-04-01

    Chimeric antigen receptor-modified T cells (CAR-T) have emerged as a new modality for cancer immunotherapy due to their potent efficacy against terminal cancers. CAR-Ts are reported to exert higher efficacy than monoclonal antibodies and antibody-drug conjugates, and act via mechanisms distinct from T cell receptor-engineered T cells. These cells are constructed by transducing genes encoding fusion proteins of cancer antigen-recognizing single-chain Fv linked to intracellular signaling domains of T cell receptors. CAR-Ts are classified as first-, second- and third-generation, depending on the intracellular signaling domain number of T cell receptors. This review covers the current status of CAR-T research, including basic proof-of-concept investigations at the cell and animal levels. Currently ongoing clinical trials of CAR-T worldwide are additionally discussed. Owing to the lack of existing approved products, several unresolved concerns remain with regard to safety, efficacy and manufacturing of CAR-T, as well as quality control issues. In particular, the cytokine release syndrome is the major side-effect impeding the successful development of CAR-T in clinical trials. Here, we have addressed the challenges and regulatory perspectives of CAR-T therapy. PMID:26895243

  6. Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells.

    Science.gov (United States)

    Levine, B L

    2015-03-01

    Performance enhancement of the immune system can now be generated through ex vivo gene modification of T cells in order to redirect native specificity to target tumor antigens. This approach combines the specificity of antibody therapy, the expanded response of cellular therapy and the memory activity of vaccine therapy. Recent clinical trials of chimeric antigen receptor (CAR) T cells directed toward CD19 as a stand-alone therapy have shown sustained complete responses in patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia. As these drug products are individually derived from a patient's own cells, a different manufacturing approach is required for this kind of personalized therapy compared with conventional drugs. Key steps in the CAR T-cell manufacturing process include the selection and activation of isolated T cells, transduction of T cells to express CARs, ex vivo expansion of modified T cells and cryopreservation in infusible media. In this review, the steps involved in isolating, genetically modifying and scaling-out the CAR T cells for use in a clinical setting are described in the context of in-process and release testing and regulatory standards. PMID:25675873

  7. Chimeric Antigen Receptor-Modified T Cells for Solid Tumors: Challenges and Prospects

    Directory of Open Access Journals (Sweden)

    Yelei Guo

    2016-01-01

    Full Text Available Recent studies have highlighted the successes of chimeric antigen receptor-modified T- (CART- cell-based therapy for B-cell malignancies, and early phase clinical trials have been launched in recent years. The few published clinical studies of CART cells in solid tumors have addressed safety and feasibility, but the clinical outcome data are limited. Although antitumor effects were confirmed in vitro and in animal models, CART-cell-based therapy still faces several challenges when directed towards solid tumors, and it has been difficult to achieve the desired outcomes in clinical practice. Many studies have struggled to improve the clinical responses to and benefits of CART-cell treatment of solid tumors. In this review, the status quo of CART cells and their clinical applications for solid tumors will be summarized first. Importantly, we will suggest improvements that could increase the therapeutic effectiveness of CART cells for solid tumors and their future clinical applications. These interventions will make treatment with CART cells an effective and routine therapy for solid tumors.

  8. Functionality of Chimeric E2 Glycoproteins of BVDV and CSFV in Virus Replication

    Directory of Open Access Journals (Sweden)

    H.G.P. van Gennip

    2008-01-01

    Full Text Available An intriguing difference between the E2 glycoprotein of CSFV and the other groups of pestiviruses (nonCSFV is a lack of two cysteine residues on positions cysteine 751 and 798. Other groups of pestivirus are not restricted to one species as swine, whereas CSFV is restricted to swine and wild boar. We constructed chimeric CSFV/BVDV E2 genes based on a 2D model of E2 proposed by van Rijn et al. (van Rijn et al. 1994, J Virol 68, 3934–42 and confirmed their expression by immunostaining of plasmid-transfected SK6 cells. No equivalents for the antigenic units B/C and A were found on E2 of BVDVII. This indicates major structural differences in E2. However, the immunodominant BVDVII domain A, containing epitopes with essential amino acids between position 760–764, showed to be dependent on the presence of the region defined by amino acids 684 to 796. As for the A domain of CSFV, the BVDVII A-like domain seemed to function as a separate unit. These combined domains in E2 proved to be the only combination which was functional in viral background of CSFV C-strain. The fitness of this virus (vfl c36BVDVII 684–796 seemed to be reduced compared to vfl c9 (with the complete antigenic region of BVDVII.

  9. Diverse hematological malignancies including hodgkin-like lymphomas develop in chimeric MHC class II transgenic mice.

    Directory of Open Access Journals (Sweden)

    Silke H Raffegerst

    Full Text Available A chimeric HLA-DR4-H2-E (DR4 homozygous transgenic mouse line spontaneously develops diverse hematological malignancies with high frequency (70%. The majority of malignancies were distributed equally between T and B cell neoplasms and included lymphoblastic T cell lymphoma (LTCL, lymphoblastic B cell lymphoma (LBCL, diffuse large B cell lymphoma (DLBCL, the histiocyte/T cell rich variant of DLBCL (DLBCL-HA/T cell rich DLBCL, splenic marginal zone lymphoma (SMZL, follicular B cell lymphoma (FBL and plasmacytoma (PCT. Most of these neoplasms were highly similar to human diseases. Also, some non-lymphoid malignancies such as acute myeloid leukemia (AML and histiocytic sarcoma were found. Interestingly, composite lymphomas, including Hodgkin-like lymphomas, were also detected that had CD30(+ Hodgkin/Reed-Sternberg (H/RS-like cells, representing a tumor type not previously described in mice. Analysis of microdissected H/RS-like cells revealed their origin as germinal center B cells bearing somatic hypermutations and, in some instances, crippled mutations, as described for human Hodgkin lymphoma (HL. Transgene integration in an oncogene was excluded as an exclusive driving force of tumorigenesis and age-related lymphoma development suggests a multi-step process. Thus, this DR4 line is a useful model to investigate common molecular mechanisms that may contribute to important neoplastic diseases in man.

  10. Modification of chimeric (2S, 3S)-butanediol dehydrogenase based on structural information.

    Science.gov (United States)

    Shimegi, Tomohito; Mochizuki, Kaito; Oyama, Takuji; Ohtsuki, Takashi; Kusunoki, Masami; Ui, Sadaharu

    2014-01-01

    A chimeric (2S, 3S)-butanediol dehydrogenase (cLBDH) was engineered to have the strict (S)-configuration specificity of the (2S, 3S)-BDH (BsLBDH) derived from Brevibacterium saccharolyticum as well as the enzymatic stability of the (2R, 3S)-BDH (KpMBDH) from Klebsiella pneumonia by swapping the domains of two native BDHs. However, while cLBDH possesses the stability, it lacks the specificity. In order to assist in the design a BDH having strict substrate specificity, an X-ray structural analysis of a cLBDH crystal was conducted at 1.58 Å. The results obtained show some readily apparent differences around the active sites of cLBDH and BsLBDH. Based on this structural information, a novel (2S, 3S)-BDH having a preferred specificity was developed by introducing a V254L mutation into cLBDH. The influence of this mutation on the stability of cLBDH was not evaluated. Nevertheless, the technique described herein is an effective method for the production of a tailor-made BDH. PMID:25612804

  11. Nanoparticles of cationic chimeric peptide and sodium polyacrylate exhibit striking antinociception activity at lower dose.

    Science.gov (United States)

    Gupta, Kshitij; Singh, Vijay P; Kurupati, Raj K; Mann, Anita; Ganguli, Munia; Gupta, Yogendra K; Singh, Yogendra; Saleem, Kishwar; Pasha, Santosh; Maiti, Souvik

    2009-02-20

    The current study investigates the performance of polyelectrolyte complexes based nanoparticles in improving the antinociceptive activity of cationic chimeric peptide-YFa at lower dose. Size, Zeta potential and morphology of the nanoparticles were determined. Size of the nanoparticles decreases and zeta potential increases with concomitant increase in charge ratio (Z(+/-)). The nanoparticles at Z(+/-)12 are spherical with 70+/-7 nm diameter in AFM and displayed positive surface charge and similar sizes (83+/-8 nm) by Zetasizer. The nanoparticles of Z(+/-) 12 are used in this study. Cytotoxicity by MTT assay on three different mammalian cell lines (liver, neuronal and kidney) revealed lower toxicity of nanoparticles. Hematological parameters were also not affected by nanoparticles compared to normal counts of water treated control group. Nanoparticles containing 10 mg/kg YFa produced increased antinociception, approximately 36%, in tail-flick latency test in mice, whereas the neat peptide at the same concentration did not show any antinociception activity. This enhancement in activity is attributed to the nanoparticle associated protection of peptide from proteolytic degradation. In vitro peptide release study in plasma also supported the antinociception profile of nanoparticles. Thus, our results suggest of a potential nanoparticle delivery system for cationic peptide drug candidates for improving their stability and bioavailability. PMID:19014986

  12. Subalpine Pyrenees received higher nitrogen deposition than predicted by EMEP and CHIMERE chemistry-transport models

    Science.gov (United States)

    Boutin, Marion; Lamaze, Thierry; Couvidat, Florian; Pornon, André

    2015-08-01

    Deposition of reactive nitrogen (N) from the atmosphere is expected to be the third greatest driver of biodiversity loss by the year 2100. Chemistry-transport models are essential tools to estimate spatially explicit N deposition but the reliability of their predictions remained to be validated in mountains. We measured N deposition and air concentration over the subalpine Pyrenees. N deposition was found to range from 797 to 1,463 mg N m-2 year-1. These values were higher than expected from model predictions, especially for nitrate, which exceeded the estimations of EMEP by a factor of 2.6 and CHIMERE by 3.6. Our observations also displayed a reversed reduced-to-oxidized ratio in N deposition compared with model predictions. The results highlight that the subalpine Pyrenees are exposed to higher levels of N deposition than expected according to standard predictions and that these levels exceed currently recognized critical loads for most high-elevation habitats. Our study reveals a need to improve the evaluation of N deposition in mountains which are home to a substantial and original part of the world’s biodiversity.

  13. Design and development of therapies using chimeric antigen receptor-expressing T cells.

    Science.gov (United States)

    Dotti, Gianpietro; Gottschalk, Stephen; Savoldo, Barbara; Brenner, Malcolm K

    2014-01-01

    Investigators developed chimeric antigen receptors (CARs) for expression on T cells more than 25 years ago. When the CAR is derived from an antibody, the resultant cell should combine the desirable targeting features of an antibody (e.g. lack of requirement for major histocompatibility complex recognition, ability to recognize non-protein antigens) with the persistence, trafficking, and effector functions of a T cell. This article describes how the past two decades have seen a crescendo of research which has now begun to translate these potential benefits into effective treatments for patients with cancer. We describe the basic design of CARs, describe how antigenic targets are selected, and the initial clinical experience with CAR-T cells. Our review then describes our own and other investigators' work aimed at improving the function of CARs and reviews the clinical studies in hematological and solid malignancies that are beginning to exploit these approaches. Finally, we show the value of adding additional engineering features to CAR-T cells, irrespective of their target, to render them better suited to function in the tumor environment, and discuss how the safety of these heavily modified cells may be maintained. PMID:24329793

  14. Synthesis and characterization of human recombinant thyrotropin (rec-hTSH) with a chimeric β-subunit (rec-hTSHβ-CTPE hCGβ)

    International Nuclear Information System (INIS)

    Recombinant hTSH is now successfully being used in clinical studies of thyroid cancer. Because of its therapeutic potential, we have constructed a longer acting analog of hTSH by fusing the carboxy terminal extension peptide (CTEP) of hCGβ onto hTSHβ. When coexpressed either with α-subunit complementary DNA or α-minigene in African green monkey (Cos-7) and human embryonic kidney (293) cells, the chimera was fully bioactive in vitro and exhibited enhanced in vivo potency associated with a prolonged plasma half-life. The addition of 29 amino acids with 4 O-linked oligosaccharide chains did not affect the assembly and secretion of chimeric TSH. Wild type (WT) and chimeric hTSH secreted by Cos-7 and 293 cells displayed wide differences in their plasma half-lives, presumably due to the difference in the terminal sialic acid and sulfate of their oligosaccharide chains. Chimeric and WT hTSH secreted by both cell lines demonstrated similar bioactivity in cAMP production, with some differences in [3 H]-thymidine incorporation. Chimeric hTSH secreted by Cos-7 appears to be more active than that secreted by 293 cells, as judged by growth assay. Cos-7 produced chimeric hTSH showed the maximum increase in half-life, indicating the importance of sialic acid in prolonging half-life and in vivo potency. Sulfation of both subunits, predominantly β and to a lesser extent α, appears to be responsible, at least in part, for the increased metabolic clearance of WT and chimeric TSH secreted by 293 cells. Apart from its therapeutic potential, chimeric TSH produced in various cell lines can be used as a tool to delineate the roles of sulfate and sialic acid in the in vivo clearance and, thereby in the in vivo bioactivity. (author). 104 refs., 23 figs., 3 tabs

  15. A fast and simple approach for the simultaneous detection of hematopoietic chimerism, NPM1, and FLT3-ITD mutations after allogeneic stem cell transplantation.

    Science.gov (United States)

    Waterhouse, Miguel; Bertz, Hartmut; Finke, Juergen

    2014-02-01

    Hematopoietic chimerism can be used as a tool for patient management after allogeneic hematopoietic stem cell transplantation (HSCT). An increase in the proportion of recipient cells after transplantation is strongly associated with relapse in chronic myeloid leukemia. However, in acute myeloid leukemia (AML) the significance of increasing mixed chimerism (MC) as a predictive marker for relapse is less clear. Several mutations frequently found in AML have been employed for minimal residual disease detection and relapse prediction. Therefore, a combined analysis of hematopoietic chimerism and of the molecular aberrations found in AML could be used to improve MC characterization. We developed a multiplex PCR for use in the simultaneous detection of hematopoietic chimerism and mutations in nucleophosmin (NPM1) and fms-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD). A total of 303 samples from 20 AML patients were analyzed after HSCT. The microsatellite markers used for hematopoietic chimerism detection were D1S80, D7S1517, D4S2366, THO1, and SE33. A total of 149 samples from 18 patients showed MC with a mean detection time of 9.7 months. From the 18 patients with MC, in 6 of the patients, no FLT3-ITD or NPM1 mutation was found at any time point tested, and these patients remained in complete hematological remission. In 12 patients with MC, FLT3-ITD and NPM1 mutations were found, and these patients showed signs of hematological relapse. Our combined analysis of NPM1/FLT3-ITD mutations and hematopoietic chimerism improved the characterization of patients with MC after HSCT. The present approach may be further expanded by combining additional mutations found in AML with hematopoietic chimerism detection. PMID:23907410

  16. Chimeric toxins inhibit growth of primary oral squamous cell carcinoma cells.

    Science.gov (United States)

    Bachran, Christopher; Heisler, Iring; Bachran, Diana; Dassler, Katrin; Ervens, Jürgen; Melzig, Matthias F; Fuchs, Hendrik

    2008-02-01

    Treatment of oral squamous cell carcinoma (OSCC) is currently based on surgery and radiotherapy. Prolongation of the survival time of patients with progressing tumors is infrequently achieved. To improve the therapeutic options, targeted therapies are a favorable alternative. Therefore, we analyzed the effect of a chimeric toxin (CT) named SE consisting of the epidermal growth factor and the plant protein toxin saporin from Saponaria officinalis. A second construct (SA2E) additionally contains a peptidic adapter designed to enhance efficacy of the CT in vivo and to reduce side effects. The IC(50) values for an OSCC cell line (BHY) were 0.27 nM and 0.73 nM for SE and SA2E, respectively, while fibroblasts remained unaffected. To investigate primary tumor cells, we developed a technique to analyze freshly prepared OSCC cells of 28 patients in a stem cell assay directly after surgery. Cells were treated for 1 h with the CTs, subsequently seeded into soft agar and colony growth determined after 1-2 weeks In spite of the short time of CT incubation, the amount of colonies was reduced to about 78% by 10 nM and to 69% by 100 nM of either toxin. A combined application of 10 nM SA2E with a saponin from Gypsophila paniculata reduced the amount of surviving cells to 68%. The results demonstrate the impact of the CTs on OSCC cells and depict that the stem cell assay is suitable to determine the potential of anti-tumor drugs before studies in vivo will be initiated. PMID:18059188

  17. Design of a novel chimeric tissue plasminogen activator with favorable Vampire bat plasminogen activator properties.

    Science.gov (United States)

    Kazemali, MohammadReza; Majidzadeh-A, Keivan; Sardari, Soroush; Saadatirad, Amir Hossein; Khalaj, Vahid; Zarei, Najmeh; Barkhordari, Farzaneh; Adeli, Ahmad; Mahboudi, Fereidoun

    2014-12-01

    Fibrinolytic agents are widely used in treatment of the thromboembolic disorders. The new generations like recombinant tissue plasminogen activator (t-PA, alteplase) are not showing promising results in clinical practice in spite of displaying specific binding to fibrin in vitro. Vampire bat plasminogen activator (b-PA) is a plasminogen activator with higher fibrin affinity and specificity in comparison to t-PA resulting in reduced probability of hemorrhage. b-PA is also resistant to plasminogen activator inhibitor-1 (PAI-1) showing higher half-life compared to other variants of t-PA. However, its non-human origin was a driving force to design a human t-PA with favorable properties of b-PA. In the present study, we designed a chimeric t-PA with desirable b-PA properties and this new molecule was called as CT-b. The construct was prepared through kringle 2 domain removal and replacement of t-PA finger domain with b-PA one. In addition, the KHRR sequence at the initial part of protease domain was replaced by four alanine residues. The novel construct was integrated in Pichia pastoris genome by electroporation. Catalytic activity was investigated in the presence and absence of fibrin. The purified protein was analyzed by western blot. Fibrin binding and PAI resistance assays were also conducted. The activity of the recombinant protein in the presence of fibrin was 1560 times more than its activity in the absence of fibrin, showing its higher specificity to fibrin. The fibrin binding of CT-b was 1.2 fold more than t-PA. In addition, it was inhibited by PAI enzyme 44% less than t-PA. Although the presented data demonstrate a promising in vitro activity, more in vivo studies are needed to confirm the therapeutic advantage of this novel plasminogen activator. PMID:25442953

  18. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo.

    Directory of Open Access Journals (Sweden)

    Binghua Xue

    Full Text Available Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.

  19. RNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteins

    KAUST Repository

    Baazim, Hatoon

    2014-05-01

    Developing targeted genome regulation approaches holds much promise for accelerating trait discovery and development in agricultural biotechnology. Clustered Regularly Interspaced Palindromic Repeats (CRISPRs)/CRISPR associated (Cas) system provides bacteria and archaea with an adaptive molecular immunity mechanism against invading nucleic acids through phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing purposes across a variety of cell types and organisms. Recently, the catalytically inactive Cas9 (dCas9) protein combined with guide RNAs (gRNAs) were used as a DNA-targeting platform to modulate the expression patterns in bacterial, yeast and human cells. Here, we employed this DNA-targeting system for targeted transcriptional regulation in planta by developing chimeric dCas9-based activators and repressors. For example, we fused to the C-terminus of dCas9 with the activation domains of EDLL and TAL effectors, respectively, to generate transcriptional activators, and the SRDX repression domain to generate transcriptional repressor. Our data demonstrate that the dCas9:EDLL and dCas9:TAD activators, guided by gRNAs complementary to promoter elements, induce strong transcriptional activation on episomal targets in plant cells. Moreover, our data suggest that the dCas9:SRDX repressor and the dCas9:EDLL and dCas9:TAD activators are capable of markedly repressing or activating, respectively, the transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9:TFs DNA targeting system can be used in plants as a functional genomic tool and for biotechnological applications.

  20. Making Better Chimeric Antigen Receptors for Adoptive T-cell Therapy.

    Science.gov (United States)

    Maus, Marcela V; June, Carl H

    2016-04-15

    Chimeric antigen receptors (CAR) are engineered fusion proteins constructed from antigen recognition, signaling, and costimulatory domains that can be expressed in cytotoxic T cells with the purpose of reprograming the T cells to specifically target tumor cells. CAR T-cell therapy uses gene transfer technology to reprogram a patient's own T cells to stably express CARs, thereby combining the specificity of an antibody with the potent cytotoxic and memory functions of a T cell. In early-phase clinical trials, CAR T cells targeting CD19 have resulted in sustained complete responses within a population of otherwise refractory patients with B-cell malignancies and, more specifically, have shown complete response rates of approximately 90% in patients with relapsed or refractory acute lymphoblastic leukemia. Given this clinical efficacy, preclinical development of CAR T-cell therapy for a number of cancer indications has been actively investigated, and the future of the CAR T-cell field is extensive and dynamic. Several approaches to increase the feasibility and safety of CAR T cells are currently being explored, including investigation into the mechanisms regulating the persistence of CAR T cells. In addition, numerous early-phase clinical trials are now investigating CAR T-cell therapy beyond targeting CD19, especially in solid tumors. Trials investigating combinations of CAR T cells with immune checkpoint blockade therapies are now beginning and results are eagerly awaited. This review evaluates several of the ongoing and future directions of CAR T-cell therapy.Clin Cancer Res; 22(8); 1875-84. ©2016 AACR SEE ALL ARTICLES IN THIS CCR FOCUS SECTION, "OPPORTUNITIES AND CHALLENGES IN CANCER IMMUNOTHERAPY". PMID:27084741

  1. Calcium-stimulated autophosphorylation site of plant chimeric calcium/calmodulin-dependent protein kinase

    Science.gov (United States)

    Sathyanarayanan, P. V.; Siems, W. F.; Jones, J. P.; Poovaiah, B. W.

    2001-01-01

    The existence of two molecular switches regulating plant chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK), namely the C-terminal visinin-like domain acting as Ca(2+)-sensitive molecular switch and calmodulin binding domain acting as Ca(2+)-stimulated autophosphorylation-sensitive molecular switch, has been described (Sathyanarayanan, P. V., Cremo, C. R., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 30417-30422). Here we report the identification of Ca(2+)-stimulated autophosphorylation site of CCaMK by matrix-assisted laser desorption ionization time of flight-mass spectrometry. Thr(267) was confirmed as the Ca(2+)-stimulated autophosphorylation site by post-source decay experiments and by site-directed mutagenesis. The purified T267A mutant form of CCaMK did not show Ca(2+)-stimulated autophosphorylation, autophosphorylation-dependent variable calmodulin affinity, or Ca(2+)/calmodulin stimulation of kinase activity. Sequence comparison of CCaMK from monocotyledonous plant (lily) and dicotyledonous plant (tobacco) suggests that the autophosphorylation site is conserved. This is the first identification of a phosphorylation site specifically responding to activation by second messenger system (Ca(2+) messenger system) in plants. Homology modeling of the kinase and calmodulin binding domain of CCaMK with the crystal structure of calcium/calmodulin-dependent protein kinase 1 suggests that the Ca(2+)-stimulated autophosphorylation site is located on the surface of the kinase and far from the catalytic site. Analysis of Ca(2+)-stimulated autophosphorylation with increasing concentration of CCaMK indicates the possibility that the Ca(2+)-stimulated phosphorylation occurs by an intermolecular mechanism.

  2. Autophosphorylation-dependent inactivation of plant chimeric calcium/calmodulin-dependent protein kinase

    Science.gov (United States)

    Sathyanarayanan, P. V.; Poovaiah, B. W.

    2002-01-01

    Chimeric calcium/calmodulin dependent protein kinase (CCaMK) is characterized by the presence of a visinin-like Ca(2+)-binding domain unlike other known calmodulin- dependent kinases. Ca(2+)-Binding to the visinin-like domain leads to autophosphorylation and changes in the affinity for calmodulin [Sathyanarayanan P.V., Cremo C.R. & Poovaiah B.W. (2000) J. Biol. Chem. 275, 30417-30422]. Here, we report that the Ca(2+)-stimulated autophosphorylation of CCaMK results in time-dependent loss of enzyme activity. This time-dependent loss of activity or self-inactivation due to autophosphorylation is also dependent on reaction pH and ATP concentration. Inactivation of the enzyme resulted in the formation of a sedimentable enzyme due to self-association. Specifically, autophosphorylation in the presence of 200 microm ATP at pH 7.5 resulted in the formation of a sedimentable enzyme with a 33% loss in enzyme activity. Under similar conditions at pH 6.5, the enzyme lost 67% of its activity and at pH 8.5, 84% enzyme activity was lost. Furthermore, autophosphorylation at either acidic or alkaline reaction pH lead to the formation of a sedimentable enzyme. Transmission electron microscopic studies on autophosphorylated kinase revealed particles that clustered into branched complexes. The autophosphorylation of wild-type kinase in the presence of AMP-PNP (an unhydrolyzable ATP analog) or the autophosphorylation-site mutant, T267A, did not show formation of branched complexes under the electron microscope. Autophosphorylation- dependent self-inactivation may be a mechanism of modulating the signal transduction pathway mediated by CCaMK.

  3. Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers

    Science.gov (United States)

    Poovaiah, B. W.; Xia, M.; Liu, Z.; Wang, W.; Yang, T.; Sathyanarayanan, P. V.; Franceschi, V. R.

    1999-01-01

    Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca(2+) and Ca(2+)/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the beta-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by beta-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca(2+) concentration in target cells, thereby controlling developmental events in the anther.

  4. Partitioning of genetically distinct cell populations in chimeric juveniles of the sponge Amphimedon queenslandica.

    Science.gov (United States)

    Gauthier, Marie; Degnan, Bernard M

    2008-01-01

    Natural chimerism, the fusion between genetically distinct conspecifics, is a process known to occur in various marine benthic invertebrates. Sponges (phylum Porifera) have proven to be a useful model to study the origin and evolution of allorecognition. Like some other invertebrates, they display an ontogenetic shift in their allorecognition response: genetically different individuals can fuse during early development, but, in most instances, not as adults. However, there is a limited understanding of the cellular organisation of sponge chimeras and the onset of this allorecognition response, which prevents integration of incompatible genotypes. Here we follow the behaviours and fates of cells derived from genetically distinct larvae of the demosponge Amphimedon queenslandica that have fused together at metamorphosis. By labelling individual larvae with different fluorescent dyes, we can follow cell movement in the postlarval chimeras. We observed that cells from the two individuals readily mixed for 2 weeks after the initial fusion. After that time, differently labelled cells began to sort into different postlarval cellular territories, with one lineage giving rise to choanocytes and the other to pinacocytes and cells of the mesohyl. These results suggest that a rapid ontogenetic shift in the allogeneic response of A. queenslandica occurs about 2 weeks after the initiation of metamorphosis and that the molecular basis of this response is also involved in creating differential cell affinities that underlie the construction of the sponge body plan. Compatible with this proposition is the observation that cells from postlarvae that are allowed to develop for 2 weeks before contact do not fuse and form a distinct boundary between genotypes. The successful chimeras remained stable for the duration of the experiment (3 weeks) raising the possibility that reproductive chimeras might persist in the natural environment, with a single genotype giving rise to germ cells

  5. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells.

    Science.gov (United States)

    Frigault, Matthew J; Lee, Jihyun; Basil, Maria Ciocca; Carpenito, Carmine; Motohashi, Shinichiro; Scholler, John; Kawalekar, Omkar U; Guedan, Sonia; McGettigan, Shannon E; Posey, Avery D; Ang, Sonny; Cooper, Laurence J N; Platt, Jesse M; Johnson, F Brad; Paulos, Chrystal M; Zhao, Yangbing; Kalos, Michael; Milone, Michael C; June, Carl H

    2015-04-01

    This study compared second-generation chimeric antigen receptors (CAR) encoding signaling domains composed of CD28, ICOS, and 4-1BB (TNFRSF9). Here, we report that certain CARs endow T cells with the ability to undergo long-term autonomous proliferation. Transduction of primary human T cells with lentiviral vectors encoding some of the CARs resulted in sustained proliferation for up to 3 months following a single stimulation through the T-cell receptor (TCR). Sustained numeric expansion was independent of cognate antigen and did not require the addition of exogenous cytokines or feeder cells after a single stimulation of the TCR and CD28. Results from gene array and functional assays linked sustained cytokine secretion and expression of T-bet (TBX21), EOMES, and GATA-3 to the effect. Sustained expression of the endogenous IL2 locus has not been reported in primary T cells. Sustained proliferation was dependent on CAR structure and high expression, the latter of which was necessary but not sufficient. The mechanism involves constitutive signaling through NF-κB, AKT, ERK, and NFAT. The propagated CAR T cells retained a diverse TCR repertoire, and cellular transformation was not observed. The CARs with a constitutive growth phenotype displayed inferior antitumor effects and engraftment in vivo. Therefore, the design of CARs that have a nonconstitutive growth phenotype may be a strategy to improve efficacy and engraftment of CAR T cells. The identification of CARs that confer constitutive or nonconstitutive growth patterns may explain observations that CAR T cells have differential survival patterns in clinical trials. PMID:25600436

  6. A novel chimeric adenoassociated virus 2/human bocavirus 1 parvovirus vector efficiently transduces human airway epithelia.

    Science.gov (United States)

    Yan, Ziying; Keiser, Nicholas W; Song, Yi; Deng, Xuefeng; Cheng, Fang; Qiu, Jianming; Engelhardt, John F

    2013-12-01

    Human bocavirus virus-1 (HBoV1), a newly discovered autonomous parvovirus with a 5,500 nt genome, efficiently infects human-polarized airway epithelia (HAE) from the apical membrane. We hypothesized that the larger genome and high airway tropism of HBoV1 would be ideal for creating a viral vector for lung gene therapy. To this end, we successfully generated recombinant HBoV1 (rHBoV1) from an open reading frames-disrupted rHBoV1 genome that efficiently transduces HAE from the apical surface. We next evaluated whether HBoV1 capsids could package oversized rAAV2 genomes. These studies created a rAAV2/HBoV1 chimeric virus (5.5 kb genome) capable of apically transducing HAE at 5.6- and 70-fold greater efficiency than rAAV1 or rAAV2 (4.7-kb genomes), respectively. Molecular studies demonstrated that viral uptake from the apical surface was significantly greater for rAAV2/HBoV1 than for rAAV2 or rAAV1, and that polarization of airway epithelial cells was required for HBoV1 capsid-mediated gene transfer. Furthermore, rAAV2/HBoV1-CFTR virus containing the full-length cystic fibrosis transmembrane conductance regulator (CFTR) gene coding sequence and the strong CBA promoter efficiently corrected CFTR-dependent chloride transport in cystic fibrosis (CF) HAE. In summary, using the combined advantages of AAV and HBoV1, we have developed a novel and promising viral vector for CF lung gene therapy and also potentially HBoV1 vaccine development. PMID:23896725

  7. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo

    Science.gov (United States)

    Xue, Binghua; Li, Yan; He, Yilong; Wei, Renyue; Sun, Ruizhen; Yin, Zhi; Bou, Gerelchimeg; Liu, Zhonghua

    2016-01-01

    Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs. PMID:26991423

  8. Adoptive immunotherapy for acute leukemia:New insights in chimeric antigen receptors

    Institute of Scientific and Technical Information of China (English)

    Ma?l; Heiblig; Mohamed; Elhamri; Mauricette; Michallet; Xavier; Thomas

    2015-01-01

    Relapses remain a major concern in acute leukemia. It is well known that leukemia stem cells(LSCs) hide in hematopoietic niches and escape to the immune system surveillance through the outgrowth of poorly immunogenic tumor-cell variants and the suppression of the active immune response. Despitethe introduction of new reagents and new therapeutic approaches, no treatment strategies have been able to definitively eradicate LSCs. However, recent adoptive immunotherapy in cancer is expected to revolutionize our way to fight against this disease, by redirecting the immune system in order to eliminate relapse issues. Initially described at the onset of the 90’s, chimeric antigen receptors(CARs) are recombinant receptors transferred in various T cell subsets, providing specific antigens binding in a non-major histocompatibility complex restricted manner, and effective on a large variety of human leukocyte antigen-divers cell populations. Once transferred, engineered T cells act like an expanding "living drug" specifically targeting the tumor-associated antigen, and ensure long-term antitumor memory. Over the last decades, substantial improvements have been made in CARs design. CAR T cells have finally reached the clinical practice and first clinical trials have shown promising results. In acute lymphoblastic leukemia, high rate of complete and prolonged clinical responses have been observed after anti-CD19 CAR T cell therapy, with specific but manageable adverse events. In this review, our goal was to describe CAR structures and functions, and to summarize recent data regarding pre-clinical studies and clinical trials in acute leukemia.

  9. Dual Regulation of a Chimeric Plant Serine/Threonine Kinase by Calcium and Calcium/Calmodulin

    Science.gov (United States)

    Takezawa, D.; Ramachandiran, S.; Paranjape, V.; Poovaiah, B. W.

    1996-01-01

    A chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) gene characterized by a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain was recently cloned from plants. The Escherichia coli-expressed CCaMK phosphorylates various protein and peptide substrates in a Ca(2+)/calmodulin-dependent manner. The calmodulin-binding region of CCAMK has similarity to the calmodulin-binding region of the alpha-subunit of multifunctional Ca(2+)/calmodulin-dependent protein kinase (CaMKII). CCaMK exhibits basal autophosphorylation at the threonine residue(s) (0.098 mol of P-32/mol) that is stimulated 3.4-fold by Ca(2+) (0.339 mol of P-32/mol), while calmodulin inhibits Ca(2+)-stimulated autophosphorylation to the basal level. A deletion mutant lacking the visinin-like domain did not show Ca(2+)-simulated autophosphorylation activity but retained Ca(2+)/calmodulin-dependent protein kinase activity at a reduced level. Ca(2+)-dependent mobility shift assays using E.coli-expressed protein from residues 358-520 revealed that Ca(2+) binds to the visinin-like domain. Studies with site-directed mutants of the visinin-like domain indicated that EF-hands II and III are crucial for Ca(2+)-induced conformational changes in the visinin-like domain. Autophosphorylation of CCaMK increases Ca(2+)/calmodulin-dependent protein kinase activity by about 5-fold, whereas it did not affect its C(2+)-independent activity. This report provides evidence for the existence of a protein kinase in plants that is modulated by Ca(2+) and Ca(2+)/calmodulin. The presence of a visinin-like Ca(2+)-binding domain in CCaMK adds an additional Ca(2+)-sensing mechanism not previously known to exist in the Ca(2+)/calmodulin-mediated signaling cascade in plants.

  10. Preubiquitinated chimeric ErbB2 is constitutively endocytosed and subsequently degraded in lysosomes

    Energy Technology Data Exchange (ETDEWEB)

    Vuong, Tram Thu [Institute of Clinical Medicine, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Berger, Christian [Department of Pathology, Oslo University Hospital, Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo (Norway); Bertelsen, Vibeke; Rødland, Marianne Skeie [Institute of Clinical Medicine, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Stang, Espen [Department of Pathology, Oslo University Hospital, Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo (Norway); Madshus, Inger Helene, E-mail: i.h.madshus@medisin.uio.no [Institute of Clinical Medicine, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Department of Pathology, Oslo University Hospital, Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo (Norway)

    2013-02-01

    The oncoprotein ErbB2 is endocytosis-deficient, probably due to its interaction with Heat shock protein 90. We previously demonstrated that clathrin-dependent endocytosis of ErbB2 is induced upon incubation of cells with Ansamycin derivatives, such as geldanamycin and its derivative 17-AAG. Furthermore, we have previously demonstrated that a preubiquitinated chimeric EGFR (EGFR-Ub{sub 4}) is constitutively endocytosed in a clathrin-dependent manner. We now demonstrate that also an ErbB2-Ub{sub 4} chimera is endocytosed constitutively and clathrin-dependently. Upon expression, the ErbB2-Ub{sub 4} was further ubiquitinated, and by Western blotting, we demonstrated the formation of both Lys48-linked and Lys63-linked polyubiquitin chains. ErbB2-Ub{sub 4} was constitutively internalized and eventually sorted to late endosomes and lysosomes where the fusion protein was degraded. ErbB2-Ub{sub 4} was not cleaved prior to internalization. Interestingly, over-expression of Ubiquitin Interaction Motif-containing dominant negative fragments of the clathrin adaptor proteins epsin1 and Eps15 negatively affected endocytosis of ErbB2. Altogether, this argues that ubiquitination is sufficient to induce clathrin-mediated endocytosis and lysosomal degradation of the otherwise plasma membrane localized ErbB2. Also, it appears that C-terminal cleavage is not required for endocytosis. -- Highlights: ► A chimera containing ErbB2 and a tetra-Ubiquitin chain internalizes constitutively. ► Receptor fragmentation is not required for endocytosis of ErbB2. ► Ubiquitination is sufficient to induce endocytosis and degradation of ErbB2. ► ErbB2-Ub4 is internalized clathrin-dependently.

  11. Sex steroids level in blood plasma and ovarian follicles of the chimeric chicken.

    Science.gov (United States)

    Sechman, A; Lakota, P; Wojtysiak, D; Hrabia, A; Mika, M; Lisowski, M; Czekalski, P; Rzasa, J; Kapkowska, E; Bednarczyk, M

    2006-12-01

    The study was performed to determine the hormonal status of mature germline chimeras obtained by blastodermal cell transfer from chicken embryos of a donor breed [Green-legged Partridgelike breed (GP) x Araucana (AR)] to those of a recipient breed [White Leghorn (WL)] being at the same stage of embryonic development. The egg-laying chimeras and WL hens (control) of the same age were used in the experiment. At first, blood samples were taken from each bird at 0.5, 5, 12.5 and 18.5 h following oviposition. Subsequently, the chimeras and the WL hens were decapitated 1-2 h after ovulation. A stroma and the following follicles were isolated from the ovary: white normal (1-4, 4-6 and 6-8 mm), white atretic and yellow preovulatory follicles (F4-F1). Sex hormones, progesterone (P4), testosterone (T) and oestradiol (E2) in blood plasma and ovarian follicles were determined radioimmunologically. The activity of the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in the granulosa and theca layers of the follicles was analysed histochemically. In chimeric chickens, a higher level of T in blood plasma during the ovulatory cycle was noticed. However, in the stroma, white prehierarchical and medium-size preovulatory ovarian follicles the level of T was significantly lower. With respect to E2, its elevated levels were found both in blood and in the ovarian follicles. There were no significant differences in P4 concentrations in blood plasma while in ovarian follicles a higher level was observed only in white 6-8 mm follicles. 3beta-HSD activity in granulosa and theca layers of the ovarian follicles in chimeras was not different from that in the WL hens. In conclusion, the results obtained indicate that germline chimeras exhibit significant alterations in sex hormone levels in the ovary and blood plasma, which in turn may affect their reproductive abilities. PMID:17105570

  12. Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides.

    Science.gov (United States)

    Viña-Gonzalez, Javier; Gonzalez-Perez, David; Ferreira, Patricia; Martinez, Angel T; Alcalde, Miguel

    2015-09-01

    Aryl-alcohol oxidase (AAO) is an extracellular flavoprotein that supplies ligninolytic peroxidases with H2O2 during natural wood decay. With a broad substrate specificity and highly stereoselective reaction mechanism, AAO is an attractive candidate for studies into organic synthesis and synthetic biology, and yet the lack of suitable heterologous expression systems has precluded its engineering by directed evolution. In this study, the native signal sequence of AAO from Pleurotus eryngii was replaced by those of the mating α-factor and the K1 killer toxin, as well as different chimeras of both prepro-leaders in order to drive secretion in Saccharomyces cerevisiae. The secretion of these AAO constructs increased in the following order: preproα-AAO > preαproK-AAO > preKproα-AAO > preproK-AAO. The chimeric preαproK-AAO was subjected to focused-directed evolution with the aid of a dual screening assay based on the Fenton reaction. Random mutagenesis and DNA recombination was concentrated on two protein segments (Met[α1]-Val109 and Phe392-Gln566), and an array of improved variants was identified, among which the FX7 mutant (harboring the H91N mutation) showed a dramatic 96-fold improvement in total activity with secretion levels of 2 mg/liter. Analysis of the N-terminal sequence of the FX7 variant confirmed the correct processing of the preαproK hybrid peptide by the KEX2 protease. FX7 showed higher stability in terms of pH and temperature, whereas the pH activity profiles and the kinetic parameters were maintained. The Asn91 lies in the flavin attachment loop motif, and it is a highly conserved residue in all members of the GMC superfamily, except for P. eryngii and P. pulmonarius AAO. The in vitro involution of the enzyme by restoring the consensus ancestor Asn91 promoted AAO expression and stability. PMID:26162870

  13. In vivo anti-tumor activity of marine hematopoietic stem cells expressing a p185HER2-specific chimeric T-cell receptor gene

    Institute of Scientific and Technical Information of China (English)

    JIAN MIN YANG; MICHAEL S FRIEDMAN; MARIANNE T HUBEN; JENNIFER FULLER; QIAO LI; ALFRED E CHANG; JAMES J MULE; KEVIN T MCDONAGH

    2006-01-01

    We have confirmed efficient anti-tumor activities of the peripheral lymphocytes transduced with a p185HER2-specific chimeric T-cell receptor gene both in murine and in human in our previous studies. To further test the feasibility of chimeric T-cell receptor in a bone marrow transplantation model, we first, made two murine tumor cell lines: MT901 and MCA-205, to express human p185HER2by retroviral gene transduction. Murine bone marrow cells were retrovirally transduced to express the chimeric T-cell receptor and gene-modified bone marrow cells were transplanted into lethally irradiated mouse. Six months post transplantation, p185HER2-positive tumor cells: MT-901/HER2 or MCA-205/HER2 was subcutaneously or intravenously injected to make mouse models simulating primary breast cancer or pulmonary metastasis. The in vivo anti-tumor effects were monitored by the size of the subcutaneous tumor or counting the tumor nodules in the lungs after India ink staining. The size of the subcutaneous tumor was significantly inhibited and the number of pulmonary nodules were significantly decreased in mouse recipients transplanted with chimeric T-cell receptor modified bone marrow cells compared with the control group. Our results suggest the efficient in vivo anti-tumor activities of chimeric T-cell receptor gene modified bone marrow cells.

  14. Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes

    Directory of Open Access Journals (Sweden)

    Alma Gedvilaite

    2015-07-01

    Full Text Available Recombinant virus-like particles (VLPs represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV viral protein 1 (VP1 was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes.

  15. PU.1-Silenced Dendritic Cells Induce Mixed Chimerism and Alleviate Intestinal Transplant Rejection in Rats via a Th1 to Th2 Shift

    Directory of Open Access Journals (Sweden)

    Xingwei Xu

    2016-01-01

    Full Text Available Background/Aims: Intestinal transplantation is an effective treatment for end-stage bowel failure; however, graft rejection and the toxicity associated with non-specific immunosuppression are major limitations of this procedure. Studies have shown that mixed chimerism can produce post-transplantation immune tolerance. Here, we demonstrate that in rat intestinal transplantation, PU.1-silenced dendritic cells (DCs plus bone marrow (BM cell transfusion results in mixed chimerism, and we investigate the mechanisms responsible for the effects of mixed chimerism rejection. Methods: In a model of intestinal transplantation, male Brown Norway rats were the donors, and female Lewis rats were the recipients that were randomly divided into 4 groups: control, BM, BM-imDCs and BM-PU.1. The dynamic changes in graft morphology, rejection scoring and serum concentrations of Th1/Th2-related cytokines were investigated on postoperative days 0, 7, 14, 21, and 30. Results: The BM-PU.1 group had better graft health, milder pathologic injuries, and lower rejection grades compared with the other groups. The rates of mixed chimerism were significantly highest in the BM-PU.1 group and correlated with decreases in serum IL-2 and increases in serum IL-10. Conclusion: Transfusion of PU.1-silenced DCs and BM cells induces stable mixed chimerism and has the potential to reduce pathologic injuries via a pro-Th2 shift in the Th1/Th2 balance.

  16. Chimerism Analysis of Cell-Free DNA in Patients Treated with Hematopoietic Stem Cell Transplantation May Predict Early Relapse in Patients with Hematologic Malignancies

    Directory of Open Access Journals (Sweden)

    Mahmoud Aljurf

    2016-01-01

    Full Text Available Background. We studied DNA chimerism in cell-free DNA (cfDNA in patients treated with HSCT. Methods. Chimerism analysis was performed on CD3+ cells, polymorphonuclear (PMN cells, and cfDNA using 16 small tandem repeat loci. The resulting labeled PCR-products were size-fractionated and quantified. Results. Analyzing samples from 191 patients treated with HSCT for nonneoplastic hematologic disorders demonstrated that the cfDNA chimerism is comparable to that seen in PMN cells. Analyzing leukemia patients (N = 126 showed that, of 84 patients with 100% donor DNA in PMN, 16 (19% had evidence of clinical relapse and >10% recipient DNA in the plasma. Additional 16 patients of the 84 (19% showed >10% recipient DNA in plasma, but without evidence of relapse. Eight patients had mixed chimerism in granulocytes, lymphocytes, and plasma, but three of these patients had >10% recipient DNA in plasma compared to PMN cells and these three patients had clinical evidence of relapse. The remaining 34 patients showed 100% donor DNA in both PMN and lymphocytes, but cfDNA showed various levels of chimerism. Of these patients 14 (41% showed laboratory or clinical evidence of relapse and all had >10% recipient DNA in cfDNA. Conclusion. Monitoring patients after HSCT using cfDNA might be more reliable than cellular DNA in predicting early relapse.

  17. Chimerism Analysis of Cell-Free DNA in Patients Treated with Hematopoietic Stem Cell Transplantation May Predict Early Relapse in Patients with Hematologic Malignancies

    Science.gov (United States)

    Aljurf, Mahmoud; Abalkhail, Hala; Alseraihy, Amal; Mohamed, Said Y.; Ayas, Mouhab; Alsharif, Fahad; Alzahrani, Hazza; Al-Jefri, Abdullah; Aldawsari, Ghuzayel; Al-Ahmari, Ali; Belgaumi, Asim F.; Walter, Claudia Ulrike; El-Solh, Hassan; Rasheed, Walid; Albitar, Maher

    2016-01-01

    Background. We studied DNA chimerism in cell-free DNA (cfDNA) in patients treated with HSCT. Methods. Chimerism analysis was performed on CD3+ cells, polymorphonuclear (PMN) cells, and cfDNA using 16 small tandem repeat loci. The resulting labeled PCR-products were size-fractionated and quantified. Results. Analyzing samples from 191 patients treated with HSCT for nonneoplastic hematologic disorders demonstrated that the cfDNA chimerism is comparable to that seen in PMN cells. Analyzing leukemia patients (N = 126) showed that, of 84 patients with 100% donor DNA in PMN, 16 (19%) had evidence of clinical relapse and >10% recipient DNA in the plasma. Additional 16 patients of the 84 (19%) showed >10% recipient DNA in plasma, but without evidence of relapse. Eight patients had mixed chimerism in granulocytes, lymphocytes, and plasma, but three of these patients had >10% recipient DNA in plasma compared to PMN cells and these three patients had clinical evidence of relapse. The remaining 34 patients showed 100% donor DNA in both PMN and lymphocytes, but cfDNA showed various levels of chimerism. Of these patients 14 (41%) showed laboratory or clinical evidence of relapse and all had >10% recipient DNA in cfDNA. Conclusion. Monitoring patients after HSCT using cfDNA might be more reliable than cellular DNA in predicting early relapse. PMID:27006832

  18. Chimeric peptide containing both B and T cells epitope of tumor-associated antigen L6 enhances anti-tumor effects in HLA-A2 transgenic mice.

    Science.gov (United States)

    Lin, Su-I; Huang, Ming-Hsi; Chang, Yu-Wen; Chen, I-Hua; Roffler, Steve; Chen, Bing-Mae; Sher, Yuh-Pyng; Liu, Shih-Jen

    2016-07-28

    Synthetic peptides are attractive for cancer immunotherapy because of their safety and flexibility. In this report, we identified a new B cell epitope of tumor-associated antigen L6 (TAL6) that could induce antibody-dependent cellular cytotoxicity (ADCC) in vivo. We incorporated the B cell epitope with a cytotoxic T lymphocyte (CTL) and a helper T (Th) epitope to form a chimeric long peptide. We formulated the chimeric peptide with different adjuvants to immunize HLA-A2 transgenic mice and evaluate their immunogenicity. The chimeric peptide formulated with an emulsion type nanoparticle (PELC) adjuvant and a toll-like receptor 9 agonist (CpG ODN) (PELC/CpG) induced the greatest ADCC and CTL responses. The induced anti-tumor immunity inhibited the growth of TAL6-positive cancer cells. Moreover, we observed that immunization with the chimeric peptide inhibited cancer cell migration in vitro and metastasis in vivo. These data suggest that a chimeric peptide containing both B and T cell epitopes of TAL6 formulated with PELC/CpG adjuvant is feasible for cancer immunotherapy. PMID:27130449

  19. Uncertainty characterization and quantification in air pollution models. Application to the CHIMERE model

    Science.gov (United States)

    Debry, Edouard; Mallet, Vivien; Garaud, Damien; Malherbe, Laure; Bessagnet, Bertrand; Rouïl, Laurence

    2010-05-01

    Prev'Air is the French operational system for air pollution forecasting. It is developed and maintained by INERIS with financial support from the French Ministry for Environment. On a daily basis it delivers forecasts up to three days ahead for ozone, nitrogene dioxide and particles over France and Europe. Maps of concentration peaks and daily averages are freely available to the general public. More accurate data can be provided to customers and modelers. Prev'Air forecasts are based on the Chemical Transport Model CHIMERE. French authorities rely more and more on this platform to alert the general public in case of high pollution events and to assess the efficiency of regulation measures when such events occur. For example the road speed limit may be reduced in given areas when the ozone level exceeds one regulatory threshold. These operational applications require INERIS to assess the quality of its forecasts and to sensitize end users about the confidence level. Indeed concentrations always remain an approximation of the true concentrations because of the high uncertainty on input data, such as meteorological fields and emissions, because of incomplete or inaccurate representation of physical processes, and because of efficiencies in numerical integration [1]. We would like to present in this communication the uncertainty analysis of the CHIMERE model led in the framework of an INERIS research project aiming, on the one hand, to assess the uncertainty of several deterministic models and, on the other hand, to propose relevant indicators describing air quality forecast and their uncertainty. There exist several methods to assess the uncertainty of one model. Under given assumptions the model may be differentiated into an adjoint model which directly provides the concentrations sensitivity to given parameters. But so far Monte Carlo methods seem to be the most widely and oftenly used [2,3] as they are relatively easy to implement. In this framework one

  20. Comparison of satellite NO2 results with mobile MAX-DOAS observations and CHIMERE model simulations for Paris

    Science.gov (United States)

    Shaiganfar, Reza; Beirle, Steffen; Petetin, Herve; Zhang, Qiji; Beekmann, Matthias; Wagner, Thomas

    2013-04-01

    Megacities are localized, heterogeneous and variable sources of various air pollutants, having great impact on air quality and ultimately on climate. Within the European project MEGAPOLI we characterise and quantify the pollution levels and emissions using spectroscopic observations from satellite and ground based instruments mounted on a car. The mobile observations are conducted on circles with different radii around megacities. From the satellite observations the link from local to regional and global scales can be made. Especially the impact of important sources like megacities on the surrounding areas and also over longer distances can be studied. The combination with the mobile measurements adds information about the heterogeneity within a satellite pixel and the diurnal cycle, which are not well captured from satellite observations. The CHIMERE model is used to produce daily 3D fields of different trace gases, ozone and aerosols. We compare the CHIMERE model with mobile MAX-DOAS and OMI satellite observations. The mobile measurements are also used for validation of the satellite observations. We compare the tropospheric NO2 from OMI (TEMIS) with our mobile MAX-DOAS vertical column densities (VCDs). In general good agreement of the spatial patterns was found between differet data sets. However, the mobile MAX-DOAS measurements usually showed much finer details of the horizontal distributions than the satellite and model data. Also differences in the absolute values were found: The Chimere data are17x% lower and 45% lower than the mobile MAX-DOAS data in summer and winter, respectively. The satellite data are about 50 % lower than mobile MAX-DOAS.

  1. Advances on the treatment of solid tumor by 131I labeled mouse-human chimeric tumor necrosis therapy monoclonal antibody

    International Nuclear Information System (INIS)

    131I labeled mouse-human chimeric tumor necrosis therapy monoclonal antibody (131I-chTNT) is a kind of new drug targeting at degenerated or necrotic nuclei in the tumor necrosis zone,and may be applicable to the majority of human solid tumors, such as lung cancer, liver cancer,colon carcinoma and glioma, while conventional tumor cell monoclonal antibody can target only tumor cell surface antigen. Enhanced effects can be achieved by 131I-chTNT in combination with other therapies, such as radiotherapy,chemotherapy or radiofrequency ablation, which may increase tumor necrosis region and expose more combinative targets. (authors)

  2. Abbreviated incubation times for human prions in mice expressing a chimeric mouse–human prion protein transgene

    OpenAIRE

    Korth, Carsten; Kaneko, Kiyotoshi; Groth, Darlene; Heye, Norbert; Telling, Glenn; Mastrianni, James; Parchi, Piero; Gambetti, Pierluigi; Will, Robert; Ironside, James; Heinrich, Cornelia; Tremblay, Patrick; Stephen J DeArmond; Prusiner, Stanley B.

    2003-01-01

    Transgenic (Tg) mouse lines that express chimeric mouse–human prion protein (PrP), designated MHu2M, are susceptible to prions from patients with sporadic Creutzfeldt–Jakob disease (sCJD). With the aim of decreasing the incubation time to fewer than 200 days, we constructed transgenes in which one or more of the nine human residues in MHu2M were changed to mouse. The construct with murine residues at positions 165 and 167 was expressed in Tg(MHu2M,M165V,E167Q) mice and resulted in shortening ...

  3. Genetically engineered T cells bearing chimeric nanoconstructed receptors harboring TAG-72-specific camelid single domain antibodies as targeting agents

    DEFF Research Database (Denmark)

    Sharifzadeh, Zahra; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad A;

    2013-01-01

    Despite the preclinical success of adoptive therapy with T cells bearing chimeric nanoconstructed antigen receptors (CARs), certain limitations of this therapeutic approach such as the immunogenicity of the antigen binding domain, the emergence of tumor cell escape variants and the blocking...... expressing tumor cells, the combination of CD3ζ, OX40, CD28 as well as the CH3-CH2-hinge-hinge domains most efficiently triggered T cell activation. Importantly, CAR mediated functions were not blocked by the soluble TAG-72 antigen at a supraphysiological concentration. Our approach may have the potential to...

  4. Chimeric antibody with human constant regions and mouse variable regions directed against carcinoma-associated antigen 17-1A

    International Nuclear Information System (INIS)

    The authors have cloned the genomic DNA fragments encoding the heavy and light chain variable regions of monoclonal antibody 17-1A, and they have inserted them into mammalian expression vectors containing genomic DNA segments encoding human γ3 and kappa constant regions. The transfer of these expression vectors containing mouse-human chimeric immunoglobulin genes into Sp2/0 mouse myeloma cells resulted in the production of functional IgG that retained the specific binding to the surface antigen 17-1A expressed on colorectal carcinoma cells

  5. A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen

    OpenAIRE

    Vijayan, Aneesh; García-Arriaza, Juan; C. Raman, Suresh; Conesa, José Javier; Chichón, Francisco Javier; Santiago, César; Sorzano, Carlos Óscar S.; Carrascosa, José L.; Esteban, Mariano

    2015-01-01

    In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size excl...

  6. Virus-Like Particles of Chimeric Recombinant Porcine Circovirus Type 2 as Antigen Vehicle Carrying Foreign Epitopes

    OpenAIRE

    2014-01-01

    Virus-like particles (VLPs) of chimeric porcine circovirus type 2 (PCV2) were generated by replacing the nuclear localization signal (NLS; at 1–39 aa) of PCV2 capsid protein (Cap) with classical swine fever virus (CSFV) T-cell epitope (1446–1460 aa), CSFV B-cell epitope (693–716 aa) and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The ab...

  7. A Molecular Model for Cocaine Binding by the Immunotherapeutic Human/Mouse Chimeric Monoclonal Antibody 2E2

    OpenAIRE

    Lape, Michael; Paula, Stefan; Ball, William J.

    2010-01-01

    Immunotherapy by cocaine-binding monoclonal antibodies (mAbs) has emerged as a promising strategy for the treatment of cocaine addiction. The human (γ1 heavy chain)/murine (λ light chain) chimeric mAb 2E2 has excellent affinity and specificity for cocaine and recent animal studies have demonstrated 2E2’s ability in vivo to reduce cocaine levels in the brain as well as alter cocaine self-administration behavior in rats. In this study, we used mAb 2E2 amino acid sequence information to create a...

  8. Construction and analysis of variants of a polyvalent Lyme disease vaccine: approaches for improving the immune response to chimeric vaccinogens

    OpenAIRE

    Earnhart, Christopher G.; Marconi, Richard T.

    2007-01-01

    There is currently no Lyme disease vaccine commercially available for use in humans. Outer surface protein C (OspC) of the Borrelia has been widely investigated as a potential vaccinogen. At least 38 OspC types have been defined. While the antibody response to OspC is protective, the range of protection is narrow due to the localization of protective epitopes within OspC type-specific domains. To develop a broadly protective vaccine, we previously constructed a tetravalent chimeric vaccinogen...

  9. Paranoid males have reduced lateralisation for processing of negative emotions: an investigation using the chimeric faces test.

    Science.gov (United States)

    Bourne, Victoria J; McKay, Ryan T

    2014-01-01

    Reduced strength of lateralisation in patients with schizophrenia has been reported in a number of studies. However the exact nature of this relationship remains unclear. In this study, lateralisation for processing emotional faces was measured using the chimeric faces test and examined in relation to paranoia in a non-clinical sample. For males only, those with higher scores on a paranoia questionnaire had reduced lateralisation for processing negative facial emotion. For females there were no significant relationships. These findings suggest that atypical patterns of lateralisation for processing emotional stimuli may be implicated in, or associated with, increased levels of paranoia. PMID:23844655

  10. Nonirradiated NOD/SCID-Human Chimeric Animal Model for Primary Human Multiple Myeloma : A Potential in Vivo Culture System

    OpenAIRE

    Huang, Shang-Yi; Tien, Hwei-Fang; Su, Fang-Hsein; Hsu, Su-Ming

    2004-01-01

    The NOD/SCID human chimeric animal model was generated by implanting of human fetal bones (FBs) into subcutaneous sites of NOD/SCID mice (NOD/SCID-hu+), followed by inoculation of primary bone marrow mononuclear cells (BMNCs) obtained from patients with multiple myeloma (MM) into the FBs. The BMNCs from 30 patients with MM were inoculated, and 28 (93%) of them revealed evidence of tumor growth of myeloma cells (MCs) in the NOD/SCID-hu+ mice. Intriguingly, 17 (61%) of the 28 patients’ BMNCs in...

  11. Functional analysis of chimeric genes obtained by exchanging homologous domains of the mouse mdr1 and mdr2 genes.

    OpenAIRE

    Buschman, E; Gros, P.

    1991-01-01

    A full-length cDNA clone for the mouse mdr1 gene can confer multidrug resistance when introduced by transfection into otherwise drug-sensitive cells. In the same assay, a full-length cDNA clone for a closely related member of the mouse mdr gene family, mdr2, fails to confer multidrug resistance. To identify the domains of mdr1 which are essential for multidrug resistance and which may be functionally distinct in mdr2, we have constructed chimeric cDNA molecules in which discrete domains of md...

  12. Chimeric TLS/FUS-CHOP Gene Expression and the Heterogeneity of its Junction in Human Myxoid and Round Cell Liposarcoma

    OpenAIRE

    Kuroda, Masahiko; Ishida, Tsuyoshi; Horiuchi, Hajime; Kida, Naotoshi; Uozaki, Hiroshi; TAKEUCHI, Hajime; Tsuji, Kaori; Imamura, Tetsuo; Mori, Shigeo; Machinami, Rikuo; Watanabe, Toshiki

    1995-01-01

    Myxoid liposarcomas have a unique and specific t(12;16)(q13;p11) chromosomal translocation. The breakpoint has recently been identified and shown to involve the TLS/FUS gene on chromosome 16 and the CHOP gene on chromosome 12. This translocation causes fusion of these genes resulting in the expression of a novel chimeric TLS/FUS-CHOP message. Using the polymerase chain reaction with primer sets derived from sequences of TLS/FUS and CHOP cDNAs, we could amplify three types of the fusion transc...

  13. Voltage-Jump Relaxation Kinetics for Wild-type and Chimeric β Subunits of Neuronal Nicotinic Receptors

    OpenAIRE

    Figl, Antonio; Labarca, Cesar; Davidson, Norman; Lester, Henry A.; Cohen, Bruce N.

    1996-01-01

    We have studied the voltage-jump relaxation currents for a series of neuronal nicotinic acetylcholine receptors resulting from the coexpression of wild-type and chimeric β4/β2 subunits with α3 subunits in Xenopus oocytes. With acetylcholine as the agonist, the wild-type α3β4 receptors displayed five- to eightfold slower voltage-jump relaxations than did the wild-type α3β2 receptors. In both cases, the relaxations could best be described by two exponential components of approximately equal amp...

  14. Chimeric hepatitis B virus (HBV)/hepatitis C virus (HCV) subviral envelope particles induce efficient anti-HCV antibody production in animals pre-immunized with HBV vaccine.

    Science.gov (United States)

    Beaumont, Elodie; Roingeard, Philippe

    2015-02-18

    The development of an effective, affordable prophylactic vaccine against hepatitis C virus (HCV) remains a medical priority. The recently described chimeric HBV-HCV subviral envelope particles could potentially be used for this purpose, as they could be produced by industrial procedures adapted from those established for the hepatitis B virus (HBV) vaccine. We show here, in an animal model, that pre-existing immunity acquired through HBV vaccination does not influence the immunogenicity of the HCV E2 protein presented by these chimeric particles. Thus, these chimeric HBV-HCV subviral envelope particles could potentially be used as a booster in individuals previously vaccinated against HBV, to induce protective immunity to HCV. PMID:25596457

  15. HLA-DR4-IE chimeric class II transgenic, murine class II-deficient mice are susceptible to experimental allergic encephalomyelitis

    OpenAIRE

    1996-01-01

    To investigate the development of HLA-DR-associated autoimmune diseases, we generated transgenic (Tg) mice with HLA-DRA-IE alpha and HLA-DRB1*0401-IE beta chimeric genes. The transgene-encoded proteins consisted of antigen-binding domains from HLA-DRA and HLA-DRB1*0401 molecules and the remaining domains from the IE(d)-alpha and IE(d)-beta chains. The chimeric molecules showed the same antigen-binding specificity as HLA-DRB1*0401 molecules, and were functional in presenting antigens to T cell...

  16. Comparison of a chimeric anti-carcinoembryonic antigen antibody conjugated with visible or near-infrared fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models

    OpenAIRE

    Maawy, Ali A.; Hiroshima, Yukihiko; Kaushal, Sharmeela; Luiken, George A.; Robert M Hoffman; Bouvet, Michael

    2013-01-01

    Abstract. The aim of this study was to evaluate a set of visible and near-infrared dyes conjugated to a tumor-specific chimeric antibody for high-resolution tumor imaging in orthotopic models of pancreatic cancer. BxPC-3 human pancreatic cancer was orthotopically implanted into pancreata of nude mice. Mice received a single intravenous injection of a chimeric anti-carcinoembryonic antigen antibody conjugated to one of the following fluorophores: 488-nm group (Alexa Fluor 488 or DyLight 488); ...

  17. Redirecting Therapeutic T Cells against Myelin-Specific T Lymphocytes Using a Humanized Myelin Basic Protein-HLA-DR2-{zeta} Chimeric Receptor

    DEFF Research Database (Denmark)

    Moisini, Ioana; Nguyen, Phuong; Fugger, Lars;

    2008-01-01

    Therapies that Ag-specifically target pathologic T lymphocytes responsible for multiple sclerosis (MS) and other autoimmune diseases would be expected to have improved therapeutic indices compared with Ag-nonspecific therapies. We have developed a cellular immunotherapy that uses chimeric receptors...... humanized mouse model system. Finally, the chimeric receptor-modified CTL ameliorated or blocked experimental allergic encephalomyelitis (EAE) disease mediated by MBP(84-102)/DR2-specific T lymphocytes. These results provide support for the further development of redirected therapeutic T cells able to...

  18. Immunogenicity of a chimeric peptide corresponding to T helper and B cell epitopes of the Chlamydia trachomatis major outer membrane protein

    OpenAIRE

    1992-01-01

    The immunogenicity of a chimeric T/B cell peptide corresponding to antigenically characterized epitopes of the Chlamydia trachomatis major outer membrane protein (MOMP) was studied in mice to further define its potential use in the development of a subunit vaccine in preventing blinding trachoma in humans. The chimeric peptide, designated A8-VDI, corresponds to a conserved MOMP T helper (Th) cell epitope(s) (A8, residues 106-130) and serovar A VDI (residues 66-80), which contains the serovar-...

  19. Biochemical characterization and evaluation of cytotoxicity of antistaphylococcal chimeric protein P128

    Directory of Open Access Journals (Sweden)

    George Shilpa E

    2012-06-01

    Full Text Available Abstract Background Antibiotic resistant S. aureus infection is a global threat. Newer approaches are required to control this organism in the current scenario. Cell wall degrading enzymes have been proposed as antibacterial agents for human therapy. P128 is a novel antistaphylococcal chimeric protein under development against S. aureus for human use which derives its bacterial cell wall degrading catalytic endopeptidase domain from ORF56, the Phage K tail-structure associated enzyme. Lead therapeutic entities have to be extensively characterized before they are assessed in animals for preclinical safety and toxicity. P128 is effective against antibiotic resistant strains as well as against a panel of isolates of global significance. Its efficacy against S. aureus in vivo has been established in our lab. Against this background, this study describes the characterization of this protein for its biochemical properties and other attributes. Results We evaluated the requirement or effect of divalent cations and the metal ion chelator, EDTA upon biological activity of P128. As the protein is intended for therapeutic use, we tested its activity in presence of body fluids and antibodies specific to P128. For the same reason, we used standard human cell lines to evaluate cytotoxic effects, if any. The divalent cations, calcium and magnesium at upto 25 mM and Zinc upto 2.5 mM neither inhibited nor enhanced P128 activity. Incubation of this protein with EDTA, human serum, plasma and blood also did not alter the antibacterial properties of the molecule. No inhibitory effect was observed in presence of hyper-immune sera raised against the protein. Finally, P128 did not show any cytotoxic effect on HEp2 and Vero cells at the highest concentration (5 mg/mL tested. Conclusions The results presented here throw light on several properties of protein P128. Taken together, these substantiate the potential of P128 for therapeutic use against S. aureus

  20. A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

    Science.gov (United States)

    Muthamilselvan, Thangarasu; Lee, Chin-Wei; Cho, Yu-Hsin; Wu, Feng-Chao; Hu, Chung-Chi; Liang, Yu-Chuan; Lin, Na-Sheng; Hsu, Yau-Heiu

    2016-01-01

    We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system. PMID:25879277

  1. {sup 99m}Tc-labeled chimeric anti-NCA 95 antigranulocyte monoclonal antibody for bone marrow imaging

    Energy Technology Data Exchange (ETDEWEB)

    Sarwar, M.; Higuchi, Tetsuya; Tomiyoshi, Katsumi [Gunma Univ., Maebashi (Japan). School of Medicine] [and others

    1998-09-01

    Chimeric mouse-human antigranulocyte monoclonal antibody (ch MAb) against non-specific cross-reacting antigen (NCA-95) was labeled with {sup 99m}Tc (using a direct method) and {sup 125}I (using the chloramine T method), and its binding to human granulocytes and LS-180 colorectal carcinoma cells expressing carcinoembryonic antigen on their surfaces, cross-reactive with anti-NCA-95 chimeric monoclonal antibody, increased in proportion to the number of cells added and reached more than 80% and 90%, respectively. In biodistribution studies, {sup 99m}Tc and {sup 125}I-labeled ch anti-NCA-95 MAb revealed high tumor uptake, and the tumor-to-blood ratio was 2.9 after 24 hours. The tumor-to-normal-organ ratio was also more than 3.0 in all organs except for the tumor-to-kidney ratio. Scintigrams of athymic nude mice confirmed the results of biodistribution studies that showed higher radioactivity in tumor and kidney of the mice administered with {sup 99m}Tc-labeled ch MAb. A normal volunteer injected with {sup 99m}Tc-labeled ch anti-NCA-95 antigranulocyte MAb showed clear bone marrow images, and a patient with aplastic anemia revealed irregular uptake in his lumbar spine, suggesting its utility for bone marrow scintigraphy and for the detection of hematological disorders, infections, and bone metastasis. (author)

  2. Structural Characterization by NMR of a Double Phosphorylated Chimeric Peptide Vaccine for Treatment of Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Stefan Berger

    2013-04-01

    Full Text Available Rational design of peptide vaccines becomes important for the treatment of some diseases such as Alzheimer’s disease (AD and related disorders. In this study, as part of a larger effort to explore correlations of structure and activity, we attempt to characterize the doubly phosphorylated chimeric peptide vaccine targeting a hyperphosphorylated epitope of the Tau protein. The 28-mer linear chimeric peptide consists of the double phosphorylated B cell epitope Tau229-237[pThr231/pSer235] and the immunomodulatory T cell epitope Ag85B241-255 originating from the well-known antigen Ag85B of the Mycobacterium tuberculosis, linked by a four amino acid sequence -GPSL-. NMR chemical shift analysis of our construct demonstrated that the synthesized peptide is essentially unfolded with a tendency to form a β-turn due to the linker. In conclusion, the -GPSL- unit presumably connects the two parts of the vaccine without transferring any structural information from one part to the other. Therefore, the double phosphorylated epitope of the Tau peptide is flexible and accessible.

  3. Virus-Like Particles of Chimeric Recombinant Porcine Circovirus Type 2 as Antigen Vehicle Carrying Foreign Epitopes

    Directory of Open Access Journals (Sweden)

    Huawei Zhang

    2014-12-01

    Full Text Available Virus-like particles (VLPs of chimeric porcine circovirus type 2 (PCV2 were generated by replacing the nuclear localization signal (NLS; at 1–39 aa of PCV2 capsid protein (Cap with classical swine fever virus (CSFV T-cell epitope (1446–1460 aa, CSFV B-cell epitope (693–716 aa and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The abilities to form PCV2 VLPs were confirmed by transmission electron microscopy. Immunogenicities of the three recombinant proteins were evaluated in mice. Our Results indicated that Cap protein NLS deletion or substitution with CSFV epitopes did not affect the VLPs assembly. Three chimeric Cap proteins could form VLPs and induce efficient humoral and cellular immunity against PCV2 and CSFV in mice. Results show that PCV2 VLPs can be used as an efficient antigen carrier for delivery of foreign epitopes, and a potential novel vaccine.

  4. Molecularly engineered live-attenuated chimeric West Nile/dengue virus vaccines protect rhesus monkeys from West Nile virus

    International Nuclear Information System (INIS)

    Two molecularly engineered, live-attenuated West Nile virus (WN) vaccine candidates were highly attenuated and protective in rhesus monkeys. The vaccine candidates are chimeric viruses (designated WN/DEN4) bearing the membrane precursor and envelope protein genes of WN on a backbone of dengue 4 virus (DEN4) with or without a deletion of 30 nucleotides (Δ30) in the 3' noncoding region of DEN4. Viremia in WN/DEN4- infected monkeys was reduced 100-fold compared to that in WN- or DEN4-infected monkeys. WN/DEN4-3'Δ30 did not cause detectable viremia, indicating that it is even more attenuated for monkeys. These findings indicate that chimerization itself and the presence of the Δ30 mutation independently contribute to the attenuation phenotype for nonhuman primates. Despite their high level of attenuation in monkeys, the chimeras induced a moderate-to-high titer of neutralizing antibodies and prevented viremia in monkeys challenged with WN. The more attenuated vaccine candidate, WN/DEN4-3'Δ30, will be evaluated first in our initial clinical studies

  5. Virus-like particles of chimeric recombinant porcine circovirus type 2 as antigen vehicle carrying foreign epitopes.

    Science.gov (United States)

    Zhang, Huawei; Qian, Ping; Liu, Lifeng; Qian, Suhong; Chen, Huanchun; Li, Xiangmin

    2014-12-01

    Virus-like particles (VLPs) of chimeric porcine circovirus type 2 (PCV2) were generated by replacing the nuclear localization signal (NLS; at 1-39 aa) of PCV2 capsid protein (Cap) with classical swine fever virus (CSFV) T-cell epitope (1446-1460 aa), CSFV B-cell epitope (693-716 aa) and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The abilities to form PCV2 VLPs were confirmed by transmission electron microscopy. Immunogenicities of the three recombinant proteins were evaluated in mice. Our Results indicated that Cap protein NLS deletion or substitution with CSFV epitopes did not affect the VLPs assembly. Three chimeric Cap proteins could form VLPs and induce efficient humoral and cellular immunity against PCV2 and CSFV in mice. Results show that PCV2 VLPs can be used as an efficient antigen carrier for delivery of foreign epitopes, and a potential novel vaccine. PMID:25490764

  6. The role of bone marrow-derived cells in bone fracture repair in a green fluorescent protein chimeric mouse model

    International Nuclear Information System (INIS)

    We investigated the role of bone marrow cells in bone fracture repair using green fluorescent protein (GFP) chimeric model mice. First, the chimeric model mice were created: bone marrow cells from GFP-transgenic C57BL/6 mice were injected into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10 Gy from a cesium source. Next, bone fracture models were created from these mice: closed transverse fractures of the left femur were produced using a specially designed device. One, three, and five weeks later, fracture lesions were extirpated for histological and immunohistochemical analyses. In the specimens collected 3 and 5 weeks after operation, we confirmed calluses showing intramembranous ossification peripheral to the fracture site. The calluses consisted of GFP- and osteocalcin-positive cells at the same site, although the femur consisted of only osteocalcin-positive cells. We suggest that bone marrow cells migrated outside of the bone marrow and differentiated into osteoblasts to make up the calluses

  7. Construction and analysis of variants of a polyvalent Lyme disease vaccine: approaches for improving the immune response to chimeric vaccinogens.

    Science.gov (United States)

    Earnhart, Christopher G; Marconi, Richard T

    2007-04-30

    There is currently no Lyme disease vaccine commercially available for use in humans. Outer surface protein C (OspC) of the Borrelia has been widely investigated as a potential vaccinogen. At least 38 OspC types have been defined. While the antibody response to OspC is protective, the range of protection is narrow due to the localization of protective epitopes within OspC type-specific domains. To develop a broadly protective vaccine, we previously constructed a tetravalent chimeric vaccinogen containing epitopes from OspC types A, B, K, and D. While this construct elicited bactericidal antibody against strains bearing each of the four OspC types, its solubility was low, and decreasing IgG titer to epitopes near the C-terminus of the construct was observed. In this report, construct solubility and immunogenicity were increased by dialysis against an Arg/Glu buffer. We also demonstrate the immunogenicity of the construct in alum. To further optimize epitope-specific immune responses, several constructs were generated with differing epitope organization or with putative C-terminal protective motifs. Analyses of murine antibody titers and isotype profiles induced by these constructs revealed that while the C-terminal tags did not enhance antibody titer, specific epitope reorganization and reiteration did. These analyses provide important information that can be exploited in the development of chimeric vaccinogens in general. PMID:17239505

  8. Iron carrier proteins facilitate engraftment of allogeneic bone marrow and enduring hemopoietic chimerism in the lethally irradiated host

    International Nuclear Information System (INIS)

    Cell-free supernatants of rabbit bone marrow were fractionated, separated, and purified by Ultrogel and Superose chromatography. A single fraction promoted engraftment of allogeneic bone marrow and enduring hemopoietic chimerism across the H-2 barrier in lethally irradiated mice. This 'bio-active' fraction, analyzed by reducing SDS-PAGE electrophoresis, and transblotted on PVDF membrane, and purified by reverse-phase HPLC and SDS-PAGE electrophoresis yielded a main prealbumin band that when examined for primary structure by Edman degradation, proved to be rabbit transferrin. This was also attested by highly specific precipitation of the prealbumin band with polyclonal antibodies to rabbit transferrin. Iron-saturated human transferrin, lactotransferrin, and egg transferrin (conalbumin) were assayed in irradiated C57BL/6 mice infused with bone marrow from histoincompatible BALB/c donors. Mice treated with iron-loaded transferrins survive and develop enduring allogeneic chimerism with no discernible signs of graft-versus-host disease. Iron carrier proteins thus provide an unique means of achieving successful engraftment of allogeneic bone marrow in immunologically hostile murine H-2 combinations

  9. Replication of chimeric tobacco mosaic viruses which carry heterologous combinations of replicase genes and 3' noncoding regions.

    Science.gov (United States)

    Ishikawa, M; Meshi, T; Watanabe, Y; Okada, Y

    1988-05-01

    Three tobacco mosaic virus (TMV)-L (tomato strain)-derived chimeras, designated OL1, LG11, or LK31, were constructed by replacing the 3' noncoding region with the corresponding sequence of TMV-OM (common strain), cucumber green mottle mosaic virus (CGMMV), or TMV-Cc (cowpea strain), respectively. The genomic RNAs of TMV-L, -OM, and CGMMV carry histidine-accepting tRNA-like structures at their 3' termini, while the genome of TMV-Cc accepts valine. The three chimeric viruses were able to multiply in both tobacco protoplasts and plants. Multiplication of OL1 in protoplasts was similar to that of the parental strain, L, but in the cases of LG11 and LK31 multiplication was decreased. Sequence analyses of progeny RNAs revealed that viruses with chimeric sequences propagated. These data suggested that TMV-L replicase recognizes the 3' terminal structures of TMV-OM, CGMMV, and TMV-Cc and can initiate minus-strand RNA synthesis. The relationship between the virus-coded component(s) of TMV replicase and the 3' terminal region may not be so stringent. PMID:2452515

  10. Exchanging murine and human immunoglobulin constant chains affects the kinetics and thermodynamics of antigen binding and chimeric antibody autoreactivity.

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    Marcela Torres

    Full Text Available Mouse-human chimeric antibodies composed of murine variable (V and human (C chains are useful therapeutic reagents. Consequently, we investigated whether heterologous C-regions from mice and humans affected specificity and affinity, and determined the contribution of C(H glycosylation to antigen binding. The interaction of a 12-mer peptide mimetic with monoclonal antibody (mAb 18B7 to Cryptococcus neoformans glucuronoxylomannan, and its chimeric (ch and deglycosylated forms were studied by surface plasmon resonance. The equilibrium and rate association constants for the chAb were higher than for mAb 18B7. V region affinity was not affected by C(H region glycosylation whereas heterologous C region of the same isotype altered the Ab binding affinity and the specificity for self-antigens. Structural models displayed local differences that implied changes on the connectivity of residues. These findings suggest that V region conformational changes can be dictated by the C(H domains through an allosteric effect involving networks of highly connected amino acids.

  11. Chimeric molecules facilitate the degradation of androgen receptors and repress the growth of LNCaP cells

    Institute of Scientific and Technical Information of China (English)

    Yue-Qing Tang; Bang-Min Han; Xin-Quan Yao; Yan Hong; Yan Wang; Fu-Jun Zhao; Sheng-Qiang Yu; Xiao-Wen Sun; Shu-Jie Xia

    2009-01-01

    Post-translational degradation of protein plays an important role in cell life.We employed chimeric molecules (dihydrotestosterone-based proteolysis-targeting chimeric molecule [DHT-PROTAC]) to facilitate androgen receptor (AR) degradation via the ubiquitin-proteasome pathway (UPP) and to investigate the role of AR in cell proliferation and viability in androgen-sensitive prostate cancer cells.Western blot analysis and immunohistochemistry were applied to analyse AR levels in LNCaP cells after DHT-PROTAC treatment.Cell counting and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell viability assay were used to evaluate cell proliferation and viability after AR elimination in both LNCaP and PC-3 cells.AR was tagged for elimination via the UPP by DHT-PROTAC,and this could be blocked by proteasome inhibitors.Degradation of AR depended on DHT-PROTAC concentration,and either DHT or an ALAPYIP-(arg)s peptide could compete with DHT-PROTAC.Inhibition of cell proliferation and decreased viability were observed in LNCaP cells,but not in PC-3 or 786-O cells after DHT-PROTAC treatment.These data indicate that AR elimination is facilitated via the UPP by DHT-PROTAC,and that the growth of LNCaP cells is repressed after AR degradation.

  12. Bioactivity assays and application of 125I labeled human mouse chimeric anti-CD22 monoclonal antibody SM03

    International Nuclear Information System (INIS)

    To investigate the bioactivity and application of 125I labeled human mouse chimeric monoclonal SM03, SM03 was labeled with 125I using Indogen method. The labeled mixture was purified by Sephacryl S-300 HR separation chromospectry. The purity and concentration of separated fractions were determined by HPLC and Protein Assay Kit, respectively. Competitive binding method and ELISA method were used for bioactivity assays. 125I-SM03 was applied to screen cell lines which express the most abundant CD22 antigen. The purity and recovery of 125I-SM03 were >99% and >47%, respectively. The bioactivity of 125I- SM03 and SM03 hasn't significant difference in statistics. Ramos cell line had the strongest special radioactivity when 125I-SM03 bound with in Raji, Daudi and Ramos cell lines. Indogen method is a good way to label Human mouse chimeric anti-CD22 monoclonal antibody SM03 and the label will not affect the activity of SM03. The 125I-SM03 not only can be used for detect agent, but also may be put into market for NHL therapy. (authors)

  13. Exploiting chimeric human antibodies to characterize a protective epitope of Neisseria adhesin A, one of the Bexsero vaccine components.

    Science.gov (United States)

    Bertoldi, Isabella; Faleri, Agnese; Galli, Barbara; Lo Surdo, Paola; Liguori, Alessia; Norais, Nathalie; Santini, Laura; Masignani, Vega; Pizza, Mariagrazia; Giuliani, Marzia Monica

    2016-01-01

    Neisseria adhesin A (NadA) is one of the antigens of Bexsero, the recently licensed multicomponent vaccine against serogroup B Neisseria meningitidis (MenB). NadA belongs to the class of oligomeric coiled-coil adhesins and is able to mediate adhesion and invasion of human epithelial cells. As a vaccine antigen, NadA has been shown to induce high levels of bactericidal antibodies; however, the domains important for protective response are still unknown. In order to further investigate its immunogenic properties, we have characterized the murine IgG1 mAb (6E3) that was able to recognize the 2 main antigenic variants of NadA on the surface of MenB strains. The epitope targeted by mAb 6E3 was mapped by hydrogen-deuterium exchange mass spectrometry and shown to be located on the coiled-coil stalk region of NadA (aa 206-249). Although no serum bactericidal activity was observed for murine IgG1 mAb 6E3, functional activity was restored when using chimeric antibodies in which the variable regions of the murine mAb 6E3 were fused to human IgG3 constant regions, thus confirming the protective nature of the mAb 6E3 epitope. The use of chimeric antibody molecules will enable future investigations of complement-mediated antibody functionality independently of the Fc-mediated differences in complement activation. PMID:26304221

  14. Immunogenicity of IMS 1113 plus soluble subunit and chimeric proteins containing Mycoplasma hyopneumoniae P97 C-terminal repeat regions.

    Science.gov (United States)

    Barate, Abhijit K; Cho, Youngjae; Truong, Quang Lam; Hahn, Tae-Wook

    2014-03-01

    The surface adhesin P97 mediates the adherence of Mycoplasma hyopneumoniae to swine cilia. Two reiterated repeats R1 and R2 are located at the C-terminus of P97. The purpose of this study was to evaluate the immunogenicity of Montanide adjuvant IMS 1113 plus soluble subunit proteins rR1, rR1R2 and their chimeric forms coupled with B subunit of the heat-labile enterotoxin of Escherichia coli (LTB). Each recombinant protein in this study was capable of eliciting anti-R1 specific humoral antibodies (IgG), mucosal antibodies (IgG and IgA) and IFN-γ production. The chimeric protein rLTBR1R2 elicited the quickest humoral antibody response among the recombinant proteins. Serum and bronchoalveolar lavage analysis revealed that each recombinant protein was capable of inducing both Th1 and Th2 responses. Importantly, all of the proteins induced an anti-R1-specific Th2-biased response in both humoral and mucosal compartments, similar to the response observed in a natural infection or vaccination process. These observations indicate that rR1, rR1R2, rLTBR1 and rLTBR1R2 with IMS 1113 might represent a promising subunit vaccine strategy against porcine enzootic pneumonia in pigs. PMID:24461070

  15. Mutation of the fiber shaft heparan sulphate binding site of a 5/3 chimeric adenovirus reduces liver tropism.

    Science.gov (United States)

    Koski, Anniina; Karli, Eerika; Kipar, Anja; Escutenaire, Sophie; Kanerva, Anna; Hemminki, Akseli

    2013-01-01

    Natural tropism to the liver is a major obstacle in systemic delivery of adenoviruses in cancer gene therapy. Adenovirus binding to soluble coagulation factors and to cellular heparan sulphate proteoglycans via the fiber shaft KKTK domain are suggested to cause liver tropism. Serotype 5 adenovirus constructs with mutated KKTK regions exhibit liver detargeting, but they also transduce tumors less efficiently, possibly due to altered fiber conformation. We constructed Ad5/3lucS*, a 5/3 chimeric adenovirus with a mutated KKTK region. The fiber knob swap was hypothesized to facilitate tumor transduction. This construct was studied with or without additional coagulation factor ablation. Ad5/3lucS* exhibited significantly reduced transduction of human hepatic cells in vitro and mouse livers in vivo. Combination of coagulation factor ablation by warfarinization to Ad5/3lucS* seemed to further enhance liver detargeting. Cancer cell transduction by Ad5/3lucS* was retained in vitro. In vivo, viral particle accumulation in M4A4-LM3 xenograft tumors was comparable to controls, but Ad5/3lucS* transgene expression was nearly abolished. Coagulation factor ablation did not affect tumor transduction. These studies set the stage for further investigations into the effects of the KKTK mutation and coagulation factor ablation in the context of 5/3 serotype chimerism. Of note, the putative disconnect between tumor transduction and transgene expression could prove useful in further understanding of adenovirus biology. PMID:23585829

  16. I-131 rituximab (chimeric anti Cd 20 mab) radioimmunotherapy of non-Hodgkins lymphoma

    International Nuclear Information System (INIS)

    radioimmunotherapy rather than antibody-targeted internal radiotherapy. Such treatment in 90 patients with relapsed/refractory follicular (grade I, II, III) (78) MALT (5) and small lymphocytic (7) lymphoma with 3 (1-8) median prior chemo-therapies, 60% stage III/IV, resulted in complete remission CR) in 51% and partial response (PR) in 23% for an overall response rate (ORR) of 74%. In contrast to radiolabelled murine antibodies, the 131I-rituximab chimeric monoclonal antibody does not cause any immunogenic HAMA host response and repeated courses of 131I-rituximab radioimmunotherapy may be given for subsequent relapse. The median duration of response was 22 months in our patients who achieved CR and retreatment was performed in 8 patients, the majority of whom responded again to the repeat 131I-rituximab radioimmunotherapy. Median progression-free survival in our patients was 13 months and the 4 year actuarial survival of all our treated patients after 131I-rituximab radioimmunotherapy for non-Hodgkins lymphoma was 63%. Non-myeloablative radioimmunotherapy of mantle cell non-Hodgkins lymphoma with 131I-rituximab showed CR in 2 of 8 patients, but the reported results of myeloablative regimens and autologous stem cell rescue demonstrate CR in 6 or 7 patients after 131I-rituximab treatment. Indolent non-Hodgkins lymphoma which transforms into more aggressive forms may also be treated with a myeloablative regimen combining standard dose 131I-rituximab radioimmunotherapy with BEAM chemotherapy for conditioning prior to stem cell autograft at 16 days. Three patients with transformed non-Hodgkins lymphoma treated with 131I-rituximab, BEAM chemotherapy and stem cell rescue all achieved CR of duration of at least 12 months. Refractory or relapsed aggressive non-Hodgkins lymphoma such as DLCBL may also be treated with non-myeloablative protocols complemented by long-term consolidation and maintenance MabThera immunotherapy, currently in clinical trial. Greater experience has been obtained with

  17. Chimeric anti-staphylococcal enterotoxin B antibodies and lovastatin act synergistically to provide in vivo protection against lethal doses of SEB.

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    Mulualem E Tilahun

    Full Text Available Staphylococcal enterotoxin B (SEB is one of a family of toxins secreted by Staphylococcus aureus that act as superantigens, activating a large fraction of the T-cell population and inducing production of high levels of inflammatory cytokines that can cause toxic shock syndrome (TSS and death. Extracellular engagement of the TCR of T-cells and class II MHC of antigen presenting cells by SEB triggers the activation of many intracellular signaling processes. We engineered chimeric antibodies to block the extracellular engagement of cellular receptors by SEB and used a statin to inhibit intracellular signaling. Chimeric human-mouse antibodies directed against different neutralizing epitopes of SEB synergistically inhibited its activation of human T-cells in vitro. In the in vivo model of lethal toxic shock syndrome (TSS in HLA-DR3 transgenic mice, two of these antibodies conferred significant partial protection when administered individually, but offered complete protection in a synergistic manner when given together. Similarly, in vivo, lovastatin alone conferred only partial protection from TSS similar to single anti-SEB antibodies. However, used in combination with one chimeric neutralizing anti-SEB antibody, lovastatin provided complete protection against lethal TSS in HLA-DR3 transgenic mice. These experiments demonstrate that in vivo protection against lethal doses of SEB can be achieved by a statin of proven clinical safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts.

  18. Chimerism analysis in clinical practice and its relevance for the detection of graft rejection and malignant relapse in pediatric hematopoietic stem cell transplant patients.

    Science.gov (United States)

    Mellgren, Karin; Arvidson, Johan; Toporski, Jacek; Winiarski, Jacek

    2015-11-01

    Chimerism and clinical outcome data from 244 hematopoietic stem cell transplants in 218 children were retrospectively analyzed to assess their relevance for the detection of graft rejection and malignant relapse. Patients transplanted for a non-malignant disease had significantly higher proportions of residual recipient T cells in peripheral blood at one, three, and six months compared with patients transplanted for malignant disease. Recipient T-cell levels were below 50% at one month after transplantation in most patients (129 of 152 transplants). Graft rejection occurred more frequently in the group of patients with high levels of recipient cells at one month (10 graft rejections in the 23 patients with recipient T cells >50% at one month as compared to seven graft rejections occurred in 129 patients with recipient T cells <50% (p < 0.001). Multilineage chimerism data in 87 children with leukemia at one, three, and six months after transplantation were not correlated with subsequent relapse of malignant disease. In conclusion, early analysis of lineage-specific chimerism in peripheral blood can be used to identify patients who are at high risk of graft rejection. However, the efficacy of early chimerism analysis for predicting leukemia relapse was limited. PMID:26290161

  19. Novel chimeric foot-and-mouth disease virus-like particles harboring serotype O VP1 protect guinea pigs against challenge.

    Science.gov (United States)

    Li, Haitao; Li, Zhiyong; Xie, Yinli; Qin, Xiaodong; Qi, Xingcai; Sun, Peng; Bai, Xingwen; Ma, Youji; Zhang, Zhidong

    2016-02-01

    Foot-and-mouth disease is a highly contagious, acute viral disease of cloven-hoofed animal species causing severe economic losses worldwide. Among the seven serotypes of foot-and-mouth disease virus (FMDV), serotype O is predominant, but its viral capsid is more acid sensitive than other serotypes, making it more difficult to produce empty serotype O VLPs in the low pH insect hemolymph. Therefore, a novel chimeric virus-like particle (VLP)-based candidate vaccine for serotype O FMDV was developed and characterized in the present study. The chimeric VLPs were composed of antigenic VP1 from serotype O and segments of viral capsid proteins from serotype Asia1. These VLPs elicited significantly higher FMDV-specific antibody levels in immunized mice than did the inactivated vaccine. Furthermore, the chimeric VLPs protected guinea pigs from FMDV challenge with an efficacy similar to that of the inactivated vaccine. These results suggest that chimeric VLPs have the potential for use in vaccines against serotype O FMDV infection. PMID:26790940

  20. Nanoscale orientation and lateral organization of chimeric metal-binding green fluorescent protein on lipid membrane determined by epifluorescence and atomic force microscopy

    International Nuclear Information System (INIS)

    Epifluorescence microscopy as well as atomic force microscopy was successfully applied to explore the orientation and lateral organization of a group of chimeric green fluorescent proteins (GFPs) on lipid membrane. Incorporation of the chimeric GFP carrying Cd-binding region (His6CdBP4GFP) to the fluid phase of DPPC monolayer resulted in a strong fluorescence intensity at the air-water interface. Meanwhile, non-specific adsorption of the GFP having hexahistidine (His6GFP) led to the perturbation of the protein structure in which very low fluorescence was observed. Specific binding of both of the chimeric GFPs to immobilized zinc ions underneath the metal-chelating lipid membrane was revealed. This specific binding could be reversibly controlled by addition of metal ions or metal chelator. Binding of the chimeric GFPs to the metal-chelating lipid membrane was proven to be the end-on orientation while the side-on adsorption was contrarily noted in the absence of metal ions. Increase of lateral mobility owing to the fluidization effect on the chelating lipid membrane subsequently facilitated crystal formation. All these findings have opened up a potential approach for a specific orientation of immobilization of protein at the membrane interface. This could have accounted for a better opportunity of sensor development

  1. Chimeric classical swine fever (CSF)-Japanese encephalitis (JE) viral particles as a non-transmissible bivalent marker vaccine candidate against CSF and JE infections

    Science.gov (United States)

    A trans-complemented CSF- JE chimeric viral replicon was constructed using an infectious cDNA clone of the CSF virus (CSFV) Alfort/187 strain. The E2 gene of CSFV Alfort/187 strain was deleted and the resultant plasmid pA187delE2 was inserted by a fragment containing the region coding for a truncate...

  2. Assessment of chimeric mice with humanized livers in new drug development: generation of pharmacokinetics, metabolism and toxicity data for selecting the final candidate compound.

    Science.gov (United States)

    Kamimura, Hidetaka; Ito, Satoshi

    2016-06-01

    1. Chimeric mice with humanized livers are expected to be a novel tool for new drug development. This review discusses four applications where these animals can be used efficiently to collect supportive data for selecting the best compound in the final stage of drug discovery. 2. The first application is selection of the final compound based on estimated pharmacokinetic parameters in humans. Since chimeric mouse livers are highly repopulated with human hepatocytes, hepatic clearance values in vivo could be used preferentially to estimate pharmacokinetic profiles for humans. 3. The second is prediction of human-specific or disproportionate metabolites. Chimeric mice reproduce human-specific metabolites of drugs under development to conform to ICH guidance M3(R2), except for compounds that were extensively eliminated by co-existing mouse hepatocytes. 4. The third is identifying metabolites with distinct pharmacokinetic profiles in humans. Slow metabolite elimination specifically in humans increases its exposure level, but if its elimination is faster in laboratory animals, the animal exposure level might not satisfy ICH guidance M3(R2). 5. Finally, two examples of reproducing acute liver toxicity in chimeric mice are introduced. Integrated pharmacokinetics, metabolism and toxicity information are expected to assist pharmaceutical scientists in selecting the best candidate compound in new drug development. PMID:26444900

  3. Short tandem repeat technology has diverse applications: Individual identification, phylogenetic reconstruction and chimerism based post haematopoietic stem cell transplantation graft monitoring

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    Agrawal Suraksha

    2004-07-01

    Full Text Available BACKGROUND: Short Tandem Repeat (STR loci are widely considered to be effective for variety of applications including forensic applications, phylogenetic reconstruction and chimerism based post Haematopoietic Stem Cell Transplantation (HSCT graft monitoring. For each application, specific sets of STR loci are used. AIMS: In the present study, we have attempted to use same set of STR loci for varied purposes based on their efficacy and informativity. SETTINGS AND DESIGN: Population and patient based study. MATERIALS AND METHODS: We have analyzed 5 STR loci - vWA, Tho1, FES, F13 and TPOX in 1000 North Indians. All five markers were also analyzed for chimerism based graft monitoring after HSCT in 42 HLA matched pair of patient-donor to predict the outcome of transplantation. STATISTICAL ANALYSIS: The analysis was done for Hardy Weinberg equilibrium (HWE, Heterozygosity, Polymorphism information content (PIC and Power of Exclusion and Phylogenetic assessment. RESULTS AND CONCLUSIONS: High allelic variability in term of Heterozygosity (0.68-0.76, PIC (0.66-0.74 and high Power of exclusion (0.28-0.38 indicating high forensic utility. The ensuing PC plots finely resolved three basal clusters corresponding to three geo-ethnic groups of African, Orientals, and Caucasians. In post HSCT chimerism analysis, it was found that together these markers were informative in 38 pairs (98% and were able to predict the chimerism status successfully. There is a possibility that these STR loci along with forensic and phylogenetic importance, can predict the outcome of HSCT successfully.

  4. Co-expression of xerophyte Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 confers enhanced salinity tolerance in chimeric sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Wu, Guo-Qiang; Feng, Rui-Jun; Wang, Suo-Min; Wang, Chun-Mei; Bao, Ai-Ke; Wei, Li; Yuan, Hui-Jun

    2015-01-01

    Salinity is one of the major abiotic stresses that limit the growth and productivity of sugar beet (Beta vulgaris L.). To improve sugar beet's salinity tolerance, the ZxNHX and ZxVP1-1 genes encoding tonoplast Na(+)/H(+) antiporter and H(+)-PPase from xerophyte Zygophyllum xanthoxylum were co-expressed by Agrobacterium tumefaciens-mediated transformation. It is showed here that co-expression of ZxNHX and ZxVP1-1 confers enhanced salinity tolerance to the transformed sugar beet plants compared with the wild-type (WT) plants. The chimeric plants grew well in the presence of high salinity (400 mM NaCl), whereas WT plants displayed chlorosis and died within 8 days. Compared to WT plants, the chimeric plants co-expressing ZxNHX and ZxVP1-1 accumulated more proline, Na(+) and K(+) in their leaves and petioles when exposed to high salinity, which caused lower solute potential, retained more water and thus subjected to lesser cell membrane damage. Interestingly, the chimeric plants accumulated higher sucrose, glucose and fructose contents in their storage roots than WT plants in the absence or presence of high salinity. Our results suggested that co-expression of ZxNHX and ZxVP1-1 improved the osmoregulatory capacity in chimeric sugar beet through increased compartmentalization of ions into the vacuoles by enhancing the activity of proton pumps and thus mitigated Na(+)-toxicity for plants. PMID:26284097

  5. Development of a chimeric Plasmodium berghei strain expressing the repeat region of the P. vivax circumsporozoite protein for in vivo evaluation of vaccine efficacy.

    Science.gov (United States)

    Espinosa, Diego A; Yadava, Anjali; Angov, Evelina; Maurizio, Paul L; Ockenhouse, Christian F; Zavala, Fidel

    2013-08-01

    The development of vaccine candidates against Plasmodium vivax-the most geographically widespread human malaria species-is challenged by technical difficulties, such as the lack of in vitro culture systems and availability of animal models. Chimeric rodent Plasmodium parasites are safe and useful tools for the preclinical evaluation of new vaccine formulations. We report the successful development and characterization of chimeric Plasmodium berghei parasites bearing the type I repeat region of P. vivax circumsporozoite protein (CSP). The P. berghei-P. vivax chimeric strain develops normally in mosquitoes and produces highly infectious sporozoites that produce patent infection in mice that are exposed to the bites of as few as 3 P. berghei-P. vivax-infected mosquitoes. Using this transgenic parasite, we demonstrate that monoclonal and polyclonal antibodies against P. vivax CSP strongly inhibit parasite infection and thus support the notion that these antibodies play an important role in protective immunity. The chimeric parasites we developed represent a robust model for evaluating protective immune responses against P. vivax vaccines based on CSP. PMID:23716612

  6. RT-SHIV, an infectious CCR5-tropic chimeric virus suitable for evaluating HIV reverse transcriptase inhibitors in macaque models

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    Emau Peter

    2009-11-01

    Full Text Available Abstract Background Non-nucleoside reverse transcriptase inhibitors (NNRTIs are an important category of drugs for both chemotherapy and prevention of human immunodeficiency virus type 1 (HIV-1 infection. However, current non-human primate (NHP models utilizing simian immunodeficiency virus (SIV or commonly used chimeric SHIV (SIV expressing HIV-1 envelope are inadequate due to the insensitivity to NNRTIs. To develop a NHP model for evaluation of NNRTI compounds, we characterized a RT-SHIV virus that was assembled by replacing the SIVmac239 reverse transcriptase (RT with that of HIV-1HXB2. Since RT-SHIV exhibited in vitro characteristics of high infectivity, CCR5-usage, and sensitivity to HIV-1 specific NNRTIs, this virus was thought to be suitable for mucosal transmission and then was used to carry out a vaginal transmission study in pigtail macaques (Macaca nemestrina. Results RT-SHIV exhibited in vitro characteristics of an infectious CCR5-tropic chimeric virus. This virus was not only highly sensitive to HIV-1 RT specific NNRTIs; its replication was also inhibited by a variety of NRTIs and protease inhibitors. For in vivo vaginal transmission studies, macaques were either pretreated with a single dose of DMPA (depot medroxyprogesterone acetate or left untreated before intravaginal inoculation with 500 or 1,000 TCID50 of RT-SHIV. All macaques became systemically infected by 2 or 3 weeks post-inoculation exhibiting persistent high viremia, marked CD4+T cell depletion, and antiviral antibody response. DMPA-pretreated macaques showed a higher mean plasma viral load after the acute infection stage, highly variable antiviral antibody response, and a higher incidence of AIDS-like disease as compared with macaques without DMPA pretreatment. Conclusion This chimeric RT-SHIV has exhibited productive replication in both macaque and human PBMCs, predominantly CCR5-coreceptor usage for viral entry, and sensitivity to NNRTIs as well as other anti

  7. HIV-1 with multiple CCR5/CXCR4 chimeric receptor use is predictive of immunological failure in infected children.

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    Mariangela Cavarelli

    Full Text Available BACKGROUND: HIV-1 R5 viruses are characterized by a large phenotypic variation, that is reflected by the mode of coreceptor use. The ability of R5 HIV-1 to infect target cells expressing chimeric receptors between CCR5 and CXCR4 (R5(broad viruses, was shown to correlate with disease stage in HIV-1 infected adults. Here, we ask the question whether phenotypic variation of R5 viruses could play a role also in mother-to-child transmission (MTCT of HIV-1 and pediatric disease progression. METHODOLOGY/PRINCIPAL FINDINGS: Viral isolates obtained from a total of 59 HIV-1 seropositive women (24 transmitting and 35 non transmitting and 28 infected newborn children, were used to infect U87.CD4 cells expressing wild type or six different CCR5/CXCR4 chimeric receptors. HIV-1 isolates obtained from newborn infants had predominantly R5(narrow phenotype (n = 20, but R5(broad and R5X4 viruses were also found in seven and one case, respectively. The presence of R5(broad and R5X4 phenotypes correlated significantly with a severe decline of the CD4+ T cells (CDC stage 3 or death within 2 years of age. Forty-three percent of the maternal R5 isolates displayed an R5(broad phenotype, however, the presence of the R5(broad virus was not predictive for MTCT of HIV-1. Of interest, while only 1 of 5 mothers with an R5X4 virus transmitted the dualtropic virus, 5 of 6 mothers carrying R5(broad viruses transmitted viruses with a similar broad chimeric coreceptor usage. Thus, the maternal R5(broad phenotype was largely preserved during transmission and could be predictive of the phenotype of the newborn's viral variant. CONCLUSIONS/SIGNIFICANCE: Our results show that R5(broad viruses are not hampered in transmission. When transmitted, immunological failure occurs earlier than in children infected with HIV-1 of R5(narrow phenotype. We believe that this finding is of utmost relevance for therapeutic interventions in pediatric HIV-1 infection.

  8. Chimeric Sex-Determining Chromosomal Regions and Dysregulation of Cell-Type Identity in a Sterile Zygosaccharomyces Allodiploid Yeast

    Science.gov (United States)

    Bizzarri, Melissa; Giudici, Paolo; Cassanelli, Stefano; Solieri, Lisa

    2016-01-01

    Allodiploidization is a fundamental yet evolutionarily poorly characterized event, which impacts genome evolution and heredity, controlling organismal development and polyploid cell-types. In this study, we investigated the sex determination system in the allodiploid and sterile ATCC 42981 yeast, a member of the Zygosaccharomyces rouxii species complex, and used it to study how a chimeric mating-type gene repertoire contributes to hybrid reproductive isolation. We found that ATCC 42981 has 7 MAT-like (MTL) loci, 3 of which encode α-idiomorph and 4 encode a-idiomorph. Two phylogenetically divergent MAT expression loci were identified on different chromosomes, accounting for a hybrid a/α genotype. Furthermore, extra a-idimorph-encoding loci (termed MTLa copies 1 to 3) were recognized, which shared the same MATa1 ORFs but diverged for MATa2 genes. Each MAT expression locus was linked to a HML silent cassette, while the corresponding HMR loci were located on another chromosome. Two putative parental sex chromosome pairs contributed to this unusual genomic architecture: one came from an as-yet-undescribed taxon, which has the NCYC 3042 strain as a unique representative, while the other did not match any MAT-HML and HMR organizations previously described in Z. rouxii species. This chimeric rearrangement produces two copies of the HO gene, which encode for putatively functional endonucleases essential for mating-type switching. Although both a and α coding sequences, which are required to obtain a functional cell-type a1-α2 regulator, were present in the allodiploid ATCC 42981 genome, the transcriptional circuit, which regulates entry into meiosis in response to meiosis-inducing salt stress, appeared to be turned off. Furthermore, haploid and α-specific genes, such as MATα1 and HO, were observed to be actively transcribed and up-regulated under hypersaline stress. Overall, these evidences demonstrate that ATCC 42981 is unable to repress haploid α-specific genes and

  9. Quadruple-component superficial circumflex iliac artery perforator (SCIP) flap: A chimeric SCIP flap for complex ankle reconstruction of an exposed artificial joint after total ankle arthroplasty.

    Science.gov (United States)

    Yamamoto, Takumi; Saito, Takafumi; Ishiura, Ryohei; Iida, Takuya

    2016-09-01

    Total ankle arthroplasty (TAA) is becoming popular in patients with rheumatoid arthritis (RA)-associated ankle joint degeneration. However, ankle wound complications can occur after TAA, which sometimes requires challenging reconstruction due to anatomical complexity of the ankle. Superficial circumflex iliac artery (SCIA) perforator (SCIP) flap has been reported to be useful for various reconstructions, but no case has been reported regarding a chimeric SCIP flap for complex ankle reconstruction. We report a case of complex ankle defect successfully reconstructed with a free quadruple-component chimeric SCIP flap. A 73-year-old female patient with RA underwent TAA, and suffered from an extensive ankle soft tissue defect (13 × 5 cm) with exposure of the implanted artificial joint and the extensor tendons. A chimeric SCIP flap was raised based on the deep branch and the superficial branch of the SCIA, which included chimeric portions of the sartorius muscle, the deep fascia, the inguinal lymph node (ILN), and the skin/fat. The flap was transferred to the recipient ankle. The sartorius muscle was used to cover the artificial joint, the deep fascia to reconstruct the extensor retinaculum, the ILN to prevent postoperative lymphedema, and the adiposal tissue to put around the extensor tendons for prevention of postoperative adhesion. Postoperatively, the patient could walk by herself without persistent leg edema or bowstringing of the extensor tendons, and was satisfied with the concealable donor scar. Although further studies are required to confirm efficacy, multicomponent chimeric SCIP has a potential to be a useful option for complex defects of the ankle. PMID:27423250

  10. Immunization with Human Papillomavirus 16 L1+E2 Chimeric Capsomers Elicits Cellular Immune Response and Antitumor Activity in a Mouse Model.

    Science.gov (United States)

    López-Toledo, Gabriela; Schädlich, Lysann; Alonso-Castro, Ángel Josabad; Monroy-García, Alberto; García-Rocha, Rosario; Guido, Miriam C; Gissmann, Lutz; García-Carrancá, Alejandro

    2016-06-01

    Development of cervical cancer is associated with persistent infections by high-risk human papillomavirus (HPV). Although current HPV L1-based prophylactic vaccines prevent infection, they do not help to eliminate prevalent infections or lesions. Our aims were (i) to generate a vaccine combining prophylactic and therapeutic properties by producing chimeric capsomers after fusion of the L1 protein to different fragments of E2 from HPV 16, and (ii) to evaluate their capacity to generate an antitumoral cellular response, while conserving L1 neutralizing epitopes. Chimeric proteins were produced in Escherichia coli and purified by glutathione S-transferase (GST)-affinity chromatography. Their structure was characterized using size exclusion chromatography, sucrose gradient centrifugation, electron microscopy, and anti-L1 enzyme-linked immunosorbent assay. All chimeric proteins form capsomers and heterogeneous aggregates. One, containing part of the carboxy-terminal domain of E2 and its hinge region (L1Δ+E2H/NC, aa 206-307), conserved the neutralizing epitope H16.V5. We then evaluated the capacity of this chimeric protein to induce a cytotoxic T-cell response against HPV 16 E2. In (51)Cr release cytotoxicity assays, splenocytes from C57BL/6 immunized mice recognized and lysed TC-1/E2 cells, which express and present endogenously processed E2 peptides. Moreover, this E2-specific cytotoxic response inhibited the growth of tumors of TC-1/E2 cells in mice. Finally, we identified an epitope (aa 292-301) of E2 involved in this cytotoxic response. We conclude that the L1Δ+E2H/NC chimeric protein produced in bacteria can be an effective and economically interesting candidate for a combined prophylactic and therapeutic vaccine that could help eliminating HPV16-positive low-grade cervical lesions and persistent viral infections, thus preventing the development of lesions and, at the same time, the establishment of new infections. PMID:27058179

  11. Free combined composite flaps using the lateral circumflex femoral system for repair of massive defects of the head and neck regions: an introduction to the chimeric flap principle.

    Science.gov (United States)

    Koshima, I; Yamamoto, H; Hosoda, M; Moriguchi, T; Orita, Y; Nagayama, H

    1993-09-01

    Chimeric composite flaps combined using microanastomoses consist of two or more flaps or tissues, each with an isolated pedicle and a single vascular source. Free combined chimeric flaps using the lateral circumflex femoral system were used to treat massive composite defects of the head and neck in 10 cases. A combined anterolateral thigh flap and vascularized iliac bone graft based on the lateral circumflex femoral system and the deep circumflex iliac system was the most commonly used combination. An anteromedial thigh flap and a paraumbilical perforator-based flap were also combined with this principal combination. The advantages of this chimeric flap over other osteocutaneous flaps are: (1) The flap is relatively thin and the pedicle vessels are up to 10 cm longer and are wider than those of other flaps for easier harvesting of the flap. (2) It is unnecessary to reposition the patient. (3) The vascular pedicle to the skin can be separated from that of the bone. (4) The donor site is not close to the recipient site. (5) The donor scar is in an unexposed area. (6) The skin territory of this flap is extremely wide. (7) A combined anterolateral and anteromedial thigh flap and vascularized iliac bone graft can be easily obtained as an extended combined osteocutaneous flap. (8) Other neighboring skin flaps, such as a groin flap, a paraumbilical perforator-based flap, or a medial thigh flap, can be combined with this chimeric flap because several major muscle branches to be anastomosed derive from the lateral circumflex femoral system. Chimeric composite flaps using the lateral circumflex femoral system are considered suitable for the repair of massive composite defects of the head and neck. PMID:8341739

  12. Possible role of minor h antigens in the persistence of donor chimerism after stem cell transplantation; relevance for sustained leukemia remission.

    Directory of Open Access Journals (Sweden)

    Cornelis R van der Torren

    Full Text Available Persistent complete donor chimerism is an important clinical indicator for remissions of hematological malignancies after HLA-matched allogeneic stem cell transplantation (SCT. However, the mechanisms mediating the persistence of complete donor chimerism are poorly understood. The frequent coincidence of complete donor chimerism with graft-versus-leukemia effects and graft-versus-host disease suggests that immune responses against minor histocompatibility antigens (mHags are playing an important role in suppressing the host hematopoiesis after allogeneic SCT. Here, we investigated a possible relationship between donor immune responses against the hematopoiesis-restricted mHag HA-1 and the long-term kinetics of host hematopoietic chimerism in a cohort of 10 patients after allogeneic HLA-matched, HA-1 mismatched SCT. Functional HA-1 specific CTLs (HA-1 CTLs were detectable in 6/10 patients lysing host-type hematopoietic cells in vitro. Presence of HA-1 CTLs in the peripheral blood coincided with low host hematopoiesis levels quantified by highly sensitive mHag specific PCR. Additionally, co-incubation of host type CD34+ cells with HA-1 CTLs isolated after allogeneic SCT prevented progenitor and cobblestone area forming cell growth in vitro and human hematopoietic engraftment in immunodeficient mice. Conversely, absence or loss of HA-1 CTLs mostly coincided with high host hematopoiesis levels and/or relapse. In summary, in this first study, presence of HA-1 CTLs paralleled low host hematopoiesis levels. This coincidence might be supported by the capacity of HA-1 CTLs isolated after allogeneic SCT to specifically eliminate host type hematopoietic stem/progenitor cells. Additional studies involving multiple mismatched mHags in more patients are required to confirm this novel characteristic of mHag CTLs as factor for the persistence of complete donor chimerism and leukemia remission after allogeneic SCT.

  13. The human application of gene therapy to re-program T-cell specificity using chimeric antigen receptors

    Institute of Scientific and Technical Information of China (English)

    Alan DGuerrero; Judy SMoyes; Laurence JN Cooper

    2014-01-01

    The adoptive transfer of T cells is a promising approach to treat cancers. Primary human T cells can be modified using viral and non-viral vectors to promote the specific targeting of cancer cells via the introduction of exogenous T-cell receptors (TCRs) or chimeric antigen receptors (CARs). This gene transfer displays the potential to increase the specificity and potency of the anticancer response while decreasing the systemic adverse effects that arise from conventional treatments that target both cancerous and healthy cells. This review highlights the generation of clinical-grade T cells expressing CARs for immunotherapy, the use of these cels to target B-cellmalignancies and, particularly, the first clinical trials deploying the Sleeping Beauty gene transfer system, which engineers T cells to target CD19+ leukemia and non-Hodgkin’s lymphoma.

  14. CD19-Chimeric Antigen Receptor T Cells for Treatment of Chronic Lymphocytic Leukaemia and Acute Lymphoblastic Leukaemia

    DEFF Research Database (Denmark)

    Lorentzen, C L; thor Straten, Per

    2015-01-01

    Adoptive cell therapy (ACT) for cancer represents a promising new treatment modality. ACT based on the administration of cytotoxic T cells genetically engineered to express a chimeric antigen receptor (CAR) recognizing CD19 expressed by B cell malignancies has been shown to induce complete lasting...... responses in patients with chronic lymphocytic leukaemia (CLL) and acute lymphoblastic leukaemia (ALL). So far, eleven clinical trials including 99 CLL and ALL patients treated with CAR T cells targeting CD19 have been published, and the results from these trials are promising with impressive clinical...... responses in heavily pretreated patients. Thus, CAR T cell therapy has induced complete responses in both CLL and ALL, and surprisingly, current results indicate that patients with ALL are more prone to respond than are CLL patients. Importantly, the majority of CAR cell studies have observed severe therapy...

  15. Cytotoxic T lymphocyte responses by chimeric thymocytes. Self-recognition is determined early in T cell development

    Energy Technology Data Exchange (ETDEWEB)

    Kruisbeek, A.M.; Hodes, R.J.; Singer, A.

    1981-01-01

    In this study the cytotoxic T lymphocyte (CTL) recognition pattern of thymocytes from recently reconstituted parent leads to F1 and F1 leads to parent radiation bone marrow chimeras was investigated. Chimeric thymocytes were entirely of donor origin approximately 4 weeks after irradiation and reconstitution but were not capable of autonomously generating either alloreactive or trinitrophenyl (TNP)-modified-self-reactive CTL responses. These experiments demonstrte that even at the earliest time CTL effectors of donor origin from the thymuses of chimeras can be studied, their self-receptor repertoire has already been restricted to recognition of host MHC determinants. These results support the cocept that the host environment influences the self-recognition capacity of T cells at the pre- or intrathymic stage of differentation.

  16. [Genetic transformation of flax (Linum usitatissimum L.) with chimeric GFP-TUA6 gene for visualisation of microtubules].

    Science.gov (United States)

    Shisha, E N; Korkhovoĭ, V I; Baer, G Ia; Guzenko, E V; Lemesh, V A; Kartel', N A; Emets, A I; Blium, Ia B

    2013-01-01

    The data of Agrobacterium-mediated transformation of some Linum usitatissimum cultivars zoned on the territories of Belarus and Ukraine with the plasmid carrying chimeric GFP-TUA6 gene and nptII gene as selectable marker conferring resistance to kanamycin are presented in this study. Transformation was affected by a number of factors including optical density (OD600), time of inoculation of explants with Agrobacterium and co-culture conditions. Transgenic nature of obtained lines was confirmed by PCR analysis. Expression of GFP-TUA6 gene was detected with confocal laser scanning microscopy. The obtained transgenic lines can be used for further functional studies the role of microtubules in the processes of building the flax fibres and resistance to wind. PMID:23745358

  17. Construction of chimeric inducible promoters by elicitors of rice fungal blast pathogen and their expression in transgenic rice

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The promoter fragments of wheat GstA1 and potato Gst1 have been amplified by PCR, cloned and fused respectively to the minimal promoter sequence of rice actin gene (Act1)) and its 5′ untranslated leader sequence together with GUS. The constructs with 2 chimeric promoters (WGA and PGA) have been transferred into rice in order to analyze their inducibility patterns in transgenic rice plants. The results show that: WGA and PGA are both inducible by elicitors of Pyricularia oryzae in transgenic rice cells; the intronⅠ of rice Act1 gene is important for the heterogenic expression of monocot and dicot promoter elements in rice; and the Act1 minimal promoter and its 5′untranslated leader sequence produced low level background expression in rice.

  18. Chimeric FimH adhesin of type 1 fimbriae: a bacterial surface display system for heterologous sequences

    DEFF Research Database (Denmark)

    Pallesen, L; Poulsen, LK; Christiansen, Gunna; Klemm, P

    1995-01-01

    The FimH adhesin of type 1 fimbriae has been tested as a display system for heterologous protein segments on the surface of Escherichia coli. This was carried out by introduction of restriction site handles (BglII sites) in two different positions in the fimH gene, followed by in-frame insertion of...... heterologous DNA segments encoding two reporter sequences. In the selected positions such insertions did not significantly alter the function of the FimH protein with regard to surface location and adhesive ability. The system seemed to be quite flexible, since chimeric versions of the FimH adhesin containing...... feasibility study point to the possibility of using the FimH adhesin as a general surface display system for sizeable protein segments....

  19. Ectopic bone formation cannot occur by hydroxyapatite/β-tricalcium phosphate bioceramics in green fluorescent protein chimeric mice

    International Nuclear Information System (INIS)

    Highlights: ► Firstly, chimeric mouse model could be established successfully by bone marrow transplantation after irradiation. ► Secondly, bone induction can occur in wild-type mice 90 days after implantation, but not occur in chimeric mice. ► Thirdly, destruction of immune function will block osteoinduction by calcium phosphate ceramics. - Abstract: Many studies have shown that calcium phosphate ceramics (CP) have osteoconductive and osteoinductive properties; however, the exact mechanism of bone induction has not yet been reported. This study was performed to investigate if destroying immunological function will influence osteogenesis, to explain the mechanism which is unclear. In this study, twenty C57BL/6 mice were divided into two groups (n = 10), in group 1, a hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ceramic was implanted into both the left and right leg muscles of each mouse; in group 2, ten mice experienced lethal irradiation, then were injected bone marrow (BM) cells from green fluorescent protein (GFP) transgenic mice by tail veil, after bone marrow transplantation (BMT), heart, liver, spleen, lung, kidney, and muscle were harvested for biological analysis, after the GFP chimera model was established successfully, the same HA/β-TCP ceramic was implanted into both leg muscles of each mouse immediately after irradiation. 45 and 90 days after implantation, the ceramics of the two groups were harvested to perform with hematoxylin and eosin (HE) and immunohistochemistry (IHC) staining; the results showed that there was no bone formation in group 2, while new bone tissues were detected in group 1. Our findings suggest that the BM cell from GFP transgenic mice is a good biomarker and it could set a good platform for chimera model; it also shows that BM cell is one of cell resources of bone induction, and destruction of immune function will impede osteoinduction by CP. Overall, our results may shed light on clear mechanism study of bone

  20. Ectopic bone formation cannot occur by hydroxyapatite/{beta}-tricalcium phosphate bioceramics in green fluorescent protein chimeric mice

    Energy Technology Data Exchange (ETDEWEB)

    Cheng Lijia [Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu (China); Duan Xin [Department of Orthopaedics, Chengdu Second People' s Hospital, Chengdu (China); Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu (China); Xiang Zhou [Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu (China); Shi Yujun; Lu Xiaofeng; Ye Feng [Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu (China); Bu Hong, E-mail: hongbu@scu.edu.cn [Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu (China); Department of Pathology, West China Hospital, Sichuan University, Chengdu (China)

    2012-12-01

    Highlights: Black-Right-Pointing-Pointer Firstly, chimeric mouse model could be established successfully by bone marrow transplantation after irradiation. Black-Right-Pointing-Pointer Secondly, bone induction can occur in wild-type mice 90 days after implantation, but not occur in chimeric mice. Black-Right-Pointing-Pointer Thirdly, destruction of immune function will block osteoinduction by calcium phosphate ceramics. - Abstract: Many studies have shown that calcium phosphate ceramics (CP) have osteoconductive and osteoinductive properties; however, the exact mechanism of bone induction has not yet been reported. This study was performed to investigate if destroying immunological function will influence osteogenesis, to explain the mechanism which is unclear. In this study, twenty C57BL/6 mice were divided into two groups (n = 10), in group 1, a hydroxyapatite/{beta}-tricalcium phosphate (HA/{beta}-TCP) ceramic was implanted into both the left and right leg muscles of each mouse; in group 2, ten mice experienced lethal irradiation, then were injected bone marrow (BM) cells from green fluorescent protein (GFP) transgenic mice by tail veil, after bone marrow transplantation (BMT), heart, liver, spleen, lung, kidney, and muscle were harvested for biological analysis, after the GFP chimera model was established successfully, the same HA/{beta}-TCP ceramic was implanted into both leg muscles of each mouse immediately after irradiation. 45 and 90 days after implantation, the ceramics of the two groups were harvested to perform with hematoxylin and eosin (HE) and immunohistochemistry (IHC) staining; the results showed that there was no bone formation in group 2, while new bone tissues were detected in group 1. Our findings suggest that the BM cell from GFP transgenic mice is a good biomarker and it could set a good platform for chimera model; it also shows that BM cell is one of cell resources of bone induction, and destruction of immune function will impede

  1. Mitochondrial DNA suggests at least 11 origins of parasitism in angiosperms and reveals genomic chimerism in parasitic plants

    Directory of Open Access Journals (Sweden)

    Croom Henrietta B

    2007-12-01

    Full Text Available Abstract Background Some of the most difficult phylogenetic questions in evolutionary biology involve identification of the free-living relatives of parasitic organisms, particularly those of parasitic flowering plants. Consequently, the number of origins of parasitism and the phylogenetic distribution of the heterotrophic lifestyle among angiosperm lineages is unclear. Results Here we report the results of a phylogenetic analysis of 102 species of seed plants designed to infer the position of all haustorial parasitic angiosperm lineages using three mitochondrial genes: atp1, coxI, and matR. Overall, the mtDNA phylogeny agrees with independent studies in terms of non-parasitic plant relationships and reveals at least 11 independent origins of parasitism in angiosperms, eight of which consist entirely of holoparasitic species that lack photosynthetic ability. From these results, it can be inferred that modern-day parasites have disproportionately evolved in certain lineages and that the endoparasitic habit has arisen by convergence in four clades. In addition, reduced taxon, single gene analyses revealed multiple horizontal transfers of atp1 from host to parasite lineage, suggesting that parasites may be important vectors of horizontal gene transfer in angiosperms. Furthermore, in Pilostyles we show evidence for a recent host-to-parasite atp1 transfer based on a chimeric gene sequence that indicates multiple historical xenologous gene acquisitions have occurred in this endoparasite. Finally, the phylogenetic relationships inferred for parasites indicate that the origins of parasitism in angiosperms are strongly correlated with horizontal acquisitions of the invasive coxI group I intron. Conclusion Collectively, these results indicate that the parasitic lifestyle has arisen repeatedly in angiosperm evolutionary history and results in increasing parasite genomic chimerism over time.

  2. reSpect: Software for Identification of High and Low Abundance Ion Species in Chimeric Tandem Mass Spectra

    Science.gov (United States)

    Shteynberg, David; Mendoza, Luis; Hoopmann, Michael R.; Sun, Zhi; Schmidt, Frank; Deutsch, Eric W.; Moritz, Robert L.

    2015-11-01

    Most shotgun proteomics data analysis workflows are based on the assumption that each fragment ion spectrum is explained by a single species of peptide ion isolated by the mass spectrometer; however, in reality mass spectrometers often isolate more than one peptide ion within the window of isolation that contribute to additional peptide fragment peaks in many spectra. We present a new tool called reSpect, implemented in the Trans-Proteomic Pipeline (TPP), which enables an iterative workflow whereby fragment ion peaks explained by a peptide ion identified in one round of sequence searching or spectral library search are attenuated based on the confidence of the identification, and then the altered spectrum is subjected to further rounds of searching. The reSpect tool is not implemented as a search engine, but rather as a post-search engine processing step where only fragment ion intensities are altered. This enables the application of any search engine combination in the iterations that follow. Thus, reSpect is compatible with all other protein sequence database search engines as well as peptide spectral library search engines that are supported by the TPP. We show that while some datasets are highly amenable to chimeric spectrum identification and lead to additional peptide identification boosts of over 30% with as many as four different peptide ions identified per spectrum, datasets with narrow precursor ion selection only benefit from such processing at the level of a few percent. We demonstrate a technique that facilitates the determination of the degree to which a dataset would benefit from chimeric spectrum analysis. The reSpect tool is free and open source, provided within the TPP and available at the TPP website.

  3. Metabolic studies of prostanozol with the uPA-SCID chimeric mouse model and human liver microsomes.

    Science.gov (United States)

    Geldof, Lore; Lootens, Leen; Decroix, Lieselot; Botrè, Francesco; Meuleman, Philip; Leroux-Roels, Geert; Deventer, Koen; Van Eenoo, Peter

    2016-03-01

    Anabolic androgenic steroids are prohibited by the World Anti-Doping Agency because of their adverse health and performance enhancing effects. Effective control of their misuse by detection in urine requires knowledge about their metabolism. In case of designer steroids, ethical objections limit the use of human volunteers to perform excretion studies. Therefore the suitability of alternative models needs to be investigated. In this study pooled human liver microsomes (HLM) and an uPA(+/+)-SCID chimeric mouse model were used to examine the metabolism of the designer steroid prostanozol as a reference standard. Metabolites were detected by GC-MS (full scan) and LC-MS/MS (precursor ion scan). In total twenty-four prostanozol metabolites were detected with the in vitro and in vivo metabolism studies, which could be grouped into two broad classes, those with a 17-hydroxy- and those with a 17-keto-substituent. Major first phase metabolic sites were tentatively identified as C-3'; C-4 and C-16. Moreover, 3'- and 16β-hydroxy-17-ketoprostanozol could be unequivocally identified, since authentic reference material was available, in both models. Comparison with published data from humans showed a good correlation, except for phase II metabolism. As metabolites were in contrast to the human studies predominantly present in the free fraction. Two types of metabolites ((di)hydroxylated prostanozol metabolites) that have not been described before could be confirmed in a real positive doping control sample. Hence, the results provide further evidence for the applicability of chimeric mice and HLM to perform metabolism studies of designer steroids. PMID:26774429

  4. Pharmacokinetics of iodine-131-labeled chimeric TNT in lung cancer patients

    International Nuclear Information System (INIS)

    Aim: mouse-human chimeric Tumor-Necrosis-Therapy (chTNT) monoclonal antibody is directed against single-stranded DNA, a universal nuclear antigen accessible in the necrotic cell within most solid tumors. The pharmacokinetics of the 1311-chTNT was evaluated in lung cancer patients. Methods: 9 lung cancer patients (age 18-74 years, mean weight (49.6±6.5) kg) were recruited for this study. All patients were administered a single intravenous dose of (29.6±3.7) MBq/kg of 1311-chTNT. Blood samples were collected at 5 min and l, 2, 4, 6, 24, 48, 72, 168 h after injection. The plasma was separated from the cellular material by centrifugation for 15 min at 4,000 rotations per min (rpm) in microcentrifuge. The kinetic parameters of plasma clearance were computed from the per cent injection dose per gram (%ID/g) of plasma using SPSS10.0 software and the sum of two exponential term models. All urine samples were collected over the 168 h period infusion of 131I-chTNT. The weight and radioactivity of urine collected at each micturition were determined specific activity was calculated. The high performance liquid chromatography (HPLC) was established to determine the stability of 131I-chTNT in serum, and analyze the metabolite of 131I-chTNT in vivo. Whole body scanning was done at a rate of 20 cm/min, keeping the camera-to-patient distance constant, while the windows of the cameras were open at 25% and adjusted on the 364 keV photoelectric peak of 1311. Anterior and posterior whole body imaging was performed at 0.5, 24, 48, 72, 120, 168 h after injection. The geometric mean of the counts in the whole body scintigram at 0.5 h after infusion was corrected for radioactive decay for the time of infusion, this value being taken as 100% injection dose (ID). The geometric mean of the anterior and posterior counts was obtained from selected region of interested (ROI) to determine activity within the critical organs after being subtracted the background activity. Counts from thyroid

  5. Expression of a Chimeric Allergen with High Rare Codons Content in Codon Bias-Adjusted Escherichia coli: Escherichia coli BL21 (DE3)-Codon Plus RIL as an Efficient Host.

    Science.gov (United States)

    Nouri, Hamid Reza; Karkhah, Ahmad; Varasteh, Abdolreza; Sankian, Mojtaba

    2016-07-01

    The expression of heterologous proteins in Escherichia coli (E. coli) is importantly affected by codon bias. Hence, the aim of the current study was to determine which codon bias-adjusted E. coli strain is sufficient for expression of a chimeric allergen coded by high rare codon content. To investigate the expression level, a chimeric protein of Chenopodium album (C. album) was used as an appropriate model. An expression construct was assembled and was transformed to four strains of codon bias-adjusted E. coli including origami, BL21 (DE3), BL21 (DE3)-codon plus RIL, and Rosetta. The level of expression and solubility of the chimeric allergen was analyzed by SDS-PAGE. In addition, the allergenicity of chimeric allergen was determined using immunoblotting. Our results showed that the chimeric allergen was expressed at high level in E. coli BL21 (DE3)-codon plus RIL and Rosetta. In detail, this recombinant allergen was isolated from soluble fraction in the codon bias-adjusted strains of E. coli BL21 (DE3)-codon plus RIL and Rosetta. Moreover, some lower molecular weight proteins were observed in Rosetta, which could be related to inappropriate expression or broken compartments of the chimeric allergen. The immunoblotting assay confirmed that the IgE-specific immune reactivity of our chimeric allergen expressed in BL21 (DE3)-codon plus RIL was significantly higher than the other strains. Our results showed that the expression of the chimeric allergen with high rare codons content in a codon bias-adjusted strain E. coli BL21 (DE3)-codon plus RIL improves the quality and solubility of the heterologous protein production. PMID:27040822

  6. Polyclonal immunoglobulins from a chronic hepatitis C virus patient protect human liver-chimeric mice from infection with a homologous hepatitis C virus strain

    DEFF Research Database (Denmark)

    Vanwolleghem, Thomas; Bukh, Jens; Meuleman, Philip;

    2008-01-01

    chimeric mice, the inoculum was pre-incubated in vitro at an IgG concentration normally observed in humans. Conclusion: Polyclonal IgG from a patient with a long-standing HCV infection not only displays neutralizing activity in vitro using the HCVpp system, but also conveys sterilizing immunity toward the......The role of the humoral immune response in the natural course of hepatitis C virus (HCV) infection is widely debated. Most chronically infected patients have immunoglobulin G (IgG) antibodies capable of neutralizing HCV pseudoparticles (HCVpp) in vitro. It is, however, not clear whether these Ig......G can prevent a de novo HCV infection in vivo and contribute to the control of viremia in infected individuals. We addressed this question with homologous in vivo protection studies in human liver-urokinase-type plasminogen activator (uPA)(+/+) severe combined immune deficient (SCID) mice. Chimeric mice...

  7. Chimeric Allografts Induced by Short-Term Treatment With Stem Cell Mobilizing Agents Result in Long-Term Kidney Transplant Survival Without Immunosuppression: II, Study in Miniature Swine.

    Science.gov (United States)

    Cameron, A M; Wesson, R N; Ahmadi, A R; Singer, A L; Hu, X; Okabayashi, T; Wang, Y; Shigoka, M; Fu, Y; Gao, W; Raccusen, L C; Montgomery, R A; Williams, G M; Sun, Z

    2016-07-01

    Transplantation is now lifesaving therapy for patients with end-stage organ failure but requires lifelong immunosuppression with resultant morbidity. Current immunosuppressive strategies inhibit T cell activation and prevent donor-recipient engagement. Therefore, it is not surprising that few host cells are demonstrated in donor grafts. However, our recent small animal studies found large numbers of recipient stem cells present after transplantation and pharmacological mobilization, resulting in a chimeric, repopulated organ. We now confirm these findings in a well-characterized large animal preclinical model. Here, we show that AMD3100 and FK506 mobilization of endogenous stem cells immediately post kidney transplantation combined with repeat therapy at 1, 2, and 3 months led to drug-free long-term survival in maximally immunologically mismatched swine. Three long-term recipients have stable chimeric transplants, preserved antidonor skin graft responses, and normal serum creatinine levels despite withdrawal of all medication for 3 years. PMID:26748958

  8. Improving bacterial cellulose for blood vessel replacement: functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide

    OpenAIRE

    Andrade, Fábia K.; Costa, Raquel; Domingues, Lucília; Soares, Raquel; Gama, F. M.

    2010-01-01

    Chimeric proteins containing a cellulose-binding module (CBM) and an adhesion peptide (RGD or GRGDY) were produced and used to improve the adhesion of human microvascular endothelial cells (HMEC) to bacterial cellulose (BC). The effect of these proteins on the HMEC–BC interaction was studied. The results obtained demonstrated that recombinant proteins containing adhesion sequences were able to significantly increase the attachment of HMEC to BC surfaces, especially the RGD sequenc...

  9. The Use of Chimeric Virus-like Particles Harbouring a Segment of Hantavirus Gc Glycoprotein to Generate a Broadly-Reactive Hantavirus-Specific Monoclonal Antibody

    Directory of Open Access Journals (Sweden)

    Aurelija Zvirbliene

    2014-02-01

    Full Text Available Monoclonal antibodies (MAbs against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have used initially a recombinant yeast-expressed full-length Puumala virus (PUUV Gc protein. However, this approach was unsuccessful. As an alternative recombinant antigen, chimeric virus-like particles (VLPs harboring a segment of PUUV Gc glycoprotein were generated in yeast Saccharomyces cerevisiae. A 99 amino acid (aa-long segment of Gc protein was inserted into the major capsid protein VP1 of hamster polyomavirus at previously defined positions: either site #1 (aa 80–89 or site #4 (aa 280–289. The chimeric proteins were found to self-assemble to VLPs as evidenced by electron microscopy. Chimeric VLPs induced an efficient insert-specific antibody response in immunized mice. Monoclonal antibody (clone #10B8 of IgG isotype specific to hantavirus Gc glycoprotein was generated. It recognized recombinant full-length PUUV Gc glycoprotein both in ELISA and Western blot assay and reacted specifically with hantavirus-infected cells in immunofluorescence assay. Epitope mapping studies revealed the N-terminally located epitope highly conserved among different hantavirus strains. In conclusion, our approach to use chimeric VLPs was proven useful for the generation of virus-reactive MAb against hantavirus Gc glycoprotein. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications.

  10. Chimeric flagellin expressed by Salmonella typhimurium induces an ESAT-6-specific Th 1-type immune response and CTL effects following intranasal immunization

    Institute of Scientific and Technical Information of China (English)

    Hui Zhang; Liu Liu; Ke Wen; Jinlin Huang; Shizhong Geng; Junsong Shen; Zhiming Pan; Xinan Jiao

    2011-01-01

    The flagellin component FliC of Salmonella typhimurium is capable of activating the innate immune system via specific interactions with TLR5 and can also act as a carrier of foreign antigen to elicit antigen-specific immune responses.Thus,we constructed an attenuated Salmonella strain SL5928(fliC/esat) expressing chimeric flagellin that contained the ESAT-6 antigen coding sequence of Mycobacterium tuberculosis inserted into the highly variable region of the Salmonella flagellin coding gene fliCi.The chimeric flagellin functioned normally,as demonstrated using a flagella swarming assay and electron microscopy.To analyze the effects of chimeric flagellin,the cell-mediated immune response and cytotoxic T lymphocyte (CTL) effects specific for ESAT-6antigen were tested after intranasal immunization of mice with flagellated Salmonella SL5928(fliC/esat).The results showed that SL5928(fliC/esat) intranasal immunization can strongly elicit an ESAT-6-specific T helper (Th) 1-type immune response in mucosal lymphoid tissues,such as nasopharynx-associated lymph nodes,lung and Peyer's patches,and a Th 1/Th2 response was elicited in spleen and mesenteric lymph nodes.Furthermore,intranasal immunization of SL5928(fliC/esat) produced efficient CTL effects,as demonstrated using a 5-and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) assay.Thus,our study revealed that Salmonella flagellin acts as a carrier for foreign antigen and triggers strong Th 1 and CTL responses during intranasal immunization.Chimeric flagellin is potentially an effective strategy for the development of novel vaccines against tuberculosis in humans and animals.

  11. A new classification of free combined or connected tissue transfers: introduction to the concept of bridge, siamese, chimeric, mosaic, and chain-circle flaps.

    OpenAIRE

    Koshima, Isao

    2001-01-01

    Recently, combined tissues or flaps have been used for the repair of extensively massive or wide defects resulting from radical wide resection. To further advance the development of combined tissue transfers, they should be reclassified. Based on our cases with free tissue transfers, we have created a new classification of combined flaps composed of "bridge", "chimeric", "siamese", "mosaic", and "chain-circle" flaps. The bridge flap is fabricated out together of separate flaps with short vasc...

  12. Protective Efficacy of a Single Immunization of a Chimeric Adenovirus Vector-Based Vaccine against Simian Immunodeficiency Virus Challenge in Rhesus Monkeys▿

    OpenAIRE

    Barouch, Dan H.; Liu, Jinyan; Lynch, Diana M; O'Brien, Kara L.; La Porte, Annalena; Simmons, Nathaniel L.; Riggs, Ambryice M.; Clark, Sarah; Abbink, Peter; Montefiori, David C.; Landucci, Gary; Forthal, Donald N.; Self, Steven G.; Carville, Angela; Mansfield, Keith

    2009-01-01

    Rare serotype and chimeric recombinant adenovirus (rAd) vectors that evade anti-Ad5 immunity are currently being evaluated as potential vaccine vectors for human immunodeficiency virus type 1 and other pathogens. We have recently reported that a heterologous rAd prime-boost regimen expressing simian immunodeficiency virus (SIV) Gag afforded durable partial immune control of an SIV challenge in rhesus monkeys. However, single-shot immunization may ultimately be preferable for global vaccine de...

  13. Risk assessment of relapse by lineage-specific monitoring of chimerism in children undergoing allogeneic stem cell transplantation for acute lymphoblastic leukemia

    Science.gov (United States)

    Preuner, Sandra; Peters, Christina; Pötschger, Ulrike; Daxberger, Helga; Fritsch, Gerhard; Geyeregger, Rene; Schrauder, André; von Stackelberg, Arend; Schrappe, Martin; Bader, Peter; Ebell, Wolfram; Eckert, Cornelia; Lang, Peter; Sykora, Karl-Walter; Schrum, Johanna; Kremens, Bernhard; Ehlert, Karoline; Albert, Michael H.; Meisel, Roland; Lawitschka, Anita; Mann, Georg; Panzer-Grümayer, Renate; Güngör, Tayfun; Holter, Wolfgang; Strahm, Brigitte; Gruhn, Bernd; Schulz, Ansgar; Woessmann, Wilhelm; Lion, Thomas

    2016-01-01

    Allogeneic hematopoietic stem cell transplantation is required as rescue therapy in about 20% of pediatric patients with acute lymphoblastic leukemia. However, the relapse rates are considerable, and relapse confers a poor outcome. Early assessment of the risk of relapse is therefore of paramount importance for the development of appropriate measures. We used the EuroChimerism approach to investigate the potential impact of lineage-specific chimerism testing for relapse-risk analysis in 162 pediatric patients with acute lymphoblastic leukemia after allogeneic stem cell transplantation in a multicenter study based on standardized transplantation protocols. Within a median observation time of 4.5 years, relapses have occurred in 41/162 patients at a median of 0.6 years after transplantation (range, 0.13–5.7 years). Prospective screening at defined consecutive time points revealed that reappearance of recipient-derived cells within the CD34+ and CD8+ cell subsets display the most significant association with the occurrence of relapses with hazard ratios of 5.2 (P=0.003) and 2.8 (P=0.008), respectively. The appearance of recipient cells after a period of pure donor chimerism in the CD34+ and CD8+ leukocyte subsets revealed dynamics indicative of a significantly elevated risk of relapse or imminent disease recurrence. Assessment of chimerism within these lineages can therefore provide complementary information for further diagnostic and, potentially, therapeutic purposes aiming at the prevention of overt relapse. This study was registered at clinical.trials.gov with the number NC01423747. PMID:26869631

  14. Short tandem repeat technology has diverse applications: Individual identification, phylogenetic reconstruction and chimerism based post haematopoietic stem cell transplantation graft monitoring

    OpenAIRE

    Agrawal Suraksha; Khan Faisal; Talwar Sudha; Nityanand Sonia

    2004-01-01

    BACKGROUND: Short Tandem Repeat (STR) loci are widely considered to be effective for variety of applications including forensic applications, phylogenetic reconstruction and chimerism based post Haematopoietic Stem Cell Transplantation (HSCT) graft monitoring. For each application, specific sets of STR loci are used. AIMS: In the present study, we have attempted to use same set of STR loci for varied purposes based on their efficacy and informativity. SETTINGS AND DESIGN: Population and patie...

  15. Solution structure of a Plasmodium falciparum AMA-1/MSP 1 chimeric protein vaccine candidate (PfCP-2.9 for malaria

    Directory of Open Access Journals (Sweden)

    Jin Changwen

    2010-03-01

    Full Text Available Abstract Background The Plasmodium falciparum chimeric protein PfCP-2.9 is a promising asexual-stage malaria vaccine evaluated in clinical trials. This chimeric protein consists of two cysteine-rich domains: domain III of the apical membrane antigen 1 (AMA-1 [III] and the C-terminal region of the merozoite surface protein 1 (MSP1-19. It has been reported that the fusion of these two antigens enhanced their immunogenicity and antibody-mediated inhibition of parasite growth in vitro. Methods The 15N-labeled and 13C/15N-labeled PfCP-2.9 was produced in Pichia pastoris for nuclear magnetic resonance (NMR structure analysis. The chemical shift assignments of PfCP-2.9 were compared with those previously reported for the individual domains (i.e., PfAMA-1(III or PfMSP 1-19. The two-dimensional spectra and transverse relaxation rates (R2 of the PfMSP1-19 alone were compared with that of the PfCP-2.9. Results Confident backbone assignments were obtained for 122 out of 241 residues of PfCP-2.9. The assigned residues in PfCP-2.9 were very similar to those previously reported for the individual domains. The conformation of the PfMSP1-19 in different constructs is essentially the same. Comparison of transverse relaxation rates (R2 strongly suggests no weak interaction between the domains. Conclusions These data indicate that the fusion of AMA-1(III and MSP1-19 as chimeric protein did not change their structures, supporting the use of the chimeric protein as a potential malaria vaccine.

  16. Characterization of the neutralization determinants of equine arteritis virus using recombinant chimeric viruses and site-specific mutagenesis of an infectious cDNA clone

    International Nuclear Information System (INIS)

    We have used an infectious cDNA clone of equine arteritis virus (EAV) and reverse genetics technology to further characterize the neutralization determinants in the GP5 envelope glycoprotein of the virus. We generated a panel of 20 recombinant viruses, including 10 chimeric viruses that each contained the ORF5 (which encodes GP5) of different laboratory, field, and vaccine strains of EAV, a chimeric virus containing the N-terminal ectodomain of GP5 of a European strain of porcine reproductive and respiratory syndrome virus, and 9 mutant viruses with site-specific substitutions in their GP5 proteins. The neutralization phenotype of each recombinant chimeric/mutant strain of EAV was determined with EAV-specific monoclonal antibodies and EAV strain-specific polyclonal equine antisera and compared to that of their parental viruses from which the substituted ORF5 was derived. The data unequivocally confirm that the GP5 ectodomain contains critical determinants of EAV neutralization. Furthermore, individual neutralization sites are conformationally interactive, and the interaction of GP5 with the unglycosylated membrane protein M is likely critical to expression of individual epitopes in neutralizing conformation. Substitution of individual amino acids within the GP5 ectodomain usually resulted in differences in neutralization phenotype of the recombinant viruses, analogous to differences in the neutralization phenotype of field strains of EAV and variants generated during persistent infection of EAV carrier stallions

  17. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yu, E-mail: xuyu1001@gmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Liu, Zhengchun, E-mail: l135027@126.com [Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Kong, Haiyan, E-mail: suppleant@163.com [Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Sun, Wenjie, E-mail: wendy11240325@163.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Liao, Zhengkai, E-mail: fastbeta@gmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Zhou, Fuxiang, E-mail: happyzhoufx@sina.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); Xie, Conghua, E-mail: chxie_65@hotmail.com [Department of Radiation and Medical Oncology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071 (China); Hubei Key Laboratory of Tumor Biological Behaviors and Hubei Cancer Clinical Study Center, 169 Donghu Road, Wuhan 430071 (China); and others

    2011-09-09

    Highlights: {yields} A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. {yields} The promoter was characterized with radiation-inducibility and tumor-specificity. {yields} Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. {yields} Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  18. Interaction analysis of chimeric metal-binding green fluorescent protein and artificial solid-supported lipid membrane by quartz crystal microbalance and atomic force microscopy

    International Nuclear Information System (INIS)

    Non-specific adsorption and specific interaction between a chimeric green fluorescent protein (GFP) carrying metal-binding region and the immobilized zinc ions on artificial solid-supported lipid membranes was investigated using the quartz crystal microbalance technique and the atomic force microscopy (AFM). Supported lipid bilayer, composed of octanethiol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-[N- (5-amino-1-carboxypentyl iminodiacetic acid)succinyl] (NTA-DOGS)-Zn2+, was formed on the gold electrode of quartz resonator (5 MHz). Binding of the chimeric GFP to zinc ions resulted in a rapid decrease of resonance frequency. Reversibility of the process was demonstrated via the removal of metal ions by EDTA. Nanoscale structural orientation of the chimeric GFP on the membrane was imaged by AFM. Association constant of the specific binding to metal ions was 2- to 3-fold higher than that of the non-specific adsorption, which was caused by the fluidization effect of the metal-chelating lipid molecules as well as the steric hindrance effect. This infers a possibility for a further development of biofunctionalized membrane. However, maximization is needed in order to attain closer advancement to a membrane-based sensor device

  19. Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

    International Nuclear Information System (INIS)

    Highlights: → A novel chimeric promoter consisting of CArG element and hTERT promoter was developed. → The promoter was characterized with radiation-inducibility and tumor-specificity. → Suicide gene system driven by the promoter showed remarkable cytotoxicity in vitro. → Co-expression of IL12 enhanced the promoter mediated suicide gene therapy in vivo. -- Abstract: The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

  20. Bifunctional chimeric SuperCD suicide gene -YCD: YUPRT fusion is highly effective in a rat hepatoma model

    Institute of Scientific and Technical Information of China (English)

    Florian Graepler; Ulrike A Lauer; Reinhard Vonthein; Michael Gregor; Sorin Armeanu; Michael Bitzer; Ulrich M. Lauer; Marie-Luise Lemken; Wolfgang A Wybranietz; Ulrike Schmidt; Irina Smirnow; Christine D Groβ; Martin Spiegel; Andrea Schenk; Hansj(o)rg Graf

    2005-01-01

    AIM: To investigate the effects of catalytically superior gene-directed enzyme prodrug therapy systems on a rat hepatoma model.METHODS: To increase hepatoma cell chemosensitivity for the prodrug 5-fluorocytosine (5-FC), we generated a chimeric bifunctional SuperCD suicide gene, a fusion of the yeast cytosine deaminase (YCD) and the yeast uracil phosphoribosyltransferase (YUPRT) gene.RESULTS: In vitro stably transduced Morris rat hepatoma cells (MH) expressing the bifunctional SuperCD suicide gene (MH SuperCD) showed a clearly marked enhancement in cell killing when incubated with 5-FC as compared with MH ceils stably expressing YCD solely (MH YCD) or the cytosine deaminase gene of bacterial origin(MH BCD), respectively. In vivo, MH SuperCD tumors implanted both subcutaneously as well as orthotopically into the livers of syngeneic ACI rats demonstrated significant tumor regressions (P<0.01) under both high dose as well as low dose systemic 5-FC application,whereas MH tumors without transgene expression (MH naive) showed rapid progression. For the first time, an order of in vivo suicide gene effectiveness (SuperCD>>YCD > > BCD > > > negative control) was defi ned as a result of a directin vivo comparison of all three suicide genes.CONCLUSION: Bifunctional SuperCD suicide gene expression is highly effective in a rat hepatoma model,thereby significantly improving both the therapeutic index and the efficacy of hepatocellular carcinoma killing by fluorocytosine.

  1. Combination of microwave ablation and 131I labeled tumor necrosis therapy chimeric antibody in the treatment of lung cancer

    International Nuclear Information System (INIS)

    Microwave ablation (MWA) has been considered as an advanced, minimally invasive technique in the treatment of lung cancer with a high local efficiency. MWA can inactivate tumor cells based on microwave heating mechanisms. It has a high local control rate of primary peripheral lung cancer and metastatic lung cancer result from percutaneous microwave antennas injection under ultrasound, CT and MRI guidance. However, for the lesions those are greater than 3.0 cm in diameter or abutting the hilars and the diffuse lesions there is less effective. 131I labeled tumor necrosis therapy chimeric antibody (131I-chTNT), a novel radioimmunotherapy which uses cell of degeneration or necrotic tumor cell nuclear antigen as target provides a new therapeutic approach for lung cancer, but the complete response is low. MWA can induce a mass tumor necrosis under the action of microwave thermal effect which greatly increases the targeting area for 131I-chTNT radioimmunotherapy. Therefore, combination of MWA and 131I-chTNT will enhance the treatment efficacy of lung cancer. (authors)

  2. T Cells Engineered With Chimeric Antigen Receptors Targeting NKG2D Ligands Display Lethal Toxicity in Mice.

    Science.gov (United States)

    VanSeggelen, Heather; Hammill, Joanne A; Dvorkin-Gheva, Anna; Tantalo, Daniela G M; Kwiecien, Jacek M; Denisova, Galina F; Rabinovich, Brian; Wan, Yonghong; Bramson, Jonathan L

    2015-10-01

    Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy targets. To assess the tumoricidal properties of T cells directed to attack NKG2D ligands, we engineered murine T cells with two distinct NKG2D-based chimeric antigen receptors (CARs): (i) a fusion between the NKG2D receptor and the CD3ζ chain and (ii) a conventional second-generation CAR, where the extracellular domain of NKG2D was fused to CD28 and CD3ζ. To enhance the CAR surface expression, we also engineered T cells to coexpress DAP10. In vitro functionality and surface expression levels of all three CARs was greater in BALB/c T cells than C57BL/6 T cells, indicating strain-specific differences. Upon adoptive transfer of NKG2D-CAR-T cells into syngeneic animals, we observed significant clinical toxicity resulting in morbidity and mortality. The severity of these toxicities varied between the CAR configurations and paralleled their in vitro NKG2D surface expression. BALB/c mice were more sensitive to these toxicities than C57BL/6 mice, consistent with the higher in vitro functionality of BALB/c T cells. Treatment with cyclophosphamide prior to adoptive transfer exacerbated the toxicity. We conclude that while NKG2D ligands may be useful targets for immunotherapy, the pursuit of NKG2D-based CAR-T cell therapies should be undertaken with caution. PMID:26122933

  3. A case of maternal-foetal chimerism identified during routine histocompatibility testing for hematopoietic stem cell transplantation.

    Science.gov (United States)

    Grubic, Z; Stingl Jankovic, K; Kelecic, J; Batinic, D; Dubravcic, K; Zunec, R

    2016-02-01

    This report describes a case of maternal-foetal chimerism identified in a boy diagnosed with SCID, who underwent HLA testing in preparation for HSCT. The first analysis was carried out on DNA from peripheral blood and included HLA-A, HLA-B, HLA-DRB1 typing using PCR-SSO. The patient's HLA-B typing results were noninterpretable. All samples were re-typed for HLA-B using PCR-SSP, again resulting in noninterpretable typing of patient's HLA-B. In both cases, several weak positive probes/reactions interfered with the interpretation when using commercial software. Next round of HLA typing, using PCR-SSP and PCR-SSO methods, included the patient's bone marrow sample and HLA-C locus, but interpretation was again not possible. The PCR-STR analysis performed on both peripheral blood and bone marrow samples revealed seven STRs for which two maternal and one paternal allele were detected. Retrospective manual interpretation of HLA-B and HLA-C typing revealed that weak positive reactions were indeed owed to paternal HLA-B and HLA-C alleles and that the patient had both maternal and one paternal allele. Retyping of HLA-B and HLA-C loci and STR analysis on the patient's buccal cells sample revealed the expected one maternal/one paternal allele pattern. In summary, the combination of several different typing methods and manual interpretation were necessary to obtain the patient's HLA typing results. PMID:26663895

  4. PRELIMINARY STUDY OF A NOVEL HUMAN PAPILLOMAVIRUS TYPE 16 L1/E6-E7 CHIMERIC RECOMBINANT DNA VACCINE

    Institute of Scientific and Technical Information of China (English)

    郑瑾; 马军; 张福萍; 杨筱凤; 董小平; 司履生; 王一理

    2004-01-01

    Objective Preparations of HPV16 L1/E6 and L1/E7 prophylactic and therapeutic DNA vaccines. Methods The nucleotides within HPV16 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by mage primer site-directed mutagenesis method. The correctly mutated E6 and E7 fragments were separately cloned into an eukaryotic expression vector pVAX1, together with HPV16 L1 gene, generating chimeric recombinants plasmids 1MpVAX1-L1E6, 2MpVAX1-L1E6, 1MpVAX1-L1E7, 2MpVAX1-L1E7 and 3MpVAX1-L1E7. CHO cells were transiently transfected with the individual DNA vaccines by calcium phosphate method. Target protein expressions in the extracts of the transfected cell lines were measured by ELISA and immunohistochemistry, with HPV16 L1 and E6 specific monoclonal antibodies. Results ELISA assays showed the P/N ratios in the cell extracts transfected with L1E6 and L1E7 plasmids were more than 2.1. Immunohistochemistry revealed brownish precipitant signal in cytoplasm and nuclei of the transfected cells. Conclusion Successful constructions of prophylactic and therapeutic DNA vaccine plasmids lay solid foundation for future animal experiment and clinical trial.

  5. The CD3-zeta chimeric antigen receptor overcomes TCR Hypo-responsiveness of human terminal late-stage T cells.

    Directory of Open Access Journals (Sweden)

    Gunter Rappl

    Full Text Available Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1(+ CD57(+ CD7(- phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8(+ T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter.

  6. Preclinical and Clinical Development of a YFV 17 D-Based Chimeric Vaccine against West Nile Virus

    Directory of Open Access Journals (Sweden)

    Gustavo H. Dayan

    2013-12-01

    Full Text Available Substantial success has been achieved in the development and implementation of West Nile (WN vaccines for horses; however, no human WN vaccines are approved. This review focuses on the construction, pre-clinical and clinical characterization of ChimeriVax-WN02 for humans, a live chimeric vaccine composed of a yellow fever (YF 17D virus in which the prM-E envelope protein genes are replaced with the corresponding genes of the WN NY99 virus. Pre-clinical studies demonstrated that ChimeriVax-WN02 was significantly less neurovirulent than YF 17D in mice and rhesus and cynomolgus monkeys. The vaccine elicited neutralizing antibody titers after inoculation in hamsters and monkeys and protected immunized animals from lethal challenge including intracerebral inoculation of high dose of WN NY99 virus. Safety, viremia and immunogenicity of ChimeriVax-WN02 were assessed in one phase I study and in two phase II clinical trials. No safety signals were detected in the three clinical trials with no remarkable differences in incidence of adverse events (AEs between vaccine and placebo recipients. Viremia was transient and the mean viremia levels were low. The vaccine elicited strong and durable neutralizing antibody and cytotoxic T cell responses. WN epidemiology impedes a classical licensure pathway; therefore, innovative licensure strategies should be explored.

  7. Antigen-specific T cell activation independently of the MHC: chimeric antigen receptor (CAR-redirected T cells.

    Directory of Open Access Journals (Sweden)

    Hinrich eAbken

    2013-11-01

    Full Text Available Adoptive T cell therapy has recently shown powerful in initiating a lasting anti-tumor response with spectacular therapeutic success in some cases. Specific T cell therapy, however, is limited since a number of cancer cells are not recognized by T cells due to various mechanisms including the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC expression of cancer cells. To make adoptive cell therapy applicable for the broad variety of cancer entities, patient's T cells are engineered ex vivo with pre-defined specificity by a recombinant chimeric antigen receptor (CAR which consists in the extracellular part of an antibody-derived domain for binding with a tumor-associated antigen and in the intracellular part of a TCR-derived signaling moiety for T cell activation. The specificity of CAR mediated T cell recognition is defined by the antibody domain, is independent of MHC presentation and can be extended to any target for which an antibody is available. We discuss the advantages and limitations of MHC-independent T cell targeting by an engineered CAR and review most significant progress recently made in early stage clinical trials to treat cancer.

  8. Molecular Pathways: Breaking the Epithelial Cancer Barrier for Chimeric Antigen Receptor and T-cell Receptor Gene Therapy.

    Science.gov (United States)

    Hinrichs, Christian S

    2016-04-01

    Adoptive transfer of T cells genetically engineered to express a tumor-targeting chimeric antigen receptor (CAR) or T-cell receptor (TCR) can mediate cancer regression in some patients. CARs are synthetic single-chain proteins that use antibody domains to target cell surface antigens. TCRs are natural heterodimeric proteins that can target intracellular antigens through recognition of peptides bound to human leukocyte antigens. CARs have shown promise in B-cell malignancies and TCRs in melanoma, but neither approach has achieved clear success in an epithelial cancer. Treatment of epithelial cancers may be particularly challenging because of a paucity of target antigens expressed by carcinomas and not by important healthy tissues. In addition, epithelial cancers may be protected by inhibitory ligands and soluble factors in the tumor microenvironment. One strategy to overcome these negative regulators is to modulate expression of T-cell genes to enhance intrinsic T-cell function. Programmable nucleases, which can suppress inhibitory genes, and inducible gene expression systems, which can enhance stimulatory genes, are entering clinical testing. Other work is delineating whether control of genes for immune checkpoint receptors (e.g.,PDCD1, CTLA4) and cytokine and TCR signaling regulators (e.g.,CBLB, CISH, IL12, IL15) can increase the antitumor activity of therapeutic T cells.Clin Cancer Res; 22(7); 1559-64. ©2016 AACR. PMID:27037253

  9. Chimeric newcastle disease virus protects chickens against avian influenza in the presence of maternally derived NDV immunity.

    Directory of Open Access Journals (Sweden)

    Constanze Steglich

    Full Text Available Newcastle disease virus (NDV, an avian paramyxovirus type 1, is a promising vector for expression of heterologous proteins from a variety of unrelated viruses including highly pathogenic avian influenza virus (HPAIV. However, pre-existing NDV antibodies may impair vector virus replication, resulting in an inefficient immune response against the foreign antigen. A chimeric NDV-based vector with functional surface glycoproteins unrelated to NDV could overcome this problem. Therefore, an NDV vector was constructed which carries the fusion (F and hemagglutinin-neuraminidase (HN proteins of avian paramyxovirus type 8 (APMV-8 instead of the corresponding NDV proteins in an NDV backbone derived from the lentogenic NDV Clone 30 and a gene expressing HPAIV H5 inserted between the F and HN genes. After successful virus rescue by reverse genetics, the resulting chNDVFHN PMV8H5 was characterized in vitro and in vivo. Expression and virion incorporation of the heterologous proteins was verified by Western blot and electron microscopy. Replication of the newly generated recombinant virus was comparable to parental NDV in embryonated chicken eggs. Immunization with chNDVFHN PMV8H5 stimulated full protection against lethal HPAIV infection in chickens without as well as with maternally derived NDV antibodies. Thus, tailored NDV vector vaccines can be provided for use in the presence or absence of routine NDV vaccination.

  10. Armed oncolytic virus enhances immune functions of chimeric antigen receptor-modified T cells in solid tumors.

    Science.gov (United States)

    Nishio, Nobuhiro; Diaconu, Iulia; Liu, Hao; Cerullo, Vincenzo; Caruana, Ignazio; Hoyos, Valentina; Bouchier-Hayes, Lisa; Savoldo, Barbara; Dotti, Gianpietro

    2014-09-15

    The clinical efficacy of chimeric antigen receptor (CAR)-redirected T cells remains marginal in solid tumors compared with leukemias. Failures have been attributed to insufficient T-cell migration and to the highly immunosuppressive milieu of solid tumors. To overcome these obstacles, we have combined CAR-T cells with an oncolytic virus armed with the chemokine RANTES and the cytokine IL15, reasoning that the modified oncolytic virus will both have a direct lytic effect on infected malignant cells and facilitate migration and survival of CAR-T cells. Using neuroblastoma as a tumor model, we found that the adenovirus Ad5Δ24 exerted a potent, dose-dependent, cytotoxic effect on tumor cells, whereas CAR-T cells specific for the tumor antigen GD2 (GD2.CAR-T cells) were not damaged. When used in combination, Ad5Δ24 directly accelerated the caspase pathways in tumor cells exposed to CAR-T cells, whereas the intratumoral release of both RANTES and IL15 attracted CAR-T cells and promoted their local survival, respectively, increasing the overall survival of tumor-bearing mice. These preclinical data support the use of this innovative biologic platform of immunotherapy for solid tumors. Cancer Res; 74(18); 5195-205. ©2014 AACR. PMID:25060519

  11. Regional scale ozone data assimilation using an Ensemble Kalman Filter and the CHIMERE Chemical-Transport Model

    Directory of Open Access Journals (Sweden)

    B. Gaubert

    2013-05-01

    Full Text Available The Ensemble Kalman Filter is an efficient algorithm for data assimilation; it allows for an estimation of forecast and analysis error by updating the model error covariance matrices at the analysis step. This algorithm has been coupled to the CHIMERE chemical transport model in order to assimilate ozone ground measurements at the regional scale. The analyzed ozone field is evaluated using a consistent set of observations and shows a reduction of the quadratic error by about a third and an improvement of the hourly correlation coefficient despite of a low ensemble size designed for operational purposes. A classification of the European observation network is derived from the ozone temporal variability in order to qualitatively determine the observation spatial representativeness. Then, an estimation of the temporal behavior of both model and observations error variances of the assimilated stations is checked using a posteriori Desroziers diagnostics. The amplitude of the additive noise applied to the ozone fields can be diagnosed and tuned online. The evaluation of the obtained background error variance distribution through the Reduced Centered Random Variable standard deviation shows improved statistics. The use of the diagnostics indicates a strong diurnal cycle of both the model and the representativeness errors. Another design of the ensemble is constructed by perturbing model parameter, but does not allow creating enough variability if used solely. Finally, the overall filter performance over evaluation stations is found to be relatively unaffected by different formulations of observation and simulation errors.

  12. T Cells Expressing CD19/CD20 Bispecific Chimeric Antigen Receptors Prevent Antigen Escape by Malignant B Cells.

    Science.gov (United States)

    Zah, Eugenia; Lin, Meng-Yin; Silva-Benedict, Anne; Jensen, Michael C; Chen, Yvonne Y

    2016-06-01

    The adoptive transfer of T cells expressing anti-CD19 chimeric antigen receptors (CARs) has shown remarkable curative potential against advanced B-cell malignancies, but multiple trials have also reported patient relapses due to the emergence of CD19-negative leukemic cells. Here, we report the design and optimization of single-chain, bispecific CARs that trigger robust cytotoxicity against target cells expressing either CD19 or CD20, two clinically validated targets for B-cell malignancies. We determined the structural parameters required for efficient dual-antigen recognition, and we demonstrate that optimized bispecific CARs can control both wild-type B-cell lymphoma and CD19(-) mutants with equal efficiency in vivo To our knowledge, this is the first bispecific CAR capable of preventing antigen escape by performing true OR-gate signal computation on a clinically relevant pair of tumor-associated antigens. The CD19-OR-CD20 CAR is fully compatible with existing T-cell manufacturing procedures and implementable by current clinical protocols. These results present an effective solution to the challenge of antigen escape in CD19 CAR T-cell therapy, and they highlight the utility of structure-based rational design in the development of receptors with higher-level complexity. Cancer Immunol Res; 4(6); 498-508. ©2016 AACRSee related Spotlight by Sadelain, p. 473. PMID:27059623

  13. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.

    Science.gov (United States)

    Kalos, Michael; Levine, Bruce L; Porter, David L; Katz, Sharyn; Grupp, Stephan A; Bagg, Adam; June, Carl H

    2011-08-10

    Tumor immunotherapy with T lymphocytes, which can recognize and destroy malignant cells, has been limited by the ability to isolate and expand T cells restricted to tumor-associated antigens. Chimeric antigen receptors (CARs) composed of antibody binding domains connected to domains that activate T cells could overcome tolerance by allowing T cells to respond to cell surface antigens; however, to date, lymphocytes engineered to express CARs have demonstrated minimal in vivo expansion and antitumor effects in clinical trials. We report that CAR T cells that target CD19 and contain a costimulatory domain from CD137 and the T cell receptor ζ chain have potent non-cross-resistant clinical activity after infusion in three of three patients treated with advanced chronic lymphocytic leukemia (CLL). The engineered T cells expanded >1000-fold in vivo, trafficked to bone marrow, and continued to express functional CARs at high levels for at least 6 months. Evidence for on-target toxicity included B cell aplasia as well as decreased numbers of plasma cells and hypogammaglobulinemia. On average, each infused CAR-expressing T cell was calculated to eradicate at least 1000 CLL cells. Furthermore, a CD19-specific immune response was demonstrated in the blood and bone marrow, accompanied by complete remission, in two of three patients. Moreover, a portion of these cells persisted as memory CAR(+) T cells and retained anti-CD19 effector functionality, indicating the potential of this major histocompatibility complex-independent approach for the effective treatment of B cell malignancies. PMID:21832238

  14. In vitro and in vivo behavior of radiolabeled chimeric anti-EGFRvIII monoclonal antibody: Comparison with its murine parent

    International Nuclear Information System (INIS)

    The mutant version of the epidermal growth factor receptor EGFRvIII has been found on gliomas and other tumors, but not on normal tissues. Radioiodinated murine (mu) L8A4 monoclonal antibody (MAb) specifically targets EGFRvIII xenografts in vivo when labeled using N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC). A chimeric (ch) MAb consisting of the variable region of muL8A4 and the constant domains of human IgG2 has been developed that has an affinity and radioiodinated immunoreactive fraction comparable to muL8A4. In vitro, both MAbs were internalized and processed by EGFRvIII expressing cell lines (U87MGΔEGFR or NR6M) at similar rates (maximum intracellular retention, 35-40%). In paired-label tissue distribution studies in athymic mice bearing U87MGΔEGFR tumor xenografts, the ch:mu L8A4 uptake ratio in normal tissues rose to greater than 2:1, whereas in tumor, the ratio remained 1:1 throughout the experiment. These results indicate that chL8A4 exhibits similar binding and internalization properties as its murine parent, but suggest different intracellular processing and/or deposition of catabolites in normal tissues for chL8A4

  15. Chimeric smooth muscle-specific enhancer/promoters: valuable tools for adenovirus-mediated cardiovascular gene therapy.

    Science.gov (United States)

    Ribault, S; Neuville, P; Méchine-Neuville, A; Augé, F; Parlakian, A; Gabbiani, G; Paulin, D; Calenda, V

    2001-03-16

    Gene transfer with adenoviral vectors is an attractive approach for the treatment of atherosclerosis and restenosis. However, because expression of a therapeutic gene in nontarget tissues may have deleterious effects, artery-specific expression is desirable. Although expression vectors containing transcriptional regulatory elements of genes expressed solely in smooth muscle cells (SMCs) have proved efficient to restrict expression of the transgene, their use in the clinical setting can be limited by their reduced strength. In the present study, we show that low levels of transgene expression are obtained with the smooth muscle (SM)-specific SM22alpha promoter compared with the viral cytomegalovirus (CMV) enhancer/promoter. We have generated chimeric transcriptional cassettes containing either a SM (SM-myosin heavy chain) or a skeletal muscle (creatine kinase) enhancer combined with the SM22alpha promoter. With both constructs we observed significantly stronger expression that remains SM-specific. In vivo, reporter gene expression was restricted to arterial SMCs with no detectable signal at remote sites. Moreover, when interferon-gamma expression was driven by one of these two chimeras, SMC growth was inhibited as efficiently as with the CMV promoter. Finally, we demonstrate that neointima formation in the rat carotid balloon injury model was reduced to the same extent by adenoviral gene transfer of interferon-gamma driven either by the SM-myosin heavy chain enhancer/SM22alpha promoter or the CMV promoter. These results indicate that such vectors can be useful for the treatment of hyperproliferative vascular disorders. PMID:11249869

  16. Recurrent cis-SAGe chimeric RNA, D2HGDH-GAL3ST2, in prostate cancer.

    Science.gov (United States)

    Qin, Fujun; Song, Zhenguo; Chang, Maxwell; Song, Yansu; Frierson, Henry; Li, Hui

    2016-09-28

    Neighboring genes transcribing in the same direction can form chimeric RNAs via cis-splicing (cis-SAGe). Previously, we reported 16 novel cis-SAGe chimeras in prostate cancer cell lines, and performed in silico validation on 14 pairs of normal and tumor samples from Chinese patients. However, whether these fusions exist in different populations, as well as their clinical implications, remains unclear. To investigate, we developed a bioinformatics pipeline using modified Spliced Transcripts Alignment to a Reference (STAR) to quantify these fusion RNAs simultaneously in silico. From RNA-Seq data of 100 paired normal and prostate cancer samples from TCGA, we find that most fusions are not specific to cancer. However, D2HGDH-GAL3ST2 is more frequently seen in cancer samples, and seems to be enriched in the African American group. Further validation with our own collection as well as from commercial sources did not detect this fusion RNA in 29 normal prostate samples, but in 19 of 93 prostate cancer samples. It is more frequently detected in late stage cancer, suggesting a role in cancer progression. Consistently, silencing this fusion resulted in dramatic reduction of cell proliferation rate and cell motility. PMID:27322736

  17. Endogenous and xenobiotic metabolite profiling of liver extracts from SCID and chimeric humanized mice following repeated oral administration of troglitazone.

    Science.gov (United States)

    Barnes, Alan J; Baker, David R; Hobby, Kirsten; Ashton, Simon; Michopoulos, Filippos; Spagou, Konstantina; Loftus, Neil J; Wilson, Ian D

    2014-01-01

    1. Metabonomic analysis, via a combination of untargeted and targeted liquid chromatography-mass spectrometry (LC-MS) and untargeted (1)H NMR spectroscopy-based metabolite profiling, was performed on aqueous (AQ) and organic liver extracts from control (SCID) and chimeric humanized (PXB) mice dosed with troglitazone at 0, 300 and 600 mg/kg/day for seven days. 2. LC-MS analysis of AQ liver extracts showed a more "human-like" profile for troglitazone metabolites for PXB, compared with SCID, mice. 3. LC-MS detected differences in endogenous metabolites, particularly lipid species in dosed mice, including elevated triacylglycerols and 1-alkyl,2-acylglycerophosphates as well as lowered diacylglycerophosphocholines and 1-alkyl,2-acylglycerophosphocholines for PXB compared with SCID mouse liver extracts. Following drug administration changes in the relative proportions of the ions for various unsaturated fatty acids were observed for both types of mouse, some of which were specific to PXB or SCID mice. 4.  (1)H NMR spectroscopy revealed that AQ PXB mouse liver extracts had elevated amounts of inosine, fumarate, creatine, aspartate, trimethylamine N-oxide, glycerophosphocholine, phosphocholine, choline, glutamine, glutamate, acetate, alanine and lactate relative to SCID mice and decreased histidine, glycogen, α- and β-glucose, taurine, and glutathione. Increased uracil and tyrosine concentrations were detected for PXB mice on troglitazone administration. 5. Metabonomic profiling thus showed clear differences between humanized and SCID mice, including after administration of troglitazone. PMID:24350779

  18. A chimeric α-amylase engineered from Bacillus acidicola and Geobacillus thermoleovorans with improved thermostability and catalytic efficiency.

    Science.gov (United States)

    Parashar, Deepak; Satyanarayana, T

    2016-04-01

    The α-amylase (Ba-amy) of Bacillus acidicola was fused with DNA fragments encoding partial N- and C-terminal region of thermostable α-amylase gene of Geobacillus thermoleovorans (Gt-amy). The chimeric enzyme (Ba-Gt-amy) expressed in Escherichia coli displays marked increase in catalytic efficiency [K cat: 4 × 10(4) s(-1) and K cat/K m: 5 × 10(4) mL(-1) mg(-1) s(-1)] and higher thermostability than Ba-amy. The melting temperature (T m) of Ba-Gt-amy (73.8 °C) is also higher than Ba-amy (62 °C), and the CD spectrum analysis revealed the stability of the former, despite minor alteration in secondary structure. Langmuir-Hinshelwood kinetic analysis suggests that the adsorption of Ba-Gt-amy onto raw starch is more favourable than Ba-amy. Ba-Gt-amy is thus a suitable biocatalyst for raw starch saccharification at sub-gelatinization temperatures because of its acid stability, thermostability and Ca(2+) independence, and better than the other known bacterial acidic α-amylases. PMID:26790418

  19. Hematopoietic microenvironment. Origin, lineage, and transplantability of the stromal cells in long-term bone marrow cultures from chimeric mice

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, S.; Fleischman, R.A.

    1988-04-01

    Studies of bone marrow transplant patients have suggested that the stromal cells of the in vitro hematopoietic microenvironment are transplantable into conditioned recipients. Moreover, in patients with myeloproliferative disorders, all of the stromal cells, which include presumptive endothelial cells, appear to be derived from hematopoietic precursors. To confirm these findings, we have constructed two chimeric mouse models: (a) traditional radiation chimeras, and (b) fetal chimeras, produced by placental injection of bone marrow into genetically anemic Wx/Wv fetuses, a technique that essentially precludes engraftment of nonhematopoietic cells. Using two-color indirect immunofluorescence, the stromal cells in long-term bone marrow culture derived from these chimeras were analyzed for donor or host origin by strain-specific H-2 antigens, and for cell lineage by a variety of other specific markers. 75-95% of the stromal cells were shown to be hematopoietic cells of the monocyte-macrophage lineage, based upon donor origin, phagocytosis, and expression of specific hematopoietic surface antigens. The remaining 5-25% of the stromal cells were exclusively host in origin. Apart from occasional fat cells, these cells uniformly expressed collagen type IV, laminin, and a surface antigen associated with endothelial cells. Since these endothelial-like cells are not transplantable into radiation or fetal chimeras, they are not derived from hematopoietic stem cells. The contrast between our findings and human studies suggests either unexpected species differences in the origin of stromal lineages or limitations in the previous methodology used to detect nonhematopoietic stromal cells.

  20. Hematopoietic microenvironment. Origin, lineage, and transplantability of the stromal cells in long-term bone marrow cultures from chimeric mice

    International Nuclear Information System (INIS)

    Studies of bone marrow transplant patients have suggested that the stromal cells of the in vitro hematopoietic microenvironment are transplantable into conditioned recipients. Moreover, in patients with myeloproliferative disorders, all of the stromal cells, which include presumptive endothelial cells, appear to be derived from hematopoietic precursors. To confirm these findings, we have constructed two chimeric mouse models: (a) traditional radiation chimeras, and (b) fetal chimeras, produced by placental injection of bone marrow into genetically anemic Wx/Wv fetuses, a technique that essentially precludes engraftment of nonhematopoietic cells. Using two-color indirect immunofluorescence, the stromal cells in long-term bone marrow culture derived from these chimeras were analyzed for donor or host origin by strain-specific H-2 antigens, and for cell lineage by a variety of other specific markers. 75-95% of the stromal cells were shown to be hematopoietic cells of the monocyte-macrophage lineage, based upon donor origin, phagocytosis, and expression of specific hematopoietic surface antigens. The remaining 5-25% of the stromal cells were exclusively host in origin. Apart from occasional fat cells, these cells uniformly expressed collagen type IV, laminin, and a surface antigen associated with endothelial cells. Since these endothelial-like cells are not transplantable into radiation or fetal chimeras, they are not derived from hematopoietic stem cells. The contrast between our findings and human studies suggests either unexpected species differences in the origin of stromal lineages or limitations in the previous methodology used to detect nonhematopoietic stromal cells

  1. Genetic manipulation of iron biomineralization enhances MR relaxivity in a ferritin-M6A chimeric complex.

    Science.gov (United States)

    Radoul, Marina; Lewin, Limor; Cohen, Batya; Oren, Roni; Popov, Stanislav; Davidov, Geula; Vandsburger, Moriel H; Harmelin, Alon; Bitton, Ronit; Greneche, Jean-Marc; Neeman, Michal; Zarivach, Raz

    2016-01-01

    Ferritin has gained significant attention as a potential reporter gene for in vivo imaging by magnetic resonance imaging (MRI). However, due to the ferritin ferrihydrite core, the relaxivity and sensitivity for detection of native ferritin is relatively low. We report here on a novel chimeric magneto-ferritin reporter gene - ferritin-M6A - in which the magnetite binding peptide from the magnetotactic bacteria magnetosome-associated Mms6 protein was fused to the C-terminal of murine h-ferritin. Biophysical experiments showed that purified ferritin-M6A assembled into a stable protein cage with the M6A protruding into the cage core, enabling magnetite biomineralisation. Ferritin-M6A-expressing C6-glioma cells showed enhanced (per iron) r2 relaxivity. MRI in vivo studies of ferritin-M6A-expressing tumour xenografts showed enhanced R2 relaxation rate in the central hypoxic region of the tumours. Such enhanced relaxivity would increase the sensitivity of ferritin as a reporter gene for non-invasive in vivo MRI-monitoring of cell delivery and differentiation in cellular or gene-based therapies. PMID:27211820

  2. Production and characterization of chimeric transferrins for the determination of the binding domains for bacterial transferrin receptors.

    Science.gov (United States)

    Retzer, M D; Kabani, A; Button, L L; Yu, R H; Schryvers, A B

    1996-01-12

    Pathogenic bacteria in the Neisseriaceae and Pasteurellaceae possess outer membrane proteins that specifically bind transferrin from the host as the first step in the iron acquisition process. As a logical progression from prior studies of the ligand-receptor interaction using biochemical approaches, we have initiated an approach involving the production of recombinant chimeric transferrins to further identify the regions of transferrin involved in receptor binding. In order to prepare bovine/human hybrids, the bovine transferrin gene was cloned, sequenced, and compared with the existing human transferrin gene sequence. After identification of potential splice sites, hybrid transferrin genes were constructed using the polymerase chain reaction-based approach of splicing by overlap extension. Five hybrid genes containing sequences from both bovine and human transferrin were constructed. Recombinant transferrins were produced in a baculovirus expression vector system and affinity-purified using concanavalin A-Sepharose. The recombinant proteins were analyzed for reactivity against polyclonal and monoclonal antibodies and assessed for binding to Neisseria meningitidis transferrin receptor proteins in solid-phase binding assays and affinity isolation experiments. These experiments enabled us to localize the regions of human transferrin predominantly involved in binding to the N. meningitidis receptor to amino acid residues 346-588. The construction of these chimeras provides unique tools for the investigation of transferrin binding to receptors from both human and bovine bacterial pathogens. PMID:8557646

  3. Chimeric relaxin peptides highlight the role of the A-chain in the function of H2 relaxin.

    Science.gov (United States)

    Hossain, Mohammed Akhter; Wade, John D; Bathgate, Ross A D

    2012-05-01

    Human gene-2 (H2) relaxin is a member of the insulin-relaxin peptide superfamily. Because of the potential clinical applications of H2 relaxin, there is a need for novel analogs that have improved biological activity and receptor specificity. In this respect, we have chemically assembled chimeric peptides consisting of the B-chain of H2 relaxin in combination with A-chains from other insulin/relaxin family members. The peptides were prepared using solid phase peptide synthesis together with regioselective disulfide bond formation and characterized by RP-HPLC, MALDI-TOF MS and amino acid analysis. Their in vitro activity was assessed in RXFP1 or RXFP2 expressing cells. Replacement of the H2 relaxin A-chain resulted in parallel losses of binding affinity and activity on RXFP1. Not surprisingly H1A-H2B demonstrated the highest activity as the H1 A-chain shares high homology with H2 relaxin whereas INSLA-H2B, which shows low homology, had very poor activity. Importantly A-chain replacements had a dramatic effect on RXFP2 activity similar to previous results demonstrating different modes of activation of A-chain variants on RXFP1 and RXFP2. H3A-H2B is particularly interesting as it displays moderate activity at RXFP1 but poor activity at RXFP2 indicating that it may be a template for specific RXFP1 agonist development. Our study confirms that the activity of H2 relaxin at both RXFP1 and RXFP2 relies on interactions with both the B- and A-chains, and also provide new biochemical insights into the mechanism of relaxin action that the A-chain needs to be in native or near-native form for strong RXFP1 or RXFP2 agonist activity. PMID:22414484

  4. A novel molecular variant of the chimeric NPM-ALK transcript in Ki-1 positive large cell lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Ladanyi, M. [Memorial Sloan-Kettering Cancer Center, New York, NY (United States)

    1994-09-01

    The breakpoints of the t(2;5)(p23;q35), reported to be present in 30-40% of Ki-1 positive large cell lymphoma (Ki-1 LCL), have recently been cloned. They involve a novel tyrosine kinase gene, ALK, at 2p23 and the nucleophosmin gene, NPM, at 5q35. Reverse transcriptase polymerase chain reaction (RT-PCR) using NPM and ALK primers detects a consistent fusion product in Ki-1 cases with the translocation. We performed NPM-ALK RT-PCR on 16 cases of Ki-1 LCL, of which 13 had informative cytogenetics. Amplifiable template was confirmed in all samples by RT-PCR using {beta}-actin primers. Ten cases showed the expected 177 base pair (bp) RT-PCR product indicative of the translocation, including all 6 cases with cytogenetic evidence of the t(2;5) and 3 cases without 5q35 abnormalities. One additional case, which had uninformative cytogenetics, showed only a novel 144 bp RT-PCR product. Sequencing of this PCR product showed an in-frame junction of NPM to ALK, 20 bp distal to the usual NPM junction site and 53 bp distal to usual ALK junction site. The predicted chimeric protein is thus shorter by 11 amino acids, but the putative ALK catalytic domain remains intact. This case was a histologically typical anaplastic Ki-1 LCL which showed a clonal rearrangement of the T-cell receptor {beta} gene. The functional significance of this molecular variant and its relation to the exon structure of both genes require further study. The overall incidence of the translocation in this series, about 70%, is higher than suspected from cytogenetic analysis alone.

  5. Chimeric antigen receptor (CAR-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

    Directory of Open Access Journals (Sweden)

    Bipulendu Jena

    Full Text Available Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63. We describe a novel anti-idiotype monoclonal antibody (mAb to detect CD19-specific CAR(+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1 was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+ tumor targets. This clone can be used to detect CD19-specific CAR(+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1 will be useful to investigators implementing CD19-specific CAR(+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

  6. Synergistic effect of chimeric antigen receptors and cytokine-induced killer cells: An innovative combination for cancer therapy

    Directory of Open Access Journals (Sweden)

    Binh Thanh Vu

    2016-06-01

    Full Text Available In recent years, the combination of gene and immunotherapy for cancer treatment has been regarded as innovative and promising; together, both therapies can help overcome limitations associated with conventional treatments. In order to augment anti-cancer efficacy and to maintain the specificity of antibody therapy, chimeric antigen receptor (CAR-modified T cells, directed toward tumor-specific antigens, have emerged as a novel and promising therapeutic platform. CARs consist of a B cell receptor (BCR-derived extracellular domain and T cell receptor (TCR-associated signaling elements. Cytokine-induced killer (CIK cells are the effector immune cells that can be activated ex vivo and possess both the anti-tumor potency of T lymphocytes and the non-major histocompatibility complex-restricted elimination of natural killer cells. With their pre-eminent ability for robust proliferation, CIK cells may overcome the main limitations of adoptive immunotherapy strategies. CIK cells have strong tumor cell killing capacity; they are effective against a wide variety of malignant tumors and have been shown to be safe in cancer patients. This review summarizes the characteristics of CARs which make them attractive for in cancer treatment strategies. In addition, the role of CIK cells and the advantages of combining CIK cells with CAR-based therapy will be discussed. Scientific evidence to support their combined therapeutic application will be highlighted, with a focus on how their innovative combination may be translated into cancer clinical trials. [Biomed Res Ther 2016; 3(6.000: 653-665

  7. High-Level Systemic Expression of Conserved Influenza Epitope in Plants on the Surface of Rod-Shaped Chimeric Particles

    Directory of Open Access Journals (Sweden)

    Natalia V. Petukhova

    2014-04-01

    Full Text Available Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMV‑based viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rod‑shaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.

  8. Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor.

    Science.gov (United States)

    Caruso, Hillary G; Torikai, Hiroki; Zhang, Ling; Maiti, Sourindra; Dai, Jianliang; Do, Kim-Anh; Singh, Harjeet; Huls, Helen; Lee, Dean A; Champlin, Richard E; Heimberger, Amy B; Cooper, Laurence J N

    2016-06-01

    Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue. PMID:27163741

  9. Design of novel chimeric melanotropin-deltorphin analogues. Discovery of the first potent human melanocortin 1 receptor antagonist.

    Science.gov (United States)

    Han, G; Quillan, J M; Carlson, K; Sadée, W; Hruby, V J

    2003-02-27

    A number of novel alpha-melanotropin (alpha-MSH) analogues have been designed, synthesized, and assayed for bioactivity at the melanocortin-1 (MC1) receptor from Xenopus frog skin, and selected potent analogues were examined at recombinant human MC1, MC3, and MC4 receptors expressed in human embryonic kidney (HEK) cells. These ligands were designed from Deltorphin-II, by a new hybrid approach, which incorporates the hydrophobic tail and the address sequence of Deltorphin-II (Glu-Val-Val-Gly-NH(2)) and key pharmacophore elements of melanotropins. Some of the ligands designed, c[Xxx-Yyy-Zzz-Arg-Trp-Glu]-Val-Val-Gly-NH(2) [XXX = nothing, Gly, beta-Ala, gamma-Abu, 6-Ahx; YYY = His, His(3-Bom), (S)-cyclopentylglycine (Cpg); ZZZ = Phe, d-Phe; d-Nal(2')], show high potency at melanocortin receptors. One ligand, GXH-32B-c[beta-Ala-His-d-Nal(2')-Arg-Trp-Glu]-Val-Val-Gly-NH(2), the most potent of the chimeric analogues tested, displayed agonist activity at each of the MC receptor subtypes analyzed, with an EC(50) of 2 nM at the amphibian MC1 receptor. In contrast, GXH-38B-c[Gly-Cpg-d-Nal(2')-Arg-Trp-Glu]-Val-Val-Gly-NH(2) (Cpg = cyclopentyl glycine) was an antagonist with a IC(50) of 43 nM at the amphibian receptor, and among the human subtypes tested, was the most potent at the MC1 receptor subtype where it also acted as an antagonist (K(i) = 53 nM), which is the first potent antagonist discovered for the human MC1 receptor. These results provide strong evidence supporting our hypothesis that ligand scaffolds for different G-protein coupled receptors (GPCRs) can be used to design ligands for other GPCRs and to design more potent ligands to treat diseases associated with the human MC1 receptor. PMID:12593660

  10. Pharmacokinetics of chimeric L6 conjugated to indium-111- and yttrium-90-DOTA-peptide in tumor-bearing mice

    International Nuclear Information System (INIS)

    A bifunctional chelating agent, DOTA-Gly3-L-(p-isothiocyanato)-phenylalanine amide (DOTA-peptide-NCS), was studied in nude mice bearing human breast cancer xenografts (HBT 3477) to determine its potential for radioimmunoconjugate therapy. Indium-111 and yttrium-90 were attached to an anti-adenocarcinoma chimeric L6 (ChL6) monoclonal antibody (MAb) after pre-chelation to the DOTA-peptide-NCS and the desired neutral radiochelates were obtained by purification. The unique characteristic of the DOTA-peptide-NCS to form neutral complexes with trivalent metals was utilized to separate the resulting 111In and 90Y radiochelates from excess chelating agent and other anionic by-products resulting from metal impurities. The purified radiochelates were then conjugated to ChL6. The paramacokinetics of 111In- and 90Y-DOTA-peptide-ChL6 were obtained for 5 days after injection in nude mice bearing HBT 3477 xenographs. The results were compared with the pharmacokinetics of 125I-ChL6 obtained in the same mouse model. The whole-body clearance of 125I-ChL6, 90Y-and 111In-DOTA-peptide-ChL6 was monoexponential with biologic half-times of 92, 104 and 160 hr, respectively. Blood clearances of the three radiopharmaceuticals were biphasic. The radiometal immunoconjugates had greater tumor uptake and slower clearances. Indium-111- and 90Y-DOTA-peptide-ChL6 can be produced at high specific activity with fewer than one chelate per MAb by using a pre-labeling method that permits radiochelate purification by charge selection. Studies in mouse xenografts indicate that tumor uptake in enhanced and a favorable therapeutic index is achieved using these agents. 29 refs., 7 figs., 2 tabs

  11. Chimeric antigen receptor (CAR)-engineered T cells redirected against hepatitis C virus (HCV) E2 glycoprotein

    Science.gov (United States)

    Sautto, Giuseppe A; Wisskirchen, Karin; Clementi, Nicola; Castelli, Matteo; Diotti, Roberta A; Graf, Julia; Clementi, Massimo; Burioni, Roberto; Protzer, Ulrike; Mancini, Nicasio

    2016-01-01

    Objective The recent availability of novel antiviral drugs has raised new hope for a more effective treatment of hepatitis C virus (HCV) infection and its severe sequelae. However, in the case of non-responding or relapsing patients, alternative strategies are needed. To this end we have used chimeric antigen receptors (CARs), a very promising approach recently used in several clinical trials to redirect primary human T cells against different tumours. In particular, we designed the first CARs against HCV targeting the HCV/E2 glycoprotein (HCV/E2). Design Anti-HCV/E2 CARs were composed of single-chain variable fragments (scFvs) obtained from a broadly cross-reactive and cross-neutralising human monoclonal antibody (mAb), e137, fused to the intracellular signalling motif of the costimulatory CD28 molecule and the CD3ζ domain. Activity of CAR-grafted T cells was evaluated in vitro against HCV/E2-transfected cells as well as hepatocytes infected with cell culture-derived HCV (HCVcc). Results In this proof-of-concept study, retrovirus-transduced human T cells expressing anti-HCV/E2 CARs were endowed with specific antigen recognition accompanied by degranulation and secretion of proinflammatory and antiviral cytokines, such as interferon γ, interleukin 2 and tumour necrosis factor α. Moreover, CAR-grafted T cells were capable of lysing target cells of both hepatic and non-hepatic origin expressing on their surface the HCV/E2 glycoproteins of the most clinically relevant genotypes, including 1a, 1b, 2a, 3a, 4 and 5. Finally, and more importantly, they were capable of lysing HCVcc-infected hepatocytes. Conclusions Clearance of HCV-infected cells is a major therapeutic goal in chronic HCV infection, and adoptive transfer of anti-HCV/E2 CARs-grafted T cells represents a promising new therapeutic tool. PMID:25661083

  12. Chimeric Plant Calcium/Calmodulin-Dependent Protein Kinase Gene with a Neural Visinin-Like Calcium-Binding Domain

    Science.gov (United States)

    Patil, Shameekumar; Takezawa, D.; Poovaiah, B. W.

    1995-01-01

    Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca-2(+) and Ca-2(+)/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca-2(+)- mediated signals. A chimeric Ca-2(+)/calmodulin-dependent protein kinase (CCaMK) gene with a visinin-like Ca-2(+)- binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca-2(+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca-2(+)/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca-2(+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approx. 56 kDa) binds calmodulin in a Ca-2(+)-dependent manner. Furthermore, Ca-45-binding assays revealed that CCaMK directly binds Ca-2(+). The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca-2(+) signaling in plants.

  13. Analysis of serpin inhibitory function by mutagenesis of ovalbumin and generation of chimeric ovalbumin/PAI-2 fusion proteins.

    Science.gov (United States)

    McCarthy, B J; Worrall, D M

    1997-04-01

    Ovalbumin is a non-inhibitory serpin which lacks the ability to undergo the S --> R transition or conformational change. Amino acid residues in the hinge region (P11 to P14) of ovalbumin and other non-inhibitory serpins differ from the concensus sequence of this region of inhibitory serpins, and have been proposed to be responsible for lack of inhibitory properties, particularly the P14 charged residue. Site directed mutagenesis using PCR overlap extension was performed on these residues in ovalbumin to create a mutant with three amino acid changes, R340T, V342A and V343A. However analysis of the mutant recombinant ovalbumin with the consensus residues failed to show inhibitory activity or decreased stability, indicating that the hinge region alone is not responsible for lack of inhibition. A series of three fusion proteins were then constructed by replacing varying C-terminal regions of ovalbumin with the corresponding region of the inhibitory ov-serpin PAI-2 in order to further analyse serpin inhibitory function. Fusion proteins F1 and F2 contained approximately 16% and 35% PAI-2, respectively. This resulted in the replacing of structural features such as the reactive site loop, hinge region and beta sheet strands 5A and 6A. However both fusion proteins showed no inhibitory activity with the PAI-2 target protease urokinase (uPA) and no decrease in stability as analysed by transverse urea gradient (TUG) gels. The third chimeric fusion protein constructed (F3) contained 64% PAI-2 and did demonstrate inhibition of uPA, SDS-PAGE stable complex formation with uPA and increased instability on TUG gels. Structural differences between the inactive F2 and active F3 include the replacement of helix F and beta sheet strand 3A of ovalbumin with those of PAI-2, suggesting that these features may have a key role in serpin beta-sheet opening and inhibitory function. PMID:9126838

  14. Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

    Science.gov (United States)

    Wang, W.; Poovaiah, B. W.

    1999-01-01

    A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

  15. Chimeric Rabies Virus-Like Particles Containing Membrane-Anchored GM-CSF Enhances the Immune Response against Rabies Virus

    Directory of Open Access Journals (Sweden)

    Hongtao Kang

    2015-03-01

    Full Text Available Rabies remains an important public health threat in most developing countries. To develop a more effective and safe vaccine against rabies, we have constructed a chimeric rabies virus-like particle (VLP, which containing glycoprotein (G and matrix protein (M of rabies virus (RABV Evelyn-Rokitnicki-Abelseth (ERA strain, and membrane-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF, and it was named of EVLP-G. The immunogenicity and protective efficacy of EVLP-G against RABV were evaluated by intramuscular administration in a mouse model. The EVLP-G was successfully produced in insect cells by coinfection with three recombinant baculoviruses expressing G, M, and GM-CSF, respectively. The membrane-anchored GM-CSF possesses a strong adjuvant activity. More B cells and dendritic cells (DCs were recruited and/or activated in inguinal lymph nodes in mice immunized with EVLP-G. EVLP-G was found to induce a significantly increased RABV-specific virus-neutralizing antibody and elicit a larger and broader antibody subclass responses compared with the standard rabies VLP (sRVLP, consisting of G and M. The EVLP-G also elicited significantly more IFN-γ- or IL-4-secreting CD4+ and CD8+ T cells than the sRVLP. Moreover, the immune responses induced by EVLP-G protect all vaccinated mice from lethal challenge with RABV. These results suggest that EVLP-G has the potential to be developed as a novel vaccine candidate for the prevention and control of animal rabies.

  16. Chimeric rabies virus-like particles containing membrane-anchored GM-CSF enhances the immune response against rabies virus.

    Science.gov (United States)

    Kang, Hongtao; Qi, Yinglin; Wang, Hualei; Zheng, Xuexing; Gao, Yuwei; Li, Nan; Yang, Songtao; Xia, Xianzhu

    2015-03-01

    Rabies remains an important public health threat in most developing countries. To develop a more effective and safe vaccine against rabies, we have constructed a chimeric rabies virus-like particle (VLP), which containing glycoprotein (G) and matrix protein (M) of rabies virus (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain, and membrane-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF), and it was named of EVLP-G. The immunogenicity and protective efficacy of EVLP-G against RABV were evaluated by intramuscular administration in a mouse model. The EVLP-G was successfully produced in insect cells by coinfection with three recombinant baculoviruses expressing G, M, and GM-CSF, respectively. The membrane-anchored GM-CSF possesses a strong adjuvant activity. More B cells and dendritic cells (DCs) were recruited and/or activated in inguinal lymph nodes in mice immunized with EVLP-G. EVLP-G was found to induce a significantly increased RABV-specific virus-neutralizing antibody and elicit a larger and broader antibody subclass responses compared with the standard rabies VLP (sRVLP, consisting of G and M). The EVLP-G also elicited significantly more IFN-γ- or IL-4-secreting CD4+ and CD8+ T cells than the sRVLP. Moreover, the immune responses induced by EVLP-G protect all vaccinated mice from lethal challenge with RABV. These results suggest that EVLP-G has the potential to be developed as a novel vaccine candidate for the prevention and control of animal rabies. PMID:25768031

  17. Chimeric Mice with Humanized Livers: A Unique Tool for in Vivo and in Vitro Enzyme Induction Studies

    Directory of Open Access Journals (Sweden)

    Masakazu Kakuni

    2013-12-01

    Full Text Available We performed in vivo and in vitro studies to determine the induction of human cytochrome P450 (CYP using chimeric mice with humanized liver (PXB-mice® and human hepatocytes isolated from the PXB-mice (PXB-cells, which were derived from the same donor. For the in vivo study, PXB-mice were injected with 3-methylcholanthrene (3-MC, 2 or 20 mg/kg or rifampicin (0.1 or 10 mg/kg for four days. For the in vitro study, PXB-cells were incubated with 3-MC (10, 50, or 250 ng/mL or with rifampicin (5 or 25 μg/mL. The CYP1A1 and 1A2, and CYP3A4 mRNA expression levels increased significantly in the PXB-mouse livers with 20 mg/kg of 3-MC (Cmax, 12.2 ng/mL, and 10 mg/kg rifampicin (Cmax, 6.9 µg/mL, respectively. The CYP1A1 mRNA expression level increased significantly in PXB-cells with 250 ng/mL of 3-MC, indicating lower sensitivity than in vivo. The CYP1A2 and CYP3A4 mRNA expression levels increased significantly with 50 ng/mL of 3-MC, and 5 μg/mL of rifampicin, respectively, which indicated that the sensitivities were similar between in vivo and in vitro studies. In conclusion, PXB-mice and PXB-cells provide a robust model as an intermediate between in vivo and in vitro human metabolic enzyme induction studies.

  18. Serotype chimeric oncolytic adenovirus coding for GM-CSF for treatment of sarcoma in rodents and humans.

    Science.gov (United States)

    Bramante, Simona; Koski, Anniina; Kipar, Anja; Diaconu, Iulia; Liikanen, Ilkka; Hemminki, Otto; Vassilev, Lotta; Parviainen, Suvi; Cerullo, Vincenzo; Pesonen, Saila K; Oksanen, Minna; Heiskanen, Raita; Rouvinen-Lagerström, Noora; Merisalo-Soikkeli, Maiju; Hakonen, Tiina; Joensuu, Timo; Kanerva, Anna; Pesonen, Sari; Hemminki, Akseli

    2014-08-01

    Sarcomas are a relatively rare cancer, but often incurable at the late metastatic stage. Oncolytic immunotherapy has gained attention over the past years, and a wide range of oncolytic viruses have been delivered via intratumoral injection with positive safety and promising efficacy data. Here, we report preclinical and clinical results from treatment of sarcoma with oncolytic adenovirus Ad5/3-D24-GMCSF (CGTG-102). Ad5/3-D24-GMCSF is a serotype chimeric oncolytic adenovirus coding for human granulocyte-macrophage colony-stimulating factor (GM-CSF). The efficacy of Ad5/3-D24-GMCSF was evaluated on a panel of soft-tissue sarcoma (STS) cell lines and in two animal models. Sarcoma specific human data were also collected from the Advanced Therapy Access Program (ATAP), in preparation for further clinical development. Efficacy was seen in both in vitro and in vivo STS models. Fifteen patients with treatment-refractory STS (13/15) or primary bone sarcoma (2/15) were treated in ATAP, and treatments appeared safe and well-tolerated. A total of 12 radiological RECIST response evaluations were performed, and two cases of minor response, six cases of stable disease and four cases of progressive disease were detected in patients progressing prior to virus treatment. Overall, the median survival time post treatment was 170 days. One patient is still alive at 1,459 days post virus treatment. In summary, Ad5/3-D24-GMCSF appears promising for the treatment of advanced STS; a clinical trial for treatment of refractory injectable solid tumors including STS is ongoing. PMID:24374597

  19. Synthetic Ciguatoxins Selectively Activate Nav1.8-derived Chimeric Sodium Channels Expressed in HEK293 Cells*

    Science.gov (United States)

    Yamaoka, Kaoru; Inoue, Masayuki; Miyazaki, Keisuke; Hirama, Masahiro; Kondo, Chie; Kinoshita, Eiji; Miyoshi, Hiroshi; Seyama, Issei

    2009-01-01

    The synthetic ciguatoxin CTX3C has been shown to activate tetrodotoxin (TTX)-sensitive sodium channels (Nav1.2, Nav1.4, and Nav1.5) by accelerating activation kinetics and shifting the activation curve toward hyperpolarization (Yamaoka, K., Inoue, M., Miyahara, H., Miyazaki, K., and Hirama, M. (2004) Br. J. Pharmacol. 142, 879–889). In this study, we further explored the effects of CTX3C on the TTX-resistant sodium channel Nav1.8. TTX-resistant channels have been shown to be involved in transducing pain and related sensations (Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257–262). Thus, we hypothesized that ciguatoxin-induced activation of the Nav1.8 current would account for the neurological symptoms of ciguatera poisoning. We found that 0.1 μm CTX3C preferentially affected the activation process of the Nav1.8 channel compared with those of the Nav1.2 and Nav1.4 channels. Importantly, without stimulation, 0.1 μm CTX3C induced a large leakage current (IL). The conductance of the IL calculated relative to the maximum conductance (Gmax) was 10 times larger than that of Nav1.2 or Nav1.4. To determine the molecular domain of Nav1.8 responsible for conferring higher sensitivity to CTX3C, we made two chimeric constructs from Nav1.4 and Nav1.8. Chimeras containing the N-terminal half of Nav1.8 exhibited a large response similar to wild-type Nav1.8, indicating that the region conferring high sensitivity to ciguatoxin action is located in the D1 or D2 domains. PMID:19164297

  20. Atomic resolution structure of a chimeric DNA-RNA Z-type duplex in complex with Ba(2+) ions: a case of complicated multi-domain twinning.

    Science.gov (United States)

    Gilski, Miroslaw; Drozdzal, Pawel; Kierzek, Ryszard; Jaskolski, Mariusz

    2016-02-01

    The self-complementary dCrGdCrGdCrG hexanucleotide, in which not only the pyrimidine/purine bases but also the ribo/deoxy sugars alternate along the sequence, was crystallized in the presence of barium cations in the form of a left-handed Z-type duplex. The asymmetric unit of the P21 crystal with a pseudohexagonal lattice contains four chimeric duplexes and 16 partial Ba(2+) sites. The chimeric (DNA-RNA)2 duplexes have novel patterns of hydration and exhibit a high degree of discrete conformational disorder of their sugar-phosphate backbones, which can at least partly be correlated with the fractional occupancies of the barium ions. The crystals of the DNA-RNA chimeric duplex in complex with Ba(2+) ions and also with Sr(2+) ions exhibit complicated twinning, which in combination with structural pseudosymmetry made structure determination difficult. The structure could be successfully solved by molecular replacement in space groups P1 and P21 but not in orthorhombic or higher symmetry and, after scrupulous twinning and packing analysis, was refined in space group P21 to an R and Rfree of 11.36 and 16.91%, respectively, using data extending to 1.09 Å resolution. With the crystal structure having monoclinic symmetry, the sixfold crystal twinning is a combination of threefold and twofold rotations. The paper describes the practical aspects of dealing with cases of complicated twinning and pseudosymmetry, and compares the available software tools for the refinement and analysis of such cases. PMID:26894669

  1. Immunomodulatory Effects of Different Cellular Therapies of Bone Marrow Origin on Chimerism Induction and Maintenance Across MHC Barriers in a Face Allotransplantation Model.

    Science.gov (United States)

    Hivelin, Mikael; Klimczak, Aleksandra; Cwykiel, Joanna; Sonmez, Erhan; Nasir, Serdar; Gatherwright, James; Siemionow, Maria

    2016-08-01

    Many more patients would benefit from vascularized composite allotransplantation if less toxic and safer immunosuppressive protocols will become available. Tolerance induction protocols with donor cells co-transplantation are one of the promising pathways to reduce maintenance immunosupressive regimens. We investigated the role of donor bone marrow cells (BMC), mesenchymal stromal cells (MSC) and in vivo created chimeric cells (CC) used as supportive therapies in a fully MHC-mismatched rat face transplantation model. Twenty-four fully MHC-mismatched hemiface transplantations were performed between ACI (RT1(a)) donors and Lewis (RT1(l)) recipients under combined seven-day immunosuppressive regimen of anti-αβ-T-cell receptor (TCR) monoclonal antibody and cyclosporin A. We studied four experimental groups-group 1: no cellular therapy; group 2: supportive therapy with BMC; group 3: supportive therapy with MSC; group 4: supportive therapy with CC generated in a primary chimera. We evaluated clinical and histological rejection grades, transplanted cells migration, donor-specific chimerism in the peripheral blood and bone marrow compartments, and CD4(+)/CD25(+) T-cell levels. Face allograft rejection was observed at 26.8 ± 0.6 days post-transplant (PT) in the absence of cellular therapy, at 34.5 ± 1.1 days for group 2, 29.3 ± 0.8 days for group 3, and 30.3 ± 1.38 PT for group 4. The longest survival was observed in allografts supported by co-transplantation of BMC. All support in cellular therapies delayed face allograft rejection by chimerism induction and/or immunomodulatory properties of co-transplanted cells. Survival time was comparable between groups, however, further studies, with different cell dosages, delivery routes and delivery times are required. PMID:26708158

  2. Novel recombinant chimeric virus-like particle is immunogenic and protective against both enterovirus 71 and coxsackievirus A16 in mice.

    Science.gov (United States)

    Zhao, Hui; Li, Hao-Yang; Han, Jian-Feng; Deng, Yong-Qiang; Zhu, Shun-Ya; Li, Xiao-Feng; Yang, Hui-Qin; Li, Yue-Xiang; Zhang, Yu; Qin, E-De; Chen, Rong; Qin, Cheng-Feng

    2015-01-01

    Hand-foot-and-mouth disease (HFMD) has been recognized as an important global public health issue, which is predominantly caused by enterovirus 71 (EV-A71) and coxsackievirus A16 (CVA16). There is no available vaccine against HFMD. An ideal HFMD vaccine should be bivalent against both EV-A71 and CVA16. Here, a novel strategy to produce bivalent HFMD vaccine based on chimeric EV-A71 virus-like particles (ChiEV-A71 VLPs) was proposed and illustrated. The neutralizing epitope SP70 within the capsid protein VP1 of EV-A71 was replaced with that of CVA16 in ChiEV-A71 VLPs. Structural modeling revealed that the replaced CVA16-SP70 epitope is well exposed on the surface of ChiEV-A71 VLPs. These VLPs produced in Saccharomyces cerevisiae exhibited similarity in both protein composition and morphology as naive EV-A71 VLPs. Immunization with ChiEV-A71 VLPs in mice elicited robust Th1/Th2 dependent immune responses against EV-A71 and CVA16. Furthermore, passive immunization with anti-ChiEV-A71 VLPs sera conferred full protection against lethal challenge of both EV-A71 and CVA16 infection in neonatal mice. These results suggested that this chimeric vaccine, ChiEV-A71 might have the potential to be further developed as a bivalent HFMD vaccine in the near future. Such chimeric enterovirus VLPs provide an alternative platform for bivalent HFMD vaccine development. PMID:25597595

  3. A new classification of free combined or connected tissue transfers: introduction to the concept of bridge, siamese, chimeric, mosaic, and chain-circle flaps.

    Directory of Open Access Journals (Sweden)

    Koshima I

    2001-12-01

    Full Text Available Recently, combined tissues or flaps have been used for the repair of extensively massive or wide defects resulting from radical wide resection. To further advance the development of combined tissue transfers, they should be reclassified. Based on our cases with free tissue transfers, we have created a new classification of combined flaps composed of "bridge", "chimeric", "siamese", "mosaic", and "chain-circle" flaps. The bridge flap is fabricated out together of separate flaps with short vascular pedicles. These form a compound flap supplied with a solitary vascular source. The chimeric flap is compounded from multiple different flaps but consists of only a single different tissue form. Each of the flaps is usually supplied by different branches from the same source vessel. It differs from the bridge flap in that the pedicle of each flap or tissue has some length for its movement for transfer. The siamese connected flap has 2 adjacent flaps that are simultaneously elevated, and a disparate vascular pedicle for each flap must be reestablished. This connected flap has double isolated pedicles. Themosaic connected flap consists of 2 adjacent flaps that are simultaneously elevated, and the pedicle of the distal flap is anastomosed to the pedicle branch of the proximal flap in the "bridge" fashion. The vascular pedicle of the proximal flap is anastomosed to a single vascular source. The chain-circle flap has 2 or more flaps like the bridge and chimeric flaps, and the distal end of the vascular source is anastomosed to the branch of the recipient vessel. Based on results with our patients, the lateral circumflex femoral system seems to be the most suitable candidate for the axial pedicle of these combined flaps, because the system has several branches of large and small caliber, and several tissue components, such as the vascularized ilium, rectus femoris muscle, gracilis muscle, lateral femoral cutaneous nerve, and fascia lata, are located nearby.

  4. RNA secondary structures located in the interchromosomal region of human ACAT1 chimeric mRNA are re-quired to produce the 56-kDa isoform

    Institute of Scientific and Technical Information of China (English)

    Jia Chen; Ta-Yuan Chang; Bo-Liang Li; Xiao-Nan Zhao; Li Yang; Guang-Jing Hu; Ming Lu; Ying Xiong; Xin-Ying Yang; Catherine CY Chang; Bao-Liang Song

    2008-01-01

    We have previously reported that the human ACAT1 gene produces a chimeric mRNAthrough the interchromosomal processing of two discontinuous RNAs transcribed from chromosomes 1 and 7. The chimeric mRNA uses AUG1397-1399 and GGC1274-1276 as translation initiation codons to produce normal 50-kDa ACATI and a novel enzymatically active 56-kDa isoform,respectively,with the latter being authentically present in human cells,including human monocyte derived macrophages. In this work,we report that RNA secondary structures located in the vicinity,of the GGC1274-1276 codon are required for production of the 56-kDa isoform. The effects of the three predicted stem-loops (nt 1255-1268,1286-1342 and 1355-1384) were tested individually by transfecting expression plasmids into cells that contained the wild-type,deleted or mutant stem-loop sequences linked to a partial ACAT1 AUG open reading frame (ORF) or to theORFs of other genes. The expression patterns were monitored by western blot analyses. We found that the upstream stem-loop1255-1268 from chromosome 7 and downstream stem-loop1286-1342 from chromosome I were needed for production of the 56-kDa isoform,whereas the last stem-ioop1355-1384 from chromosome I was dispensable. The results of experi ments using both monocistronic and bicistronic vectors with a stable hairpin showed that translation initiation from the GGC1274-1276 codon was mediated by an internal ribosome entry site (IRES). Further experiments revealed that translation initiation from the GGC1274-1276 codon requires the upstream AU-constituted RNA secondary structure and the downstream GC-rich structure. This mechanistic work provides further support for the biological significance of the chimeric nature of the human ACATI transcript.

  5. Radiolabeling and biotinylation of internalizing monoclonal antibody chimeric BR96: Potential use of extracorporeal immunoadsorption with enhanced tumor radioactivity retention of Iodine, Indium and Rhenium

    Energy Technology Data Exchange (ETDEWEB)

    Chen, JianQing

    1997-01-01

    In this thesis, methodology of radiolabeling and simultaneous biotinylation for internalizing monoclonal antibody chimeric BR96 have been investigated by using three element groups of potential therapeutic radionuclides iodine, indium and rhenium, and their different labeling methods. The biodistribution and kinetics of biotinylated and radiolabeled chiBR96 have been studied in colon carcinoma isografted rats. The potential use of ECIA, based on the biotin-avidin concept, has been evaluated and compared with the approach of avidin `chase` in the same animal tumor model with respect to an enhancement of tumor-to-normal tissue (T/N) activity ratio. 131 refs.

  6. Radiolabeling and biotinylation of internalizing monoclonal antibody chimeric BR96: Potential use of extracorporeal immunoadsorption with enhanced tumor radioactivity retention of Iodine, Indium and Rhenium

    International Nuclear Information System (INIS)

    In this thesis, methodology of radiolabeling and simultaneous biotinylation for internalizing monoclonal antibody chimeric BR96 have been investigated by using three element groups of potential therapeutic radionuclides iodine, indium and rhenium, and their different labeling methods. The biodistribution and kinetics of biotinylated and radiolabeled chiBR96 have been studied in colon carcinoma isografted rats. The potential use of ECIA, based on the biotin-avidin concept, has been evaluated and compared with the approach of avidin 'chase' in the same animal tumor model with respect to an enhancement of tumor-to-normal tissue (T/N) activity ratio. 131 refs

  7. Novel recombinant chimeric virus-like particle is immunogenic and protective against both enterovirus 71 and coxsackievirus A16 in mice

    OpenAIRE

    Hui Zhao; Hao-Yang Li; Jian-Feng Han; Yong-Qiang Deng; Shun-Ya Zhu; Xiao-Feng Li; Hui-Qin Yang; Yue-Xiang Li; Yu Zhang; E-De Qin; Rong Chen; Cheng-Feng Qin

    2015-01-01

    Hand-foot-and-mouth disease (HFMD) has been recognized as an important global public health issue, which is predominantly caused by enterovirus 71 (EV-A71) and coxsackievirus A16 (CVA16). There is no available vaccine against HFMD. An ideal HFMD vaccine should be bivalent against both EV-A71 and CVA16. Here, a novel strategy to produce bivalent HFMD vaccine based on chimeric EV-A71 virus-like particles (ChiEV-A71 VLPs) was proposed and illustrated. The neutralizing epitope SP70 within the cap...

  8. Molecularly Targeted Therapy of Human Hepatocellular Carcinoma Xenografts with Radio-iodinated Anti-VEGFR2 Murine-Human Chimeric Fab

    OpenAIRE

    Jianfei Huang; Qi Tang; Changjun Wang; Huixin Yu; Zhenqing Feng; Jin Zhu

    2015-01-01

    Vascular endothelial growth factor receptor 2 (VEGFR2) is traditionally regarded as an important therapeutic target in a wide variety of malignancies, such as hepatocellular carcinoma (HCC). We previously generated a murine-human anti-VEGFR2 chimeric Fab (cFab), named FA8H1, which has the potential to treat VEGFR2-overexpressing solid tumors. Here, we investigated whether FA8H1 can be used as a carrier in molecularly targeted therapy in HCC xenograft models. FA8H1 was labeled with 131I, and t...

  9. Construction of exogenous multiple epitopes of helper T lymphocytes and DNA immunization of its chimeric plasmid with HBV pre-S2/S gene

    Institute of Scientific and Technical Information of China (English)

    Wen-Jun Gao; Xiao-Mou Peng; Dong-Ying Xie; Qi-Feng Xie; Zhi-Liang Gao; Ji-Lu Yao

    2004-01-01

    AIM: To design and construct an exogenous multiple epitope of helper T lymphocytes (HTL), and to evaluate its effect on anti-HBs response through DNA immunization.METHODS: Artificial HTL epitope, PADRE and four other HTL epitopes from different proteins were linked together using splicing by overlap extension to generate exogenous multiple epitopes of HTL, MTE5. pcMTE5 and pcHB weregenerated by cloning MTE5 and fragments of HBV pre-S2/S gene into mammalian expression plasmid pcDNA3. Four chimeric plasmids were constructed by cloning MTE5 into the region of pre-S2 gene (Bam HI), 5′ terminal of S gene (HincⅡ, Xba Ⅰ) and 3′ terminal of S gene (Acc Ⅰ) of pcHB respectively. BALB/c mice were used in DNA immunization of the recombinant plasmids. Anti-HBs was detected using Abbott IMx AUSAB test kits.RESULTS: The sequences of MTE5 and the 6 constructs of recombinant plasmids were confirmed to be correct by DNA sequencing. The anti-HBs response of the coinoculation of pcHB and pcMTE5 was much higher than that of the inoculation of pcHB only (136.7±69.1 mIU/mL vs 27.6±17.3 mIU/mL, P<0.01, t = -6.56). Among the 4 chimeric plasmids, only the plasmid in which MTE5 was inserted into the pre-S2 region had good anti-HBs response (57.54±7.68 mIU/mL), and had no significant difference compared with those of pcHB and the co-inoculation of pcHB and pcMTE5.CONCLUSION: Exogenous multiple epitopes of HTL had immune enhancement when they were co-inoculated with pre-S2/S gene or inoculated in the chimeric form at a proper site of pre-S2/S gene of HBV. It might suggest that it was possible to improve hepatitis B vaccine using exogenous multiple epitopes of HTL. The antibody responses were very low using DNA immunization in the study. Thus, the immune enhancement effect of exogenous multiple epitopes of HTL has to be confirmed and the effect on overcoming the drawback of the polymorphism of HLA Ⅱ antigens should also be evaluated after these chimeric plasmids are expressed

  10. A DNA vaccine encoding a chimeric allergen derived from major group 1 allergens of dust mite can be used for specific immunotherapy

    OpenAIRE

    Sun, Tong; Yin, Kang; Wu, Lu-Yi; Jin, Wen-Jie; Li, Yang; Sheng, Bin; Jiang, Yu-Xin

    2014-01-01

    Immunization with DNA-based constructs has been shown to be against the antigen and the response is skewed in such a way as to ameliorate the symptoms of allergic disease. This approach is particularly useful in the treatment of allergic inflammatory diseases, such as asthma. The major group 1 allergen from house dust mites is one of the triggers of allergic asthma. This study explores whether a chimeric gene R8, derived from the major group 1 allergen of house dust mite species (Dermatophago...

  11. Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells

    OpenAIRE

    Kochenderfer, James N.; Yu, Zhiya; Frasheri, Dorina; Restifo, Nicholas P; Rosenberg, Steven A.

    2010-01-01

    Adoptive T-cell therapy with anti-CD19 chimeric antigen receptor (CAR)–expressing T cells is a new approach for treating advanced B-cell malignancies. To evaluate anti-CD19–CAR-transduced T cells in a murine model of adoptive T-cell therapy, we developed a CAR that specifically recognized murine CD19. We used T cells that were retrovirally transduced with this CAR to treat mice bearing a syngeneic lymphoma that naturally expressed the self-antigen murine CD19. One infusion of anti-CD19–CAR-tr...

  12. A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27 Protein as an HIV Immunogen.

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    Aneesh Vijayan

    Full Text Available In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K by fusing gp120 from clade B with the vaccinia virus (VACV 14K oligomeric protein (derived from A27L gene. Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs, gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES. Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1

  13. A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen

    Science.gov (United States)

    Vijayan, Aneesh; García-Arriaza, Juan; C. Raman, Suresh; Conesa, José Javier; Chichón, Francisco Javier; Santiago, César; Sorzano, Carlos Óscar S.; Carrascosa, José L.; Esteban, Mariano

    2015-01-01

    In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive

  14. Effect of natural and chimeric haemagglutinin genes on influenza A virus replication in baby hamster kidney cells.

    Science.gov (United States)

    van Wielink, R; Harmsen, M M; Martens, D E; de Leeuw, O S; Peeters, B P H; Wijffels, R H; Moormann, R J M

    2012-12-31

    Baby hamster kidney (BHK21) cells are used to produce vaccines against various viral veterinary diseases, including rabies and foot-and-mouth-disease. Although particular influenza virus strains replicate efficiently in BHK21 cells the general use of these cells for influenza vaccine production is prohibited by the poor replication of most strains, including model strain A/PR/8/34 [H1N1] (PR8). We now show that in contrast to PR8, the related strain A/WSN/33 [H1N1] (WSN) replicates efficiently in BHK21 cells. This difference is determined by the haemagglutinin (HA) protein since reciprocal reassortant viruses with swapped HAs behave similarly with respect to growth on BHK21 cells as the parental virus from which their HA gene is derived. The ability or inability of six other influenza virus strains to grow on BHK21 cells appears to be similarly dependent on the nature of the HA gene since reassortant PR8 viruses containing the HA of these strains grow to similar titres as the parental virus from which the HA gene was derived. However, the growth to low titres of a seventh influenza strain was not due to the nature of the HA gene since a reassortant PR8 virus containing this HA grew efficiently on BHK21 cells. Taken together, these results suggest that the HA gene often primarily determines influenza replication efficiency on BHK21 cells but that in some strains other genes are also involved. High virus titres could be obtained with reassortant PR8 strains that contained a chimeric HA consisting of the HA1 domain of PR8 and the HA2 domain of WSN. HA1 contains most antigenic sites and is therefore important for vaccine efficacy. This method of producing the HA1 domain as fusion to a heterologous HA2 domain could possibly also be used for the production of HA1 domains of other viruses to enable the use of BHK21 cells as a generic platform for veterinary influenza vaccine production. PMID:23079079

  15. Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR expression by flow cytometry

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    Zheng Zhili

    2012-02-01

    Full Text Available Abstract Background There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymphocytes (PBL with CARs directed against a variety of tumor associated antigens. Despite the improvements in the design of CARs and expansion of the number of target antigens, there is no universal flow cytometric method available to detect the expression of CARs on the surface of transduced lymphocytes. Methods Currently anti-fragment antigen binding (Fab conjugates are most widely used to determine the expression of CARs on gene-modified lymphocytes by flow cytometry. The limitations of these reagents are that many of them are not commercially available, generally they are polyclonal antibodies and often the results are inconsistent. In an effort to develop a simple universal flow cytometric method to detect the expression of CARs, we employed protein L to determine the expression of CARs on transduced lymphocytes. Protein L is an immunoglobulin (Ig-binding protein that binds to the variable light chains (kappa chain of Ig without interfering with antigen binding site. Protein L binds to most classes of Ig and also binds to single-chain antibody fragments (scFv and Fab fragments. Results We used CARs derived from both human and murine antibodies to validate this novel protein L based flow cytometric method and the results correlated well with other established methods. Activated human PBLs were transduced with retroviral vectors expressing two human antibody based CARs (anti-EGFRvIII, and anti-VEGFR2, two murine antibody derived CARs (anti-CSPG4, and anti

  16. Modulating p56Lck in T-Cells by a Chimeric Peptide Comprising Two Functionally Different Motifs of Tip from Herpesvirus saimiri

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    Jean-Paul Vernot

    2015-01-01

    Full Text Available The Lck interacting protein Tip of Herpesvirus saimiri is responsible for T-cell transformation both in vitro and in vivo. Here we designed the chimeric peptide hTip-CSKH, comprising the Lck specific interacting motif CSKH of Tip and its hydrophobic transmembrane sequence (hTip, the latter as a vector targeting lipid rafts. We found that hTip-CSKH can induce a fivefold increase in proliferation of human and Aotus sp. T-cells. Costimulation with PMA did not enhance this proliferation rate, suggesting that hTip-CSKH is sufficient and independent of further PKC stimulation. We also found that human Lck phosphorylation was increased earlier after stimulation when T-cells were incubated previously with hTip-CSKH, supporting a strong signalling and proliferative effect of the chimeric peptide. Additionally, Lck downstream signalling was evident with hTip-CSKH but not with control peptides. Importantly, hTip-CSKH could be identified in heavy lipid rafts membrane fractions, a compartment where important T-cell signalling molecules (LAT, Ras, and Lck are present during T-cell activation. Interestingly, hTip-CSKH was inhibitory to Jurkat cells, in total agreement with the different signalling pathways and activation requirements of this leukemic cell line. These results provide the basis for the development of new compounds capable of modulating therapeutic targets present in lipid rafts.

  17. Chimeric avian paramyxovirus-based vector immunization against highly pathogenic avian influenza followed by conventional Newcastle disease vaccination eliminates lack of protection from virulent ND virus

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    C. Steglich

    2014-01-01

    Full Text Available Recently, we described a chimeric, hemagglutinin of highly pathogenic avian influenza virus (HPAIV H5 expressing Newcastle disease virus (NDV-based vector vaccine (chNDVFHNPMV8H5 in which NDV envelope glycoproteins were replaced by those of avian paramyxovirus-8 (APMV-8. This chimeric vaccine induced solid protection against lethal HPAIV H5N1 even in chickens with maternal antibodies against NDV (MDA+. However, due to the absence of the major NDV immunogens it failed to induce protection against Newcastle disease (ND. Here, we report on protection of MDA+ chickens against HPAI H5N1 and ND, by vaccination with chNDVFHNPMV8H5 either on day 1 or day seven after hatch, and subsequent immunization with live attenuated NDV seven days later. Vaccination was well tolerated and three weeks after immunization, challenge infections with highly pathogenic NDV as well as HPAIV H5N1 were carried out. All animals remained healthy without exhibiting any clinical signs, whereas non-vaccinated animals showed morbidity and mortality. Therefore, vaccination with chNDVFHNPMV8H5 can be followed by NDV vaccination to protect chickens from HPAIV as well as NDV, indicating that the antibody response against chNDVFHNPMV8H5 does not interfere with live ND vaccination.

  18. In vitro and in vivo characterization of chimeric duck Tembusu virus based on Japanese encephalitis live vaccine strain SA14-14-2.

    Science.gov (United States)

    Wang, Hong-Jiang; Liu, Long; Li, Xiao-Feng; Ye, Qing; Deng, Yong-Qiang; Qin, E-De; Qin, Cheng-Feng

    2016-07-01

    Duck Tembusu virus (DTMUV), a newly identified flavivirus, has rapidly spread to China, Malaysia and Thailand. The potential threats to public health have been well-highlighted; however its virulence and pathogenesis remain largely unknown. Here, by using reverse genetics, a recombinant chimeric DTMUV based on Japanese encephalitis live vaccine strain SA14-14-2 was obtained by substituting the corresponding prM and E genes (named ChinDTMUV). In vitro characterization demonstrated that ChinDTMUV replicated efficiently in mammalian cells with small-plaque phenotype in comparison with its parental viruses. Mouse tests showed ChinDTMUV exhibited avirulent phenotype in terms of neuroinvasiveness, while it retained neurovirulence from its parental virus DTMUV. Furthermore, immunization with ChinDTMUV was evidenced to elicit robust IgG and neutralizing antibody responses in mice. Overall, we successfully developed a viable chimeric DTMUV, and these results provide a useful platform for further investigation of the pathogenesis of DTMUV and development of a live attenuated DTMUV vaccine candidate. PMID:27100268

  19. Boosting BCG-primed mice with chimeric DNA vaccine HG856A induces potent multifunctional T cell responses and enhanced protection against Mycobacterium tuberculosis.

    Science.gov (United States)

    Ji, Ping; Hu, Zhi-Dong; Kang, Han; Yuan, Qin; Ma, Hui; Wen, Han-Li; Wu, Juan; Li, Zhong-Ming; Lowrie, Douglas B; Fan, Xiao-Yong

    2016-02-01

    The tuberculosis pandemic continues to rampage despite widespread use of the current Bacillus Calmette-Guerin (BCG) vaccine. Because DNA vaccines can elicit effective antigen-specific immune responses, including potent T cell-mediated immunity, they are promising vehicles for antigen delivery. In a prime-boost approach, they can supplement the inadequate anti-TB immunological memory induced by BCG. Based on this, a chimeric DNA vaccine HG856A encoding Mycobacterium tuberculosis (M. tuberculosis) immunodominant antigen Ag85A plus two copies of ESAT-6 was constructed. Potent humoral immune responses, as well as therapeutic effects induced by this DNA vaccine, were observed previously in M. tuberculosis-infected mice. In this study, we further evaluated the antigen-specific T cell immune responses and showed that repeated immunization with HG856A gave modest protection against M. tuberculosis challenge infection and significantly boosted the immune protection primed by BCG vaccination. Enhanced protection was accompanied by increased multifunctional Th1 CD4(+) T cell responses, most notably by an elevated frequency of M. tuberculosis antigen-specific IL-2-producing CD4(+) T cells post-vaccination. These data confirm the potential of chimeric DNA vaccine HG856A as an anti-TB vaccine candidate. PMID:26111521

  20. Sequencing and characterisation of rearrangements in three S. pastorianus strains reveals the presence of chimeric genes and gives evidence of breakpoint reuse.

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    Sarah K Hewitt

    Full Text Available Gross chromosomal rearrangements have the potential to be evolutionarily advantageous to an adapting organism. The generation of a hybrid species increases opportunity for recombination by bringing together two homologous genomes. We sought to define the location of genomic rearrangements in three strains of Saccharomyces pastorianus, a natural lager-brewing yeast hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus, using whole genome shotgun sequencing. Each strain of S. pastorianus has lost species-specific portions of its genome and has undergone extensive recombination, producing chimeric chromosomes. We predicted 30 breakpoints that we confirmed at the single nucleotide level by designing species-specific primers that flank each breakpoint, and then sequencing the PCR product. These rearrangements are the result of recombination between areas of homology between the two subgenomes, rather than repetitive elements such as transposons or tRNAs. Interestingly, 28/30 S. cerevisiae-S. eubayanus recombination breakpoints are located within genic regions, generating chimeric genes. Furthermore we show evidence for the reuse of two breakpoints, located in HSP82 and KEM1, in strains of proposed independent origin.

  1. Analysis of the atmospheric composition during the summer 2013 over the Mediterranean area using the CHARMEX measurements and the CHIMERE model

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    L. Menut

    2014-09-01

    Full Text Available The ADRIMED campaign provides measurements of all key parameters regarding atmospheric composition in the Mediterranean area during the summer 2013. This is an opportunity to quantify the ability of current models to adequately represent the atmospheric composition in this complex region, which is influenced by anthropogenic emissions from Europe, Africa, the Middle-East and from shipping activities as well as mineral dust emissions mostly from the arid areas in Africa, sea-salt emissions, biomass burning emissions and biogenic emissions from the vegetation. The CHIMERE model in its present version is a chemistry-transport model which takes into account all these processes. We show here by simulating the period from 5 June to 15 July 2013 with the CHIMERE model and comparing the results to both routine and specific ADRIMED measurements that this model allows an adequate representation the atmospheric composition over the western Mediterranean, in terms of ozone concentration, particulate matter (PM and aerosol optical depth (AOD. It is also shown that the concentrations of PM on all the considered area is dominated by mineral dust, even though local dust emissions in Europe are certainly overestimated by the model. A comparison with sulphate concentrations at Cape Corsica exhibits some discrepancies related to the regridding of shipping emissions.

  2. Eliciting neutralizing antibodies against the membrane proximal external region of HIV-1 Env by chimeric live attenuated influenza A virus vaccines.

    Science.gov (United States)

    Zang, Yang; Du, Dongchuan; Li, Na; Su, Weiheng; Liu, Xintao; Zhang, Yan; Nie, Jianhui; Wang, Youchun; Kong, Wei; Jiang, Chunlai

    2015-07-31

    Despite significant efforts directed toward research on HIV-1 vaccines, a truly effective immunogen has not been achieved. However, the broadly neutralizing antibodies (BnAbs) 2F5 and 4E10, targeting the highly conserved membrane proximal external region (MPER) of HIV-1, are two promising tools for vaccine development. Here we engrafted the MPER into the linker domain between the trimeric core structure and the transmembrane domain of influenza A virus HA2 to investigate the potential of such chimeric viruses to elicit HIV-1 neutralizing antibodies. In the context of proliferating attenuated influenza A viruses, these HIV-1 neutralizing antibody epitopes could be continuously expressed and mimicked their native conformation to induce humoral immune responses. While MPER-specific antibodies could be detected in serum of guinea pigs vaccinated with the chimeric viruses, they exhibited only weakly neutralizing activities. These antisera from vaccinated animals neutralized viruses of clades B and BC (tier 1), but not of clades AE (tier 1) and C (tier 2). These results suggest that influenza A virus can be used as a vehicle for displaying MPER and inducing BnAbs, but it provides limited protection against HIV-1 infection. In the future development of HIV-1 vaccines by rational design, a more effective live virus vector or multiple antigens should be chosen to facilitate the process of neutralizing antibody maturation. PMID:26126669

  3. Exploiting the yeast L-A viral capsid for the in vivo assembly of chimeric VLPs as platform in vaccine development and foreign protein expression.

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    Frank Powilleit

    Full Text Available A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8(+ memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i a heterologous model protein (GFP, (ii a per se toxic protein (K28 alpha-subunit, and (iii a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A. Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.

  4. Comparison of a chimeric anti-carcinoembryonic antigen antibody conjugated with visible or near-infrared fluorescent dyes for imaging pancreatic cancer in orthotopic nude mouse models

    Science.gov (United States)

    Maawy, Ali A.; Hiroshima, Yukihiko; Kaushal, Sharmeela; Luiken, George A.; Hoffman, Robert M.; Bouvet, Michael

    2013-12-01

    The aim of this study was to evaluate a set of visible and near-infrared dyes conjugated to a tumor-specific chimeric antibody for high-resolution tumor imaging in orthotopic models of pancreatic cancer. BxPC-3 human pancreatic cancer was orthotopically implanted into pancreata of nude mice. Mice received a single intravenous injection of a chimeric anti-carcinoembryonic antigen antibody conjugated to one of the following fluorophores: 488-nm group (Alexa Fluor 488 or DyLight 488); 550-nm group (Alexa Fluor 555 or DyLight 550); 650-nm group (Alexa Fluor 660 or DyLight 650), or the 750-nm group (Alexa Fluor 750 or DyLight 755). After 24 h, the Olympus OV100 small-animal imaging system was used for noninvasive and intravital fluorescence imaging of mice. Dyes were compared with respect to depth of imaging, resolution, tumor-to-background ratio (TBR), photobleaching, and hemoglobin quenching. The longer wavelength dyes had increased depth of penetration and ability to detect the smallest tumor deposits and provided the highest TBRs, resistance to hemoglobin quenching, and specificity. The shorter wavelength dyes were more photostable. This study showed unique advantages of each dye for specific cancer imaging in a clinically relevant orthotopic model.

  5. Chimeric viruses containing the N-terminal ectodomains of GP5 and M proteins of porcine reproductive and respiratory syndrome do not change the cellular tropism of equine arteritis virus

    Science.gov (United States)

    Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) are members of family Arteriviridae; they share many biological properties but differ significantly in cellular tropism. Using an infectious cDNA clone of EAV, we engineered a panel of six chimeric viruses b...

  6. Generation and Characterization of a Human/Mouse Chimeric GD2-Mimicking Anti-Idiotype Antibody Ganglidiximab for Active Immunotherapy against Neuroblastoma.

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    Christin Eger

    Full Text Available Vaccination with proteins mimicking GD2 that is highly expressed on neuroblastoma (NB cells is a promising strategy in treatment of NB, a pediatric malignancy with poor prognosis. We previously showed efficacy of ganglidiomab in vivo, a murine anti-idiotype (anti-Id IgG1. In order to tailor immune responses to variable regions, we generated a new human/mouse chimeric anti-Id antibody (Ab ganglidiximab by replacing murine constant fragments with corresponding human IgG1 regions. DNA sequences encoding for variable regions of heavy (VH and light chains (VL were synthesized by RT-PCR from total RNA of ganglidiomab-producing hybridoma cells and further ligated into mammalian expression plasmids with coding sequences for constant regions of human IgG1 heavy and light chains, respectively. We established a stable production cell line using Chinese hamster ovarian (CHO cells co-transfected with two expression plasmids driving the expression of either ganglidiximab heavy or light chain. After purification from supernatants, anti-idiotypic characteristics of ganglidiximab were demonstrated. Binding of ganglidiximab to anti-GD2 Abs of the 14.18 family as well as to NK-92tr cells expressing a GD2-specific chimeric antigen receptor (scFv(ch14.18-zeta was shown using standard ELISA and flow cytometry analysis, respectively. Ganglidiximab binding affinities to anti-GD2 Abs were further determined by surface plasmon resonance technique. Moreover, binding of anti-GD2 Abs to the nominal antigen GD2 as well as GD2-specific Ab-mediated cytotoxicity (ADCC, CDC was competitively inhibited by ganglidiximab. Finally, ganglidiximab was successfully used as a protein vaccine in vivo to induce a GD2-specific humoral immune response. In summary, we report generation and characterization of a new human/mouse chimeric anti-Id Ab ganglidiximab for active immunotherapy against NB. This Ab may be useful to tailor immune responses to the paratope regions mimicking GD2

  7. Up-regulating CYP3A4 expression in C3A cells by transfection with a novel chimeric regulator of hPXR-p53-AD.

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    Feng Chen

    Full Text Available Most hepatoma cell lines lack proper expression and induction of CYP3A4 enzyme, which limits their use for predicting drug metabolism and toxicity. Nuclear receptor pregnane X receptor (PXR has been well recognized for its critical role in regulating expression of CYP3A4 gene. However, its physiological activity of binding to the particular site of promoter is significantly weakened in hepatic cell lines. To address this problem, we created "chimeric PXR" constructs by appending a strong activation domain (AD from p53 subunit to either N- or C- termini of the human PXR (hPXR, that is, hPXR-p53 and p53-hPXR. C3A, a hepatoma cell line, was used as the cell model to test the regulation effect of chimeric hPXR over wild type (WT hPXR on CYP3A4 expression at gene, protein, and metabolism levels, respectively. Compared with C3A cells transiently transfected with WT hPXR, the activity of CYP3A4.XREM.luc reporter gene in C3A cells transfected with hPXR-p53 or p53-hPXR increased 5- and 9-fold respectively, and the levels of CYP3A4 mRNA expression increased 3.5- and 2.6-fold, respectively. C3A cells stably transfected with hPXR-p53-AD exhibited an improved expression of CYP3A4 at both gene (2-fold and protein (1.5-fold levels compared to WT C3A cells. Testosterone, a CYP3A4-specific substrate, was used for detecting the metabolism activity of CYP3A4. No testosterone metabolite could be detected in microsomes from WT C3A cells and WT C3A cells-based array, while the formation of 6β-hydroxytestosterone metabolite in the transfected cells was 714 and 55 pmol/mg protein/min, respectively. In addition, all the above expression levels in the transfected cell models could be further induced with additional treatment of Rifampicin, a specific inducer for CYP3A4. In conclusion, our study established a proof-of-principle example that genetic modification with chimeric hPXR-p53-AD could improve CYP3A4 metabolism ability in hepatic cell line.

  8. 猪繁殖与呼吸综合征病毒强弱毒株嵌合感染性克隆的构建及鉴定%The Construction of Chimeric Clone of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus and Attenuated PRRSV Strain and Identification of Chimeric Viruses Rescued

    Institute of Scientific and Technical Information of China (English)

    吕健; 韦祖樟; 高飞; 郑海红; 童光志; 袁世山

    2012-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of the ongoing "porcine high fever syndrome" in China, is capable of genetic and antigenic mutations at high frequency. How to design vaccine rationally to keep up with the ever-changing prevalent PRRSV variant is of great interest. In this study, based on an infectious cDNA clone of an attenuated Typell PRRSV strain pAPRRS and the highly pathogenic PRRSV cDNA clone pJX143, we replaced the coding sequence of pAPRRS nsp2 with those of the HP PRRSV to develop a series of chimeric clones. Upon transfection of chimeric clones into MA104 cells, typical PRRSV cyto-pathic effects were observed. This study provided a valuable tool to develop the chimeric PRRSV as vaccine candidate offering cross-protection to HP PRRSV strains. Furthermore, the infectious chimeric cDNA clone provides a powerful tool to molecular dissection of the mechanism of patho-genesis of the increasing-virulence of the on-going prevalent PRRSV in China.%查明猪繁殖与呼吸综合征病毒(PRRSV)致病性大幅增高的机制,进而研制用于防治流行PRRSV变异株的高效疫苗无疑是兽医工作者的当务之急.在弱毒株APRRS的全长感染性克隆pAPRRS以及我室构建的高致病性HP PRRSV感染性克隆pJX143的基础上,构建了nsp2替换的强弱毒PRRSV嵌合感染性克隆.将构建的嵌合克隆转染MA104,4d后观察到典型的CPE.通过RT-PCR和免疫荧光证明获得了一系列强弱毒株之间的嵌合病毒.这些嵌合病毒的构建成功和相应反向遗传操作平台的建立及应用,为研发预防HP PRRSV的高效嵌合疫苗奠定了基础.该类嵌合感染性cDNA克隆也为解析目前流行的HP PRRSV毒力因子和高致病力机制奠定了基础.

  9. Young T Cells Age During a Redirected Anti-Tumor Attack: Chimeric Antigen Receptor-Provided Dual Costimulation is Half the Battle.

    Science.gov (United States)

    Hombach, Andreas A; Abken, Hinrich

    2013-01-01

    Adoptive therapy with chimeric antigen receptor (CAR)-redirected T cells showed spectacular efficacy in the treatment of leukemia in recent early phase trials. Patient's T cells were ex vivo genetically engineered with a CAR, amplified and re-administered to the patient. While T cells mediating the primary response were predominantly of young effector and central memory phenotype, repetitive antigen engagement irreversible triggers T cell maturation leaving late memory cells with the KLRG1(+) CD57(+) CD7(-) CCR7(-) phenotype in the long-term. These cells preferentially accumulate in the periphery, are hypo-responsive upon TCR engagement and prone to activation-induced cell death. A recent report indicates that those T cells can be rescued by CAR provided CD28 and OX40 (CD134) stimulation. We discuss the strategy with respect to prolong the anti-tumor response and to improve the over-all efficacy of adoptive cell therapy. PMID:23761793

  10. Review of Cancer Immunotherapy: Application of Chimeric Antigen Receptor T Cells and Programmed Death 1/Programmed Death-ligand 1 Antibodies

    Directory of Open Access Journals (Sweden)

    Tengfei Zhang

    2015-01-01

    Full Text Available Cancer immunotherapy strategies based on chimeric antigen receptor (CAR transduced T cells or antibodies against immune checkpoints, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4 and programmed death 1 (PD-1, achieved significant successes from bench to clinic in the past 2 years. CARs are artificial engineered receptors that can specifically target tumor cell surface antigen, activate T cell and further enhance T cell function, independent of major histocompatibility complex. CAR T cells have shown promising outcomes in cancers, especially in hematologic malignancies. CTLA-4 and PD-1 are two important immune checkpoints negatively regulating T cell activation. Clinical benefits of CTLA-4/PD-1 antibodies are significant in melanoma and other solid tumors. PD-1 is predicted to have fewer side effects and greater antitumor activity than CTLA-4. In this review, we will summarize current immunotherapies based on CAR T cells and PD-1.

  11. New advances in leukaemia immunotherapy by the use of Chimeric Artificial Antigen Receptors (CARs: state of the art and perspectives for the near future

    Directory of Open Access Journals (Sweden)

    Cribioli Elisabetta

    2011-09-01

    Full Text Available Abstract Leukaemia immunotherapy represents a fascinating and promising field of translational research, particularly as an integrative approach of bone marrow transplantation. Adoptive immunotherapy by the use of donor-derived expanded leukaemia-specific T cells has showed some kind of clinical response, but the major advance is nowadays represented by gene manipulation of donor immune cells, so that they acquire strict specificity towards the tumour target and potent lytic activity, followed by significant proliferation, increased survival and possibly anti-tumour memory state. This is achieved by gene insertion of Chimeric T-cell Antigen Receptors (CARs, which are artificial molecules containing antibody-derived fragments (to bind the specific target, joined with potent signalling T-Cell Receptor (TCR-derived domains that activate the manipulated cells. This review will discuss the main application of this approach particularly focusing on the paediatric setting, raising advantages and disadvantages and discussing relevant perspectives of use in the nearest future.

  12. Young T cells age during a redirected anti-tumour attack: chimeric antigen receptor (CAR-provided dual costimulation is half the battle.

    Directory of Open Access Journals (Sweden)

    Andreas A Hombach

    2013-06-01

    Full Text Available Adoptive therapy with chimeric antigen receptor (CAR-redirected T cells showed spectacular efficacy in the treatment of leukaemia in recent early phase trials. Patient's T cells were ex vivo genetically engineered with a CAR, amplified and re-administered to the patient. While T cells mediating the primary response were predominantly of young effector and central memory phenotype, repetitive antigen engagement irreversible triggers T cell maturation leaving late memory cells with the KLRG-1+ CD57+ CD7- CCR7- phenotype in the long-term. These cells preferentially accumulate in the periphery, are hypo-responsive upon TCR engagement and prone to activation-induced cell death. A recent report indicates that those T cells can be rescued by CAR provided CD28 and OX40 (CD134 stimulation. We discuss the strategy with respect to prolong the anti-tumour response and to improve the over-all efficacy of adoptive cell therapy.

  13. 放射诱导的肿瘤特异性嵌合启动子研究%Study of cancer-specific chimeric promoters induced by irradiation

    Institute of Scientific and Technical Information of China (English)

    熊杰; 周云峰; 孙文洁; 王伟峰; 廖正凯; 周福祥; 谢丛华

    2010-01-01

    Objective To combine the radio-inducible CArG element with cancer-specific human telomerase reverse transcriptase(hTERT) gene promoter,and to construct the novel chimeric promoters.Methods The synthetic hTERT promoters containing different number of radio-inducible CArG elements were constructed.and the activities of the promoters in the cancer cells (HeLa,A549,and MHCC97 cells) and nomal cells(hEL cells) were detected by using luciferase-reporter assays afer the treatment of irradiation(a single or fractionated irradiation dose).Results Synthetic promoter containing 6 repeated CArG units was better in radio-inducibility than any other promoters containing different number of CArG units.and nearly maximum levels obtained at 4-6 Gy.The very low activities of the chimeric promoters could be detected in normal hEL cells.A similar level of reporter gene expression was observed after 3 fractionated doses of 2 Gy compared with a single dose of 6 Gy in cancer cells.Conclusions The cancerspecific chimeric promoter containing 6 CArG elements showes the best radio-response,and the chimeric promoter system has the potential in cancer gene therapy.%目的 将放射敏感性的CArG元件与肿瘤特异性的人端粒酶反转录酶(hTERT)基因启动子相结合,构建新的嵌合启动子.方法 构建含不同CArG元件数量的嵌合hTERT启动子,在放射线诱导下(单剂量或分割照射),利用荧光素酶报告基因检测其在肿瘤细胞(HeLa、A549、MHCC97细胞)及正常细胞(hEL细胞)中的活性.结果 包含6个CArG元件的hTERT嵌合启动子较含其他数量CArG的嵌合启动子显示了更好的放射诱导性,并在4~6 Gy时达到顶峰,而构建的嵌合启动子在正常的hEL细胞中活性很低.在肿瘤细胞中,3次2 Gy与单次6 Gy剂量照射相比较,报告基因的表达相当.结论 包含6个CArG元件的肿瘤特异性嵌合启动子具备良好的放射诱导性,这种嵌合的启动子系统具有肿瘤基因治疗的潜力.

  14. Efficiently Production of Chimeric Mice Competent for Germline Transmitted%如何高效制备生殖系传递的嵌合体小鼠

    Institute of Scientific and Technical Information of China (English)

    杨冠恒; 施皖生; 曾凡一

    2012-01-01

    Chimeric mice capable of germline transmission are important tools for studying gene function, mechanisms of development, and disease pathogenesis. Chimeras are indispensable for research on embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). When planning an experiment that includes production of chimeric mice, several factors should be considered that may have an impact on the molecular, cellular, or tissue/ organ levels. Recipient embryos at different stages of development have different efficiencies for donor cell engraft-ment and survival, leading to a wide range of levels of chimerism. Capability for tetraploid complementation is key, but so are other recipient embryo factors such as mitochondrial quantity and quality. Whether the recipient strain is inbred or out-breeding needs to also be considered. In the donor stem cells, genetic background is critically important, as are the cell culture conditions such as the KSR system used to engineer and expand donor cells before transplantation. The injection and aggregation methods are the two primary methods for producing chimeras, and both have advantages and disadvantages. This review is intended to assist investigators with a comprehensive assessment of what factors will be most important for meeting their specific goals in a chimeric mouse project.%目前,嵌合体技术已成为胚胎干细胞(ES细胞)和诱导多能性干细胞(iPS细胞)研究不可或缺的一部分,嵌合体小鼠模型主要用于基因功能、调控机制、致病机理等各方面的研究.要安全可靠地进行ES细胞和iPS细胞的各种应用性研究,首先需要验证细胞在体内向各种组织定向分化的能力,特别是生殖系传递的能力.而高效制备多能性干细胞生殖系传递的嵌合体小鼠,需要在分子水平、细胞水平、动物个体水平以及制备方法等各方面全面综合地分析考虑,才能达到实验设计的最优化.制备嵌合体小鼠

  15. Therapeutic Potential of T Cell Chimeric Antigen Receptors (CARs) in Cancer Treatment: Counteracting Off-Tumor Toxicities for Safe CAR T Cell Therapy.

    Science.gov (United States)

    Gross, Gideon; Eshhar, Zelig

    2016-01-01

    A chimeric antigen receptor (CAR) is a recombinant fusion protein combining an antibody-derived targeting fragment with signaling domains capable of activating T cells. Recent early-phase clinical trials have demonstrated the remarkable ability of CAR-modified T cells to eliminate B cell malignancies. This review describes the choice of target antigens and CAR manipulations to maximize antitumor specificity. Benefits and current limitations of CAR-modified T cells are discussed, with a special focus on the distribution of tumor antigens on normal tissues and the risk of on-target, off-tumor toxicities in the clinical setting. We present current methodologies for pre-evaluating these risks and review the strategies for counteracting potential off-tumor effects. Successful implementation of these approaches will improve the safety and efficacy of CAR T cell therapy and extend the range of cancer patients who may be treated. PMID:26738472

  16. A convenient method for europium-labeling of a recombinant chimeric relaxin family peptide R3/I5 for receptor-binding assays.

    Science.gov (United States)

    Zhang, Wei-Jie; Jiang, Qian; Wang, Xin-Yi; Song, Ge; Shao, Xiao-Xia; Guo, Zhan-Yun

    2013-06-01

    Relaxin family peptides have important biological functions, and so far, four G-protein-coupled receptors have been identified as their receptors (RXFP1-4). A chimeric relaxin family peptide R3/I5, containing the B-chain of relaxin-3 and the A-chain of INSL5, is a selective agonist for both RXFP3 and RXFP4. In a previous study, europium-labeled R3/I5, as a nonradioactive and low-background receptor-binding tracer, was prepared through a chemical synthesis approach. In the present study, we established a convenient alternative approach for preparing the europium-labeled R3/I5 tracer based on a recombinant R3/I5 designed to carry a solubilizing tag at the A-chain N-terminus and a pyroglutamate residue at the B-chain N-terminus. Because of the presence of a single primary amine moiety, the recombinant R3/I5 peptide was site-specifically mono-labeled at the A-chain N-terminus by a diethylenetriaminepentaacetic acid/europium moiety through a convenient one-step procedure. The diethylenetriaminepentaacetic acid/Eu3+-labeled R3/I5 bound both receptors RXFP3 and RXFP4 with high binding affinities and low nonspecific binding. Thus, we have presented a valuable nonradioactive tracer for future interaction studies on RXFP3 and RXFP4 with various natural or designed ligands. The present approach could also be adapted for preparing and labeling of other chimeric relaxin family peptides. PMID:23526726

  17. Target antigen expression on a professional antigen-presenting cell induces superior proliferative antitumor T-cell responses via chimeric T-cell receptors.

    Science.gov (United States)

    Rossig, Claudia; Bär, Annette; Pscherer, Sibylle; Altvater, Bianca; Pule, Martin; Rooney, Cliona M; Brenner, Malcolm K; Jürgens, Heribert; Vormoor, Josef

    2006-01-01

    Human T cells expressing tumor antigen-specific chimeric receptors fail to sustain their growth and activation in vivo, which greatly reduces their therapeutic value. The defective proliferative response to tumor cells in vitro can partly be overcome by concomitant CD28 costimulatory signaling. We investigated whether T-cell activation via chimeric receptors (chRec) can be further improved by ligand expression on antigen-presenting cells of B-cell origin. We generated Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) expressing a CD19-specific chRec. These CTLs are provided with native receptor stimulation by autologous EBV-transformed B-lymphoblastoid cell lines (LCLs) but exclusively with chRec (CD19-specific) stimulation by allogeneic, human leukocyte antigen (HLA)-mismatched CD19+ LCLs. CD19zeta-transduced EBV-specific CTLs specifically lysed both allogeneic EBV targets and CD19+ tumor cells through the chRec in a major histocompatibility complex-independent manner, while maintaining their ability to recognize autologous EBV targets through the native T-cell receptor. The transduced CTLs failed to proliferate in response to CD19+ tumor targets even in the presence of CD28 costimulatory signaling. By contrast, CD19 expressed on HLA-mismatched LCL-induced T-cell activation and long-term proliferation that essentially duplicated the result from native receptor stimulation with autologous LCLs, suggesting that a deficit of costimulatory molecules on target cells in addition to CD28 is indeed responsible for inadequate chRec-mediated T-cell function. Hence, effective tumor immunotherapy may be favored if engagement of the chRec on modified T cells is complemented by interaction with multiple costimulator molecules. The use of T cells with native specificity for EBV may be one means of attaining this objective. PMID:16365597

  18. Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells.

    Science.gov (United States)

    Morton, H C; Atkin, J D; Owens, R J; Woof, J M

    1993-11-01

    Ag-specific chimeric human IgA molecules, of the two human subclasses, IgA1 and IgA2, have been expressed in two mammalian cell systems. Analysis of the secreted IgA molecules, purified in milligram quantities from stable Chinese hamster ovary transfectants by Ag affinity chromatography, has allowed a direct comparison of the biologic properties of the two subclasses. HPLC gel filtration analysis revealed that in both subclasses, the IgA molecules associate predominantly into dimers. The monomer units are presumed to interact noncovalently, inasmuch as no dimers are evident when the antibodies are subjected to SDS-PAGE. The recombinant antibodies are glycosylated, inasmuch as a lectin blotting procedure revealed that the H chains of both subclasses are recognized by Con A. When subjected to digestion by preparations of IgA1-specific proteases secreted by two pathogenic streptococcal strains, Streptococcus sanguis and Streptococcus oralis, the recombinant IgA molecules behave just as their natural equivalents. Thus, only the chimeric IgA1 molecule is cleaved, with the IgA2 remaining intact. In terms of interaction with natural effector molecules, both recombinant IgA isotypes were shown to interact with Fc alpha receptors on calcitriol-stimulated HL-60 cells with similar affinity, but neither antibody was found to interact with human C1q. The expression system described readily permits manipulation of the human IgA genes, which should lead to a fuller molecular understanding of how this important antibody mediates its function. PMID:8409433

  19. Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2

    DEFF Research Database (Denmark)

    Elster, L; Kristiansen, U; Pickering, D S;

    2001-01-01

    Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma...... opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important...... for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch-clamp recordings, which was independent of whether the chimera was expressed...

  20. New concepts for the comparison of tropospheric NO2 column densities derived from car-MAX-DOAS observations, OMI satellite observations and the regional model CHIMERE during two MEGAPOLI campaigns in Paris 2009/10

    OpenAIRE

    Shaiganfar, R.; S. Beirle; H. Petetin; Q. Zhang; M. Beekmann; Wagner, T.

    2015-01-01

    We compare tropospheric column densities (vertically integrated concentrations) of NO2 from three data sets for the metropolitan area of Paris during two extensive measurement campaigns (25 days in summer 2009 and 29 days in winter 2010) within the European research project MEGAPOLI. The selected data sets comprise a regional chemical transport model (CHIMERE) as well as two observational data sets: ground-based mobile Multi-AXis-Differential Optical Absorption Spectroscopy ...

  1. New concepts for the comparison of tropospheric NO2 column densities derived from car-MAX-DOAS observations, OMI satellite observations and the regional model CHIMERE during two MEGAPOLI campaigns in Paris 2009/10

    OpenAIRE

    R. Shaiganfar; Beirle, S.; H. Petetin; Zhang, Q.; M. Beekmann; Wagner, T

    2015-01-01

    We compare tropospheric column densities (vertically integrated concentrations) of NO2 from three data sets for the metropolitan area of Paris during two extensive measurement campaigns (25 days in summer 2009 and 29 days in winter 2010) within the European research project MEGAPOLI. The selected data sets comprise a regional chemical transport model (CHIMERE) as well as two observational data sets: ground based mobile Multi-AXis-Differential Optical Absorpt...

  2. Formation of organic aerosol in the Paris region during the MEGAPOLI summer campaign: evaluation of the Volatility-Basis-Set approach within the CHIMERE model

    Directory of Open Access Journals (Sweden)

    Q. J. Zhang

    2012-11-01

    Full Text Available Results of the chemistry transport model CHIMERE are compared with the measurements performed during the MEGAPOLI summer campaign in the Greater Paris Region in July, 2009. The Volatility-Basis-Set approach (VBS is implemented into this model, taking into account the volatility of primary organic aerosol (POA and the chemical aging of semi-volatile organic species. Organic aerosol is the main focus and is simulated with three different configurations related to the volatility of POA and the scheme of secondary organic aerosol (SOA formation. In addition, two types of emission inventories are used as model input in order to test the uncertainty related to the emissions. Predictions of basic meteorological parameters and primary and secondary pollutant concentrations are evaluated and four pollution regimes according to the air mass origin are defined. Primary pollutants are generally overestimated, while ozone is consistent with observations. Sulfate is generally overestimated, while ammonium and nitrate levels are well simulated with the refined emission data set. As expected, the simulation with non-volatile POA and a single-step SOA formation mechanism largely overestimates POA and underestimates SOA. Simulation of organic aerosol with the VBS approach taking into account the aging of semi-volatile organic compounds (SVOC shows the best correlation with measurements. All observed high concentration events are reproduced by the model mostly after long range transport, indicating that long range transport of SOA to Paris is well reproduced. Depending on the emission inventory used, simulated POA levels are either reasonable or underestimated, while SOA levels tend to be overestimated. Several uncertainties related to the VBS scheme (POA volatility, SOA yields, the aging parameterization, to emission input data, and to simulated OH levels can be responsible for this behavior. Despite these uncertainties, the implementation of the VBS scheme into

  3. Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli.

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    Xolani Henry Makhoba

    Full Text Available S-adenosylmethionine decarboxylase (PfAdoMetDC from Plasmodium falciparum is a prospective antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical validation as a drug target is important. The production of PfAdoMetDC in Escherichia coli has been reported to result in unsatisfactory yields and poor quality product. The co-expression of recombinant proteins with molecular chaperones has been proposed as one way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK, GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf, which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain of P. falciparum Hsp70 (PfHsp70 has been previously shown to exhibit chaperone function when it was expressed in E. coli cells whose resident Hsp70 (DnaK function was impaired. We proposed that because of its domain constitution, KPf would most likely be recognised by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC expressed in E. coli. First, using site-directed mutagenesis, followed by complementation assays, we established that KPf with a mutation in the hydrophobic residue located in its substrate binding cavity was functionally compromised. We further co-expressed PfAdoMetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdoMetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant

  4. Genetic Construction of Truncated and Chimeric Metalloproteins Derived from the Alpha Subunit of Acetyl-CoA Synthase from Clostridium thermoaceticum

    Energy Technology Data Exchange (ETDEWEB)

    Huay-Keng Loke; Xiangshi Tan; Paul A. Lindahl

    2002-06-28

    In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an alpha2beta2 tetrameric 310 kda bifunctional enzyme. ACS catalyzes the reversible reduction of CO2 to CO and the synthesis of acetyl-CoA from CO (or CO2 in the presence of low-potential reductants), CoA, and a methyl group bound to a corrinoid-iron sulfur protein (CoFeSP). ACS contains 7 metal-sulfur clusters of 4 different types called A, B, C, and D. The B, C, and D clusters are located in the 72 kda beta subunit while the A-cluster, a Ni-X-Fe4S4 cluster that serves as the active site for acetyl-CoA synthase activity, is located in the 82 kda alpha subunit. The extent to which the essential properties of the cluster, including catalytic, redox, spectroscopic, and substrate-binding properties, were retained as ACS was progressively truncated was determined. Acetyl-CoA synthase catalytic activity remained when the entire alpha subunit was removed, as long as CO, rather than CO2 and a low-potential reductant, was used as a substrate. Truncating an {approx} 30 kda region from the N-terminus of the alpha subunit yielded a 49 kda protein that lacked catalytic activity but exhibited A-cluster-like spectroscopic, redox, and CO binding properties. Further truncation afforded a 23 kda protein that lacked recognizable A-cluster properties except for UV-vis spectra typical of [Fe4S4]2+ clusters. Two chimeric proteins were constructed by fusing the gene encoding a ferredoxin from Chromatium vinosum to genes encoding the 49 kda and 82 kda fragments of the alpha subunit. The chimeric proteins exhibited EPR signals that were not the simple sum of the signals from the separate proteins, suggesting magnetic interactions between clusters. This study highlights the potential for using genetics to simplify the study of complex multi-centered metalloenzymes and to generate new complex metalloenzymes with interesting properties.

  5. Overexpression of the Synthetic Chimeric Native-T-phylloplanin-GFP Genes Optimized for Monocot and Dicot Plants Renders Enhanced Resistance to Blue Mold Disease in Tobacco (N. tabacum L.

    Directory of Open Access Journals (Sweden)

    Dipak K. Sahoo

    2014-01-01

    Full Text Available To enhance the natural plant resistance and to evaluate the antimicrobial properties of phylloplanin against blue mold, we have expressed a synthetic chimeric native-phylloplanin-GFP protein fusion in transgenic Nicotiana tabacum cv. KY14, a cultivar that is highly susceptible to infection by Peronospora tabacina. The coding sequence of the tobacco phylloplanin gene along with its native signal peptide was fused with GFP at the carboxy terminus. The synthetic chimeric gene (native-phylloplanin-GFP was placed between the modified Mirabilis mosaic virus full-length transcript promoter with duplicated enhancer domains and the terminator sequence from the rbcSE9 gene. The chimeric gene, expressed in transgenic tobacco, was stably inherited in successive plant generations as shown by molecular characterization, GFP quantification, and confocal fluorescent microscopy. Transgenic plants were morphologically similar to wild-type plants and showed no deleterious effects due to transgene expression. Blue mold-sensitivity assays of tobacco lines were performed by applying P. tabacina sporangia to the upper leaf surface. Transgenic lines expressing the fused synthetic native-phyllopanin-GFP gene in the leaf apoplast showed resistance to infection. Our results demonstrate that in vivo expression of a synthetic fused native-phylloplanin-GFP gene in plants can potentially achieve natural protection against microbial plant pathogens, including P. tabacina in tobacco.

  6. Persistent replication of a hepatitis C virus genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B in a New World monkey.

    Science.gov (United States)

    Suzuki, Saori; Mori, Ken-Ichi; Higashino, Atsunori; Iwasaki, Yuki; Yasutomi, Yasuhiro; Maki, Noboru; Akari, Hirofumi

    2016-01-01

    The development of effective hepatitis C virus (HCV) vaccines is essential for the prevention of further HCV dissemination, especially in developing countries. Therefore the aim of this study is to establish a feasible and immunocompetent surrogate animal model of HCV infection that will help in evaluation of the protective efficacy of newly developing HCV vaccine candidates. To circumvent the narrow host range of HCV, an HCV genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B (GBV-B), which is closely related to HCV, was generated. The chimera between HCV and GBV-B, named HCV/G, replicated more efficiently as compared with the HCV clone in primary marmoset hepatocytes. Furthermore, it was found that the chimera persistently replicated in a tamarin for more than 2 years after intrahepatic inoculation of the chimeric RNA. Although relatively low (production of the chimeric virus. Our results will help establish a novel non-human primate model for HCV infection on the basis of the HCV/G chimera in the major framework of the HCV genome. PMID:26634303

  7. Overexpression of the synthetic chimeric native-T-phylloplanin-GFP genes optimized for monocot and dicot plants renders enhanced resistance to blue mold disease in tobacco (N. tabacum L.).

    Science.gov (United States)

    Sahoo, Dipak K; Raha, Sumita; Hall, James T; Maiti, Indu B

    2014-01-01

    To enhance the natural plant resistance and to evaluate the antimicrobial properties of phylloplanin against blue mold, we have expressed a synthetic chimeric native-phylloplanin-GFP protein fusion in transgenic Nicotiana tabacum cv. KY14, a cultivar that is highly susceptible to infection by Peronospora tabacina. The coding sequence of the tobacco phylloplanin gene along with its native signal peptide was fused with GFP at the carboxy terminus. The synthetic chimeric gene (native-phylloplanin-GFP) was placed between the modified Mirabilis mosaic virus full-length transcript promoter with duplicated enhancer domains and the terminator sequence from the rbcSE9 gene. The chimeric gene, expressed in transgenic tobacco, was stably inherited in successive plant generations as shown by molecular characterization, GFP quantification, and confocal fluorescent microscopy. Transgenic plants were morphologically similar to wild-type plants and showed no deleterious effects due to transgene expression. Blue mold-sensitivity assays of tobacco lines were performed by applying P. tabacina sporangia to the upper leaf surface. Transgenic lines expressing the fused synthetic native-phyllopanin-GFP gene in the leaf apoplast showed resistance to infection. Our results demonstrate that in vivo expression of a synthetic fused native-phylloplanin-GFP gene in plants can potentially achieve natural protection against microbial plant pathogens, including P. tabacina in tobacco. PMID:24778589

  8. L1 Cell Adhesion Molecule-Specific Chimeric Antigen Receptor-Redirected Human T Cells Exhibit Specific and Efficient Antitumor Activity against Human Ovarian Cancer in Mice.

    Directory of Open Access Journals (Sweden)

    Hao Hong

    Full Text Available New therapeutic modalities are needed for ovarian cancer, the most lethal gynecologic malignancy. Recent clinical trials have demonstrated the impressive therapeutic potential of adoptive therapy using chimeric antigen receptor (CAR-redirected T cells to target hematological cancers, and emerging studies suggest a similar impact may be achieved for solid cancers. We sought determine whether genetically-modified T cells targeting the CE7-epitope of L1-CAM, a cell adhesion molecule aberrantly expressed in several cancers, have promise as an immunotherapy for ovarian cancer, first demonstrating that L1-CAM was highly over-expressed on a panel of ovarian cancer cell lines, primary ovarian tumor tissue specimens, and ascites-derived primary cancer cells. Human central memory derived T cells (TCM were then genetically modified to express an anti-L1-CAM CAR (CE7R, which directed effector function upon tumor antigen stimulation as assessed by in vitro cytokine secretion and cytotoxicity assays. We also found that CE7R+ T cells were able to target primary ovarian cancer cells. Intraperitoneal (i.p. administration of CE7R+ TCM induced a significant regression of i.p. established SK-OV-3 xenograft tumors in mice, inhibited ascites formation, and conferred a significant survival advantage compared with control-treated animals. Taken together, these studies indicate that adoptive transfer of L1-CAM-specific CE7R+ T cells may offer a novel and effective immunotherapy strategy for advanced ovarian cancer.

  9. In Vitro and In Vivo Antitumor Effect of Anti-CD33 Chimeric Receptor-Expressing EBV-CTL against CD33+ Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    A. Dutour

    2012-01-01

    Full Text Available Genetic engineering of T cells with chimeric T-cell receptors (CARs is an attractive strategy to treat malignancies. It extends the range of antigens for adoptive T-cell immunotherapy, and major mechanisms of tumor escape are bypassed. With this strategy we redirected immune responses towards the CD33 antigen to target acute myeloid leukemia. To improve in vivo T-cell persistence, we modified human Epstein Barr Virus-(EBV- specific cytotoxic T cells with an anti-CD33.CAR. Genetically modified T cells displayed EBV and HLA-unrestricted CD33 bispecificity in vitro. In addition, though showing a myeloablative activity, they did not irreversibly impair the clonogenic potential of normal CD34+ hematopoietic progenitors. Moreover, after intravenous administration into CD33+ human acute myeloid leukemia-bearing NOD-SCID mice, anti-CD33-EBV-specific T cells reached the tumor sites exerting antitumor activity in vivo. In conclusion, targeting CD33 by CAR-modified EBV-specific T cells may provide additional therapeutic benefit to AML patients as compared to conventional chemotherapy or transplantation regimens alone.

  10. Combining a CD20 chimeric antigen receptor and an inducible caspase 9 suicide switch to improve the efficacy and safety of T cell adoptive immunotherapy for lymphoma.

    Directory of Open Access Journals (Sweden)

    Lihua E Budde

    Full Text Available Modification of T cells with chimeric antigen receptors (CAR has emerged as a promising treatment modality for human malignancies. Integration of co-stimulatory domains into CARs can augment the activation and function of genetically targeted T cells against tumors. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe methods for removing transferred cells an important consideration. We have genetically modified human T cells with a lentiviral vector to express a CD20-CAR containing both CD28 and CD137 co-stimulatory domains, a "suicide gene" relying on inducible activation of caspase 9 (iC9, and a truncated CD19 selectable marker. Rapid expansion (2000 fold of the transduced T cells was achieved in 28 days after stimulation with artificial antigen presenting cells. Transduced T cells exhibited effective CD20-specific cytotoxic activity in vitro and in a mouse xenograft tumor model. Activation of the iC9 suicide switch resulted in efficient removal of transduced T cells both in vitro and in vivo. Our work demonstrates the feasibility and promise of this approach for treating CD20(+ malignancies in a safe and more efficient manner. A phase I clinical trial using this approach in patients with relapsed indolent B-NHL is planned.

  11. Tumor Antigen Specific Activation of Primary Human T-Cells Expressing a Virally Encoded Chimeric T-Cell Receptor Specific for p185HER2

    Institute of Scientific and Technical Information of China (English)

    杨建民; MichaelSFRIEDMAN; ChristopherMREYNOLDS; MarianneTHUBEN; LeeWILKE; JenniferFULLER; 李桥; ZeligESHHAR; JamesJMULE; KevimTMCDONAGH

    2004-01-01

    We have developed and tested chimeric T-cell receptors (TCR) specific for p185HER2. In these experiments,retroviral vectors expressing the N297 or N29ξ receptors were constructed in pRET6. Amphotropic viral producer cells were established in the GALV-based PG13 packaging cell line. Ficoll purified human peripheral blood lymphocytes (PBL) were vitally transduced using an optimized protocol incorporating activation with immobilized anti-CD3/anti-CD28 monoclonal antibodies, followed by viral infection in the presence of fibronectin fragment CH296. Transduced cells were co-cultured with human tumor cell lines that overexpress (SK-OV-3) or underexpress (MCF7) p185HER2 to assay for antigen specific immune responses. Both CD4+ and CD8+ T-cells transduced with the N297 or N29ξ chTCR demonstrated HER2-specific antigen responses, as determined by release of Th1 like cytokines, and cellular cytotoxicity assays. Our results support the feasibility of adoptive immunothempy with genetically modified T-cells expressing a chTCR specific for p185HER2.

  12. Virus-Specific Read-Through Codon Preference Affects Infectivity of Chimeric Cucumber Green Mottle Mosaic Viruses Displaying a Dengue Virus Epitope

    Directory of Open Access Journals (Sweden)

    Pak-Guan Teoh

    2009-01-01

    Full Text Available A Cucumber green mottle mosaic virus (CGMMV was used to present a truncated dengue virus type 2 envelope (E protein binding region from amino acids 379 to 423 (EB4. The EB4 gene was inserted at the terminal end of the CGMMV coat protein (CP open reading frame (ORF. Read-through sequences of TMV or CGMMV, CAA-UAG-CAA-UUA, or AAA-UAG-CAA-UUA were, respectively, inserted in between the CP and the EB4 genes. The chimeric clones, pRT, pRG, and pCG+FSRTRE, were transcribed into full-length capped recombinant CGMMV transcripts. Only constructs with the wild-type CGMMV read-through sequence yielded infectious viruses following infection of host plant, muskmelon (Cucumis melo leaves. The ratio of modified to unmodified CP for the read-through expression clone developed was also found to be approximately 1:1, higher than what has been previously reported. It was also observed that infectivity was not affected by differences in pI between the chimera and its wild counterpart. Analysis of recombinant viruses after 21-days-postinculation (dpi revealed that deletions occurred resulting in partial reversions of the viral population to near wild type and suggesting that this would be the limiting harvest period for obtaining true to type recombinants with this construct.

  13. Efficient amplification of chimeric adenovirus 5/40S vectors carrying the short fiber protein of Ad40 in suspension cell cultures.

    Directory of Open Access Journals (Sweden)

    Marta Miralles

    Full Text Available The human adenovirus 40 (Ad40 is a promising tool for gene therapy of intestinal diseases. Since the production of Ad40 in vitro is extremely inefficient, chimeric Adenovirus 5/40S vectors carrying the Ad40 short fiber on the Ad5 capsid have been developed. However, Ad5/40S productivity is low. We hypothesized that low productivity was a result of inefficient viral entry into producer cells during amplification. To this end, we have developed a production strategy based on using 211B cells (expressing Ad5 fiber during amplification steps, while Ad5/40S infectivity is further improved by adding polybrene during infections. In addition, the optimal harvesting time was determined by evaluating the Ad5/40S viral cycle. The developed production strategy significantly reduces the number of amplification cycles and duration of the process. Finally, to further facilitate Ad5/40S production, 211B cells were adapted to suspension thus allowing to easily upscale the production process in bioreactors.

  14. Pathogenesis of Alfalfa mosaic virus in Soybean (Glycine max) and Expression of Chimeric Rabies Peptide in Virus-Infected Soybean Plants.

    Science.gov (United States)

    Fleysh, N; Deka, D; Drath, M; Koprowski, H; Yusibov, V

    2001-10-01

    ABSTRACT Infection of soybean (Glycine max) plants inoculated with particles of Alfalfa mosaic virus (AlMV) isolate 425 at 12 days after germination was monitored throughout the life cycle of the plant (vegetative growth, flowering, seed formation, and seed maturation) by western blot analysis of tissue samples. At 8 to 10 days after inoculation, the upper uninoculated leaves showed symptoms of virus infection and accumulation of viral coat protein (CP). Virus CP was detectable in leaves, stem, roots, seedpods, and seed coat up to 45 days postinoculation (dpi), but only in the seedpod and seed coat at 65 dpi. No virus accumulation was detected in embryos and cotyledons at any time during infection, and no seed transmission of virus was observed. Soybean plants inoculated with recombinant AlMV passaged from upper uninoculated leaves of infected plants showed accumulation of full-length chimeric AlMV CP containing rabies antigen in systemically infected leaves and seed coat. These results suggest the potential usefulness of plants and plant viruses as vehicles for producing proteins of biomedical importance in a safe and inexpensive manner. Moreover, even the soybean seed coat, treated as waste tissue during conventional processing for oil and other products, may be utilized for the expression of value-added proteins. PMID:18944120

  15. Prospects for chimeric antigen receptor (CAR) γδ T cells: A potential game changer for adoptive T cell cancer immunotherapy.

    Science.gov (United States)

    Mirzaei, Hamid Reza; Mirzaei, Hamed; Lee, Sang Yun; Hadjati, Jamshid; Till, Brian G

    2016-10-01

    Excitement is growing for therapies that harness the power of patients' immune systems to combat their diseases. One approach to immunotherapy involves engineering patients' own T cells to express a chimeric antigen receptor (CAR) to treat advanced cancers, particularly those refractory to conventional therapeutic agents. Although these engineered immune cells have made remarkable strides in the treatment of patients with certain hematologic malignancies, success with solid tumors has been limited, probably due to immunosuppressive mechanisms in the tumor niche. In nearly all studies to date, T cells bearing αβ receptors have been used to generate CAR T cells. In this review, we highlight biological characteristics of γδ T cells that are distinct from those of αβ T cells, including homing to epithelial and mucosal tissues and unique functions such as direct antigen recognition, lack of alloreactivity, and ability to present antigens. We offer our perspective that these features make γδ T cells promising for use in cellular therapy against several types of solid tumors, including melanoma and gastrointestinal cancers. Engineered γδ T cells should be considered as a new platform for adoptive T cell cancer therapy for mucosal tumors. PMID:27392648

  16. Vaccination of dogs with six different candidate leishmaniasis vaccines composed of a chimerical recombinant protein containing ribosomal and histone protein epitopes in combination with different adjuvants.

    Science.gov (United States)

    Poot, J; Janssen, L H M; van Kasteren-Westerneng, T J; van der Heijden-Liefkens, K H A; Schijns, V E J C; Heckeroth, A

    2009-07-16

    Chimerical protein "Q", composed of antigenic ribosomal and histone sequences, in combination with live BCG is a promising canine leishmaniasis vaccine candidate; one of the few vaccine candidates that have been tested successfully in dogs. Unfortunately, live BCG is not an appropriate adjuvant for commercial application due to safety problems in dogs. In order to find a safe adjuvant with similar efficacy to live BCG, muramyl dipeptide, aluminium hydroxide, Matrix C and killed Propionibacterium acnes in combination with either E. coli- or baculovirus-produced recombinant JPCM5_Q protein were tested. Groups of five or seven dogs were vaccinated with six different adjuvant-antigen combinations and challenged with a high dose intravenous injection of Leishmania infantum JPC strain promastigotes. All candidate vaccines proved to be safe, and both humoral and cellular responses to the recombinant proteins were detected at the end of the prime-boost vaccination scheme. However, clinical and parasitological data obtained during the 10 month follow-up period indicated that protection was not induced by either of the six candidate vaccines. Although no direct evidence was obtained, our data suggest that live BCG may have a significant protective effect against challenge with L. infantum in dogs. PMID:19500553

  17. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy.

    Science.gov (United States)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku; Okada, Naoki

    2016-04-22

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. PMID:26993168

  18. Benefit of STR-based chimerism analysis to identify TA-GVHD as a cause of death: Utility of various biological specimens.

    Science.gov (United States)

    Raina, Anupuma; Chaudhary, Garima; Dogra, Tirath Das; Khandelwal, Deepchand; Balayan, Ajay; Jain, Vandana; Kanga, Uma; Seth, Tulika

    2016-04-01

    Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare condition. It can occur after blood transfusion in immune-compromised and occasionally even in immune-competent patients, and is associated with a mortality rate of >90%. The diagnosis of TA-GVHD is often delayed because of its non-specific clinical features. A case of an immune-competent child who developed TA-GVHD is reported here. DNA profiling (short tandem repeat analysis), a technique that has a wide application in forensic medicine, was performed to detect the presence of donor cells in this patient. The findings suggest that more studies are needed with this tool, and the diagnostic potential of using other multiple biological specimens for DNA profiling such as the hair follicle and buccal swab should be evaluated. This is the first case report where the donor's DNA fingerprinting pattern was substantiated from a patient's hair follicle sample. Chimerism was also present in the blood and buccal swab specimens. PMID:25852093

  19. The novel anti-CD19 chimeric antigen receptors with humanized scFv (single-chain variable fragment) trigger leukemia cell killing.

    Science.gov (United States)

    Qian, Liren; Li, Dan; Ma, Lie; He, Ting; Qi, Feifei; Shen, Jianliang; Lu, Xin-An

    2016-01-01

    The molecular design of CARs (Chimeric Antigen Receptors), especially the scFv, has been a major part to use of CAR-T cells for targeted adoptive immunotherapy. To address this issue, we chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR. Next, we generated a panel of humanized scFvs and tested in vitro for their ability to direct CAR-T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. Furthermore, in a xenograft model of lymphoma, human T cells expressing humanized scFvs exhibited the same anti-tumor efficacy as those expressing murine scFv and prolonged survival compared with cells expressing control CAR. Therefore, we uncovered CARs expressing humanized scFv domain that contribute the similar enhanced antileukemic efficacy and survival in tumor bearing mice. These results provide the basis for the future clinical studies of CAR-T cells transduced with humanized scFv directed to CD19. PMID:26996927

  20. STAT1, STAT3 and p38MAPK are involved in the apoptotic effect induced by a chimeric cyclic interferon-{alpha}2b peptide

    Energy Technology Data Exchange (ETDEWEB)

    Blank, Viviana C.; Pena, Clara [Institute of Biochemistry and Biophysics (UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, Junin 956-C1113AAD Buenos Aires (Argentina); Roguin, Leonor P., E-mail: rvroguin@qb.ffyb.uba.ar [Institute of Biochemistry and Biophysics (UBA-CONICET), School of Pharmacy and Biochemistry, University of Buenos Aires, Junin 956-C1113AAD Buenos Aires (Argentina)

    2010-02-15

    In the search of mimetic peptides of the interferon-{alpha}2b molecule (IFN-{alpha}2b), we have previously designed and synthesized a chimeric cyclic peptide of the IFN-{alpha}2b that inhibits WISH cell proliferation by inducing an apoptotic response. Here, we first studied the ability of this peptide to activate intracellular signaling pathways and then evaluated the participation of some signals in the induction of apoptosis. Stimulation of WISH cells with the cyclic peptide showed tyrosine phosphorylation of Jak1 and Tyk2 kinases, tyrosine and serine phosphorylation of STAT1 and STAT3 transcription factors and activation of p38 MAPK pathway, although phosphorylation levels or kinetics were in some conditions different to those obtained under IFN-{alpha}2b stimulus. JNK and p44/42 pathways were not activated by the peptide in WISH cells. We also showed that STAT1 and STAT3 downregulation by RNA interference decreased the antiproliferative activity and the amount of apoptotic cells induced by the peptide. Pharmacological inhibition of p38 MAPK also reduced the peptide growth inhibitory activity and the apoptotic effect. Thus, we demonstrated that the cyclic peptide regulates WISH cell proliferation through the activation of Jak/STAT signaling pathway. In addition, our results indicate that p38 MAPK may also be involved in cell growth regulation. This study suggests that STAT1, STAT3 and p38 MAPK would be mediating the antitumor and apoptotic response triggered by the cyclic peptide in WISH cells.

  1. High-yield nontoxic gene transfer through conjugation of the CM₁₈-Tat₁₁ chimeric peptide with nanosecond electric pulses.

    Science.gov (United States)

    Salomone, Fabrizio; Breton, Marie; Leray, Isabelle; Cardarelli, Francesco; Boccardi, Claudia; Bonhenry, Daniel; Tarek, Mounir; Mir, Lluis M; Beltram, Fabio

    2014-07-01

    We report a novel nontoxic, high-yield, gene delivery system based on the synergistic use of nanosecond electric pulses (NPs) and nanomolar doses of the recently introduced CM18-Tat11 chimeric peptide (sequence of KWKLFKKIGAVLKVLTTGYGRKKRRQRRR, residues 1-7 of cecropin-A, 2-12 of melittin, and 47-57 of HIV-1 Tat protein). This combined use makes it possible to drastically reduce the required CM18-Tat11 concentration and confines stable nanopore formation to vesicle membranes followed by DNA release, while no detectable perturbation of the plasma membrane is observed. Two different experimental assays are exploited to quantitatively evaluate the details of NPs and CM18-Tat11 cooperation: (i) cytofluorimetric analysis of the integrity of synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles exposed to CM18-Tat11 and NPs and (ii) the in vitro transfection efficiency of a green fluorescent protein-encoding plasmid conjugated to CM18-Tat11 in the presence of NPs. Data support a model in which NPs induce membrane perturbation in the form of transient pores on all cellular membranes, while the peptide stabilizes membrane defects selectively within endosomes. Interestingly, atomistic molecular dynamics simulations show that the latter activity can be specifically attributed to the CM18 module, while Tat11 remains essential for cargo binding and vector subcellular localization. We argue that this result represents a paradigmatic example that can open the way to other targeted delivery protocols. PMID:24865174

  2. Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge

    International Nuclear Information System (INIS)

    We generated a new live-attenuated vaccine against Ebola virus (EBOV) based on a chimeric virus HPIV3/ΔF-HN/EboGP that contains the EBOV glycoprotein (GP) as the sole transmembrane envelope protein combined with the internal proteins of human parainfluenza virus type 3 (HPIV3). Electron microscopy analysis of the virus particles showed that they have an envelope and surface spikes resembling those of EBOV and a particle size and shape resembling those of HPIV3. When HPIV3/ΔF-HN/EboGP was inoculated via apical surface of an in vitro model of human ciliated airway epithelium, the virus was released from the apical surface; when applied to basolateral surface, the virus infected basolateral cells but did not spread through the tissue. Following intranasal (IN) inoculation of guinea pigs, scattered infected cells were detected in the lungs by immunohistochemistry, but infectious HPIV3/ΔF-HN/EboGP could not be recovered from the lungs, blood, or other tissues. Despite the attenuation, the virus was highly immunogenic, and a single IN dose completely protected the animals against a highly lethal intraperitoneal challenge of guinea pig-adapted EBOV

  3. 2B4 (CD244) signaling via chimeric receptors costimulates tumor-antigen specific proliferation and in vitro expansion of human T cells.

    Science.gov (United States)

    Altvater, Bianca; Landmeier, Silke; Pscherer, Sibylle; Temme, Jaane; Juergens, Heribert; Pule, Martin; Rossig, Claudia

    2009-12-01

    Regulatory NK cell receptors can contribute to antigen-specific adaptive immune responses by modulating T cell receptor (TCR)-induced T cell activation. We investigated the potential of the NK cell receptor 2B4 (CD244) to enhance tumor antigen-induced activation of human T cells. 2B4 is a member of the CD2 receptor subfamily with both activating and inhibitory functions in NK cells. In T cells, its expression is positively associated with the acquisition of a cytolytic effector memory phenotype. Recombinant chimeric receptors that link extracellular single-chain Fv fragments specific for the tumor-associated surface antigens CD19 and G(D2) to the signaling domains of human 2B4 and/or TCRzeta were expressed in non-specifically activated peripheral blood T cells by retroviral gene transfer. While 2B4 signaling alone failed to induce T cell effector functions or proliferation, it significantly augmented the antigen-specific activation responses induced by TCRzeta. 2B4 costimulation did not affect the predominant effector memory phenotype of expanding T cells, nor did it increase the proportion of T cells with regulatory phenotype (CD4+CD25(hi)FoxP3+). These data support a costimulatory role for 2B4 in human T cell subpopulations. As an amplifier of TCR-mediated signals, 2B4 may provide a powerful new tool for immunotherapy of cancer, promoting sustained activation and proliferation of gene-modified antitumor T cells. PMID:19360406

  4. In silico analysis of chimeric espA, eae and tir fragments of Escherichia coli O157:H7 for oral immunogenic applications

    Directory of Open Access Journals (Sweden)

    Rafati Sima

    2009-12-01

    Full Text Available Abstract Background In silico techniques are highly suited for both the discovery of new and development of existing vaccines. Enterohemorrhagic Escherichia coli O157:H7 (EHEC exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological lesion (attaching/effacing. The genes encoding the products responsible for this phenotype are clustered on a 35-kb pathogenicity island. Among these proteins, Intimin, Tir, and EspA, which are expressed by attaching-effacing genes, are responsible for the attachment to epithelial cell that leads to lesions. Results We designed synthetic genes encoding the carboxy-terminal fragment of Intimin, the middle region of Tir and the carboxy-terminal part of EspA. These multi genes were synthesized with codon optimization for a plant host and were fused together by the application of four repeats of five hydrophobic amino acids as linkers. The structure of the synthetic construct gene, its mRNA and deduced protein and their stabilities were analyzed by bioinformatic software. Furthermore, the immunogenicity of this multimeric recombinant protein consisting of three different domains was predicted. Conclusion a structural model for a chimeric gene from LEE antigenic determinants of EHEC is presented. It may define accessibility, solubility and immunogenecity.

  5. A Novel Chimeric Antigen Receptor Against Prostate Stem Cell Antigen Mediates Tumor Destruction in a Humanized Mouse Model of Pancreatic Cancer

    Science.gov (United States)

    Lagisetty, Kiran H.; Tran, Eric; Zheng, Zhili; Gattinoni, Luca; Yu, Zhiya; Burns, William R.; Miermont, Anne M.; Teper, Yaroslav; Rudloff, Udo; Restifo, Nicholas P.; Feldman, Steven A.; Rosenberg, Steven A.; Morgan, Richard A.

    2014-01-01

    Abstract Despite advances in the understanding of its molecular pathophysiology, pancreatic cancer remains largely incurable, highlighting the need for novel therapies. We developed a chimeric antigen receptor (CAR) specific for prostate stem cell antigen (PSCA), a glycoprotein that is overexpressed in pancreatic cancer starting at early stages of malignant transformation. To optimize the CAR design, we used antigen-recognition domains derived from mouse or human antibodies, and intracellular signaling domains containing one or two T cell costimulatory elements, in addition to CD3zeta. Comparing multiple constructs established that the CAR based on human monoclonal antibody Ha1-4.117 had the greatest reactivity in vitro. To further analyze this CAR, we developed a human pancreatic cancer xenograft model and adoptively transferred CAR-engineered T cells into animals with established tumors. CAR-engineered human lymphocytes induced significant antitumor activity, and unlike what has been described for other CARs, a second-generation CAR (containing CD28 cosignaling domain) induced a more potent antitumor effect than a third-generation CAR (containing CD28 and 41BB cosignaling domains). While our results provide evidence to support PSCA as a target antigen for CAR-based immunotherapy of pancreatic cancer, the expression of PSCA on selected normal tissues could be a source of limiting toxicity. PMID:24694017

  6. Transduction and selection of human T cells with novel CD34/thymidine kinase chimeric suicide genes for the treatment of graft-versus-host disease.

    Science.gov (United States)

    Rettig, Michael P; Ritchey, Julie K; Meyerrose, Todd E; Haug, Jeffrey S; DiPersio, John F

    2003-07-01

    Clinical trials evaluating the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) suicide gene therapy system for the control of graft-versus-host disease (GVHD) have been limited by low transduction efficiencies and inefficient selection procedures. In this study, we designed and evaluated a novel chimeric suicide gene consisting of the extracellular and transmembrane domains of human CD34 and full-length HSV-tk (DeltaCD34-tk). High-efficiency transfer of DeltaCD34-tk to primary human T cells was accomplished after a single exposure to VSV-G-pseudotyped, Moloney murine leukemia virus-based retrovirus 48 h after activation of human PBMCs with anti-CD3 and anti-CD28 antibodies immobilized on magnetic beads. Using an optimized 5-day transduction and selection procedure, transduction efficiencies averaged 71%, with isolation purities greater than 95% and yields exceeding 90%. The immunoselected T cells were selectively eliminated by GCV (IC(50) approximately 3 nM), maintained a normal subset composition, exhibited a polyclonal TCR Vbeta family repertoire, and contained 5 or 6 vector copies per transduced cell when optimally transduced. No increase in GCV sensitivity was observed upon incorporation of highly active mutant HSV-tk enzymes into the DeltaCD34-tk suicide gene. T cells modified with the DeltaCD34-tk gene using the optimized protocol should improve the overall efficacy of the HSV-tk/GCV suicide gene therapy method of GVHD control. PMID:12842426

  7. Effects of RING-SH2Grb², a chimeric protein containing the E3 ligase domain of Cbl, on the EGFR pathway.

    Science.gov (United States)

    Lee, Wei-Hao; Wang, Pei-Yu; Lin, Yu-Hung; Chou, He-Yen; Lee, Yen-Hsien; Lee, Chien-Kuo; Pai, Li-Mei

    2014-12-31

    The E3 ubiquitin-protein ligase Casitas B-lineage lymphoma protein (Cbl) negatively regulates epidermal growth factor receptor (EGFR) signaling pathway in many organisms, and has crucial roles in cell growth, development and human pathologies, including lung cancers. RING-SH2Grb² a chimeric protein of 215 amino acids containing the RING domain of Cbl that provides E3 ligase activity, and the SH2 domain of Grb2 that serves as an adaptor for EGFR. In this study, we demonstrated that RING-SH2Grb² could promote the ubiquitinylation and degradation of EGFR in a human non-small cell lung carcinoma cell line H1299. Moreover, we discovered that the RING-SH2Grb² chimera promoted the internalization of ligand-bound EGFR, inhibited the growth of H1299 cells, and significantly suppressed tumor growth in a xenograft mouse model. In summary, our results revealed a potential new cancer therapeutic approach for non-small cell lung cancer. PMID:25575524

  8. Immunogenicity of retransplanted rat kidney allografts. Effect of inducing chimerism in the first recipient and quantitative studies on immunosuppression of the second recipient

    International Nuclear Information System (INIS)

    It has been previously shown that long surviving, enhanced (AS X AUG)F1 rat kidneys residing in a primary AS recipient are not acutely rejected if transferred into a second AS recipient. The reduced immunogenicity of the retransplanted graft was attributed to a depletion of incompatible passenger cells. It is shown here that if the primary AS recipient is made chimeric by x irradiation and injection of (AS X AUG)F1 bone marrow cells, transfer of the long surviving, enhanced graft into a second AS recipient provokes acute graft rejection comparable to that observed when normal (AS X AUG)F1 kidneys are transplanted into untreated AS recipients. Transplantation of passenger cell-depleted AUG kidneys into AS recipients leads to graft rejection, with a median survival time of 22 d. Treatment of these recipients with as little as 1.5 mg/kg cyclophosphamide for 14 d induces prolonged graft survival. By contrast, five times as much cyclophosphamide treatment is required to induce prolonged survival of normal AUG kidneys (i.e., containing incompatible passenger cells) transplanted to AS recipients. These results confirm that the major alloimmunogenic stimulus of rat kidney grafts is provided by the incompatible passenger cells

  9. Bleomycin-Treated Chimeric Thy1-Deficient Mice with Thy1-Deficient Myofibroblasts and Thy-Positive Lymphocytes Resolve Inflammation without Affecting the Fibrotic Response

    Directory of Open Access Journals (Sweden)

    Pazit Y. Cohen

    2015-01-01

    Full Text Available Lung fibrosis is characterized by abnormal accumulation of fibroblasts in the interstitium of the alveolar space. Two populations of myofibroblasts, distinguished by Thy1 expression, are detected in human and murine lungs. Accumulation of Thy1-negative (Thy1− myofibroblasts was shown in the lungs of humans with idiopathic pulmonary fibrosis (IPF and of bleomycin-treated mice. We aimed to identify genetic changes in lung myofibroblasts following Thy1 crosslinking and assess the impact of specific lung myofibroblast Thy1-deficiency, in vivo, in bleomycin-injured mouse lungs. Thy1 increased in mouse lung lymphocytes following bleomycin injury but decreased in myofibroblasts when fibrosis was at the highest point (14 days, as assessed by immunohistochemistry. Using gene chip analysis, we detected that myofibroblast Thy1 crosslinking mediates downregulation of genes promoting cell proliferation, survival, and differentiation, and reduces production of extracellular matrix (ECM components, while concurrently mediating the upregulation of genes known to foster inflammation and immunological functions. Chimeric Thy1-deficient mice with Thy1+ lymphocytes and Thy1− myofibroblasts showed fibrosis similar to wild-type mice and an increased number of CD4/CD25 regulatory T cells, with a concomitant decrease in inflammation. Lung myofibroblasts downregulate Thy1 expression to increase their proliferation but to diminish the in vivo inflammatory milieu. Inflammation is not essential for evolution of fibrosis as was previously stated.

  10. An adenine-to-guanine nucleotide change in the IRES SL-IV domain of picornavirus/hepatitis C chimeric viruses leads to a nonviable phenotype

    International Nuclear Information System (INIS)

    The inability for the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) to be readily studied in the context of viral replication has been circumvented by constructing chimeras such as with poliovirus (PV), in which translation of the genome polyprotein is under control of the HCV IRES. During our attempts to configure the PV/HCV chimera for our drug discovery efforts, we discovered that an adenine- (A) to-guanine (G) change at nt 350 in domain IV of the HCV IRES resulted in a nonviable phenotype. Similarly, a mengovirus (MV)/HCV chimera using the same configuration with a G at nt 350 (G-350) was found to be nonviable. In contrast, a bovine viral diarrhea virus (BVDV)/HCV chimera remained viable with G-350 in the HCV IRES insert. Second-site, resuscitating mutations were identified from the G-350 PV/HCV and MV/HCV viruses after blind passaging. For both viruses, the resuscitating mutations involved destabilization of domain IV in the HCV IRES. The nonviability of G-350 in the picornavirus/HCV chimeric background might be linked to translation efficiency as indicated by analyses with dual reporter and PV/HCV replicon constructs

  11. An HPV 16 L1-based chimeric human papilloma virus-like particles containing a string of epitopes produced in plants is able to elicit humoral and cytotoxic T-cell activity in mice

    Directory of Open Access Journals (Sweden)

    Weiss-Steider Benny

    2009-01-01

    Full Text Available Abstract Background Even though two prophylactic vaccines against HPV are currently licensed, infections by the virus continue to be a major health problem mainly in developing countries. The cost of the vaccines limits wide-scale application in poor countries. A promising strategy for producing affordable and efficient vaccines involves the expression of recombinant immunogens in plants. Several HPV genes have been expressed in plants, including L1, which can self-assemble into virus-like particles. A plant-based, dual prophylactic/therapeutic vaccine remains an attractive possibility. Results We sought to express in tomato plants chimeric HPV 16 VLPs containing L1 fused to a string of epitopes from HPV 16 E6 and E7 proteins. The L1 employed had been modified to eliminate a strong inhibitory region at the 5' end of the molecule to increase expression levels. Several tomato lines were obtained expressing either L1 alone or L1-E6/E7 from 0.05% to 0.1% of total soluble protein. Stable integration of the transgenes was verified by Southern blot. Northern and western blot revealed successful expression of the transgenes at the mRNA and protein level. The chimeric VLPs were able to assemble adequately in tomato cells. Intraperitoneal administration in mice was able to elicit both neutralizing antibodies against the viral particle and cytotoxic T-lymphocytes activity against the epitopes. Conclusion In this work, we report for the first time the expression in plants of a chimeric particle containing the HPV 16 L1 sequence and a string of T-cell epitopes from HPV 16 E6 and E7 fused to the C-terminus. The particles were able to induce a significant antibody and cytotoxic T-lymphocytes response. Experiments in vivo are in progress to determine whether the chimeric particles are able to induce regression of disease and resolution of viral infection in mice. Chimeric particles of the type described in this work may potentially be the basis for developing

  12. Reduction of porcine circovirus type 2 (PCV2 viremia by a reformulated inactivated chimeric PCV1-2 vaccine-induced humoral and cellular immunity after experimental PCV2 challenge

    Directory of Open Access Journals (Sweden)

    Seo Hwi

    2012-10-01

    Full Text Available Abstract Background The objective of the present study was to elucidate the humoral and cellular immune response mechanisms by which a reformulated inactivated chimeric PCV1-2 vaccine reduces the PCV2 viremia. Forty PCV2 seronegative 3-week-old pigs were randomly divided into the following four groups: vaccinated challenged (T01, vaccinated non-challenged (T02, non-vaccinated challenged (T03, and non-vaccinated non-challenged (T04 animals. The pigs in groups T01 and T02 were immunized with a reformulated inactivated chimeric PCV1-2 vaccine (Fostera™ PCV; Pfizer Animal Health administered as a 2.0 ml dose at 21 days of age. At 35 days of age (0 days post-challenge, the pigs in groups T01 and T03 were inoculated intranasally with 2 ml each of PCV2b. Results A reduction of PCV2 viremia coincided with the appearance of both PCV2-specific neutralizing antibodies (NA and interferon-γ-secreting cells (IFN-γ-SCs in the vaccinated animals. However, the presence of anti-PCV2 IgG antibodies did not correlate with the reduction of PCV2 viremia. Lymphocyte subset analysis indicated that the numbers of CD3+ and CD4+ cells increased in vaccinated animals but the numbers of CD4+ cells decreased transiently in non-vaccinated animals. The observation of a delayed type hypersensitivity response in only the vaccinated animals also supports a CD4+ cell-associated protective cellular immune response induced by the reformulated inactivated chimeric PCV1-2 vaccine. Conclusions The induction of PCV2-specific NA and IFN-γ-SCs, and CD4+ cells by the reformulated inactivated chimeric PCV1-2 vaccine is the important protective immune response leading to reduction of the PCV2 viremia and control of the PCV2 infection. To our knowledge this is the first demonstration of protective humoral and cellular immunity induced by the reformulated inactivated chimeric PCV1-2 vaccine and its effect on reduction of PCV2 viremia by vaccination.

  13. Crystal Structures of Yeast-Produced Enterovirus 71 and Enterovirus 71/Coxsackievirus A16 Chimeric Virus-Like Particles Provide the Structural Basis for Novel Vaccine Design against Hand-Foot-and-Mouth Disease

    Science.gov (United States)

    Lyu, Ke; He, Ya-Ling; Li, Hao-Yang

    2015-01-01

    ABSTRACT Human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two major causative agents for hand-foot-and-mouth disease (HFMD). Previously, we demonstrated that a virus-like particle (VLP) for EV71 produced from Saccharomyces cerevisiae is a potential vaccine candidate against EV71 infection, and an EV71/CVA16 chimeric VLP can elicit protective immune responses against both virus infections. Here, we presented the crystal structures of both VLPs, showing that both the linear and conformational neutralization epitopes identified in EV71 are mostly preserved on both VLPs. The replacement of only 4 residues in the VP1 GH loop converted strongly negatively charged surface patches formed by portions of the SP70 epitope in EV71 VLP into a relatively neutral surface in the chimeric VLP, which likely accounted for the additional neutralization capability of the chimeric VLP against CVA16 infection. Such local variations in the amino acid sequences and the surface charge potential are also present in different types of polioviruses. In comparison to EV71 VLP, the chimeric VLP exhibits structural changes at the local site of amino acid replacement and the surface loops of all capsid proteins. This is consistent with the observation that the VP1 GH loop located near the pseudo-3-fold junction is involved in extensive interactions with other capsid regions. Furthermore, portions of VP0 and VP1 in EV71 VLP are at least transiently exposed, revealing the structural flexibility of the VLP. Together, our structural analysis provided insights into the structural basis of enterovirus neutralization and novel vaccine design against HFMD and other enterovirus-associated diseases. IMPORTANCE Our previous studies demonstrated that the enterovirus 71 (EV71) virus-like particle (VLP) produced from yeast is a vaccine candidate against EV71 infection and that a chimeric EV71/coxsackievirus A16 (CVA16) VLP with the replacement of 4 amino acids in the VP1 GH loop can confer

  14. Use of retroviral-mediated gene transfer to deliver and test function of chimeric antigen receptors in human T-cells

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    Ana C. Parente-Pereira

    2014-07-01

    Full Text Available Chimeric antigen receptors (CARs are genetically delivered fusion molecules that elicit T-cell activation upon binding of a native cell surface molecule. These molecules can be used to generate a large number of memory and effector T-cells that are capable of recognizing and attacking tumor cells. Most commonly, stable CAR expression is achieved in T-cells using retroviral vectors. In the method described here, retroviral vectors are packaged in a two-step procedure. First, H29D human retroviral packaging cells (a derivative of 293 cells are transfected with the vector of interest, which is packaged transiently in vesicular stomatitis virus (VSV G pseudotyped particles. These particles are used to deliver the vector to PG13 cells, which achieve stable packaging of gibbon ape leukaemia virus (GALV-pseudotyped particles that are suitable for infection of human T-cells. The key advantage of the method reported here is that it robustly generates polyclonal PG13 cells that are 100% positive for the vector of interest. This means that efficient gene transfer may be repeatedly achieved without the need to clone individual PG13 cells for experimental pre-clinical testing. To achieve T-cell transduction, cells must first be activated using a non-specific mitogen. Phytohemagglutinin (PHA provides an economic and robust stimulus to achieve this. After 48-72 h, activated T-cells and virus-conditioned medium are mixed in RetroNectin-coated plasticware, which enhances transduction efficiency. Transduced cells are analyzed for gene transfer efficiency by flow cytometry 48 h following transduction and may then be tested in several assays to evaluate CAR function, including target-dependent cytotoxicity, cytokine production and proliferation.

  15. Hepatitis C virus dynamics and cellular gene expression in uPA-SCID chimeric mice with humanized livers during intravenous silibinin monotherapy.

    Science.gov (United States)

    DebRoy, S; Hiraga, N; Imamura, M; Hayes, C N; Akamatsu, S; Canini, L; Perelson, A S; Pohl, R T; Persiani, S; Uprichard, S L; Tateno, C; Dahari, H; Chayama, K

    2016-09-01

    Legalon SIL (SIL) is a chemically hydrophilized version of silibinin, an extract of milk thistle (Silybum marianum) seeds that has exhibited hepatoprotective and antiviral effectiveness against hepatitis C virus (HCV) in patients leading to viral clearance in combination with ribavirin. To elucidate the incompletely understood mode of action of SIL against HCV, mathematical modelling of HCV kinetics and human hepatocyte gene expression studies were performed in uPA-SCID-chimeric mice with humanized livers. Chronically HCV-infected mice (n = 15) were treated for 14 days with daily intravenous SIL at 469, 265 or 61.5 mg/kg. Serum HCV and human albumin (hAlb) were measured frequently, and liver HCV RNA was analysed at days 3 and 14. Microarray analysis of human hepatocyte gene expression was performed at days 0, 3 and 14 of treatment. While hAlb remained constant, a biphasic viral decline in serum was observed consisting of a rapid 1st phase followed by a second slower phase (or plateau with the two lower SIL dosings). SIL effectiveness in blocking viral production was similar among dosing groups (median ε = 77%). However, the rate of HCV-infected hepatocyte decline, δ, was dose-dependent. Intracellular HCV RNA levels correlated (r = 0.66, P = 0.01) with serum HCV RNA. Pathway analysis revealed increased anti-inflammatory and antiproliferative gene expression in human hepatocytes in SIL-treated mice. The results suggest that SIL could lead to a continuous second-phase viral decline, that is potentially viral clearance, in the absence of adaptive immune response along with increased anti-inflammatory and antiproliferative gene expression in human hepatocytes. PMID:27272497

  16. Treatment of melanoma with a serotype 5/3 chimeric oncolytic adenovirus coding for GM-CSF: Results in vitro, in rodents and in humans.

    Science.gov (United States)

    Bramante, Simona; Kaufmann, Johanna K; Veckman, Ville; Liikanen, Ilkka; Nettelbeck, Dirk M; Hemminki, Otto; Vassilev, Lotta; Cerullo, Vincenzo; Oksanen, Minna; Heiskanen, Raita; Joensuu, Timo; Kanerva, Anna; Pesonen, Sari; Matikainen, Sampsa; Vähä-Koskela, Markus; Koski, Anniina; Hemminki, Akseli

    2015-10-01

    Metastatic melanoma is refractory to irradiation and chemotherapy, but amenable to immunological approaches such as immune-checkpoint-inhibiting antibodies or adoptive cell therapies. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules. Therefore, oncolytic immuno-virotherapy of malignant melanoma is an appealing approach, which was recently validated by a positive phase 3 trial. We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a panel of melanoma cell lines and animal models, and summarized the melanoma-specific human data from the Advanced Therapy Access Program (ATAP). The virus effectively eradicated human melanoma cells in vitro and subcutaneous SK-MEL-28 melanoma xenografts in nude mice when combined with low-dose cyclophosphamide. Furthermore, virally-expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated the differentiation of human monocytes into macrophages. In contrast to human cells, RPMI 1846 hamster melanoma cells exhibited no response to oncolytic viruses and the chimeric 5/3 fiber failed to increase the efficacy of transduction, suggesting limited utility of the hamster model in the context of viruses with this capsid. In ATAP, treatments appeared safe and well-tolerated. Four out of nine melanoma patients treated were evaluable for possible therapy benefit with modified RECIST criteria: one patient had minor response, two had stable disease, and one had progressive disease. Two patients were alive at 559 and 2,149 days after treatment. Ad5/3-D24-GMCSF showed promising efficacy in preclinical studies and possible antitumor activity in melanoma patients refractory to other forms of therapy. This data supports continuing the clinical development of oncolytic adenoviruses for treatment of malignant melanoma. PMID:25821063

  17. Gene editing by co-transformation of TALEN and chimeric RNA/DNA oligonucleotides on the rice OsEPSPS gene and the inheritance of mutations.

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    Mugui Wang

    Full Text Available Although several site-specific nucleases (SSNs, such as zinc-finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs, and the clustered regularly interspaced short palindromic repeat (CRISPR/Cas, have emerged as powerful tools for targeted gene editing in many organisms, to date, gene targeting (GT in plants remains a formidable challenge. In the present study, we attempted to substitute a single base in situ on the rice OsEPSPS gene by co-transformation of TALEN with chimeric RNA/DNA oligonucleotides (COs, including different strand composition such as RNA/DNA (C1 or DNA/RNA (C2 but contained the same target base to be substituted. In contrast to zero GT event obtained by the co-transformation of TALEN with homologous recombination plasmid (HRP, we obtained one mutant showing target base substitution although accompanied by undesired deletion of 12 bases downstream the target site from the co-transformation of TALEN and C1. In addition to this typical event, we also obtained 16 mutants with different length of base deletions around the target site among 105 calli lines derived from transformation of TALEN alone (4/19 as well as co-transformation of TELAN with either HRP (5/30 or C1 (2/25 or C2 (5/31. Further analysis demonstrated that the homozygous gene-edited mutants without foreign gene insertion could be obtained in one generation. The induced mutations in transgenic generation were also capable to pass to the next generation stably. However, the genotypes of mutants did not segregate normally in T1 population, probably due to lethal mutations. Phenotypic assessments in T1 generation showed that the heterozygous plants with either one or three bases deletion on target sequence, called d1 and d3, were more sensitive to glyphosate and the heterozygous d1 plants had significantly lower seed-setting rate than wild-type.

  18. Immunotherapy of non-Hodgkin's lymphoma with a defined ratio of CD8+ and CD4+ CD19-specific chimeric antigen receptor-modified T cells.

    Science.gov (United States)

    Turtle, Cameron J; Hanafi, Laïla-Aïcha; Berger, Carolina; Hudecek, Michael; Pender, Barbara; Robinson, Emily; Hawkins, Reed; Chaney, Colette; Cherian, Sindhu; Chen, Xueyan; Soma, Lorinda; Wood, Brent; Li, Daniel; Heimfeld, Shelly; Riddell, Stanley R; Maloney, David G

    2016-09-01

    CD19-specific chimeric antigen receptor (CAR)-modified T cells have antitumor activity in B cell malignancies, but factors that affect toxicity and efficacy have been difficult to define because of differences in lymphodepletion and heterogeneity of CAR-T cells administered to individual patients. We conducted a clinical trial in which CD19 CAR-T cells were manufactured from defined T cell subsets and administered in a 1:1 CD4(+)/CD8(+) ratio of CAR-T cells to 32 adults with relapsed and/or refractory B cell non-Hodgkin's lymphoma after cyclophosphamide (Cy)-based lymphodepletion chemotherapy with or without fludarabine (Flu). Patients who received Cy/Flu lymphodepletion had increased CAR-T cell expansion and persistence, and higher response rates [50% complete remission (CR), 72% overall response rate (ORR)] than patients who received Cy-based lymphodepletion without Flu (8% CR, 50% ORR). The CR rate in patients treated with Cy/Flu at the maximally tolerated dose was 64% (82% ORR; n = 11). Cy/Flu minimized the effects of an immune response to the murine single-chain variable fragment component of the CAR, which limited CAR-T cell expansion and clinical efficacy in patients who received Cy-based lymphodepletion without Flu. Severe cytokine release syndrome (sCRS) and grade ≥3 neurotoxicity were observed in 13 and 28% of all patients, respectively. Serum biomarkers, one day after CAR-T cell infusion, correlated with subsequent sCRS and neurotoxicity. Immunotherapy with CD19 CAR-T cells in a defined CD4(+)/CD8(+) ratio allowed identification of correlative factors for CAR-T cell expansion, persistence, and toxicity, and facilitated optimization of lymphodepletion that improved disease response and overall and progression-free survival. PMID:27605551

  19. Immunogenicity of a virosomally-formulated Plasmodium falciparum GLURP-MSP3 chimeric protein-based malaria vaccine candidate in comparison to adjuvanted formulations

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    Tamborrini Marco

    2011-12-01

    Full Text Available Abstract Background In clinical trials, immunopotentiating reconstituted influenza virosomes (IRIVs have shown great potential as a versatile antigen delivery platform for synthetic peptides derived from Plasmodium falciparum antigens. This study describes the immunogenicity of a virosomally-formulated recombinant fusion protein comprising domains of the two malaria vaccine candidate antigens MSP3 and GLURP. Methods The highly purified recombinant protein GMZ2 was coupled to phosphatidylethanolamine and the conjugates incorporated into the membrane of IRIVs. The immunogenicity of this adjuvant-free virosomal formulation was compared to GMZ2 formulated with the adjuvants Montanide ISA 720 and Alum in three mouse strains with different genetic backgrounds. Results Intramuscular injections of all three candidate vaccine formulations induced GMZ2-specific antibody responses in all mice tested. In general, the humoral immune response in outbred NMRI mice was stronger than that in inbred BALB/c and C57BL/6 mice. ELISA with the recombinant antigens demonstrated immunodominance of the GLURP component over the MSP3 component. However, compared to the Al(OH3-adjuvanted formulation the two other formulations elicited in NMRI mice a larger proportion of anti-MSP3 antibodies. Analyses of the induced GMZ2-specific IgG subclass profiles showed for all three formulations a predominance of the IgG1 isotype. Immune sera against all three formulations exhibited cross-reactivity with in vitro cultivated blood-stage parasites. Immunofluorescence and immunoblot competition experiments showed that both components of the hybrid protein induced IgG cross-reactive with the corresponding native proteins. Conclusion A virosomal formulation of the chimeric protein GMZ2 induced P. falciparum blood stage parasite cross-reactive IgG responses specific for both MSP3 and GLURP. GMZ2 thus represents a candidate component suitable for inclusion into a multi-valent virosomal

  20. Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

    Science.gov (United States)

    Sathyanarayanan, P. V.; Cremo, C. R.; Poovaiah, B. W.

    2000-01-01

    Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.

  1. EGFRvIII-specific chimeric antigen receptor T cells migrate to and kill tumor deposits infiltrating the brain parenchyma in an invasive xenograft model of glioblastoma.

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    Hongsheng Miao

    Full Text Available Glioblastoma (GBM is the most common primary malignant brain tumor in adults and is uniformly lethal. T-cell-based immunotherapy offers a promising platform for treatment given its potential to specifically target tumor tissue while sparing the normal brain. However, the diffuse and infiltrative nature of these tumors in the brain parenchyma may pose an exceptional hurdle to successful immunotherapy in patients. Areas of invasive tumor are thought to reside behind an intact blood brain barrier, isolating them from effective immunosurveillance and thereby predisposing the development of "immunologically silent" tumor peninsulas. Therefore, it remains unclear if adoptively transferred T cells can migrate to and mediate regression in areas of invasive GBM. One barrier has been the lack of a preclinical mouse model that accurately recapitulates the growth patterns of human GBM in vivo. Here, we demonstrate that D-270 MG xenografts exhibit the classical features of GBM and produce the diffuse and invasive tumors seen in patients. Using this model, we designed experiments to assess whether T cells expressing third-generation chimeric antigen receptors (CARs targeting the tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, would localize to and treat invasive intracerebral GBM. EGFRvIII-targeted CAR (EGFRvIII+ CAR T cells demonstrated in vitro EGFRvIII antigen-specific recognition and reactivity to the D-270 MG cell line, which naturally expresses EGFRvIII. Moreover, when administered systemically, EGFRvIII+ CAR T cells localized to areas of invasive tumor, suppressed tumor growth, and enhanced survival of mice with established intracranial D-270 MG tumors. Together, these data demonstrate that systemically administered T cells are capable of migrating to the invasive edges of GBM to mediate antitumor efficacy and tumor regression.

  2. Chimeric antigen receptor-engineered cytokine-induced killer cells overcome treatment resistance of pre-B-cell acute lymphoblastic leukemia and enhance survival.

    Science.gov (United States)

    Oelsner, Sarah; Wagner, Juliane; Friede, Miriam E; Pfirrmann, Verena; Genßler, Sabrina; Rettinger, Eva; Buchholz, Christian J; Pfeifer, Heike; Schubert, Ralf; Ottmann, Oliver G; Ullrich, Evelyn; Bader, Peter; Wels, Winfried S

    2016-10-15

    Pre-emptive cancer immunotherapy by donor lymphocyte infusion (DLI) using cytokine-induced killer (CIK) cells may be beneficial to prevent relapse with a reduced risk of causing graft-versus-host-disease. CIK cells are a heterogeneous effector cell population including T cells (CD3(+) CD56(-) ), natural killer (NK) cells (CD3(-) CD56(+) ) and natural killer T (T-NK) cells (CD3(+) CD56(+) ) that exhibit non-major histocompatibility complex (MHC)-restricted cytotoxicity and are generated by ex vivo expansion of peripheral blood mononuclear cells in the presence of interferon (IFN)-γ, anti-CD3 antibody, interleukin-2 (IL-2) and interleukin-15 (IL-15). To facilitate selective target-cell recognition and enhance specific cytotoxicity against B-cell acute lymphoblastic leukemia (B-ALL), we transduced CIK cells with a lentiviral vector encoding a chimeric antigen receptor (CAR) that carries a composite CD28-CD3ζ domain for signaling and a CD19-specific scFv antibody fragment for cell binding (CAR 63.28.z). In vitro analysis revealed high and specific cell killing activity of CD19-targeted CIK/63.28.z cells against otherwise CIK-resistant cancer cell lines and primary B-ALL blasts, which was dependent on CD19 expression and CAR signaling. In a xenograft model in immunodeficient mice, treatment with CIK/63.28.z cells in contrast to therapy with unmodified CIK cells resulted in complete and durable molecular remissions of established primary pre-B-ALL. Our results demonstrate potent antileukemic activity of CAR-engineered CIK cells in vitro and in vivo, and suggest this strategy as a promising approach for adoptive immunotherapy of refractory pre-B-ALL. PMID:27253354

  3. Sleeping Beauty Transposition of Chimeric Antigen Receptors Targeting Receptor Tyrosine Kinase-Like Orphan Receptor-1 (ROR1 into Diverse Memory T-Cell Populations.

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    Drew C Deniger

    Full Text Available T cells modified with chimeric antigen receptors (CARs targeting CD19 demonstrated clinical activity against some B-cell malignancies. However, this is often accompanied by a loss of normal CD19+ B cells and humoral immunity. Receptor tyrosine kinase-like orphan receptor-1 (ROR1 is expressed on sub-populations of B-cell malignancies and solid tumors, but not by healthy B cells or normal post-partum tissues. Thus, adoptive transfer of T cells specific for ROR1 has potential to eliminate tumor cells and spare healthy tissues. To test this hypothesis, we developed CARs targeting ROR1 in order to generate T cells specific for malignant cells. Two Sleeping Beauty transposons were constructed with 2nd generation ROR1-specific CARs signaling through CD3ζ and either CD28 (designated ROR1RCD28 or CD137 (designated ROR1RCD137 and were introduced into T cells. We selected for T cells expressing CAR through co-culture with γ-irradiated activating and propagating cells (AaPC, which co-expressed ROR1 and co-stimulatory molecules. Numeric expansion over one month of co-culture on AaPC in presence of soluble interleukin (IL-2 and IL-21 occurred and resulted in a diverse memory phenotype of CAR+ T cells as measured by non-enzymatic digital array (NanoString and multi-panel flow cytometry. Such T cells produced interferon-γ and had specific cytotoxic activity against ROR1+ tumors. Moreover, such cells could eliminate ROR1+ tumor xenografts, especially T cells expressing ROR1RCD137. Clinical trials will investigate the ability of ROR1-specific CAR+ T cells to specifically eliminate tumor cells while maintaining normal B-cell repertoire.

  4. Safety and immunogenicity of a malaria vaccine, Plasmodium falciparum AMA-1/MSP-1 chimeric protein formulated in montanide ISA 720 in healthy adults.

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    Jinhong Hu

    Full Text Available BACKGROUND: The P. falciparum chimeric protein 2.9 (PfCP-2.9 consisting of the sequences of MSP1-19 and AMA-1 (III is a malaria vaccine candidate that was found to induce inhibitory antibodies in rabbits and monkeys. This was a phase I randomized, single-blind, placebo-controlled, dose-escalation study to evaluate the safety and immunogenicity of the PfCP-2.9 formulated with a novel adjuvant Montanide ISA720. Fifty-two subjects were randomly assigned to 4 dose groups of 10 participants, each receiving the test vaccine of 20, 50, 100, or 200 microg respectively, and 1 placebo group of 12 participants receiving the adjuvant only. METHODS AND FINDINGS: The vaccine formulation was shown to be safe and well-tolerated, and none of the participants withdrew. The total incidence of local adverse events (AEs was 75%, distributed among 58% of the placebo group and 80% of those vaccinated. Among the vaccinated, 65% had events that were mild and 15% experienced moderate AEs. Almost all systemic adverse reactions observed in this study were graded as mild and required no therapy. The participants receiving the test vaccine developed detectable antibody responses which were boosted by the repeated vaccinations. Sixty percent of the vaccinated participants had high ELISA titers (>1:10,000 of antigen-specific antibodies which could also recognize native parasite proteins in an immunofluorescence assay (IFA. CONCLUSION: This study is the first clinical trial for this candidate and builds on previous investigations supporting PfCP-2.9/ISA720 as a promising blood-stage malaria vaccine. Results demonstrate safety, tolerability (particularly at the lower doses tested and immunogenicity of the formulation. Further clinical development is ongoing to explore optimizing the dose and schedule of the formulation to decrease reactogenicity without compromising immunogenicity. TRIAL REGISTRATION: Chinese State Food and Drug Administration (SFDA 2002SL0046; Controlled

  5. N-terminal fusion of a toll-like receptor 2-ligand to a Neospora caninum chimeric antigen efficiently modifies the properties of the specific immune response.

    Science.gov (United States)

    Aguado-Martínez, Adriana; Basto, Afonso P; Müller, Joachim; Balmer, Vreni; Manser, Vera; Leitão, Alexandre; Hemphill, Andrew

    2016-04-01

    Immunoprophylactic products against neosporosis during pregnancy should induce an appropriately balanced immune response. In this respect, OprI, a bacterial lipoprotein targeting toll like receptor (TLR)2, provides promising adjuvant properties. We report on the manipulation of the innate and the T-cell immune response through the fusion of OprI with the Neospora caninum chimeric protein Mic3-1-R. In contrast to Mic3-1-R, OprI-MIC3-1-R significantly activated bone-marrow dendritic cells from naïve mice. Mice immunized with OprI-Mic3-1-R induced an immune response with mixed T helper (Th)1 and Th2 properties (high levels of both immunoglobulin (Ig)G1 and IgG2a and of interleukin (IL)-10, IL-12(p70) and interferon-γ responses) whereas Mic3-1-R+saponin induced a clear Th2-biased response (low IgG2a and high IL-4 and IL-10). After mating and challenge with N. caninum, increased expression of interferon-γ was only found in placentas from OprI-Mic3-1-R immunized dams. However, no protection against vertical transmission and neonatal mortality was observed in either of the two groups. These results indicated that more exhaustive studies must be done to elucidate the immune mechanisms associated with transplacental transmission. Antigen linkage to TLR2-ligands, such as OprI, is a useful tool to investigate this enigma by reorienting the innate and adaptive immune responses against other candidate antigens in future studies. PMID:26932317

  6. Radioimmunotherapy in refractory b-cell nonhodgkins lymphoma with I-131-labeled chimeric anti cd-20 c2b8 (I-131 rituximab): preliminary result

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Hye Jin; Park, Yeon Hee; Kim, Sung Eun and others [Korea University Medical School, Seoul (Korea, Republic of)

    2005-07-01

    Recently, the native chimeric human-mouse anti CD-20 antibody IDEC-C2B8 (Rituximab) has been widely applied in NHL. This ongoing phase study was to evaluate whether radioimmunotherapy (RIT) with I-131 rituximab is effective in refractory B-cell NHL. Inclusion criteria were as follows: B-cell NHL with relapsed or refractory to primary standard therapy, measurable disease, adequate hematologic, renal, and hepatic function, informed consent. The rituximab (Mabthera, Roach) was radiolabeled with iodine-131(I-131) using a modified chloramine T method with high radiochemical purity (95%) and preservation of immuno-reactivity. All patients received loading doses of unlabeled rituximab (median, 40 mg: range, 20{approx}70 mg) immediately prior to administration of therapeutic dose (51.4{approx}152.2 MBq/kg), and then underwent gamma camera scan. 11 patients were enrolled (4 low-grade B-cell NHL, 7 DLBCL, median age 63 years). Patients had received a median of three prior chemotherapy regimens. The objective response rate was 36.4% (1 CR, 3 PRs). These all responses were observed in low-grade B-cell NHL, except one with DLBCL. Adverse events were primarily hematologic toxicities; the incidence of grade 3/4 neutropenia, thrombocytopenia, and anemia was 27.3%, 45.5%, and 18.2%, respectively. The treatment-related mortality was observed in one patient, who had been previously treated with high-dose chemotherapy plus TBI with autologous stem cell transplantation. RIT with I-131 rituximab seems to be effective tolerable in refractory low-grade B-cell NHL, although modest activity in refractory DLBCL. Further studies to define the efficacy of I-131 rituximab in DLBCL are warranted.

  7. Quantitative PET of EGFR expression in xenograft-bearing mice using {sup 64}Cu-labeled cetuximab, a chimeric anti-EGFR monoclonal antibody

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    Cai, Weibo; Chen, Kai; He, Lina; Cao, Qizhen; Chen, Xiaoyuan [Stanford University School of Medicine, The Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Stanford, CA (United States); Koong, Albert [Stanford University School of Medicine, Department of Radiation Oncology, Stanford, CA (United States)

    2007-06-15

    Cetuximab, a chimeric monoclonal antibody targeting epidermal growth factor receptor (EGFR) on the surface of cancer cells, was approved by the FDA to treat patients with metastatic colorectal cancer. It is currently also in advanced-stage development for the treatment of several other solid tumors. Here we report for the first time the quantitative positron emission tomography (PET) imaging of EGFR expression in xenograft-bearing mice using {sup 64}Cu-labeled cetuximab. We conjugated cetuximab with macrocyclic chelating agent 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA), labeled with {sup 64}Cu, and tested the resulting {sup 64}Cu-DOTA-cetuximab in seven xenograft tumor models. The tracer uptake measured by PET was correlated with the EGFR expression quantified by western blotting. The estimated human dosimetry based on the PET data in Sprague-Dawley rats was also calculated. MicroPET imaging showed that {sup 64}Cu-DOTA-cetuximab had increasing tumor activity accumulation over time in EGFR-positive tumors but relatively low uptake in EGFR-negative tumors at all times examined (<5%ID/g). There was a good correlation (R {sup 2} = 0.80) between the tracer uptake (measured by PET) and the EGFR expression level (measured by western blotting). Human dosimetry estimation indicated that the tracer may be safely administered to human patients for tumor diagnosis, with the dose-limiting organ being the liver. The success of EGFR-positive tumor imaging using {sup 64}Cu-DOTA-cetuximab can be translated into the clinic to characterize the pharmacokinetics, to select the right population of patients for EGFR-targeted therapy, to monitor the therapeutic efficacy of anti-EGFR treatment, and to optimize the dosage of either cetuximab alone or cetuximab in combination with other therapeutic agents. (orig.)

  8. Evaluation of the immunogenicity and protective effects of a trivalent chimeric norovirus P particle immunogen displaying influenza HA2 from subtypes H1, H3 and B

    Science.gov (United States)

    Gong, Xin; Yin, He; Shi, Yuhua; He, Xiaoqiu; Yu, Yongjiao; Guan, Shanshan; Kuai, Ziyu; Haji, Nasteha M; Haji, Nafisa M; Kong, Wei; Shan, Yaming

    2016-01-01

    The ectodomain of the influenza A virus (IAV) hemagglutinin (HA) stem is highly conserved across strains and has shown promise as a universal influenza vaccine in a mouse model. In this study, potential B-cell epitopes were found through sequence alignment and epitope prediction in a stem fragment, HA2:90-105, which is highly conserved among virus subtypes H1, H3 and B. A norovirus (NoV) P particle platform was used to express the HA2:90-105 sequences from subtypes H1, H3 and B in loops 1, 2 and 3 of the protrusion (P) domain, respectively. Through mouse immunization and microneutralization assays, the immunogenicity and protective efficacy of the chimeric NoV P particle (trivalent HA2-PP) were tested against infection with three subtypes (H1N1, H3N2 and B) of IAV in Madin–Darby canine kidney cells. The protective efficacy of the trivalent HA2-PP was also evaluated preliminarily in vivo by virus challenge in the mouse model. The trivalent HA2-PP immunogen induced significant IgG antibody responses, which could be enhanced by a virus booster vaccination. Moreover, the trivalent HA2-PP immunogen also demonstrated in vitro neutralization of the H3 and B viruses, and in vivo protection against the H3 virus. Our results support the notion that a broadly protective vaccine approach using an HA2-based NoV P particle platform can provide cross-protection against challenge viruses of different IAV subtypes. The efficacy of the immunogen should be further enhanced for practicality, and a better understanding of the protective immune mechanism will be critical for the development of HA2-based multivalent vaccines. PMID:27222326

  9. Establishment of human-rhesus chimeric liver using adult bone marrow mesenchymal stem cells%应用成人骨髓间充质干细胞建立人-猴肝脏嵌合体

    Institute of Scientific and Technical Information of China (English)

    何保丽; 马丽花; 陈丽玲; 刘汝文; 杨仁华

    2013-01-01

    BACKGROUND:Human-mammal chimeric liver chimera has been a vital significance for the proliferation and differentiation of bone marrow mesenchymal stem cells. OBJECTIVE:To establish an animal model of human-rhesus chimeric liver using adult bone marrow mesenchymal stem cells. METHODS:Adult bone marrow mesenchymal stem cells were isolated, purified and cultured for the sixth generation. The number of bone marrow mesenchymal stem cells was no less than 5×108. Bone marrow mesenchymal stem cells labeled with green fluorescent protein were transplanted into the liver of the embryo rhesus with pregnancy of 10 weeks under guided by type-B ultrasound. At the 1st and 3rd months of birth, the liver tissue of the infant rhesus was taken for biopsy. After routine pathological section, histological specimens were observed under fluorescence microscope to confirm if there were adult bone marrow mesenchymal stem cells positive for green fluorescent protein and their distribution, and detected by immunohistochemical staining to identify if human albumin expressed in the liver of infant rhesus. RESULTS AND CONCLUSION:Fluorescence microscope observation indicated that at the 1st and 3rd months after birth, there were surviving bone marrow mesenchymal stem cells derived from human with green fluorescence in the liver of infant rhesus, and these cells migrated to form more concentrated distribution. The immunohistochemical results demonstrated that functional liver cells expressing human albumin were observed in the liver of infant rhesus at the 1st and 3rd months after birth, and their distribution was in accordance with bone marrow mesenchymal stem cells with green fluorescence. Human-rhesus chimeric liver can be established using adult bone marrow mesenchymal stem cells, which can generate functional liver cells in the liver of infant rhesus.%BACKGROUND:Human-mammal chimeric liver chimera has been a vital significance for the proliferation and differentiation of bone marrow

  10. Bioprocess development for the production of mouse-human chimeric anti-epidermal growth factor receptor vIII antibody C12 by suspension culture of recombinant Chinese hamster ovary cells

    OpenAIRE

    Hu, Suwen; Deng, Lei; Wang, Huamao; Zhuang, Yingping; Chu, Ju; Zhang, Siliang; Li, Zhonghai; Guo, Meijin

    2011-01-01

    The mouse-human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 is a promising candidate for the diagnosis of hepatocellular carcinoma (HCC). In this study, 3 processes were successfully developed to produce C12 by cultivation of recombinant Chinese hamster ovary (CHO-DG44) cells in serum-free medium. The effect of inoculum density was evaluated in batch cultures of shaker flasks to obtain the optimal inoculum density of 5 × 105 cells/mL. Then, the basic metabolic ...

  11. Close-up of the alpha-1,3-Gal epitope as defined by a monoclonal chimeric IgE and human serum using saturation transfer difference (STD) NMR

    DEFF Research Database (Denmark)

    Plum, Melanie; Michel, Yvonne; Wallach, Katharina;

    2011-01-01

    epitopes in allergy. The aim of this study was to dissect immunoreactivity, interaction and fine epitope of alpha-Gal-specific antibodies to obtain insights into the recognition of carbohydrate epitopes by IgE antibodies and their consequences on a molecular and cellular level. The antigen binding moiety...... by mediator release assays, surface plasmon resonance (SPR) and STD NMR analyses. The alpha-Gal-specific chimeric IgE and IgG antibodies were proven functional regarding interaction with antigen and Fc receptors. SPR measurements demonstrated affinities in the micromolar range. In contrast to a reference...

  12. Two RNAs or DNAs May Artificially Fuse Together at a Short Homologous Sequence (SHS) during Reverse Transcription or Polymerase Chain Reactions, and Thus Reporting an SHS-Containing Chimeric RNA Requires Extra Caution.

    Science.gov (United States)

    Xie, Bingkun; Yang, Wei; Ouyang, Yongchang; Chen, Lichan; Jiang, Hesheng; Liao, Yuying; Liao, D Joshua

    2016-01-01

    Tens of thousands of chimeric RNAs have been reported. Most of them contain a short homologous sequence (SHS) at the joining site of the two partner genes but are not associated with a fusion gene. We hypothesize that many of these chimeras may be technical artifacts derived from SHS-caused mis-priming in reverse transcription (RT) or polymerase chain reactions (PCR). We cloned six chimeric complementary DNAs (cDNAs) formed by human mitochondrial (mt) 16S rRNA sequences at an SHS, which were similar to several expression sequence tags (ESTs).These chimeras, which could not be detected with cDNA protection assay, were likely formed because some regions of the 16S rRNA are reversely complementary to another region to form an SHS, which allows the downstream sequence to loop back and anneal at the SHS to prime the synthesis of its complementary strand, yielding a palindromic sequence that can form a hairpin-like structure.We identified a 16S rRNA that ended at the 4th nucleotide(nt) of the mt-tRNA-leu was dominant and thus should be the wild type. We also cloned a mouse Bcl2-Nek9 chimeric cDNA that contained a 5-nt unmatchable sequence between the two partners, contained two copies of the reverse primer in the same direction but did not contain the forward primer, making it unclear how this Bcl2-Nek9 was formed and amplified. Moreover, a cDNA was amplified because one primer has 4 nts matched to the template, suggesting that there may be many more artificial cDNAs than we have realized, because the nuclear and mt genomes have many more 4-nt than 5-nt or longer homologues. Altogether, the chimeric cDNAs we cloned are good examples suggesting that many cDNAs may be artifacts due to SHS-caused mis-priming and thus greater caution should be taken when new sequence is obtained from a technique involving DNA polymerization. PMID:27148738

  13. Tribute to dr louis keith: twin and physician extraordinaire/twin research reports: influences on asthma severity; chimerism revisited; DNA strand break repair/media reports: twins born apart; elevated twin frequencies; celebrity father of twins; conjoined twinning.

    Science.gov (United States)

    Segal, Nancy L

    2014-10-01

    The International Society for Twin Studies has lost a valued friend and colleague. Dr Louis Keith, Emeritus Professor of Obstetrics and Gynecology at Northwestern University, in Chicago, passed away on Sunday, July 6, 2014. His life and work with twins will be acknowledged at the November 2014 International Twin Congress in Budapest, Hungary. Next, twin research reports on the severity of asthma symptoms, a case of chimerism, and factors affecting DNA breakage and repair mechanisms are reviewed. Media reports cover twins born apart, elevated twin frequencies, a celebrity father of twins, and a family's decision to keep conjoined twins together. PMID:25213730

  14. Two RNAs or DNAs May Artificially Fuse Together at a Short Homologous Sequence (SHS) during Reverse Transcription or Polymerase Chain Reactions, and Thus Reporting an SHS-Containing Chimeric RNA Requires Extra Caution

    Science.gov (United States)

    Xie, Bingkun; Yang, Wei; Ouyang, Yongchang; Chen, Lichan; Jiang, Hesheng; Liao, Yuying; Liao, D. Joshua

    2016-01-01

    Tens of thousands of chimeric RNAs have been reported. Most of them contain a short homologous sequence (SHS) at the joining site of the two partner genes but are not associated with a fusion gene. We hypothesize that many of these chimeras may be technical artifacts derived from SHS-caused mis-priming in reverse transcription (RT) or polymerase chain reactions (PCR). We cloned six chimeric complementary DNAs (cDNAs) formed by human mitochondrial (mt) 16S rRNA sequences at an SHS, which were similar to several expression sequence tags (ESTs).These chimeras, which could not be detected with cDNA protection assay, were likely formed because some regions of the 16S rRNA are reversely complementary to another region to form an SHS, which allows the downstream sequence to loop back and anneal at the SHS to prime the synthesis of its complementary strand, yielding a palindromic sequence that can form a hairpin-like structure.We identified a 16S rRNA that ended at the 4th nucleotide(nt) of the mt-tRNA-leu was dominant and thus should be the wild type. We also cloned a mouse Bcl2-Nek9 chimeric cDNA that contained a 5-nt unmatchable sequence between the two partners, contained two copies of the reverse primer in the same direction but did not contain the forward primer, making it unclear how this Bcl2-Nek9 was formed and amplified. Moreover, a cDNA was amplified because one primer has 4 nts matched to the template, suggesting that there may be many more artificial cDNAs than we have realized, because the nuclear and mt genomes have many more 4-nt than 5-nt or longer homologues. Altogether, the chimeric cDNAs we cloned are good examples suggesting that many cDNAs may be artifacts due to SHS-caused mis-priming and thus greater caution should be taken when new sequence is obtained from a technique involving DNA polymerization. PMID:27148738

  15. Novel Chimeric β-Lactamase CTX-M-64, a Hybrid of CTX-M-15-Like and CTX-M-14 β-Lactamases, Found in a Shigella sonnei Strain Resistant to Various Oxyimino-Cephalosporins, Including Ceftazidime▿

    OpenAIRE

    Nagano, Yukiko; Nagano, Noriyuki; Wachino, Jun-ichi; Ishikawa, Keiko; Arakawa, Yoshichika

    2008-01-01

    The plasmid-mediated novel β-lactamase CTX-M-64 was first identified in Shigella sonnei strain UIH-1, which exhibited resistance to cefotaxime (MIC, 1,024 μg/ml) and ceftazidime (MIC, 32 μg/ml). The amino acid sequence of CTX-M-64 showed a chimeric structure of a CTX-M-15-like β-lactamase (N- and C-terminal moieties) and a CTX-M-14-like β-lactamase (central portion, amino acids 63 to 226), suggesting that it originated by homologous recombination between the corresponding genes. The introduct...

  16. Characterization of Hepatitis C Virus Recombinants with Chimeric E1/E2 Envelope Proteins and Identification of Single Amino Acids in the E2 Stem Region Important for Entry

    OpenAIRE

    Carlsen, Thomas H. R.; Scheel, Troels K. H.; Ramirez, Santseharay; Foung, Steven K. H.; Bukh, Jens

    2013-01-01

    The hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexes in vitro. Using genotype 1a/2a JFH1-based recombinants expressing 1a core-NS2, we exchanged E2 with functional isolate sequences of genotypes 1a (alternative isolate), 1b, and 2a. While the 1a-E2 exchange did not impact virus viability, the 2a-E2 recombinant was nonviab...

  17. Tissue-Specific Replicating Capacity of a Chimeric Poliovirus That Carries the Internal Ribosome Entry Site of Hepatitis C Virus in a New Mouse Model Transgenic for the Human Poliovirus Receptor

    OpenAIRE

    Yanagiya, Akiko; Ohka, Seii; Hashida, Noriyasu; Okamura, Masahito; Taya, Choji; Kamoshita, Nobuhiko; Iwasaki, Kuniko; Sasaki, Yukari; Yonekawa, Hiromichi; Nomoto, Akio

    2003-01-01

    Nucleotides (nt) 108 to 742 of an infectious cDNA clone of poliovirus (PV) Mahoney strain, including the corresponding region of the internal ribosome entry site (IRES), was replaced by nt 28 to 710 of hepatitis C virus (HCV) cDNA corresponding to the whole HCV IRES. A chimeric PV (2A-369) was generated by transfecting mammalian cells with an RNA transcribed in vitro from the cDNA. To examine replicating capacity of virus 2A-369 in the brain and liver of a mouse model for poliomyelitis, a new...

  18. The chimeric genes AML1/MDS1 and AML1/EAP inhibit AML1B activation at the CSF1R promoter, but only AML1/MDS1 has tumor-promoter properties.

    OpenAIRE

    Zent, C S; Mathieu, C.; Claxton, D F; Zhang, D E; Tenen, D.G.; Rowley, J D; Nucifora, G

    1996-01-01

    The (3;21)(q26;q22) translocation associated with treatment-related myelodysplastic syndrome, treatment-related acute myeloid leukemia, and blast crisis of chronic myeloid leukemia results in the expression of the chimeric genes AML1/EAP, AML1/MDS1, and AML1/EVI1. AML1 (CBFA2), which codes for the alpha subunit of the heterodimeric transcription factor CBF, is also involved in the t(8;21), and the gene coding for the beta subunit (CBFB) is involved in the inv(16). These are two of the most co...

  19. 鸡胚胎干细胞的分离及其嵌合体制备条件探索%Isolation of Chicken Embryonic Stem Cell and Exploring the Preparation Conditions of Chicken Chimeric

    Institute of Scientific and Technical Information of China (English)

    张亚妮; 杨海燕; 施青青; 张振韬; 郑蒙蒙; 王丹; 李碧春

    2012-01-01

    分离培养鸡胚胎干细胞(ESCs),体外培养传代后,进行碱性磷酸酶活性检测和SSEA-1染色鉴定;并用线性化的质粒pEGFP-N1转染鸡ESCs.受体种蛋经赤道面开窗后,将经转染的ESCs注射到受体鸡胚胚下腔,以便制作嵌合体鸡;对获得的嵌合体鸡分别提取血液和组织DNA后进行PCR检测.结果表明:5只存活的的嵌合体鸡血液中没有发生EGFP基因嵌合,但有4只嵌合体鸡的部分器官表达了EGFP基因,其中有2只鸡的性腺发生嵌合,表明利用赤道面开窗后对受体鸡进行胚下腔显微注射可以生产嵌合体鸡.%Chicken embryonic stem cells (ESCs) were separated and cultured in vitro. Alkaline phosphatase activity (AKP) and SSEA-1 staining was conducted to detected ESCs. Then, chicken ESCs were transfected with linearized plasmid pEGFP-Nl. The receptor eggs were drilled a window at the lateral shell of egg, then, the ESCs transfected with pEGFP-Nl was injected into chick embryo cavity so as to make chimeric chicken. The chimeric chicken was detected for both of blood and different tissue by PCR. The results showed five chimeric chicken were obtained in this study and found that four of them expressed EGFP gene in some organs, only two of them expressed EGFP gene in the gonad, indicating the chimeric chicken could be obtained through chick embryo cavity micro-injection by drilling a window at the lateral shell of egg.

  20. Chimeric RXFP1 and RXFP2 receptors highlight the similar mechanism of activation utilizing their N-terminal low density lipoprotein class A modules

    Directory of Open Access Journals (Sweden)

    Shoni eBruell

    2013-11-01

    Full Text Available Relaxin family peptide (RXFP receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low density lipoprotein type A (LDLa module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP based signalling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a

  1. Chimeric RXFP1 and RXFP2 Receptors Highlight the Similar Mechanism of Activation Utilizing Their N-Terminal Low-Density Lipoprotein Class A Modules.

    Science.gov (United States)

    Bruell, Shoni; Kong, Roy C K; Petrie, Emma J; Hoare, Brad; Wade, John D; Scott, Daniel J; Gooley, Paul R; Bathgate, Ross A D

    2013-01-01

    Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP-based signaling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand-mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a similar mechanism. PMID

  2. Combination therapy using chimeric monoclonal antibodies protects mice from lethal H5N1 infection and prevents formation of escape mutants.

    Directory of Open Access Journals (Sweden)

    Mookkan Prabakaran

    Full Text Available BACKGROUND: Given that there is a possibility of a human H5N1 pandemic and the fact that the recent H5N1 viruses are resistant to the anti-viral drugs, newer strategies for effective therapy are warranted. Previous studies show that single mAbs in immune prophylaxis can be protective against H5N1 infection. But a single mAb may not be effective in neutralization of a broad range of different strains of H5N1 and control of potential neutralization escape mutants. METHODS/PRINCIPAL FINDINGS: We selected two mAbs which recognized different epitopes on the hemagglutinin molecule. These two mAbs could each neutralize in vitro escape mutants to the other and in combination could effectively neutralize viruses from clades 0, 1, 2.1, 2.2, 2.3, 4, 7 and 8 of influenza A H5N1 viruses. This combination of chimeric mAbs when administered passively, pre or post challenge with 10 MLD50 (50% mouse lethal dose HPAI H5N1 influenza A viruses could protect 100% of the mice from two different clades of viruses (clades 1 and 2.1. We also tested the efficacy of a single dose of the combination of mAbs versus two doses. Two doses of the combination therapy not only affected early clearance of the virus from the lung but could completely prevent lung pathology of the H5N1 infected mice. No escape variants were detected after therapy. CONCLUSIONS/SIGNIFICANCE: Our studies provide proof of concept that the synergistic action of two or more mAbs in combination is required for preventing the generation of escape mutants and also to enhance the therapeutic efficacy of passive therapy against H5N1 infection. Combination therapy may allow for a lower dose of antibody to be administered for passive therapy of influenza infection and hence can be made available at reduced economic costs during an outbreak.

  3. Synergy of Taxol and radioimmunotherapy with yttrium-90-labeled chimeric L6 antibody: Efficacy and toxicity in breast cancer xenografts

    Science.gov (United States)

    DeNardo, Sally J.; Kukis, David L.; Kroger, Linda A.; O’Donnell, Robert T.; Lamborn, Kathleen R.; Miers, Laird A.; DeNardo, David G.; Meares, Claude F.; DeNardo, Gerald L.

    1997-01-01

    Synergistic multimodality therapy is needed for breast cancer. Breast cancer frequently has p53 mutations that result in cells less likely to undergo apoptosis when exposed to DNA damaging therapies. Taxol (paclitaxel) is more effective in the presence of mutant p53. 90Y-labeled DOTA-peptide-ChL6 (90Y-ChL6, where ChL6 is chimeric L6 antibody and DOTA is 1,4,7,10-tetraazacyclododecane-N,N′,N",N‴-tetraacetic acid) is a novel radioimmunoconjugate for targeting radiation to cancer. It has a stable metal chelator and a peptide linker that can be catabolized by hepatic lysozymes. This study was designed to assess potential synergism between Taxol and 90Y-ChL6 in a highly anaplastic breast cancer model, HBT 3477. There was no tumor response in mice receiving ChL6 or Taxol alone. In mice receiving 90Y-ChL6 alone, 79% (15 of 19) of tumors responded although none were cured. If Taxol was administered 24–72 hours before 90Y-ChL6, again, 79% (23 of 29) of tumors responded but 21% were cured. When Taxol was administered 6 or 24 hours after 90Y-ChL6, 100% (46 of 46) of tumors responded and 48% were cured. Taxol given with 90Y-ChL6 did not substantially increase toxicity. Enhancement of the therapeutic effect when Taxol was added to 90Y-ChL6 therapy for HBT 3477 xenografts was striking. The synergistic therapeutic effect of Taxol with 90Y-ChL6 may relate to the p53 mutant status and BCL2 expression in HBT 3477 cells, observations that increase the likelihood that the results of this study are relevant to therapy for breast cancer in patients. In conclusion, Taxol seemed to be synergistic with 90Y-ChL6 in this human breast cancer model. Up to 50% of these anaplastic breast cancer xenografts were cured by combined modality therapy. PMID:9108094

  4. Labelling of anti-human bladder tumor chimeric antibody with 99Tcm and radioimmunoimaging of bladder carcinoma xenograft in nude mice

    International Nuclear Information System (INIS)

    Objective: To study the in vitro immunoreactivity and in vivo tissue distribution, tumor targeting property of anti-human bladder tumor human-murine chimeric antibody (ch-BDI) labeled with 99Tcm and to investigate its possibility for being used in guiding diagnosis and guiding therapy of bladder cancer. Methods: The ch-BDI was labeled with 99Tcm by improved Schwarz method and the labeled antibody was purified by Sephadex G-50. Labeling yield and radiochemical purity were measured by paper chromatography. The immunoreactive fraction and association constant (Ka) were measured by Lindmo method and Scatchard analysis, respectively. 11.1 MBq (30 μg) 99Tcm-ch-BDI was intravenously injected into nude mice bearing human bladder cancer xenografts in the right thigh and radioimmunoimaging (RII) was performed 2, 6, 20 and 24 h postinjection. The images were processed by region of interest (ROI) method to acquire the counts of whole body and the tumor and the counts ratios of tumor to contralateral normal tissue or to tissues of other non-tumor bearing organs. The mice were killed after 24 h postinjection imaging and tissue distribution was measured. %ID/g and target to nontarget (T/NT) ratios were calculated. Results: The labeling yield and radiochemical purity of 99Tcm-ch-BDI were (66.5±7.3)% and >90%, respectively. The immunoreactive fraction was 76% and Ka was 3.56 x 109 L/mol. RII showed that the tumor was clearly visualized 6 h postinjection and becoming clearer along with time prolonging. The radioactivity of whole body decreased rapidly with time, whereas the radioactivity of the tumor decreased slowly. The T/NT ratios was increased with time. Biodistribution results showed that tumor uptake was 17.4%ID/g 24 h postinjection. T/NT ratios were very high except for the kidney. T/NT ratios for brain, muscle, intestinal wall, bone and heart wall were 136.0, 55.1, 39.3, 29.7 and 27.9, respectively. Conclusion: 99Tcm-ch-BDI exhibits excellent immunoreactivity and tumor

  5. Evaluation of hematopoietic chimerism fol-lowing allogenetic stem cell transplantation%异基因干细胞移植患者基因状态研究

    Institute of Scientific and Technical Information of China (English)

    张艳; 夏冬景; 何凤琴; 陈玲; 杨玉玲

    2013-01-01

    O bjective: To investigate the genotype diversity of the periph-eral blood cells,epithelial cells from oral mucosa and root-hair cells in re-cipients following transplantation of hematopoietic stem cells from the pe-ripheral blood.Methods: Polymerase chain reaction of short tandem re-peat sequences ( PCR-STR ) assay was performed for genotype of the blood,samples by oral swab and root-hair cells in 44 recipients.Results:The genotype of donor origin was commonly identified in 44 patients in samples from both peripheral blood and root-hair cells.In addition,16 of 44 subjects were appearance of recipient′s genotype for the mucosal epi-thelial cells and 28,coexistence of donor and host hematopoiesis ( mixed chimerism) .Conclusion: The transplanted allogeneic peripheral blood stem cells may convert into buccal epithelial cells , and this finding is of high value in diagnosis of engraftment failure in patients with malignancy after allogenetic stem cell transplantation and forensic case-work.%目的:应用PCR-STR技术,研究干细胞移植患者外周血、口腔黏膜上皮细胞及毛根的基因型变化。方法:对44例干细胞移植患者的血液、口腔拭子及毛根进行分型。结果:44例患者移植后外周血检出单一供者基因分型,毛根均检出单一受者基因分型,口腔黏膜上皮细胞有16例检出单一受者的基因分型,28例检出供者、受者的混合基因分型。结论:移植后外周血干细胞可以转变为口腔黏膜上皮细胞,这对干细胞治疗各种血液恶性疾病及法医学应用具有较高的参考价值。

  6. A chimeric 18L1-45RG1 virus-like particle vaccine cross-protects against oncogenic alpha-7 human papillomavirus types.

    Directory of Open Access Journals (Sweden)

    Bettina Huber

    Full Text Available Persistent infection with oncogenic human papillomaviruses (HPV types causes all cervical and a subset of other anogenital and oropharyngeal carcinomas. Four high-risk (hr mucosal types HPV16, 18, 45, or 59 cause almost all cervical adenocarcinomas (AC, a subset of cervical cancer (CxC. Although the incidence of cervical squamous cell carcinoma (SCC has dramatically decreased following introduction of Papanicolaou (PAP screening, the proportion of AC has relatively increased. Cervical SCC arise mainly from the ectocervix, whereas AC originate primarily from the endocervical canal, which is less accessible to obtain viable PAP smears. Licensed (bivalent and quadrivalent HPV vaccines comprise virus-like particles (VLP of the most important hr HPV16 and 18, self-assembled from the major capsid protein L1. Due to mainly type-restricted efficacy, both vaccines do not target 13 additional hr mucosal types causing 30% of CxC. The papillomavirus genus alpha species 7 (α7 includes a group of hr types of which HPV18, 45, 59 are proportionally overrepresented in cervical AC and only partially (HPV18 targeted by current vaccines. To target these types, we generated a chimeric vaccine antigen that consists of a cross-neutralizing epitope (homologue of HPV16 RG1 of the L2 minor capsid protein of HPV45 genetically inserted into a surface loop of HPV18 L1 VLP (18L1-45RG1. Vaccination of NZW rabbits with 18L1-45RG1 VLP plus alum-MPL adjuvant induced high-titer neutralizing antibodies against homologous HPV18, that cross-neutralized non-cognate hr α7 types HPV39, 45, 68, but not HPV59, and low risk HPV70 in vitro, and induced a robust L1-specific cellular immune response. Passive immunization protected mice against experimental vaginal challenge with pseudovirions of HPV18, 39, 45 and 68, but not HPV59 or the distantly related α9 type HPV16. 18L1-45RG1 VLP might be combined with our previously described 16L1-16RG1 VLP to develop a second generation bivalent

  7. Neurocognitive outcome of children exposed to perinatal mother-to-child Chikungunya virus infection: the CHIMERE cohort study on Reunion Island.

    Directory of Open Access Journals (Sweden)

    Patrick Gérardin

    2014-07-01

    Full Text Available Little is known about the neurocognitive outcome in children exposed to perinatal mother-to-child Chikungunya virus (p-CHIKV infection.The CHIMERE ambispective cohort study compared the neurocognitive function of 33 p-CHIKV-infected children (all but one enrolled retrospectively at around two years of age with 135 uninfected peers (all enrolled prospectively. Psychomotor development was assessed using the revised Brunet-Lezine scale, examiners blinded to infectious status. Development quotients (DQ with subscores covering movement/posture, coordination, language, sociability skills were calculated. Predictors of global neurodevelopmental delay (GND, DQ ≤ 85, were investigated using multivariate Poisson regression modeling. Neuroradiologic follow-up using magnetic resonance imaging (MRI scans was proposed for most of the children with severe forms.The mean DQ score was 86.3 (95%CI: 81.0-91.5 in infected children compared to 100.2 (95%CI: 98.0-102.5 in uninfected peers (P<0.001. Fifty-one percent (n = 17 of infected children had a GND compared to 15% (n = 21 of uninfected children (P<0.001. Specific neurocognitive delays in p-CHIKV-infected children were as follows: coordination and language (57%, sociability (36%, movement/posture (27%. After adjustment for maternal social situation, small for gestational age, and head circumference, p-CHIKV infection was found associated with GND (incidence rate ratio: 2.79, 95%CI: 1.45-5.34. Further adjustments on gestational age or breastfeeding did not change the independent effect of CHIKV infection on neurocognitive outcome. The mean DQ of p-CHIKV-infected children was lower in severe encephalopathic children than in non-severe children (77.6 versus 91.2, P<0.001. Of the 12 cases of CHIKV neonatal encephalopathy, five developed a microcephaly (head circumference <-2 standard deviations and four matched the definition of cerebral palsy. MRI scans showed severe restrictions of white matter areas

  8. Tumor-specific accumulation of 125I-labeled mouse-human chimeric anti-CEA antibody in a xenografted human cancer model demonstrated by whole-body autoradiography and immunostaining

    International Nuclear Information System (INIS)

    Whole-body autoradiography (WBAR) was used to study the biodistribution of 125I-labeled mouse-human chimeric antibody (Ch F11-39) to carcinoembryonic antigen (CEA) in athymic nude mice bearing the CEA-producing MKN-45 human gastric carcinoma xenografts. Significantly high uptake of 125I-Ch F11-39 in the tumors obtained by tissue-counting technique was confirmed by WBAR of mice of 12, 24, 48, and 96 h postinjection of 125I-Ch F11-39. When compared with histochemical or immunohistochemical staining results of the tumor tissue sections, imaging profiles of 125I-Ch F11-39 obtained by WBARs were topographically correlated with histopathological findings of tissues and immunohistochemical localization of CEA in the tumor tissues, indicating that the accumulation of 125I-Ch F11-39 at the tumor site is based on its specificity for CEA. These results demonstrate that this chimeric antibody may serve as a potential useful diagnostic and/or therapeutic reagent for human CEA-producing cancers

  9. Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors.

    Science.gov (United States)

    Thokala, Radhika; Olivares, Simon; Mi, Tiejuan; Maiti, Sourindra; Deniger, Drew; Huls, Helen; Torikai, Hiroki; Singh, Harjeet; Champlin, Richard E; Laskowski, Tamara; McNamara, George; Cooper, Laurence J N

    2016-01-01

    Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR's in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies. PMID:27548616

  10. Effects of T cell depletion in radiation bone marrow chimeras. I. Evidence for a donor cell population which increases allogeneic chimerism but which lacks the potential to produce GVHD

    International Nuclear Information System (INIS)

    The opposing problems of graft-vs-host disease (GVHD) and failure of alloengraftment present major obstacles to the application of bone marrow transplantation (BMT) across complete MHC barriers. The addition of syngeneic T-cell-depleted (TCD) bone marrow (BM) to untreated fully allogeneic marrow inocula in lethally irradiated mice has been previously shown to provide protection from GVHD. We have used this model to study the effects of allogeneic T cells on levels of chimerism in recipients of mixed marrow inocula. The results indicate that T cells in allogeneic BM inocula eliminate both coadministered recipient-strain and radioresistant host hematopoietic elements to produce complete allogeneic chimerism without clinical GVHD. To determine the role of GVH reactivity in this phenomenon, we performed similar studies in an F1 into parent combination, in which the genetic potential for GVHD is lacking. The presence of T cells in F1 marrow inocula led to predominant repopulation with F1 lymphocytes in such chimeras, even when coadministered with TCD-recipient-strain BM. These results imply that the ability of allogeneic BM cells removed by T cell depletion to increase levels of allochimerism may be mediated by a population which is distinct from that which produces GVHD. These results may have implications for clinical BM transplantation

  11. Chimeric virus-like particles containing a conserved region of the G protein in combination with a single peptide of the M2 protein confer protection against respiratory syncytial virus infection.

    Science.gov (United States)

    Qiao, Lei; Zhang, Yuan; Chai, Feng; Tan, Yiluo; Huo, Chunling; Pan, Zishu

    2016-07-01

    To investigate the feasibility and efficacy of a virus-like particle (VLP) vaccine composed of the conserved antigenic epitopes of respiratory syncytial virus (RSV), the chimeric RSV VLPs HBcΔ-tG and HBcΔ-tG/M282-90 were generated based on the truncated hepatitis B virus core protein (HBcΔ). HBcΔ-tG consisted of HBcΔ, the conserved region (aa 144-204) of the RSV G protein. HBcΔ-tG was combined with a single peptide (aa 82-90) of the M2 protein to generate HBcΔ-tG/M282-90. Immunization of mice with the HBcΔ-tG or HBcΔ-tG/M282-90 VLPs elicited RSV-specific IgG and neutralizing antibody production and conferred protection against RSV infection. Compared with HBcΔ-tG, HBcΔ-tG/M282-90 induced decreased Th2 cytokine production (IL-4 and IL-5), increased Th1 cytokine response (IFN-γ, TNF-α, and IL-2), and increased ratios of IgG2a/IgG1 antibodies, thereby relieving pulmonary pathology upon subsequent RSV infection. Our results demonstrated that chimeric HBcΔ-tG/M282-90 VLPs represented an effective RSV subunit vaccine candidate. PMID:27154395

  12. Tyrosine kinase activity of a chimeric insulin-like-growth-factor-1 receptor containing the insulin receptor C-terminal domain. Comparison with the tyrosine kinase activities of the insulin and insulin-like-growth-factor-1 receptors using a cell-free system.

    Science.gov (United States)

    Mothe, I; Tartare, S; Kowalski-Chauvel, A; Kaliman, P; Van Obberghen, E; Ballotti, R

    1995-03-15

    In a previous study, we showed that a chimeric insulin-like-growth-factor-1 (IGF-1) receptor, with the beta subunit C-terminal part of the insulin receptor was more efficient in stimulating glycogen synthesis and p44mapk activity compared to the wild-type IFG-1 receptor [Tartare, S., Mothe, I., Kowalski-Chauvel, A., Breittmayer, J.-P., Ballotti, R. & Van Obberghen, E. (1994) J. Biol. Chem. 269, 11449-11455]. These data indicate that the receptor C-terminal domain plays an important role in the transmission of biological effects. To understand the molecular basis of the differences in receptor specificity, we studied the characteristics of insulin, IGF-1 and chimeric receptor tyrosine kinase activities in a cell-free system. We found that, compared to wild-type insulin and IGF-1 receptors, the chimeric receptor showed a decrease in (a) autophosphorylation, (b) tyrosine kinase activity towards insulin receptor substrate-1 and the insulin receptor-(1142-1158)-peptide, and (c) the ability to activate phosphatidylinositol 3-kinase. However, for all the effects measured in a cell-free system, the chimeric receptor displayed an increased response to IGF-1 compared to the native IGF-1 receptor. Concerning the cation dependence of the tyrosine kinase activity, we showed that, at 10 mM Mg2+, the ligand-stimulated phosphorylation of poly(Glu80Tyr20) by both insulin receptor and chimeric receptor was increased by Mn2+. Conversely at 50 mM Mg2+, the chimeric receptor behaved like the IGF-1 receptor, since the presence of Mn2+ decreased the stimulatory effect of IGF-1 on their kinase activity. Furthermore, the Km of the chimeric receptor for ATP was increased compared to the wild-type receptors. These data demonstrate that the replacement of the C-terminal tail of the IGF-1 receptor by that of the insulin receptor has changed the receptor characteristics studied in a cell-free system. Our findings indicate that the C-terminal domain of the insulin receptor beta subunit plays a

  13. New concepts for the comparison of tropospheric NO2 column densities derived from car-MAX-DOAS observations, OMI satellite observations and the regional model CHIMERE during two MEGAPOLI campaigns in Paris 2009/10

    Directory of Open Access Journals (Sweden)

    R. Shaiganfar

    2015-03-01

    Full Text Available We compare tropospheric column densities (vertically integrated concentrations of NO2 from three data sets for the metropolitan area of Paris during two extensive measurement campaigns (25 days in summer 2009 and 29 days in winter 2010 within the European research project MEGAPOLI. The selected data sets comprise a regional chemical transport model (CHIMERE as well as two observational data sets: ground based mobile Multi-AXis-Differential Optical Absorption Spectroscopy (car-MAX-DOAS measurements and satellite measurements from the Ozone Monitoring Instrument (OMI. On most days, car-MAX-DOAS measurements were carried out along large circles (diameter ~35 km around Paris. The car-MAX-DOAS results are compared to coincident data from CHIMERE and OMI. All three data sets have their specific strengths and weaknesses, especially with respect to their spatio-temporal resolution and coverage as well as their uncertainties. Thus we compare them in two different ways: first, we simply consider the original data sets. Second, we compare modified versions making synergistic use of the complementary information from different data sets. For example, profile information from the regional model is used to improve the satellite data, observations of the horizontal trace gas distribution are used to adjust the respective spatial patterns of the model simulations, or the model is used as a transfer tool to bridge the spatial scales between car-MAX-DOAS and satellite observations. Using the modified versions of the data sets, the comparison results substantially improve compared to the original versions. In general, good agreement between the data sets is found outside the emission plume, but inside the emission plumes the tropospheric NO2 VCDs are systematically underestimated by the CHIMERE model and the satellite observations (compared to the car-MAX-DOAS observations. One major result from our study is that for satellite validation close to strong emission

  14. New concepts for the comparison of tropospheric NO2 column densities derived from car-MAX-DOAS observations, OMI satellite observations and the regional model CHIMERE during two MEGAPOLI campaigns in Paris 2009/10

    Science.gov (United States)

    Shaiganfar, R.; Beirle, S.; Petetin, H.; Zhang, Q.; Beekmann, M.; Wagner, T.

    2015-07-01

    We compare tropospheric column densities (vertically integrated concentrations) of NO2 from three data sets for the metropolitan area of Paris during two extensive measurement campaigns (25 days in summer 2009 and 29 days in winter 2010) within the European research project MEGAPOLI. The selected data sets comprise a regional chemical transport model (CHIMERE) as well as two observational data sets: ground-based mobile Multi-AXis-Differential Optical Absorption Spectroscopy (car-MAX-DOAS) measurements and satellite measurements from the Ozone Monitoring Instrument (OMI). On most days, car-MAX-DOAS measurements were carried out along large circles (diameter ~ 35 km) around Paris. The car-MAX-DOAS results are compared to coincident data from CHIMERE and OMI. All three data sets have their specific strengths and weaknesses, especially with respect to their spatiotemporal resolution and coverage as well as their uncertainties. Thus we compare them in two different ways: first, we simply consider the original data sets. Second, we compare modified versions making synergistic use of the complementary information from different data sets. For example, profile information from the regional model is used to improve the satellite data, observations of the horizontal trace gas distribution are used to adjust the respective spatial patterns of the model simulations, or the model is used as a transfer tool to bridge the spatial scales between car-MAX-DOAS and satellite observations. Using the modified versions of the data sets, the comparison results substantially improve compared to the original versions. In general, good agreement between the data sets is found outside the emission plume, but inside the emission plumes the tropospheric NO2 vertical column densities (VCDs). are systematically underestimated by the CHIMERE model and the satellite observations (compared to the car-MAX-DOAS observations). One major result from our study is that for satellite validation close to

  15. Characterization of hepatitis C virus recombinants with chimeric E1/E2 envelope proteins and identification of single amino acids in the E2 stem region important for entry

    DEFF Research Database (Denmark)

    Carlsen, Thomas H R; Scheel, Troels K H; Ramirez, Santseharay;

    2013-01-01

    . For recovered 1b-E2 recombinants, single E2 stem region amino acid changes were identified at residues 706, 707, and 710. In reverse genetic studies, these mutations increased infectivity titers by ~100-fold, apparently without influencing particle stability or cell binding although introducing slight...... decrease in particle density. In addition, the 1b-E2 exchange led to a decrease in secreted core protein of 25 to 50%, which was further reduced by the E2 stem region mutations. These findings indicated that compensatory mutations permitted robust infectious virus production, without increasing assembly....../release. Studies of E1/E2 heterodimerization showed no differences in intracellular E1/E2 interaction for chimeric constructs with or without E2 stem region mutations. Interestingly, the E2 stem region mutations allowed efficient entry, which was verified in 1a-E1/1b-E2 HCV pseudoparticle assays. A CD81 inhibition...

  16. In Vitro and In Vivo Comparison of Lymphocytes Transduced with a Human CD16 or with a Chimeric Antigen Receptor Reveals Potential Off-Target Interactions due to the IgG2 CH2-CH3 CAR-Spacer

    Directory of Open Access Journals (Sweden)

    Béatrice Clémenceau

    2015-01-01

    Full Text Available The present work was designed to compare two mechanisms of cellular recognition based on Ab specificity: firstly, when the anti-HER2 mAb trastuzumab bridges target cells and cytotoxic lymphocytes armed with a Fc receptor (ADCC and, secondly, when HER2 positive target cells are directly recognized by cytotoxic lymphocytes armed with a chimeric antigen receptor (CAR. To compare these two mechanisms, we used the same cellular effector (NK-92 and the same signaling domain (FcεRIγ. The NK-92 cytotoxic cell line was transfected with either a FcγRIIIa-FcεRIγ (NK-92CD16 or a trastuzumab-based scFv-FcεRIγ chimeric receptor (NK-92CAR. In vitro, the cytotoxic activity against HER2 positive target cells after indirect recognition by NK-92CD16 was always inferior to that observed after direct recognition by NK-92CAR. In contrast, and somehow unexpectedly, in vivo, adoptive transfer of NK-92CD16 + trastuzumab but not of NK-92CAR induced tumor regression. Analysis of the in vivo xenogeneic system suggested that the human CH2-CH3 IgG2 used as a spacer in our construct was able to interact with the FcR present at the cell surface of the few NSG-FcR+ remaining immune cells. This interaction, leading to blockage of the NK-92CAR in the periphery of the engrafted tumor cells, stresses the critical role of the composition of the spacer domain.

  17. The chimeric ubiquitin ligase SH2-U-box inhibits the growth of imatinib-sensitive and resistant CML by targeting the native and T315I-mutant BCR-ABL.

    Science.gov (United States)

    Ru, Yi; Wang, Qinhao; Liu, Xiping; Zhang, Mei; Zhong, Daixing; Ye, Mingxiang; Li, Yuanchun; Han, Hua; Yao, Libo; Li, Xia

    2016-01-01

    Chronic myeloid leukemia (CML) is characterized by constitutively active fusion protein tyrosine kinase BCR-ABL. Although the tyrosine kinase inhibitor (TKI) against BCR-ABL, imatinib, is the first-line therapy for CML, acquired resistance almost inevitably emerges. The underlying mechanism are point mutations within the BCR-ABL gene, among which T315I is notorious because it resists to almost all currently available inhibitors. Here we took use of a previously generated chimeric ubiquitin ligase, SH2-U-box, in which SH2 from the adaptor protein Grb2 acts as a binding domain for activated BCR-ABL, while U-box from CHIP functions as an E3 ubiquitin ligase domain, so as to target the ubiquitination and degradation of both native and T315I-mutant BCR-ABL. As such, SH2-U-box significantly inhibited proliferation and induced apoptosis in CML cells harboring either the wild-type or T315I-mutant BCR-ABL (K562 or K562R), with BCR-ABL-dependent signaling pathways being repressed. Moreover, SH2-U-box worked in concert with imatinib in K562 cells. Importantly, SH2-U-box-carrying lentivirus could markedly suppress the growth of K562-xenografts in nude mice or K562R-xenografts in SCID mice, as well as that of primary CML cells. Collectively, by degrading the native and T315I-mutant BCR-ABL, the chimeric ubiquitin ligase SH2-U-box may serve as a potential therapy for both imatinib-sensitive and resistant CML. PMID:27329306

  18. CD19 chimeric antigen receptor (CD19 CAR)-redirected adoptive T-cell immunotherapy for the treatment of relapsed or refractory B-cell Non-Hodgkin’s Lymphomas

    Science.gov (United States)

    Onea, Alexandra S; Jazirehi, Ali R

    2016-01-01

    Recovery rates for B-cell Non-Hodgkin’s Lymphoma (NHL) are up to 70% with current standard-of-care treatments including rituximab (chimeric anti-CD20 monoclonal antibody) in combination with chemotherapy (R-CHOP). However, patients who do not respond to first-line treatment or develop resistance have a very poor prognosis. This signifies the need for the development of an optimal treatment approach for relapsed/refractory B-NHL. Novel CD19- chimeric antigen receptor (CAR) T-cell redirected immunotherapy is an attractive option for this subset of patients. Anti-CD19 CAR T-cell therapy has already had remarkable efficacy in various leukemias as well as encouraging outcomes in phase I clinical trials of relapsed/refractory NHL. In going forward with additional clinical trials, complementary treatments that may circumvent potential resistance mechanisms should be used alongside anti-CD19 T-cells in order to prevent relapse with resistant strains of disease. Some such supplementary tactics include conditioning with lymphodepletion agents, sensitizing with kinase inhibitors and Bcl-2 inhibitors, enhancing function with multispecific CAR T-cells and CD40 ligand-expressing CAR T-cells, and safeguarding with lymphoma stem cell-targeted treatments. A therapy regimen involving anti-CD19 CAR T-cells and one or more auxiliary treatments could dramatically improve prognoses for patients with relapsed/refractory B-cell NHL. This approach has the potential to revolutionize B-NHL salvage therapy in much the same way rituximab did for first-line treatments. PMID:27186412

  19. High-dose radioimmunotherapy in refractory b-celI non-Hodgikin's lymphoma with I-131-labeled chimeric anti CD-20 C2B8 (I-131 rituximab): pilot trial

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sung Eun; Park, Yeon Hee; Cheon, Gi Jeong; Ryoo, Baek Yeol; Lee, Seung Sook; Choi, Chang Woon; Lim, Sang Moo [Korea Institute of Radiological and Medical Science, Seoul (Korea, Republic of)

    2004-07-01

    The native chimeric human-mouse anti CD-20 antibody IDEC-C2B8 (rituximab) is therapeutically applied in relapsed or refractory NHL. This ongoing pilot study was to evaluate whether high-dose radioimmunotherapy (RIT) with I-131 rituximab is therapeutically effective in refractory B-cell NHL. 5 patients (5 male, aged 50.89{+-}16.89) with chemorefractory NHL of B-cell origin (2 diffuse large B cell, 1 burkitt's lymphoma, and 2 mantle cell Iymphoma) oe, with a life expectancy of at least 3 months, and with a Kamofsky performance score of 60 and above were studied. The chimeric IgG1 anti CD 20 monoclonal antibody rituximab (mabthera, Roche) was radiolabelled with iodine-131 (I-131) using a modified chloaramine T method with high radiochemical purity (95%{+-}0.9) and preservation of immunoreactivity. All patients received therapeutic loading doses of unlabelled rituximab (18.5 MBq/kg) immediately prior to administration of therapeutic dose (3.7 GBq-8.5 GBq), and then underwent gamma camera scan and pre-and post-RIT FDG PET (within 7 day and day 30). Blood cell nadirs were reached at 2-3 weeks after therapy infusion, but all patients recovered at 6 weeks after treatment. Non hematologic toxicity was restricted to mild-to moderate nausea, fever, transient bilirubin, or liver enzyme elevation. Two (8.5 GBq) with mantle cell lymphoma and one with burkitt's lymphoma experienced good partial remissions, and one (5.5 GBq, DLBL) with bulky disease had a partial remission, and one patient (3.7 GBq, DLBL) with bulky disease had a mixed response. High-dose RIT with I-131 labelled rituximab seems to be effective and moderate toxicity. Further follow-up to monitor the long-term outcome are indicated.

  20. Reversion of the Arabidopsis rpn12a-1 exon-trap mutation by an intragenic suppressor that weakens the chimeric 5’ splice site [v1; ref status: indexed, http://f1000r.es/vz

    Directory of Open Access Journals (Sweden)

    Jasmina Kurepa

    2013-02-01

    Full Text Available Background: In the Arabidopsis 26S proteasome mutant rpn12a-1, an exon-trap T-DNA is inserted 531 base pairs downstream of the RPN12a STOP codon. We have previously shown that this insertion activates a STOP codon-associated latent 5' splice site that competes with the polyadenylation signal during processing of the pre-mRNA. As a result of this dual input from splicing and polyadenylation in the rpn12a-1 mutant, two RPN12a transcripts are produced and they encode the wild-type RPN12a and a chimeric RPN12a-NPTII protein. Both proteins form complexes with other proteasome subunits leading to the formation of wild-type and mutant proteasome versions. The net result of this heterogeneity of proteasome particles is a reduction of total cellular proteasome activity. One of the consequences of reduced proteasomal activity is decreased sensitivity to the major plant hormone cytokinin. Methods: We performed ethyl methanesulfonate mutagenesis of rpn12a-1 and isolated revertants with wild-type cytokinin sensitivity. Results: We describe the isolation and analyses of suppressor of rpn12a-1 (sor1. The sor1 mutation is intragenic and located at the fifth position of the chimeric intron. This mutation weakens the activated 5' splice site associated with the STOP codon and tilts the processing of the RPN12a mRNA back towards polyadenylation. Conclusions: These results validate our earlier interpretation of the unusual nature of the rpn12a-1 mutation. Furthermore, the data show that optimal 26S proteasome activity requires RPN12a accumulation beyond a critical threshold. Finally, this finding reinforces our previous conclusion that proteasome function is critical for the cytokinin regulation of plant growth.