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Sample records for chimeric mice provide

  1. 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). ...

  2. Chimeric mice with a humanized liver as an animal model of troglitazone-induced liver injury.

    Science.gov (United States)

    Kakuni, Masakazu; Morita, Mayu; Matsuo, Kentaro; Katoh, Yumiko; Nakajima, Miki; Tateno, Chise; Yokoi, Tsuyoshi

    2012-10-01

    Troglitazone (Tro) is a thiazolidinedione antidiabetic drug that was withdrawn from the market due to its association with idiosyncratic severe liver injury. Tro has never induced liver injury in experimental animals in vivo. It was assumed that the species differences between human and experimental animals in the pharmaco- or toxicokinetics of Tro might be associated with these observations. In this study, we investigated whether a chimeric mouse with a humanized liver that we previously established, whose replacement index with human hepatocytes is up to 92% can reproduce Tro-induced liver injury. When the chimeric mice were orally administered Tro for 14 or 23 days (1000mg/kg/day), serum alanine aminotransferase (ALT) was significantly increased by 2.1- and 3.6-fold, respectively. Co-administration of l-buthionine sulfoximine (10mM in drinking water), an inhibitor of glutathione (GSH) synthesis, unexpectedly prevented the Tro-dependent increase of ALT, which suggests that the GSH scavenging pathway will not be involved in Tro-induced liver injury. To elucidate the mechanism of the onset of liver injury, hepatic GSH content, the level of oxidative stress markers and phase I and phase II drug metabolizing enzymes were determined. However, these factors were not associated with Tro-induced liver injury. An immune-mediated reaction may be associated with Tro-induced liver toxicity in vivo, because the chimeric mouse is derived from an immunodeficient SCID mouse. In conclusion, we successfully reproduced Tro-induced liver injury using chimeric mice with a humanized liver, which provides a new animal model for studying idiosyncratic drug-induced liver injury.

  3. Fialuridine induces acute liver failure in chimeric TK-NOG mice: a model for detecting hepatic drug toxicity prior to human testing.

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    Dan Xu

    2014-04-01

    Full Text Available BACKGROUND: Seven of 15 clinical trial participants treated with a nucleoside analogue (fialuridine [FIAU] developed acute liver failure. Five treated participants died, and two required a liver transplant. Preclinical toxicology studies in mice, rats, dogs, and primates did not provide any indication that FIAU would be hepatotoxic in humans. Therefore, we investigated whether FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice with humanized livers. METHODS AND FINDINGS: Control and chimeric TK-NOG mice with humanized livers were treated orally with FIAU 400, 100, 25, or 2.5 mg/kg/d. The response to drug treatment was evaluated by measuring plasma lactate and liver enzymes, by assessing liver histology, and by electron microscopy. After treatment with FIAU 400 mg/kg/d for 4 d, chimeric mice developed clinical and serologic evidence of liver failure and lactic acidosis. Analysis of liver tissue revealed steatosis in regions with human, but not mouse, hepatocytes. Electron micrographs revealed lipid and mitochondrial abnormalities in the human hepatocytes in FIAU-treated chimeric mice. Dose-dependent liver toxicity was detected in chimeric mice treated with FIAU 100, 25, or 2.5 mg/kg/d for 14 d. Liver toxicity did not develop in control mice that were treated with the same FIAU doses for 14 d. In contrast, treatment with another nucleotide analogue (sofosbuvir 440 or 44 mg/kg/d po for 14 d, which did not cause liver toxicity in human trial participants, did not cause liver toxicity in mice with humanized livers. CONCLUSIONS: FIAU-induced liver toxicity could be readily detected using chimeric TK-NOG mice with humanized livers, even when the mice were treated with a FIAU dose that was only 10-fold above the dose used in human participants. The clinical features, laboratory abnormalities, liver histology, and ultra-structural changes observed in FIAU-treated chimeric mice mirrored those of FIAU-treated human participants. The use

  4. Modeling cognition and disease using human glial chimeric mice

    DEFF Research Database (Denmark)

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

    2015-01-01

    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......As new methods for producing and isolating human glial progenitor cells (hGPCs) have been developed, the disorders of myelin have become especially compelling targets for cell-based therapy. Yet as animal modeling of glial progenitor cell-based therapies has progressed, it has become clear...... that transplanted hGPCs not only engraft and expand within murine hosts, but dynamically outcompete the resident progenitors so as to ultimately dominate the host brain. The engrafted human progenitor cells proceed to generate parenchymal astrocytes, and when faced with a hypomyelinated environment...

  5. 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...

  6. Pharmacokinetics and effects on serum cholinesterase activities of organophosphorus pesticides acephate and chlorpyrifos in chimeric mice transplanted with human hepatocytes.

    Science.gov (United States)

    Suemizu, Hiroshi; Sota, Shigeto; Kuronuma, Miyuki; Shimizu, Makiko; Yamazaki, Hiroshi

    2014-11-01

    Organophosphorus pesticides acephate and chlorpyrifos in foods have potential to impact human health. The aim of the current study was to investigate the pharmacokinetics of acephate and chlorpyrifos orally administered at lowest-observed-adverse-effect-level doses in chimeric mice transplanted with human hepatocytes. Absorbed acephate and its metabolite methamidophos were detected in serum from wild type mice and chimeric mice orally administered 150mg/kg. Approximately 70% inhibition of cholinesterase was evident in plasma of chimeric mice with humanized liver (which have higher serum cholinesterase activities than wild type mice) 1day after oral administrations of acephate. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated plasma concentrations of acephate and chlorpyrifos in humans were consistent with reported concentrations. Acephate cleared similarly in humans and chimeric mice but accidental/incidental overdose levels of chlorpyrifos cleared (dependent on liver metabolism) more slowly from plasma in humans than it did in mice. The data presented here illustrate how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of toxicological potential of organophosphorus pesticides.

  7. Generation of Novel Chimeric Mice with Humanized Livers by Using Hemizygous cDNA-uPA/SCID Mice.

    Science.gov (United States)

    Tateno, Chise; Kawase, Yosuke; Tobita, Yoshimi; Hamamura, Satoko; Ohshita, Hiroki; Yokomichi, Hiroshi; Sanada, Harumi; Kakuni, Masakazu; Shiota, Akira; Kojima, Yuha; Ishida, Yuji; Shitara, Hiroshi; Wada, Naoko A; Tateishi, Hiromi; Sudoh, Masayuki; Nagatsuka, Shin-Ichiro; Jishage, Kou-Ichi; Kohara, Michinori

    2015-01-01

    We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression-not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for

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

    Directory of Open Access Journals (Sweden)

    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.

  9. 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.

  10. Mice chronically infected with chimeric HIV resist peripheral and brain superinfection: a model of protective immunity to HIV.

    Science.gov (United States)

    Kelschenbach, Jennifer L; Saini, Manisha; Hadas, Eran; Gu, Chao-Jiang; Chao, Wei; Bentsman, Galina; Hong, Jessie P; Hanke, Tomas; Sharer, Leroy R; Potash, Mary Jane; Volsky, David J

    2012-06-01

    Infection by some viruses induces immunity to reinfection, providing a means to identify protective epitopes. To investigate resistance to reinfection in an animal model of HIV disease and its control, we employed infection of mice with chimeric HIV, EcoHIV. When immunocompetent mice were infected by intraperitoneal (IP) injection of EcoHIV, they resisted subsequent secondary infection by IP injection, consistent with a systemic antiviral immune response. To investigate the potential role of these responses in restricting neurotropic HIV infection, we established a protocol for efficient EcoHIV expression in the brain following intracranial (IC) inoculation of virus. When mice were inoculated by IP injection and secondarily by IC injection, they also controlled EcoHIV replication in the brain. To investigate their role in EcoHIV antiviral responses, CD8+ T lymphocytes were isolated from spleens of EcoHIV infected and uninfected mice and adoptively transferred to isogenic recipients. Recipients of EcoHIV primed CD8+ cells resisted subsequent EcoHIV infection compared to recipients of cells from uninfected donors. CD8+ spleen cells from EcoHIV-infected mice also mounted modest but significant interferon-γ responses to two HIV Gag peptide pools. These findings suggest EcoHIV-infected mice may serve as a useful system to investigate the induction of anti-HIV protective immunity for eventual translation to human beings.

  11. 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

  12. 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.

  13. 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

  14. An H1-H3 chimeric influenza virosome confers complete protection against lethal challenge with PR8 (H1N1) and X47 (H3N2) viruses in mice.

    Science.gov (United States)

    Abdoli, Asghar; Soleimanjahi, Hoorieh; Tavassoti Kheiri, Masoumeh; Jamali, Abbas; Mazaheri, Vahideh; Abdollahpour Alitappeh, Meghdad

    2014-12-01

    Annual health threats and economic damages caused by influenza virus are still a main concern of the World Health Organization and other health departments all over the world. An influenza virosome is a highly efficient immunomodulating carrier mimicking the natural antigen presentation pathway and has shown an excellent tolerability profile due to its biocompatibility and purity. The major purpose of this study was to construct a new chimeric virosome influenza vaccine containing hemagglutinin (HA) and neuraminidase (NA) proteins derived from the A/PR/8/1934 (H1N1) (PR8) and A/X/47 (H3N2) (X47) viruses, and to evaluate its efficacy as a vaccine candidate in mice. A single intramuscular vaccination with the chimeric virosomes provided complete protection against lethal challenge with the PR8 and X47 viruses. The chimeric virosomes induced high IgG antibody responses as well as hemagglutination inhibition (HAI) titers. HAI titers following the chimeric virosome vaccination were at the same level as the whole inactivated influenza vaccine. Mice immunized with the chimeric virosomes displayed considerably less weight loss and exhibited significantly reduced viral load in their lungs compared with the controls. The chimeric virosomes can be used as an innovative vaccine formulation to confer protection against a broad range of influenza viruses.

  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. Immunogenicity and therapeutic effects of Ag85A/B chimeric DNA vaccine in mice infected with Mycobacterium tuberculosis.

    Science.gov (United States)

    Liang, Yan; Wu, Xueqiong; Zhang, Junxian; Xiao, Li; Yang, Yourong; Bai, Xuejuan; Yu, Qi; Li, Zhongming; Bi, Lan; Li, Ning; Wu, Xiaoli

    2012-12-01

    The situation of tuberculosis (TB) is very severe in China. New therapeutic agents or regimens to treat TB are urgently needed. In this study, Mycobacterium tuberculosis-infected mice were given immunotherapy intramuscularly with Ag85A/B chimeric DNA or saline, plasmid vector pVAX1, or Mycobacterium vaccae vaccine. The mice treated with Ag85A/B chimeric DNA showed significantly higher numbers of T cells secreting interferon-gamma (IFN-γ), more IFN-γ in splenocyte culture supernatant, more Th1 and Tc1 cells, and higher ratios of Th1/Th2 and Tc1/Tc2 cells in whole blood, indicating a predominant Th1 immune response to treatment. Infected mice treated with doses of 100 μg Ag85A/B chimeric DNA had an extended time until death of 50% of the animals that was markedly longer than the saline and vector control groups, and the death rate at 1 month after the last dose was lower than that in the other groups. Compared with the saline group, 100 μg Ag85A/B chimeric DNA and 100 μg Ag85A DNA reduced the pulmonary bacterial loads by 0.79 and 0.45 logs, and the liver bacterial loads by 0.52 and 0.50 logs, respectively. Pathological changes in the lungs were less, and the lesions were more limited. These results show that Ag85A/B chimeric DNA was effective for the treatment of TB, significantly increasing the cellular immune response and inhibiting the growth of M. tuberculosis.

  17. HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice.

    Science.gov (United States)

    Holloway, Terrell; Moreno, Jose L; González-Maeso, Javier

    2016-01-01

    The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis(1,2). Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice(3,4). Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs(3). Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice(5). These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2ΔTM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs(6). PMID:27501227

  18. 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...... 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 immunization of the CVPs...... also generated CPMV- and FnBP-specific serum IgG; however, these titers were significantly lower and more variable than those generated by the intranasal route, and FnBP-specific intestinal Ig A was undetectable. Neither the ISCOM matrix nor cholera toxin enhanced these responses. These studies...

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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 ...

  5. 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

  6. A competitive advantage by neonatally engrafted human glial progenitors yields mice whose brains are chimeric for human glia

    DEFF Research Database (Denmark)

    Windrem, Martha S; Schanz, Steven J; Morrow, Carolyn;

    2014-01-01

    Neonatally transplanted human glial progenitor cells (hGPCs) densely engraft and myelinate the hypomyelinated shiverer mouse. We found that, in hGPC-xenografted mice, the human donor cells continue to expand throughout the forebrain, systematically replacing the host murine glia. The differentiat......Neonatally transplanted human glial progenitor cells (hGPCs) densely engraft and myelinate the hypomyelinated shiverer mouse. We found that, in hGPC-xenografted mice, the human donor cells continue to expand throughout the forebrain, systematically replacing the host murine glia....... The differentiation of the donor cells is influenced by the host environment, such that more donor cells differentiated as oligodendrocytes in the hypomyelinated shiverer brain than in myelin wild-types, in which hGPCs were more likely to remain as progenitors. Yet in each recipient, both the number and relative...... and ultimately replaced the host population of mouse GPCs, ultimately generating mice with a humanized glial progenitor population. These human glial chimeric mice should permit us to define the specific contributions of glia to a broad variety of neurological disorders, using human cells in vivo....

  7. 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)...

  8. 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

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

    OpenAIRE

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

    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...

  10. The peripheral chimerism of bone marrow-derived stem cells after transplantation: regeneration of gastrointestinal tissues in lethally irradiated mice.

    Science.gov (United States)

    Filip, Stanislav; Mokrý, Jaroslav; Vávrová, Jiřina; Sinkorová, Zuzana; Mičuda, Stanislav; Sponer, Pavel; Filipová, Alžběta; Hrebíková, Hana; Dayanithi, Govindan

    2014-05-01

    Bone marrow-derived cells represent a heterogeneous cell population containing haematopoietic stem and progenitor cells. These cells have been identified as potential candidates for use in cell therapy for the regeneration of damaged tissues caused by trauma, degenerative diseases, ischaemia and inflammation or cancer treatment. In our study, we examined a model using whole-body irradiation and the transplantation of bone marrow (BM) or haematopoietic stem cells (HSCs) to study the repair of haematopoiesis, extramedullary haematopoiesis and the migration of green fluorescent protein (GFP(+)) transplanted cells into non-haematopoietic tissues. We investigated the repair of damage to the BM, peripheral blood, spleen and thymus and assessed the ability of this treatment to induce the entry of BM cells or GFP(+) lin(-) Sca-1(+) cells into non-haematopoietic tissues. The transplantation of BM cells or GFP(+) lin(-) Sca-1(+) cells from GFP transgenic mice successfully repopulated haematopoiesis and the haematopoietic niche in haematopoietic tissues, specifically the BM, spleen and thymus. The transplanted GFP(+) cells also entered the gastrointestinal tract (GIT) following whole-body irradiation. Our results demonstrate that whole-body irradiation does not significantly alter the integrity of tissues such as those in the small intestine and liver. Whole-body irradiation also induced myeloablation and chimerism in tissues, and induced the entry of transplanted cells into the small intestine and liver. This result demonstrates that grafted BM cells or GFP(+) lin(-) Sca-1(+) cells are not transient in the GIT. Thus, these transplanted cells could be used for the long-term treatment of various pathologies or as a one-time treatment option if myeloablation-induced chimerism alone is not sufficient to induce the entry of transplanted cells into non-haematopoietic tissues.

  11. Construction and characterization of a recombinant fowlpox virus containing HIV-1 multi-epitope-p24 chimeric gene in mice

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The epidemic of HIV/AIDS is sweeping across the world. It is of great importance to figure out new ways to curb this disease. Epitope-based vaccine is one of these solutions. In this study, a chimeric gene was obtained by combination of a designed HIV-1 multi-epitope gene (MEG) and HIV-1 p24 gene. A re- combinant plasmid pUTA2-MEGp24 was then constructed by inserting MEGp24 gene into the down- stream of the promoter (ATI-P7.5×20) of fowlpox virus (FPV) transfer vector pUTA2. The recombinant plasmid and wild-type FPV 282E4 strain were then co-transfected into CEF cells and homologous re- combination occurred. A recombinant virus expressing HIV-1 protein MEGp24 was screened by ge- nome PCR and Western blot assay. Large scale preparation and purification of the recombinant fowl- pox virus (rFPV) were then carried out. BALB/c mice were immunized intramuscularly with the rFPV for three times on day 0, 14 and 42. Mice were executed and sampled one week after the third inoculation. Anti-HIV-1 antibody in serum and Th1 cytokines in the supernatant of cultured spleen cells were as- sayed by ELISA. The count of T lymphocyte subsets and the CTL activity of spleen lymphocytes were analyzed by flow cytometry and lactate dehydrogenase (LDH) release assay, respectively. The results showed that HIV-1 specific antibody in serum and increased T lymphocyte subsets (CD4+ T, CD8+ T) were detected in the immunization group. CTL target-killing activity and higher secretion of Th1 cyto- kines (IFN-γ and IL-2) of spleen lymphocytes stimulated by H-2d-restricted CTL peptide were observed in immunized mice. We concluded that the rFPV may induce HIV-1 specific immunity especially cellular immunity in mice.

  12. Construction and characterization of a recombinant fowlpox virus containing HIV-1 multi-epitope-p24 chimeric gene in mice

    Institute of Scientific and Technical Information of China (English)

    ZHANG LiShu; JIN NingYi; SONG YingJin; WANG Hong; MA HeWen; LI ZiJian; JIANG WenZheng

    2007-01-01

    The epidemic of HIV/AIDS is sweeping across the world. It is of great importance to figure out new ways to curb this disease. Epitope-based vaccine is one of these solutions. In this study, a chimeric gene was obtained by combination of a designed HIV-1 multi-epitope gene (MEG) and HIV-1 p24 gene. A recombinant plasmid pUTA2-MEGp24 was then constructed by inserting MEGp24 gene into the down-stream of the promoter (ATI-P7.5×20) of fowlpox virus (FPV) transfer vector pUTA2. The recombinant plasmid and wild-type FPV 282E4 strain were then co-transfected into CEF cells and homologous recombination occurred. A recombinant virus expressing HIV-1 protein MEGp24 was screened by genome PCR and Western blot assay. Large scale preparation and purification of the recombinant fowlpox virus (rFPV) were then carried out. BALB/c mice were immunized intramuscularly with the rFPV for three times on day 0, 14 and 42. Mice were executed and sampled one week after the third inoculation.Anti-HIV-1 antibody in serum and Th1 cytokines in the supernatant of cultured spleen cells were assayed by ELISA. The count of T lymphocyte subsets and the CTL activity of spleen lymphocytes were analyzed by flow cytometry and lactate dehydrogenase (LDH) release assay, respectively. The results showed that HIV-1 specific antibody in serum and increased T lymphocyte subsets (CD4+ T, CD8+ T)were detected in the immunization group. CTL target-killing activity and higher secretion of Th1 cytokines (IFN-Y and IL-2) of spleen lymphocytes stimulated by H-2d-restricted CTL peptide were observed in immunized mice.We concluded that the rFPV may induce HIV-1 specific immunity especially cellular immunity in mice.

  13. Pain regulation of endokinin A/B or endokinin C/D on chimeric peptide MCRT in mice.

    Science.gov (United States)

    He, Chunbo; Gong, Junbin; Yang, Lixia; Zhang, Hongwei; Dong, Shouliang; Zhou, Lanxia

    2016-09-01

    The present study focused on the interactive pain regulation of endokinin A/B (EKA/B, the common C-terminal decapeptide in EKA and EKB) or endokinin C/D (EKC/D, the common C-terminal duodecapeptide in EKC and EKD) on chimeric peptide MCRT (YPFPFRTic-NH2, based on YPFP-NH2 and PFRTic-NH2) at the supraspinal level in mice. Results demonstrated that the co-injection of nanomolar EKA/B and MCRT showed moderate regulation, whereas 30 pmol EKA/B had no effect on MCRT. The combination of EKC/D and MCRT produced enhanced antinociception, which was nearly equal to the sum of the mathematical values of single EKC/D and MCRT. Mechanism studies revealed that pre-injected naloxone attenuated the combination significantly compared with the equivalent analgesic effects of EKC/D alone, suggesting that EKC/D and MCRT might act on two totally independent pathways. Moreover, based on the above results and previous reports, we made two reasonable hypotheses to explain the cocktail-induced analgesia, which may potentially pave the way to explore the respective regulatory mechanisms of EKA/B, EKC/D, and MCRT and to better understand the complicated pain regulation of NK1 and μ opioid receptors, as follows: (1) MCRT and endomorphin-1 possibly activated different μ subtypes; and (2) picomolar EKA/B might motivate the endogenous NPFF system after NK1 activation.

  14. 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

  15. Repopulation of adult and neonatal mice with human hepatocytes: A chimeric animal model

    OpenAIRE

    Bissig, Karl-Dimiter; Le, Tam T.; Woods, Niels-Bjarne; Verma, Inder M.

    2007-01-01

    We report the successful transplantation of human hepatocytes in immunodeficient, fumarylacetoacetate hydrolase-deficient (fah−/−) mice. Engraftment occurs over the entire liver acinus upon transplantation. A few weeks after transplantation, increasing concentrations of human proteins (e.g., human albumin and human C3a) can be measured in the blood of the recipient mouse. No fusion between mouse and human hepatocytes can be detected. Three months after transplantation, up to 20% of the mouse ...

  16. Bone marrow phenotype determines genetic resistance to RadLV-induced leukemia in radiation chimeric mice

    Energy Technology Data Exchange (ETDEWEB)

    St. Pierre, Y.; Lussier, G.; Potworowski, E.F.

    1987-01-01

    The development of RadLV-induced T-cell leukemia is a multistep process which evolves along the bone marrow-thymus axis. This process has been shown to be under the control of resistance and susceptibility genes. The relative importance of bone marrow and thymic phenotypes in this genetic control have not been established. We have constructed radiation chimeras with bone marrow from susceptible C57BL/Ka and thymus from resistant B10.A(5R) mice (and vice versa). The rate of leukemia development in the various groups indicates that the phenotype of the bone marrow and not that of the thymus determines the expression of resistance or susceptibility.

  17. Immunization with an HPV-16 L1-based chimeric virus-like particle containing HPV-16 E6 and E7 epitopes elicits long-lasting prophylactic and therapeutic efficacy in an HPV-16 tumor mice model.

    Science.gov (United States)

    Monroy-García, Alberto; Gómez-Lim, Miguel Angel; Weiss-Steider, Benny; Hernández-Montes, Jorge; Huerta-Yepez, Sara; Rangel-Santiago, Jesús F; Santiago-Osorio, Edelmiro; Mora García, María de Lourdes

    2014-02-01

    HPV L1-based virus-like particles vaccines (VLPs) efficiently induce temporary prophylactic activity through the induction of neutralizing antibodies; however, VLPs that can provide prophylactic as well as therapeutic properties for longer periods of time are needed. For this purpose, we generated a novel HPV 16 L1-based chimeric virus-like particle (cVLP) produced in plants that contains a string of T-cell epitopes from HPV 16 E6 and E7 fused to its C-terminus. In the present study, we analyzed the persistence of specific IgG antibodies with neutralizing activity induced by immunization with these cVLPs, as well as their therapeutic potential in a tumor model of C57BL/6 mice. We observed that these cVLPs induced persistent IgG antibodies for over 12 months, with reactivity and neutralizing activity for VLPs composed of only the HPV-16 L1 protein. Efficient protection for long periods of time and inhibition of tumor growth induced by TC-1 tumor cells expressing HPV-16 E6/E7 oncoproteins, as well as significant tumor reduction (57 %), were observed in mice immunized with these cVLPs. Finally, we discuss the possibility that chimeric particles of the type described in this work may be the basis for developing HPV prophylactic and therapeutic vaccines with high efficacy.

  18. Human biofluid concentrations of mono(2-ethylhexyl)phthalate extrapolated from pharmacokinetics in chimeric mice with humanized liver administered with di(2-ethylhexyl)phthalate and physiologically based pharmacokinetic modeling.

    Science.gov (United States)

    Adachi, Koichiro; Suemizu, Hiroshi; Murayama, Norie; Shimizu, Makiko; Yamazaki, Hiroshi

    2015-05-01

    Di(2-ethylhexyl)phthalate (DEHP) is a reproductive toxicant in male rodents. The aim of the current study was to extrapolate the pharmacokinetics and toxicokinetics of mono(2-ethylhexyl)phthalate (MEHP, a primary metabolite of DEHP) in humans by using data from oral administration of DEHP to chimeric mice transplanted with human hepatocytes. MEHP and its glucuronide were detected in plasma from control mice and chimeric mice after single oral doses of 250mg DEHP/kg body weight. Biphasic plasma concentration-time curves of MEHP and its glucuronide were seen only in control mice. MEHP and its glucuronide were extensively excreted in urine within 24h in mice with humanized liver. In contrast, fecal excretion levels of MEHP glucuronide were high in control mice compared with those with humanized liver. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated urine MEHP concentrations in humans were consistent with reported concentrations. This research illustrates how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of pharmacokinetics or toxicokinetics of the primary or secondary metabolites of DEHP.

  19. 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

    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...... were loaded with chronic phase polyclonal IgG and challenged 3 days later with a 100% infectious dose of the acute phase H77C virus, both originating from patient H. Passive immunization induced sterilizing immunity in five of eight challenged animals. In the three nonprotected animals, the HCV...... infection was attenuated, as evidenced by altered viral kinetics in comparison with five control IgG-treated animals. Plasma samples obtained from the mice at viral challenge neutralized H77C-HCVpp at dilutions as high as 1/400. Infection was completely prevented when, before administration to naïve...

  20. Human plasma concentrations of herbicidal carbamate molinate extrapolated from the pharmacokinetics established in in vivo experiments with chimeric mice with humanized liver and physiologically based pharmacokinetic modeling.

    Science.gov (United States)

    Yamashita, Masanao; Suemizu, Hiroshi; Murayama, Norie; Nishiyama, Sayako; Shimizu, Makiko; Yamazaki, Hiroshi

    2014-10-01

    To predict concentrations in humans of the herbicidal carbamate molinate, used exclusively in rice cultivation, a forward dosimetry approach was carried out using data from lowest-observed-adverse-effect-level doses orally administered to rats, wild type mice, and chimeric mice with humanized liver and from in vitro human and rodent experiments. Human liver microsomes preferentially mediated hydroxylation of molinate, but rat livers additionally produced molinate sulfoxide and an unidentified metabolite. Adjusted animal biomonitoring equivalents for molinate and its primary sulfoxide from animal studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and human metabolic data with a simple physiologically based pharmacokinetic (PBPK) model. The slower disposition of molinate and accumulation of molinate sulfoxide in humans were estimated by modeling after single and multiple doses compared with elimination in rodents. The results from simplified PBPK modeling in combination with chimeric mice with humanized liver suggest that ratios of estimated parameters of molinate sulfoxide exposure in humans to those in rats were three times as many as general safety factor of 10 for species difference in toxicokinetics. Thus, careful regulatory decision is needed when evaluating the human risk resulting from exposure to low doses of molinate and related carbamates based on data obtained from rats.

  1. Expression of c-kit receptor in human cholangiocarcinoma and in vivo treatment with imatinib mesilate in chimeric mice

    Institute of Scientific and Technical Information of China (English)

    Thomas Kamenz; Karel Caca; Thilo Blüthner; Andrea Tannapfel; Joachim M(o)ssner; Marcus Wiedmann

    2006-01-01

    treatment with imatinib mesilate, using the chimeric mouse model, c-kit positive Mz-ChA-2 tumors had a significantly reduced volume and mass as compared to NS treatment (P<0.05). In contrast to that,treatment of mice bearing c-kit negative EGI-1 tumors did not result in any change of tumor volume and mass as compared to NS treatment.CONCLUSION: c-kit expression is detectable at a moderate to low protein level in biliary tract cancer.Imatinib mesilate exerts marked effects on tumor growth in vitro and in vitro dependent on the level of c-kit expression.

  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. 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

  4. 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.

  5. 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...

  6. BPI700-Fcγ1700 chimeric gene expression and its protective effect in a mice model of the lethal E. coli infection

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background Infections caused by gram-negative bacteria (GNB) often lead to high mortality in common clinical settings. The effect of traditional antibiotic therapy is hindered by drug-resistant bacteria and unneutralizable endotoxin. Few effective methods can protect high risk patients from bacterial infection. This study explored the protection of adeno-associated virus 2 (AAV2)-bacteriacidal permeability increasing protein 700 (BPI700) -fragment crystallizable gamma one 700 (Fcγ1700) chimeric gene transferred mice against the minimal lethal dose (MLD) of E.coli and application of gene therapy for bacterial infection.Methods After AAV2-BPI700-Fcγ1700 virus transfection,dot blotting and Western blotting were used to detect the target gene products in Chinese hamster ovary-K1 cells (CHO-K1cells). Reverse transcription-polymerase chain reaction and immunohistochemical assay were carried out to show the target gene expression in mice. Modified BPI-enzyme linked immunosorbent assay was used to identify the target gene products in murine serum. The protection of BPI700-Fcγ1700 gene transferred mice was examined by survival rate after MLD E. coli challenge. Colony forming unit (CFU) count, limulus amebocyte lysate kit and cytokine kit were used to quantify the bacteria, the level of endotoxin, and proinflammatory cytokine.Results BPI1-199-Fc(1 protein was identified in the CHO-K1 cell culture supernatant, injected muscles and serum of the gene transferred mice. After MLD E. coli challenge, the survival rate of AAV2-BPI700-Fc(1700 gene transferred mice (36.7%) was significantly higher than that of AAV2-enhanced green fluorescent protein (AAV2- EGFP) gene transferred mice (3.3%) and PBS control mice (5.6%). The survival rate of AAV2-BPI700-Fc(1700 gene transferred mice treated with cefuroxime sodium was 65.0%. The bacterium number in main viscera, the levels of endotoxin and proinflammatory cytokine (tumor necrcsis factor-α and interleukin-1β) in serum of the AAV2-BPI

  7. 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.

  8. 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

  9. Vaccine-induced protection from infection of mice by chimeric human immunodeficiency virus type 1, EcoHIV/NL4-3.

    Science.gov (United States)

    Saini, Manisha; Hadas, Eran; Volsky, David J; Potash, Mary Jane

    2007-12-17

    EcoHIV/NL4-3 is a chimeric human immunodeficiency virus type 1 (HIV-1) that can productively infect mice. This study tests the utility of EcoHIV/NL4-3 infection to reveal protective immune responses to an HIV-1 vaccine. Immunocompetent mice were first immunized with VRC 4306 which encodes subtype B consensus sequences of gag, pol, and nef and then were infected by EcoHIV/NL4-3. Anti-Gag antibodies were sampled during immunization and infection. The extent of EcoHIV/NL4-3 infection in spleen cells and peritoneal macrophages was determined by quantitative real-time PCR (QPCR). Although antibody titres were not significantly different in control and vaccinated groups, VRC 4306 immunization induced protective responses that significantly reduced virus burden in both lymphocyte and macrophage compartments. These results indicate that EcoHIV/NL4-3 infection can be controlled by HIV-1 vaccine-induced responses, introducing a small animal model to test vaccine efficacy against HIV-1 infection.

  10. Clinical significance of chimerism.

    Science.gov (United States)

    Abuelo, Dianne

    2009-05-15

    Twins have been previously classified as either monozygotic or dizygotic. In recent years, fascinating, non-traditional mechanisms of twinning have been uncovered. We define chimerism versus mosaicism, touch on chimerism in the animal world, and explain timing of chimerism in humans. In addition, we discuss when to suspect chimerism in patients, and how to proceed with diagnostic evaluation and confirmation.

  11. 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

  12. 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.

  13. 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

  14. Transmission of chimeric HIV by mating in conventional mice: prevention by pre-exposure antiretroviral therapy and reduced susceptibility during estrus.

    Science.gov (United States)

    Hadas, Eran; Chao, Wei; He, Hongxia; Saini, Manisha; Daley, Eleen; Saifuddin, Mohammed; Bentsman, Galina; Ganz, Eric; Volsky, David J; Potash, Mary Jane

    2013-09-01

    Heterosexual transmission accounts for the majority of new human immunodeficiency virus (HIV) cases worldwide. The current approach to investigate HIV heterosexual transmission in animals involves application of virus stock to the vaginal surface, a method that does not reproduce the physiological conditions of vaginal intercourse that influence the rate of transmission. We have previously described efficient infection of conventional mice using EcoHIV/NL4-3 and EcoHIV/NDK, chimeric HIV molecular clones constructed to express all HIV structural and regulatory genes except envelope, which is replaced by a rodent-tropic envelope gene. Here we investigated whether EcoHIV/NDK-infected male mice transmit virus to females during coitus, and the sensitivity of this transmission to HIV pre-exposure prophylaxis and the estrus state. Our general approach was to allow mating between EcoHIV/NDK-infected male mice and uninfected females for 1-7 nights. At 1-6 weeks after mating, mice were euthanized and virus burdens were measured by quantitative PCR (qPCR) amplification of HIV RNA or DNA in peritoneal macrophages, inguinal lymph node cells, spleen cells or vas deferens, or by ELISA for antibodies to HIV Gag. We found that 70-100% of female mice mated to EcoHIV/NDK-infected males acquired infection. Pericoital treatment of females with either 2',3'-dideoxcytidine (ddC) or tenofovir largely prevented their EcoHIV/NDK infection by mating (P<0.05 and P<0.003, respectively). In males, T cells were dispensable for virus transmission. The rate of EcoHIV/NDK sexual transmission to females in estrus declined sharply (P=0.003) but their infection by injection was unaffected, indicating that the local environment in the female reproductive tract influences susceptibility to HIV. We conclude that this system of EcoHIV/NDK transmission during mouse mating reproduces key features of heterosexual transmission of HIV in humans and can be used to investigate its biology and control.

  15. Transmission of chimeric HIV by mating in conventional mice: prevention by pre-exposure antiretroviral therapy and reduced susceptibility during estrus

    Directory of Open Access Journals (Sweden)

    Eran Hadas

    2013-09-01

    Heterosexual transmission accounts for the majority of new human immunodeficiency virus (HIV cases worldwide. The current approach to investigate HIV heterosexual transmission in animals involves application of virus stock to the vaginal surface, a method that does not reproduce the physiological conditions of vaginal intercourse that influence the rate of transmission. We have previously described efficient infection of conventional mice using EcoHIV/NL4-3 and EcoHIV/NDK, chimeric HIV molecular clones constructed to express all HIV structural and regulatory genes except envelope, which is replaced by a rodent-tropic envelope gene. Here we investigated whether EcoHIV/NDK-infected male mice transmit virus to females during coitus, and the sensitivity of this transmission to HIV pre-exposure prophylaxis and the estrus state. Our general approach was to allow mating between EcoHIV/NDK-infected male mice and uninfected females for 1–7 nights. At 1–6 weeks after mating, mice were euthanized and virus burdens were measured by quantitative PCR (qPCR amplification of HIV RNA or DNA in peritoneal macrophages, inguinal lymph node cells, spleen cells or vas deferens, or by ELISA for antibodies to HIV Gag. We found that 70–100% of female mice mated to EcoHIV/NDK-infected males acquired infection. Pericoital treatment of females with either 2′,3′-dideoxcytidine (ddC or tenofovir largely prevented their EcoHIV/NDK infection by mating (P<0.05 and P<0.003, respectively. In males, T cells were dispensable for virus transmission. The rate of EcoHIV/NDK sexual transmission to females in estrus declined sharply (P=0.003 but their infection by injection was unaffected, indicating that the local environment in the female reproductive tract influences susceptibility to HIV. We conclude that this system of EcoHIV/NDK transmission during mouse mating reproduces key features of heterosexual transmission of HIV in humans and can be used to investigate its biology and control.

  16. Pathogenesis of Lassa fever virus infection: I. Susceptibility of mice to recombinant Lassa Gp/LCMV chimeric virus.

    Science.gov (United States)

    Lee, Andrew M; Cruite, Justin; Welch, Megan J; Sullivan, Brian; Oldstone, Michael B A

    2013-08-01

    Lassa virus (LASV) is a BSL-4 restricted agent. To allow study of infection by LASV under BSL-2 conditions, we generated a recombinant virus in which the LASV glycoprotein (Gp) was placed on the backbone of lymphocytic choriomeningitis virus (LCMV) Cl13 nucleoprotein, Z and polymerase genes (rLCMV Cl13/LASV Gp). The recombinant virus displayed high tropism for dendritic cells following in vitro or in vivo infection. Inoculation of immunocompetent adults resulted in an acute infection, generation of virus-specific CD8(+) T cells and clearance of the infection. Inoculation of newborn mice with rLCMV Cl13/LASV Gp resulted in a life-long persistent infection. Interestingly, adoptive transfer of rLCMV Cl13/LASV Gp immune memory cells into such persistently infected mice failed to purge virus but, in contrast, cleared virus from mice persistently infected with wt LCMV Cl13. PMID:23684417

  17. Pathogenesis of Lassa Fever Virus Infection: I. Susceptibility of Mice to Recombinant Lassa Gp/LCMV Chimeric Virus

    Science.gov (United States)

    Lee, Andrew M.; Cruite, Justin; Welch, Megan J.; Sullivan, Brian; Oldstone, Michael B.A.

    2013-01-01

    Lassa virus (LASV) is a BSL-4 restricted agent. To allow study of infection by LASV under BSL-2 conditions, we generated a recombinant virus in which the LASV glycoprotein (Gp) was placed on the backbone of lymphocytic choriomeningitis virus (LCMV) Cl13 nucleoprotein, Z and polymerase genes (rLCMV Cl13/LASV Gp). The recombinant virus displayed high tropism for dendritic cells following in vitro or in vivo infection. Inoculation of immunocompetent adults resulted in an acute infection, generation of virus-specific CD8+ T cells and clearance of the infection. Inoculation of newborn mice with rLCMV Cl13/LASV Gp resulted in a life-long persistent infection. Interestingly, adoptive transfer of rLCMV Cl13/LASV Gp immune memory cells into such persistently infected mice failed to purge virus but, in contrast, cleared virus from mice persistently infected with wt LCMV Cl13. PMID:23684417

  18. Ectopic bone formation cannot occur by hydroxyapatite/β-tricalcium phosphate bioceramics in green fluorescent protein chimeric mice

    Science.gov (United States)

    Cheng, Lijia; Duan, Xin; Xiang, Zhou; Shi, Yujun; Lu, Xiaofeng; Ye, Feng; Bu, Hong

    2012-12-01

    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 induction in the future.

  19. Quantitative PET of EGFR expression in xenograft-bearing mice using {sup 64}Cu-labeled cetuximab, a chimeric anti-EGFR monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    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.)

  20. 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

  1. 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

  2. Tritium distribution in newborn mice after providing mother mice with drinking water containing tritiated thymidine

    International Nuclear Information System (INIS)

    Throughout gestation pregnant mice received drinking water which contained [methyl-3H]thymidine (18.5 kBq/ml). The newborn mice were divided into two groups. One group was nursed by their own mothers, which were further supplied with tritiated thymidine until 4 weeks after delivery (Experiment I). The other group was nursed by ''nonradioactive mothers'' which were given no tritiated thymidine (Experiment II). Tritium incorporation into the small molecular components of the acid-soluble fraction, lipid, RNA, DNA, and protein was analyzed for the newborn mice at various ages. In Experiment II, total radioactivity per gram tissue decreased initially after birth with a half life of 2.5-2.9 days in spleen, liver, intestine, stomach, thymus, lung, kidney, heart, and brain. At about 2 weeks after birth, a slower component of tritium elimination due mainly to the DNA-bound tritium appeared. Specific activity of DNA at birth was organ specific, highest in heart and lowest in thymus. Cumulative absorbed dose in various organs was estimated for the first 4 weeks after birth based upon an assumption that total and DNA-bound tritium are uniformly distributed. The result showed that organ specificity of dose accumulation is obvious for DNA-bound tritium, highest in spleen (1.15 mGy) and lowest in brain (0.13 mGy). It was also shown that the tritium supply from mother's milk is of minor importance for dose accumulation of DNA-bound tritium in the cell nuclei of organs of suckling mice

  3. Tritium distribution in newborn mice after providing mother mice with drinking water containing tritiated thymidine

    International Nuclear Information System (INIS)

    Throughout gestation pregnant mice received drinking water which contained [methyl-3H]thymidine (18.5 kBq/ml). The newborn mice were divided into two groups. One group was nursed by their own mothers, which were further supplied with tritiated thymidine until 4 weeks after delivery (Experiment I). The other group was nursed by nonradioactive mothers which were given no tritiated thymidine (Experiment II). Tritium incorporation into the small molecular components of the acid-soluble fraction, lipid, RNA, DNA, and protein was analyzed for the newborn mice at various ages. In Experiment II, total radioactivity per gram tissue decreased initially after birth with a half life of 2.5 to 2.9 days in spleen, liver, intestine, stomach, thymus, lung, kidney, heart, and brain. At about 2 weeks after birth, a slower component of tritium elimination due mainly to the DNA-bound tritium appeared. Specific activity of DNA at birth was organ specific, highest in heart and lowest in thymus. Cumulative absorbed dose in various organs was estimated for the first 4 weeks after birth based upon an assumption that total and DNA-bound tritium are uniformly distributed. The result showed that organ specificity of dose accumulation is obvious for DNA-bound tritium, highest in spleen (1.15 mGy) and lowest in brain (0.13 mGy). It was also shown that the tritium supply from mother's milk is of minor importance for dose accumulation of DNA-bound tritium in the cell nuclei of organs of suckling mice

  4. A novel recombinant 6Aβ15-THc-C chimeric vaccine (rCV02) mitigates Alzheimer’s disease-like pathology, cognitive decline and synaptic loss in aged 3 × Tg-AD mice

    Science.gov (United States)

    Yu, Yun-Zhou; Liu, Si; Wang, Hai-Chao; Shi, Dan-Yang; Xu, Qing; Zhou, Xiao-Wei; Sun, Zhi-Wei; Huang, Pei-Tang

    2016-01-01

    Alzheimer’s disease (AD) is a neurodegenerative disorder that impairs memory and cognition. Targeting amyloid-β (Aβ) may be currently the most promising immunotherapeutic strategy for AD. In this study, a recombinant chimeric 6Aβ15-THc-C immunogen was formulated with alum adjuvant as a novel Aβ B-cell epitope candidate vaccine (rCV02) for AD. We examined its efficacy in preventing the cognitive deficit and synaptic impairment in 3 × Tg-AD mice. Using a toxin-derived carrier protein, the rCV02 vaccine elicited robust Aβ-specific antibodies that markedly reduced AD-like pathology and improved behavioral performance in 3 × Tg-AD mice. Along with the behavioral improvement in aged 3 × Tg-AD mice, rCV02 significantly decreased calpain activation concurrent with reduced soluble Aβ or oligomeric forms of Aβ, probably by preventing dynamin 1 and PSD-95 degradation. Our data support the hypothesis that reducing Aβ levels in rCV02-immunized AD mice increases the levels of presynaptic dynamin 1 and postsynaptic PSD-95 allowing functional recovery of cognition. In conclusion, this novel and highly immunogenic rCV02 shows promise as a new candidate prophylactic vaccine for AD and may be useful for generating rapid and strong Aβ-specific antibodies in AD patients with pre-existing memory Th cells generated after immunization with conventional tetanus toxoid vaccine. PMID:27255752

  5. A novel recombinant 6Aβ15-THc-C chimeric vaccine (rCV02) mitigates Alzheimer's disease-like pathology, cognitive decline and synaptic loss in aged 3 × Tg-AD mice.

    Science.gov (United States)

    Yu, Yun-Zhou; Liu, Si; Wang, Hai-Chao; Shi, Dan-Yang; Xu, Qing; Zhou, Xiao-Wei; Sun, Zhi-Wei; Huang, Pei-Tang

    2016-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder that impairs memory and cognition. Targeting amyloid-β (Aβ) may be currently the most promising immunotherapeutic strategy for AD. In this study, a recombinant chimeric 6Aβ15-THc-C immunogen was formulated with alum adjuvant as a novel Aβ B-cell epitope candidate vaccine (rCV02) for AD. We examined its efficacy in preventing the cognitive deficit and synaptic impairment in 3 × Tg-AD mice. Using a toxin-derived carrier protein, the rCV02 vaccine elicited robust Aβ-specific antibodies that markedly reduced AD-like pathology and improved behavioral performance in 3 × Tg-AD mice. Along with the behavioral improvement in aged 3 × Tg-AD mice, rCV02 significantly decreased calpain activation concurrent with reduced soluble Aβ or oligomeric forms of Aβ, probably by preventing dynamin 1 and PSD-95 degradation. Our data support the hypothesis that reducing Aβ levels in rCV02-immunized AD mice increases the levels of presynaptic dynamin 1 and postsynaptic PSD-95 allowing functional recovery of cognition. In conclusion, this novel and highly immunogenic rCV02 shows promise as a new candidate prophylactic vaccine for AD and may be useful for generating rapid and strong Aβ-specific antibodies in AD patients with pre-existing memory Th cells generated after immunization with conventional tetanus toxoid vaccine. PMID:27255752

  6. 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...

  7. 利用敲除PLCξ基因的JM8细胞制备嵌合体小鼠%Chimeric mice production by PLCξ-deletant JM8 cells

    Institute of Scientific and Technical Information of China (English)

    潘晓靖; 王鹏博; 胡春超; 徐继强; 崔丽芳; 李相运

    2013-01-01

    为了研究PLCξ对公鼠生殖能力的影响,试验采用显微注射的方法将敲除PLCξ基因的JM8胚胎干细胞注入8-细胞胚胎获得嵌合体小鼠,并通过微卫星分析检测公鼠生殖系嵌合的情况及能否通过生殖系传递.共移植562枚胚胎,出生小鼠127只,出生率为22.60%,其中成年的毛色嵌合体小鼠数为46只,嵌合率为36.22%,对10只成年嵌合体公鼠的12种组织(脑,心脏,膀胱,肺脏,肝脏,胃,肾脏,睾丸,胰腺,肠,肌肉,脾脏)进行检验,有4只小鼠睾丸发生了嵌合,说明通过显微注射获得了生殖系嵌合的公鼠,但是生殖系嵌合的公鼠和CD-1母鼠交配后没有生下毛色嵌合小鼠,说明这些公鼠没有生殖系传递的痕迹,表明敲除了PLCξ基因影响了嵌合体公鼠的生精能力.%To explore the effect of PLCξ on reproductive capacity,chimeras were obtained by injecting the PLCξ-deletant JM8 cells into 8-cell embryos.We detected whether these chimeras germline were derived partly from JM8 cells by microsatellite analysis.A total of 562 embryos were transplanted and 127 mice were born,which included 46 adult chimeras.The birth rate was 22.60%.and chimeric rate was 36.22%.We detected twelve tissues in 10 adult chimeras (brain,heart,bladder,lungs,liver,stomach,kidney,testes,pancreas,intestine,muscle,spleen) by microsatellite analysis.We found four testes chimeric mice and after mating with CD-1 female mice,coat color chimeras were not born.The results indicated that the male mice whose germlines were derived partly from JM8 cells could not get JM8 fetus,which proved that the PLCξ influence the reproduction of male chimera mice.

  8. Mouse-hamster chimeric prion protein (PrP) devoid of N-terminal residues 23-88 restores susceptibility to 22L prions, but not to RML prions in PrP-knockout mice.

    Science.gov (United States)

    Uchiyama, Keiji; Miyata, Hironori; Yano, Masashi; Yamaguchi, Yoshitaka; Imamura, Morikazu; Muramatsu, Naomi; Das, Nandita Rani; Chida, Junji; Hara, Hideyuki; Sakaguchi, Suehiro

    2014-01-01

    Prion infection induces conformational conversion of the normal prion protein PrPC, into the pathogenic isoform PrPSc, in prion diseases. It has been shown that PrP-knockout (Prnp0/0) mice transgenically reconstituted with a mouse-hamster chimeric PrP lacking N-terminal residues 23-88, or Tg(MHM2Δ23-88)/Prnp 0/0 mice, neither developed the disease nor accumulated MHM2ScΔ23-88 in their brains after inoculation with RML prions. In contrast, RML-inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice developed the disease with abundant accumulation of MHM2ScΔ23-88 in their brains. These results indicate that MHM2Δ23-88 itself might either lose or greatly reduce the converting capacity to MHM2ScΔ23-88, and that the co-expressing wild-type PrPC can stimulate the conversion of MHM2Δ23-88 to MHM2ScΔ23-88 in trans. In the present study, we confirmed that Tg(MHM2Δ23-88)/Prnp 0/0 mice remained resistant to RML prions for up to 730 days after inoculation. However, we found that Tg(MHM2Δ23-88)/Prnp 0/0 mice were susceptible to 22L prions, developing the disease with prolonged incubation times and accumulating MHM2ScΔ23-88 in their brains. We also found accelerated conversion of MHM2Δ23-88 into MHM2ScΔ23-88 in the brains of RML- and 22L-inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice. However, wild-type PrPSc accumulated less in the brains of these inoculated Tg(MHM2Δ23-88)/Prnp 0/+ mice, compared with RML- and 22L-inoculated Prnp 0/+ mice. These results show that MHM2Δ23-88 itself can convert into MHM2ScΔ23-88 without the help of the trans-acting PrPC, and that, irrespective of prion strains inoculated, the co-expressing wild-type PrPC stimulates the conversion of MHM2Δ23-88 into MHM2ScΔ23-88, but to the contrary, the co-expressing MHM2Δ23-88 disturbs the conversion of wild-type PrPC into PrPSc.

  9. 转增强型绿色荧光蛋白的脐血干细胞制备嵌合体小鼠的研究%The chimeric mice derived from umbilical cord blood stem cells of EGFP-transgenic mouse

    Institute of Scientific and Technical Information of China (English)

    段彪; 杜海燕; 张荣

    2013-01-01

    Objective The chimeric mice were prepared by microinjection of blastocyst cavity using umbilical cord blood stem cells(UCBSCs) of Enhanced Green Fluorescent Protein(EGFP)-transgenic mouse, which was expected to provide a theoretical and experimental basis for the study of in-vivo differentiation of adult stem cells. Methods Mouse UCBSCs expressing green fluorescence was microinjected into blastocyst cavity and several blastocysts were transferred into uterus of pseudo pregnant mouse. First of all, new-born candidate chimeric mouse were observed through feather color. Secondly, the genomic DNA and total RNA were extracted to analyze chimeric rate in several tissues. Finally, flow cytometry was used to detect percentage of green fluorescent cells mice in several tissues. Results The UCBSCs expressing green fluorescent protein were successfully isolated. After flow cytometry analysis, the proportion of cells expressing green fluorescence was 80.25%. Through microinjection and embryo transfer, we got five white new-born mice and no chimeric feather color was observed. The analyses of PCR and RT-PCR were carried out to detect EGFP gene using six tissues including, heart muscle, liver, lung, skin, leg muscle and adipose tissue. The results showed that the leg muscle and adipose tissue of two mice were positive and the other tissues and six tissues of the other 3 mice were all negative. The leg muscle and adipose tissue of two positive mice were digested into single-cells suspension and were carried out flow cytometry analysis. The Results showed that the average chimeric rates of leg muscle and adipose tissue of two positive mice were 9.87% and 5.78%, respectively. Conclusion The results demonstrated that adult UCBSCs could differentiate into leg muscle and adipose tissue in vivo.%目的:本研究通过囊胚腔显微注射转增强型绿色荧光蛋白(EGFP)基因的脐血干细胞的方法制备嵌合体小鼠,以期为研究成体干细胞的体内分化提

  10. 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

  11. 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

  12. Human COL2A1-directed SV40 T antigen expression in transgenic and chimeric mice results in abnormal skeletal development

    OpenAIRE

    1995-01-01

    The ability of SV40 T antigen to cause abnormalities in cartilage development in transgenic mice and chimeras has been tested. The cis- regulatory elements of the COL2A1 gene were used to target expression of SV40 T antigen to differentiating chondrocytes in transgenic mice and chimeras derived from embryonal stem (ES) cells bearing the same transgene. The major phenotypic consequences of transgenic (pAL21) expression are malformed skeleton, disproportionate dwarfism, and perinatal/neonatal d...

  13. Impaired virus control and severe CD8+ T-cell-mediated immunopathology in chimeric mice deficient in gamma interferon receptor expression on both parenchymal and hematopoietic cells

    DEFF Research Database (Denmark)

    Henrichsen, Pernille; Bartholdy, Christina; Christensen, Jan Pravsgaard;

    2005-01-01

    Bone marrow chimeras were used to determine the cellular target(s) for the antiviral activity of gamma interferon (IFN-gamma). By transfusing such mice with high numbers of naive virus-specific CD8(+) T cells, a system was created in which the majority of virus-specific CD8(+) T cells would be...

  14. Electromagnetic treatment to old Alzheimer's mice reverses β-amyloid deposition, modifies cerebral blood flow, and provides selected cognitive benefit.

    Directory of Open Access Journals (Sweden)

    Gary W Arendash

    Full Text Available Few studies have investigated physiologic and cognitive effects of "long-term" electromagnetic field (EMF exposure in humans or animals. Our recent studies have provided initial insight into the long-term impact of adulthood EMF exposure (GSM, pulsed/modulated, 918 MHz, 0.25-1.05 W/kg by showing 6+ months of daily EMF treatment protects against or reverses cognitive impairment in Alzheimer's transgenic (Tg mice, while even having cognitive benefit to normal mice. Mechanistically, EMF-induced cognitive benefits involve suppression of brain β-amyloid (Aβ aggregation/deposition in Tg mice and brain mitochondrial enhancement in both Tg and normal mice. The present study extends this work by showing that daily EMF treatment given to very old (21-27 month Tg mice over a 2-month period reverses their very advanced brain Aβ aggregation/deposition. These very old Tg mice and their normal littermates together showed an increase in general memory function in the Y-maze task, although not in more complex tasks. Measurement of both body and brain temperature at intervals during the 2-month EMF treatment, as well as in a separate group of Tg mice during a 12-day treatment period, revealed no appreciable increases in brain temperature (and no/slight increases in body temperature during EMF "ON" periods. Thus, the neuropathologic/cognitive benefits of EMF treatment occur without brain hyperthermia. Finally, regional cerebral blood flow in cerebral cortex was determined to be reduced in both Tg and normal mice after 2 months of EMF treatment, most probably through cerebrovascular constriction induced by freed/disaggregated Aβ (Tg mice and slight body hyperthermia during "ON" periods. These results demonstrate that long-term EMF treatment can provide general cognitive benefit to very old Alzheimer's Tg mice and normal mice, as well as reversal of advanced Aβ neuropathology in Tg mice without brain heating. Results further underscore the potential for EMF

  15. 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.

  16. Immunization of mice with YscF provides protection from Yersinia pestis infections

    Directory of Open Access Journals (Sweden)

    Bradley David S

    2005-06-01

    mice with YscF resulted in a high anti-YscF titer and provided protection against i.v. challenge with Y. pestis. This is the first report to our knowledge utilizing a conserved protein from the type III secretion complex of a gram-negative pathogen as a candidate for vaccine development.

  17. 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.

  18. Studies of tolerance induction through mixed chimerism in cynomolgus monkeys. Method for detection of chimeric cells and effect of thymic irradiation on induction of tolerance

    Energy Technology Data Exchange (ETDEWEB)

    Hoshino, Tomoaki; Kawai, Tatsuo; Ota, Kazuo [Tokyo Women`s Medical Coll. (Japan)

    1996-12-01

    To establish the method for the detection of chimerism in cynomologus monkeys, we tested cross reactivity of various anti-HLA monoclonal antibodies (mAb) to cynomolgus monkeys. In 29 mAb we tested, only three monoclonal anti-HLA antibodies crossreacted with lymphocytes of monkeys. With these mAb, chimeric cell can be detected up to 1% by flow cytometric analysis (study 1). Utilizing the method we developed in study 1, we applied the regimen that induces mixed chimerism and skin graft tolerance in mice to renal allotransplantation of cynomolgus monkey. Regimen A includes non-lethal dose of total body irradiation (TBI), administration of anti-thymocyte globulin (ATG) for 3 days, donor bone marrow infusion and 45 days course of cyclosporine (CYA) administration. We added 7 Gy of thymic irradiation on day-6 in regimen B and on day-1 in regimen C. Although all monkeys in regimen A and B consistently developed chimerism, they rejected kidney allografts soon after stopping CYA. In contrast, 4 monkeys out of 5 failed to develop chimerism in regimen C, but renal allograft tolerance was induced in one monkey who developed chimerism in regimen C. In conclusion, the induction of chimerism is considered necessary but not sufficient for tolerance induction. (author)

  19. 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

  20. A Novel Contraceptive Vaccine:Design and Synthesis of the Chimeric Peptide Containing Multivalent Sperm-Specific Epitopes

    Institute of Scientific and Technical Information of China (English)

    何畏; 梁志清; 史常旭; 李玉清

    2001-01-01

    Objective To develop a novel multivalent chimeric peptide vaccine for bisexual fertility regulation Materials & Methods On the basis of the amino acid sequence of the two peptides respectively selected from the mouse sperm/testis-specific proteins SP17 and Cyritestin, and one T cell epitope in bovine ribonuclease (RNase), a novel chimeric peptide consisting of 35 amino acid was designed and subsequently synthesized on the 430A peptide synthesizer. After being emulified with the equivalence Freund's adjuvant, the peptide with 35 amino acid residues was used to immunogenity the female BALB/c mice to investigate its immunity.Results The peptide was successfully synthesized, after being purified in high performance liquid chromatography (HPLC), its purity reached 95%. The specific antisera collected from the immunogenity mice could identify the corresponding proteins of testis tissues of mice, rats and human. The highest specific IgG titer in serum was 1:6 000, while the IgA titer in the washing of vaginal mucous membrane was 1:300.Conclusion The antibodies from the peptide with specific amino acid sequence can identify the original antigens, and stimulate powerful specific humoral immunity in mice. It provides a experimental bases for polyvalent contraceptive vaccine study.

  1. 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 ...

  2. 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.)

  3. 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.

  4. A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Tomioka, Yukiko, E-mail: ytomi@muses.tottori-u.ac.jp [Division of Disease Model Innovation, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815 (Japan); Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, Tottori 680-8553 (Japan); Morimatsu, Masami, E-mail: mmorimat@vetmed.hokudai.ac.jp [Division of Disease Model Innovation, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815 (Japan); Laboratory of Laboratory Animal Science and Medicine, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan); Nishijima, Ken-ichi, E-mail: nishijma@nubio.nagoya-u.ac.jp [Department of Biotechnology, Graduate School of Engineering, Nagoya University, Nagoya 464-8603 (Japan); Usui, Tatsufumi, E-mail: usutatsu@muses.tottori-u.ac.jp [Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, Tottori 680-8553 (Japan); Yamamoto, Sayo, E-mail: ysayo@anim.med.kyushu-u.ac.jp [Center of Biomedical Research, Research Center for Human Disease Modeling, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Suyama, Haruka, E-mail: sharuka@anim.med.kyushu-u.ac.jp [Center of Biomedical Research, Research Center for Human Disease Modeling, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Ozaki, Kinuyo, E-mail: k-ozaki@anim.med.kyushu-u.ac.jp [Center of Biomedical Research, Research Center for Human Disease Modeling, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582 (Japan); Ito, Toshihiro, E-mail: toshiito@muses.tottori-u.ac.jp [Avian Zoonosis Research Center, Faculty of Agriculture, Tottori University, Tottori 680-8553 (Japan); and others

    2014-07-18

    Highlights: • Tumor-associated antigen MUC1 binds to Siglec-9. • Soluble Siglec-9 reduced proliferation of MUC1-positive tumor in transgenic mice. • Soluble Siglec-9 and MUC1 on tumor cells were colocalized in transgenic mice. • MUC1 expression on tumor cells were reduced in soluble Siglec-9 transgenic mice. - Abstract: Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9 (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation.

  5. A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice

    International Nuclear Information System (INIS)

    Highlights: • Tumor-associated antigen MUC1 binds to Siglec-9. • Soluble Siglec-9 reduced proliferation of MUC1-positive tumor in transgenic mice. • Soluble Siglec-9 and MUC1 on tumor cells were colocalized in transgenic mice. • MUC1 expression on tumor cells were reduced in soluble Siglec-9 transgenic mice. - Abstract: Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9 (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation

  6. Direct observation of failing fibers in muscles of dystrophic mice provides mechanistic insight into muscular dystrophy.

    Science.gov (United States)

    Claflin, Dennis R; Brooks, Susan V

    2008-02-01

    Duchenne muscular dystrophy is caused by the absence of the protein dystrophin. Dystrophin's function is not known, but its cellular location and associations with both the force-generating contractile core and membrane-spanning entities suggest a role in mechanically coupling force from its intracellular origins to the fiber membrane and beyond. We report here the presence of destructive contractile activity in lumbrical muscles from dystrophin-deficient (mdx) mice during nominally quiescent periods following exposure to mechanical stress. The ectopic activity, which was observable microscopically, resulted in longitudinal separation and clotting of fiber myoplasm and was absent when calcium (Ca(2+)) was removed from the bathing medium. Separation and clotting of myoplasm were also produced in dystrophin-deficient muscles by local application of a Ca(2+) ionophore to create membrane breaches in the absence of mechanical stress, whereas muscles from control mice tolerated ionophore-induced entry of Ca(2+) without damage. These observations suggest a failure cascade in dystrophin-deficient fibers that 1) is initiated by a stress-induced influx of extracellular Ca(2+), causing localized activation to continue after cessation of stimulation, and 2) proceeds as the persistent local activation, combined with reduced lateral mechanical coupling between the contractile core and the extracellular matrix, results in longitudinal separation of myoplasm in nonactivated regions of the fiber. This mechanism invokes both the membrane stabilization and the mechanical coupling functions frequently proposed for dystrophin and suggests that, whereas the absence of either function alone is not sufficient to cause fiber failure, their combined absence is catastrophic.

  7. 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.

  8. Immunization of mice with cells from juvenile worms of Schistosoma japonicum provides immunoprotection against schistosomiasis

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To validate the protective efficacy against schistosomiasis by immunization with cells from juvenile Schistosoma japonicum in a murine model and to analyze possible factors related to protection, in this study, two independent repeated vaccination trials were performed. After three subcutaneous vaccinations, in trial one, in the absence of adjuvant, primary juvenile worm cells (pJCs) from S. japonicum induced remarkable average reductions in worm burden (54.3%), liver eggs per gram (LEPG) load (59.8%) as well as egg granulomas size (66.5%) compared to PBS control group (P<0.01), which were significantly higher than those elicited by fractions of juvenile worm cells (JCFs) or fractions of juvenile worms (JWFs) (P<0.05). Non-cell components of worms (WNCs) showed no significant protection. In trial two, compared to PBS control group, significant protective effect was also observed for cultured juvenile worm cells (cJCs) from S. japonicum with 58.4% worm reduction and 68.1% LEPG reduction (P<0.01). However, cultured adult worms cells (cACs) showed significantly higher worm burden (P<0.05) and egg burden (P<0.01) when compared to cJCs. Immunological analysis of trial two revealed that cJCs engendered a Th1-biased mixed Th1/Th2 type of immune response while cACs elicited a Th2-type response. Our data indicated that immunization with both primary and cultured cells from S. japonicum juvenile worms provided high immunoprotection, for which the physical character of immunogens, stage-specific parasite and the type of immune response induced might be responsible, suggesting that vaccination with whole cells from S. japonicum larvae is a promising approach to produce protective immunity against schistosomiasis.

  9. Live, attenuated influenza A H5N1 candidate vaccines provide broad cross-protection in mice and ferrets.

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    Amorsolo L Suguitan

    2006-09-01

    Full Text Available BACKGROUND: Recent outbreaks of highly pathogenic influenza A H5N1 viruses in humans and avian species that began in Asia and have spread to other continents underscore an urgent need to develop vaccines that would protect the human population in the event of a pandemic. METHODS AND FINDINGS: Live, attenuated candidate vaccines possessing genes encoding a modified H5 hemagglutinin (HA and a wild-type (wt N1 neuraminidase from influenza A H5N1 viruses isolated in Hong Kong and Vietnam in 1997, 2003, and 2004, and remaining gene segments derived from the cold-adapted (ca influenza A vaccine donor strain, influenza A/Ann Arbor/6/60 ca (H2N2, were generated by reverse genetics. The H5N1 ca vaccine viruses required trypsin for efficient growth in vitro, as predicted by the modification engineered in the gene encoding the HA, and possessed the temperature-sensitive and attenuation phenotypes specified by the internal protein genes of the ca vaccine donor strain. More importantly, the candidate vaccines were immunogenic in mice. Four weeks after receiving a single dose of 10(6 50% tissue culture infectious doses of intranasally administered vaccines, mice were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wt H5N1 viruses from different genetic sublineages (clades 1, 2, and 3 that were isolated in Asia between 1997 and 2005. Four weeks after receiving two doses of the vaccines, mice and ferrets were fully protected against pulmonary replication of homologous and heterologous wt H5N1 viruses. CONCLUSIONS: The promising findings in these preclinical studies of safety, immunogenicity, and efficacy of the H5N1 ca vaccines against antigenically diverse H5N1 vaccines provide support for their careful evaluation in Phase 1 clinical trials in humans.

  10. Gamma-irradiated influenza A virus provides adjuvant activity to a co-administered poorly immunogenic SFV vaccine in mice.

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    Rachelle eBabb

    2014-06-01

    Full Text Available Many currently available inactivated vaccines require 'adjuvants' to maximise the protective immune responses generated against the antigens of interest. Recent studies in mice with gamma-irradiated influenza A virus (γ-FLU have shown its superior efficacy compared to other forms of inactivated FLU vaccines and its ability to induce both potent type-I interferon (IFN-I responses and the IFN-I associated partial lymphocyte activation. Commonly, IFN-I responses induced by adjuvants, combined in vaccine preparations, have been shown to effectively enhance the immunogenicity of the antigens of interest. Therefore, we investigated the potential adjuvant activity of γ-FLU and the possible effect on antibody responses against co-administrated antigens, using gamma-irradiated Semliki Forest Virus (γ-SFV as the experimental vaccine in mice. Our data show that co-vaccination with γ-FLU and γ-SFV resulted in enhanced SFV-specific antibody responses in terms of increased titres by 6 fold and greater neutralisation efficacy, when compared to vaccination with γ-SFV alone. This study provides promising evidence related to the possible use of γ-FLU as an adjuvant to poorly immunogenic vaccines without compromising the vaccine efficacy of γ-FLU.

  11. 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.

  12. 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.

  13. 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.

  14. Immunogenicity and antigenicity of a recombinant chimeric protein containing epitopes of poliovirus type 1.

    Science.gov (United States)

    Pan, X-X; Wang, J; Xia, W-Y; Li, X-F; Yang, L-J; Huang, C; Chen, Y-D

    2016-01-01

    To design a vaccine that simultaneously prevents both rotavirus (RV) and poliovirus (PV), a PV type 1 (PV1) chimeric protein using RV VP6 as a vector (VP6F) was constructed, expressed in Escherichia coli expression system and characterized by SDS-PAGE, Western blot, immunofluorescence assay and neutralization test. The results showed that the chimeric protein reacted with anti-VP6F and anti-PV1 antibodies and elicited production of serum antibodies against the chimeric protein in guinea pigs. Antibodies against the chimeric protein neutralized RV Wa and PV1 infection in vitro. The results provided a relevant possibility of developing novel approaches in the rational design of vaccines effective against both RV and PV. PMID:27640433

  15. Late Miocene insular mice from the Tusco-Sardinian palaeobioprovince provide new insights on the palaeoecology of the Oreopithecus faunas.

    Science.gov (United States)

    Casanovas-Vilar, Isaac; van Dam, Jan A; Moyà-Solà, Salvador; Rook, Lorenzo

    2011-07-01

    Oreopithecus bambolii is one of the few hominoids that evolved under insular conditions, resulting in the development of unique adaptations that have fueled an intensive debate. The palaeoenvironment associated with this great ape has been the subject of great controversy as well. On the one hand, palaeobotanical data indicate that Oreopithecus likely inhabited mixed mesophytic forests interrupted by swamps; on the other hand, an abundance of hypsodont bovids points towards the existence of dry and open environments. Here, we provide a new approach based on the ecomorphology of the extinct endemic Muridae (rats and mice) of the so-called Oreopithecus faunas. Our results show that the successive species of endemic insular murids (Huerzelerimys and Anthracomys) evolved a number of adaptations observed only in extant family members that include significant proportions of grass in their diet. While this fits the pattern exhibited by large mammals, it contrasts with the available palaeobotanical information, which indicates that grasses were minor components of the vegetation. This contradiction may be explained because these endemic murids may have been adapted to the consumption of particular food items such as hard parts of aquatic plants (as shown by some extant murid species). However, because it is unlikely that the remaining herbivore mammals were adapted to this diet as well, we favour an alternative hypothesis that takes into account the peculiar ecological conditions of insular ecosystems leading to a density-dependent selective regime with strong competition. Such a regime would promote the selection of dental adaptations to increase feeding efficiency and durability of the dentition (such as hypsodonty) as seen in some fossil insular ruminants. This hypothesis requires further testing, but may partly account for parallel evolution of dental traits in phylogenetically unrelated insular mammals. PMID:21371736

  16. 输注分子嵌合小鼠前体T细胞抑制异源小鼠T细胞增殖反应的研究%Research on molecular chimeric mice pre-T cells infusion to reduce response to allogeneic T cells stimulation

    Institute of Scientific and Technical Information of China (English)

    陆礼; 宋东旭; 李卫东; 付蔚华

    2014-01-01

    目的 研究分子嵌合前体T细胞(pre-T细胞)对异基因T淋巴细胞增殖能力的影响,旨在为研究同种异体器官移植诱导免疫耐受提供新策略.方法 RT-PCR获取C57BL/6小鼠MHC-Ⅰ等位基因H-2Kb和H-2Db基因,并与pIRES质粒连接,构建重组质粒并转染体外培养BALB/c小鼠pre-T细胞构建分子嵌合细胞,并设立未转染及转染空质粒对照组.各组细胞回输BALB/c小鼠,7d后提取各组小鼠脾脏T细胞,将其与C57BL/6小鼠T淋巴细胞混合培养,观察刺激指数(SI).结果 构建质粒测序表明,插入序列的结果包含GenBank检索的C57BL/6小鼠H-2Kb和H-2Db序列,流式细胞仪检测质粒pIRES-H-2Kb和pIRES-H-2Db转染后,pre-T细胞表面H-2Kb及H-2Db蛋白的表达增高,与未转染组及转染空质粒组相比,其差异均具有统计学意义(P<0.05).单向混合淋巴细胞培养结果显示:2质粒共注射pre-T细胞组的SI值(0.764±0.074)较空质粒组(0.983±0.081)和未转染组(0.994±0.142)明显下降,其差异有统计学意义(P<0.05).结论 分子嵌合细胞可降低受体T细胞对异基因T淋巴细胞刺激的增殖反应,有望应用于体内诱导免疫耐受.%Objective To research the effect of molecular chimeric mice pre-T cells on proliferation ability of allogeneic mouse T cells.Methods The MHC-Ⅰ gene (H-2Kband H-2Db gene) were extracted and amplified by RT-PCR,the identified pre-T cells were transfected by the constructed eukaryotic expression vector of C57BL/6mouse MHC-I (pIRES-H-2Db and pIRES-H-2Kb),non-transfected group and sham pIRES-transfected control group were set.The molecular chimeric cells were transfused back to BALB/c mouse.After 7 days,T lymphocyte cells of each group were extracted,the ability of molecular chimeric cells inducing spleen T lymphocyte response to allogeneic T cells was observed through mixed lymphocyte culture (MLC).Results Sequencing of the plasmid we have constructed showed that insertion sequence contained C57BL/6 mice

  17. Chimerism and xenotransplantation. New concepts.

    Science.gov (United States)

    Starzl, T E; Rao, A S; Murase, N; Demetris, A J; Thomson, A; Fung, J J

    1999-02-01

    In both transplant and infectious circumstances, the immune response is governed by migration and localization of the antigen. If the antigenic epitopes of transgenic xenografts are sufficiently altered to avoid evoking the destructive force of innate immunity, the mechanisms of engraftment should be the same as those that permit the chimerism-dependent immunologic confrontation and resolution that is the basis of allograft acceptance. In addition to "humanizing" the epitopes, one of the unanswered questions is whether the species restriction of complement described in 1994 by Valdivia and colleagues also necessitates the introduction of human complement regulatory genes in animal donors. Because the liver is the principal or sole source of most complement components, the complement quickly is transformed to that of the donor after hepatic transplantation. Thus, the need for complementary regulatory transgenes may vary according to the kind of xenograft used. Much evidence shows that physiologically important peptides produced by xenografts (e.g., insulin, clotting factors, and enzymes) are incorporated into the metabolic machinery of the recipient body. To the extent that this is not true, xenotransplantation could result in the production of diseases that are analogous to inborn errors of metabolism. In the climate of pessimism that followed the failures of baboon to human liver xenotransplantation in 1992-1993, it seemed inconceivable that the use of even more discordant donors, such as the pig, could ever be seriously entertained; however, this preceded insight into the xenogeneic and allogeneic barriers that has brought transplantation infectious immunity to common ground. With this new insight and the increasing ease of producing transgenic donors, the goal of clinical xenotransplantation may not be so distant.

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

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    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.

  19. Hepatitis C virus infection suppresses the interferon response in the liver of the human hepatocyte chimeric mouse.

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    Masataka Tsuge

    Full Text Available BACKGROUND AND AIMS: Recent studies indicate that hepatitis C virus (HCV can modulate the expression of various genes including those involved in interferon signaling, and up-regulation of interferon-stimulated genes by HCV was reported to be strongly associated with treatment outcome. To expand our understanding of the molecular mechanism underlying treatment resistance, we analyzed the direct effects of interferon and/or HCV infection under immunodeficient conditions using cDNA microarray analysis of human hepatocyte chimeric mice. METHODS: Human serum containing HCV genotype 1b was injected into human hepatocyte chimeric mice. IFN-α was administered 8 weeks after inoculation, and 6 hours later human hepatocytes in the mouse livers were collected for microarray analysis. RESULTS: HCV infection induced a more than 3-fold change in the expression of 181 genes, especially genes related to Organismal Injury and Abnormalities, such as fibrosis or injury of the liver (P = 5.90E-16∼3.66E-03. IFN administration induced more than 3-fold up-regulation in the expression of 152 genes. Marked induction was observed in the anti-fibrotic chemokines such as CXCL9, suggesting that IFN treatment might lead not only to HCV eradication but also prevention and repair of liver fibrosis. HCV infection appeared to suppress interferon signaling via significant reduction in interferon-induced gene expression in several genes of the IFN signaling pathway, including Mx1, STAT1, and several members of the CXCL and IFI families (P = 6.0E-12. Genes associated with Antimicrobial Response and Inflammatory Response were also significantly repressed (P = 5.22×10(-10∼1.95×10(-2. CONCLUSIONS: These results provide molecular insights into possible mechanisms used by HCV to evade innate immune responses, as well as novel therapeutic targets and a potential new indication for interferon therapy.

  20. Arabinoxylan rice bran (MGN-3/Biobran) provides protection against whole-body γ-irradiation in mice via restoration of hematopoietic tissues

    International Nuclear Information System (INIS)

    The aim of the current study is to examine the protective effect of MGN-3 on overall maintenance of hematopoietic tissue after γ-irradiation. MGN-3 is an arabinoxylan from rice bran that has been shown to be a powerful antioxidant and immune modulator. Swiss albino mice were treated with MGN-3 prior to irradiation and continued to receive MGN-3 for 1 or 4 weeks. Results were compared with mice that received radiation (5 Gy γ rays) only, MGN-3 (40 mg/kg) only and control mice (receiving neither radiation nor MGN-3). At 1 and 4 weeks post-irradiation, different hematological, histopathological and biochemical parameters were examined. Mice exposed to irradiation alone showed significant depression in their complete blood count (CBC) except for neutrophilia. Additionally, histopathological studies showed hypocellularity of their bone marrow, as well as a remarkable decrease in splenic weight/relative size and in number of megakaryocytes. In contrast, pre-treatment with MGN-3 resulted in protection against irradiation-induced damage to the CBC parameters associated with complete bone marrow cellularity, as well as protection of the aforementioned splenic changes. Furthermore, MGN-3 exerted antioxidative activity in whole-body irradiated mice, and provided protection from irradiation-induced loss of body and organ weight. In conclusion, MGN-3 has the potential to protect progenitor cells in the bone marrow, which suggests the possible use of MGN-3/Biobran as an adjuvant treatment to counteract the severe adverse side effects associated with radiation therapy

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

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    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.

  2. Faith-based perspectives on the use of chimeric organisms for medical research.

    Science.gov (United States)

    Degeling, Chris; Irvine, Rob; Kerridge, Ian

    2014-04-01

    Efforts to advance our understanding of neurodegenerative diseases involve the creation chimeric organisms from human neural stem cells and primate embryos--known as prenatal chimeras. The existence of potential mentally complex beings with human and non-human neural apparatus raises fundamental questions as to the ethical permissibility of chimeric research and the moral status of the creatures it creates. Even as bioethicists find fewer reasons to be troubled by most types of chimeric organisms, social attitudes towards the non-human world are often influenced by religious beliefs. In this paper scholars representing eight major religious traditions provide a brief commentary on a hypothetical case concerning the development and use of prenatal human-animal chimeric primates in medical research. These commentaries reflect the plurality and complexity within and between religious discourses of our relationships with other species. Views on the moral status and permissibility of research on neural human animal chimeras vary. The authors provide an introduction to those who seek a better understanding of how faith-based perspectives might enter into biomedical ethics and public discourse towards forms of biomedical research that involves chimeric organisms.

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    Yukiko eMishina

    2014-09-01

    Full Text Available Deciphering how the brain generates cognitive function from patterns of electrical signals is one of the ultimate challenges in neuroscience. To this end, it would be highly desirable to monitor the activities of very large numbers of neurons while an animal engages in complex behaviours. Optical imaging of electrical activity using genetically encoded voltage indicators (GEVIs has the potential to meet this challenge. Currently prevalent GEVIs are based on the voltage-sensitive fluorescent protein (VSFP prototypical design or on the voltage dependent state transitions of microbial opsins.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.

  5. 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

  6. HBV和HIV的复合型DNA免疫可诱发BALB/c小鼠的细胞免疫应答%Vaccination of BALB/c Mice with Chimeric HBV and HIV DNA can Induce Cellular Immune Responses

    Institute of Scientific and Technical Information of China (English)

    和绍清; 董承红; 孙明; 李琦涵

    2001-01-01

    本研究是通过细胞免疫检测来研究含有两种不同病毒基因的复合型DNA免疫的效果,同时和单一病毒基因的DNA免疫作比较。在本实验中,将HBV的S基因和(或)HⅣ-1的gp120基因插入到真核表达载体pcDNA3中,得到能表达HBsAg和gp120融合蛋白的重组体pcDNA-S-120,和能表达HBsAg的重组体pcDNA-S。在实验中,将重组质粒DNA直接注射到BALB/c小鼠股四头肌内,按每只小鼠100μg/100μl的剂量经3次免疫后,在T细胞增殖实验中,用HBsAg蛋白刺激体外培养的被免疫小鼠T细胞后,出现了明显的T细胞增殖。采用”cr释放法检测特异性CTL杀伤作用时,发现致敏的CTL对HIV gp120多肽孵育后的靶细胞P815有明显的杀伤作用。%Recent studies support the notion that vaccination with plasmid DNA encording genes for viral or bacterial antigens can elicite both humoral and cellular immune responses in rodents and non-human primates, but their major advantage at immunologic level are their capacity to induce antigen-specific CD8 + T cell responses. In the present study,BALB/c mice were immunilized intramuscularly with recombinant plasmid pcDNA-S-120 in which the S gene of HBV and gp120 gene of HIV-1 were cloned into pcDNA3 in fusion form, and also with pcDNA-S in which the S gene of HBV had been cloned into pcDNA3. Mice were immunilized 3 times with 100 μg/100 μi per mouse. In the T cell proliferation assays, it was showed that T cells proliferated in vitro obviously after stimulation with pcDNA-S and pcDNA-S-120. The sensitized CTL could specifically kill the target cell P815 which were pulsed by 10-AA peptide of HIV. It concludes that cellular immune responses could be elicited in the experimental animals after direct injections of recombinatant plasmid DNA in which the S gene and gp120 gene were cloned in fusion form.

  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. Induction of pluripotent protective immunity following immunisation with a chimeric vaccine against human cytomegalovirus.

    Directory of Open Access Journals (Sweden)

    Jie Zhong

    Full Text Available Based on the life-time cost to the health care system, the Institute of Medicine has assigned the highest priority for a vaccine to control human cytomegalovirus (HCMV disease in transplant patients and new born babies. In spite of numerous attempts successful licensure of a HCMV vaccine formulation remains elusive. Here we have developed a novel chimeric vaccine strategy based on a replication-deficient adenovirus which encodes the extracellular domain of gB protein and multiple HLA class I & II-restricted CTL epitopes from HCMV as a contiguous polypeptide. Immunisation with this chimeric vaccine consistently generated strong HCMV-specific CD8(+ and CD4(+ T-cells which co-expressed IFN-gamma and TNF-alpha, while the humoral response induced by this vaccine showed strong virus neutralizing capacity. More importantly, immunization with adenoviral chimeric vaccine also afforded protection against challenge with recombinant vaccinia virus encoding HCMV antigens and this protection was associated with the induction of a pluripotent antigen-specific cellular and antibody response. Furthermore, in vitro stimulation with this adenoviral chimeric vaccine rapidly expanded multiple antigen-specific human CD8(+ and CD4(+ T-cells from healthy virus carriers. These studies demonstrate that the adenovirus chimeric HCMV vaccine provides an excellent platform for reconstituting protective immunity to prevent HCMV diseases in different clinical settings.

  9. Chimeric hERG channels containing a tetramerization domain are functional and stable.

    Science.gov (United States)

    Hausammann, Georg J; Grütter, Markus G

    2013-12-23

    Biochemical and detailed structural information of human ether-a-go-go-related gene (hERG) potassium channels are scarce but are a prerequisite to understand the unwanted interactions of hERG with drugs and the effect of mutations that lead to long QT syndrome. Despite the huge interest in hERG, to our knowledge, procedures that provide a purified, functional, and tetrameric hERG channel are not available. Here, we describe hybrid hERG molecules, termed chimeric hERG channels, in which the N-terminal Per-Arnt-Sim (PAS) domain is deleted and the C-terminal C-linker as well as the cyclic nucleotide binding domain (CNBD) portion is replaced by an artificial tetramerization domain. These chimeric hERG channels can be overexpressed in HEK cells, solubilized in detergent, and purified as tetramers. When expressed in Xenopus laevis oocytes, the chimeric channels exhibit efficient trafficking to the cell surface, whereas a hERG construct lacking the PAS and C-linker/CNBD domains is retained in the cytoplasm. The chimeric hERG channels retain essential hERG functions such as voltage-dependent gating and inhibition by astemizole and the scorpion toxin BeKm-1. The chimeric channels are thus powerful tools for helping to understand the contribution of the cytoplasmic hERG domains to the gating process and are suitable for in vitro biochemical and structural studies. PMID:24325597

  10. 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

  11. Analysis of mice with targeted deletion of AQP9 gene provides conclusive evidence for expression of AQP9 in neurons

    DEFF Research Database (Denmark)

    Mylonakou, Maria N; Petersen, Petur H; Rinvik, Eric;

    2009-01-01

    AQP9 is an aquaglyceroporin that serves important functions in peripheral organs, including the liver. Reflecting the lack of AQP9 knockout mice, uncertainties still prevail regarding the localization and roles of AQP9 in the central nervous system. Here we present a comprehensive analysis of AQP9...... and mouse liver, the organ with the highest level of AQP9. By blue native gel analysis it could be demonstrated that the brain contains tetrameric AQP9, corresponding to the functional form of AQP9. The band corresponding to the AQP9 tetramer was absent in AQP9 knockout brain and liver. Immunocytochemistry...... and in situ hybridization analyses with AQP9 knockout controls show that subpopulations of nigral neurons express AQP9 both at the mRNA and at the protein levels and that populations of cortical cells (including hilar neurons in the hippocampus) contain AQP9 mRNA but no detectable AQP9 immunosignal...

  12. Adenoviral Expression of a Bispecific VHH-Based Neutralizing Agent That Targets Protective Antigen Provides Prophylactic Protection from Anthrax in Mice.

    Science.gov (United States)

    Moayeri, Mahtab; Tremblay, Jacqueline M; Debatis, Michelle; Dmitriev, Igor P; Kashentseva, Elena A; Yeh, Anthony J; Cheung, Gordon Y C; Curiel, David T; Leppla, Stephen; Shoemaker, Charles B

    2016-03-01

    Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 μg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 μg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 μg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors. PMID:26740390

  13. Construction of a Chimeric Secretory IgA and Its Neutralization Activity against Avian Influenza Virus H5N1

    Directory of Open Access Journals (Sweden)

    Cun Li

    2014-01-01

    Full Text Available Secretory immunoglobulin A (SIgA acts as the first line of defense against respiratory pathogens. In this assay, the variable regions of heavy chain (VH and Light chain (VL genes from a mouse monoclonal antibody against H5N1 were cloned and fused with human IgA constant regions. The full-length chimeric light and heavy chains were inserted into a eukaryotic expressing vector and then transfected into CHO/dhfr-cells. The chimeric monomeric IgA antibody expression was confirmed by using ELISA, SDS-PAGE, and Western blot. In order to obtain a dimeric secretory IgA, another two expressing plasmids, namely, pcDNA4/His A-IgJ and pcDNA4/His A-SC, were cotransfected into the CHO/dhfr-cells. The expression of dimeric SIgA was confirmed by using ELISA assay and native gel electrophoresis. In microneutralization assay on 96-well immunoplate, the chimeric SIgA showed neutralization activity against H5N1 virus on MDCK cells and the titer was determined to be 1 : 64. On preadministrating intranasally, the chimeric SIgA could prevent mice from lethal attack by using A/Vietnam/1194/04 H5N1 with a survival rate of 80%. So we concluded that the constructed recombinant chimeric SIgA has a neutralization capability targeting avian influenza virus H5N1 infection in vitro and in vivo.

  14. 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.

  15. Oral administration of Lactobacillus plantarum strain AYA enhances IgA secretion and provides survival protection against influenza virus infection in mice.

    Directory of Open Access Journals (Sweden)

    Yosuke Kikuchi

    Full Text Available The mucosal immune system provides the first line of defense against inhaled and ingested pathogenic microbacteria and viruses. This defense system, to a large extent, is mediated by the actions of secretory IgA. In this study, we screened 140 strains of lactic acid bacteria for induction of IgA production by murine Peyer's patch cells. We selected one strain and named it Lactobacillus plantarum AYA. We found that L. plantarum AYA-induced production of IL-6 in Peyer's patch dendritic cells, with this production promoting IgA(+ B cells to differentiate into IgA-secreting plasma cells. We also observed that oral administration of L. plantarum AYA in mice caused an increase in IgA production in the small intestine and lung. This production of IgA correlated strongly with protective ability, with the treated mice surviving longer than the control mice after lethal influenza virus infection. Our data therefore reveals a novel immunoregulatory role of the L. plantarum AYA strain which enhances mucosal IgA production and provides protection against respiratory influenza virus infection.

  16. CHIMERE 2013: a model for regional atmospheric composition modelling

    Directory of Open Access Journals (Sweden)

    L. Menut

    2013-07-01

    Full Text Available Tropospheric trace gas and aerosol pollutants have adverse effects on health, environment and climate. In order to quantify and mitigate such effects, a wide range of processes leading to the formation and transport of pollutants must be considered, understood and represented in numerical models. Regional scale pollution episodes result from the combination of several factors: high emissions (from anthropogenic or natural sources, stagnant meteorological conditions, kinetics and efficiency of the chemistry and the deposition. All these processes are highly variable in time and space, and their relative contribution to the pollutants budgets can be quantified with chemistry-transport models. The CHIMERE chemistry-transport model is dedicated to regional atmospheric pollution event studies. Since it has now reached a certain level a maturity, the new stable version, CHIMERE 2013, is described to provide a reference model paper. The successive developments of the model are reviewed on the basis of published investigations that are referenced in order to discuss the scientific choices and to provide an overview of the main results.

  17. 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...

  18. Human antibody expression in transgenic rats: comparison of chimeric IgH loci with human VH, D and JH but bearing different rat C-gene regions.

    Science.gov (United States)

    Ma, Biao; Osborn, Michael J; Avis, Suzanne; Ouisse, Laure-Hélène; Ménoret, Séverine; Anegon, Ignacio; Buelow, Roland; Brüggemann, Marianne

    2013-12-31

    Expression of human antibody repertoires in transgenic animals has been accomplished by introducing large human Ig loci into mice and, more recently, a chimeric IgH locus into rats. With human VH, D and JH genes linked to the rat C-region antibody expression was significantly increased, similar to wild-type levels not found with fully human constructs. Here we compare four rat-lines containing the same human VH-region (comprising 22 VHs, all Ds and all JHs in natural configuration) but linked to different rat CH-genes and regulatory sequences. The endogenous IgH locus was silenced by zinc-finger nucleases. After breeding, all lines produced exclusively chimeric human H-chain with near normal IgM levels. However, in two lines poor IgG expression and inefficient immune responses were observed, implying that high expression, class-switching and hypermutation are linked to optimal enhancer function provided by the large regulatory region at the 3' end of the IgH locus. Furthermore, exclusion of Cδ and its downstream interval region may assist recombination. Highly diverse IgG and immune responses similar to normal rats were identified in two strains carrying diverse and differently spaced C-genes.

  19. 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

  20. 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

  1. Adult hematopoietic progenitors are pluripotent in chimeric mice

    OpenAIRE

    Pessac, Bernard; K. Nimmagadda, Vamshi; Makar, Tapas; S. Fishman, Paul; T. Bever Jr., Christopher; Trisler, David

    2012-01-01

    18 pages, 7 figures. Embryonic stem cells (ESCs) and adult somatic cells, induced to pluripotency (iPSCs) by genetic manipulation, display high self-­‐renewal potential and the capacity to differentiate into multiple cell lineages. We asked whether there are in adult mammals natural stem cells that are pluripotent. We previously reported that normal adult mammalian bone marrow contains a sub-­‐population of CD34+ cells, that naturally expresses genes characteristic of ESCs and those requir...

  2. 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...

  3. Does brain slices from pentylenetetrazole-kindled mice provide a more predictive screening model for antiepileptic drugs?

    DEFF Research Database (Denmark)

    Hansen, Suzanne L.; Sterjev, Zoran; Werngreen, Marie;

    2012-01-01

    The cortical wedge is a commonly applied model for in vitro screening of new antiepileptic drugs (AEDs) and has been extensively used in characterization of well-known AEDs. However, the predictive validity of this model as a screening model has been questioned as, e.g., carbamazepine has been...... reported to lack effect in this model. The neuroplastic changes induced in acute and chronic animal models of epilepsy are known to affect the pharmacological profile of AEDs in vivo. Hence, we investigated whether brain slices from pentylenetetrazole (PTZ)-kindled animals could provide a more predictive...... screening model for AEDs. To this end, we compared the in vitro and in vivo pharmacological profile of several selected AEDs (phenobarbital, phenytoin, tiagabine, fosphenytoin, valproate, and carbamazepine) along with citalopram using the PTZ-kindled model and brain slices from naïve, saline...

  4. Immunization with Cytomegalovirus Envelope Glycoprotein M and Glycoprotein N DNA Vaccines can Provide Mice with Complete Protection against a Lethal Murine Cytomegalovirus Challenge

    Institute of Scientific and Technical Information of China (English)

    Huadong Wang; Yanfeng Yao; Chaoyang Huang; Quanjiao Chen; Jianjun Chen; Ze Chen

    2013-01-01

    Human cytomegalovirus virions contain three major glycoprotein complexes (gC Ⅰ,Ⅱ,Ⅲ),all of which are required for CMV infectivity.These complexes also represent major antigenic targets for anti-viral immune responses.The gC Ⅱ complex consists of two glycoproteins,gM and gN.In the current study,DNA vaccines expressing the murine cytomegalovirus (MCMV) homologs of the gM and gN proteins were evaluated for protection against lethal MCMV infection in a mouse model.Humoral and cellular immune responses,spleen viral titers,and mice survival and body-weight changes were examined.The results showed that immunization with gM or gN DNA vaccine alone was not able to offer good protection,whereas co-immunization with both gM and gN induced an effective neutralizing antibody response and cellular immune response,and provided mice with complete protection against a lethal MCMV challenge.This study provides the first in vivo evidence that the gC Ⅱ (gM-gN) complex may be able to serve as a protective subunit antigen for future HCMV vaccine development.

  5. A human/mouse chimeric monoclonal antibody against intercellular adhesion molecule-1 for tumor radioimmunoimaging

    Energy Technology Data Exchange (ETDEWEB)

    Yamamura, Miyuki; Hinoda, Yuji; Sasaki, Shigeru; Tsujisaki, Masayuki; Imai, Kohzoh [Sapporo Medical Univ. (Japan); Oriuchi, Noboru; Endo, Keigo

    1996-04-01

    A mouse-human chimeric antibody for intercellular adhesion molecule-1 (ICAM-1) was established by using heavy chain loss mouse mutant hybridoma and human immunoglobulin expression vector. The HA58 hybridoma secreted anti-ICAM-1 monoclonal antibody (MoAb) (IgG1,{kappa}). The gene of the mouse variable region of heavy chain was amplified and cloned by the polymerase chain reaction technique directly from the HA58 hybridoma RNA. The variable region of heavy chain was joined with an expression vector which contains human {gamma}1 constant gene. The expression vector was transfected into heavy chain loss mutant cells HA58-7, which produced only murine immunoglobulin light chains. The resultant chimeric MoAb HA58, chHA58, retained full-binding reactivity to ICAM-1 compared with murine HA58 parental antibody. The chimeric MoAb chHA58 showed little antibody dependent cell-mediated cytotoxic activity against cultured tumor cells. Biodistribution studies with {sup 99m}Tc-labeled chHA58 in nude mice bearing human gastric carcinoma JRST cells, demonstrated that the tumor-blood ratio was 1.55 at 18 h after injection, when the tumors were clearly visible in gamma scintigraphy. These data suggest that chHA58 may be of practical use for radioimmunoimaging of a wide variety of tumors. (author).

  6. 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.

  7. In silico and experimental characterization of chimeric Bacillus thermocatenulatus lipase with the complete conserved pentapeptide of Candida rugosa lipase.

    Science.gov (United States)

    Hosseini, Mostafa; Karkhane, Ali Asghar; Yakhchali, Bagher; Shamsara, Mehdi; Aminzadeh, Saeed; Morshedi, Dena; Haghbeen, Kamahldin; Torktaz, Ibrahim; Karimi, Esmat; Safari, Zahra

    2013-02-01

    Lipases are one of the highest value commercial enzymes as they have broad applications in detergent, food, pharmaceutical, and dairy industries. To provide chimeric Bacillus thermocatenulatus lipase (BTL2), the completely conserved pentapeptide (¹¹²Ala-His-Ser-Gln-Gly¹¹⁶) was replaced with similar sequences (²⁰⁷Gly-Glu-Ser-Ala-Gly²¹¹) of Candida rugosa lipase (CLR) at the nucleophilic elbow region. For this purpose, three mutations including A112G, H113E, and Q115A were inserted in the conserved pentapeptide sequence of btl2 gene. Based on the crystal structures of 2W22, the best structure of opened form of the chimeric lipases were garnered using the MODELLER v9.10 software. The native and chimeric lipases were docked to a set of ligands, and a trial version of Molegro Virtual Docker (MVD) software was used to obtain the energy values. Docking results confirmed chimeric lipase to be better than the native lipase. Following the in silico study, cloning experiments were conducted and expression of native and chimeric btl2 gene in Pichia pastoris was performed. The native and chimeric lipases were purified, and the effect of these mutations on characteristics of chimeric lipase studied and then compared with those of native lipase. Chimeric lipase exhibited 1.6-fold higher activity than the native lipase at 55 °C. The highest percentage of both lipases activity was observed at 60 °C and pH of 8.0. The ion Ca²⁺ slightly inhibited the activity of both lipases, whereas the organic solvent enhanced the lipase stability of chimeric lipase as compared with the native lipase. According to the results, the presence of two glycine residues at the conserved pentapeptide region of this chimeric lipase (¹¹²Gly-Glu-Ser-Ala-Gly¹¹⁶) may increase the flexibility of the nucleophilic elbow region and affect the enzyme activity level. PMID:23274720

  8. 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.

  9. Regional atmospheric composition modeling with CHIMERE

    Science.gov (United States)

    Menut, L.; Bessagnet, B.; Khvorostyanov, D.; Beekmann, M.; Colette, A.; Coll, I.; Curci, G.; Foret, G.; Hodzic, A.; Mailler, S.; Meleux, F.; Monge, J.-L.; Pison, I.; Turquety, S.; Valari, M.; Vautard, R.; Vivanco, M. G.

    2013-01-01

    Tropospheric trace gas and aerosol pollutants have adverse effects on health, environment and climate. In order to quantify and mitigate such effects, a wide range of processes leading to the formation and transport of pollutants must be considered, understood and represented in numerical models. Regional scale pollution episodes result from the combination of several factors: high emissions (from anthropogenic or natural sources), stagnant meteorological conditions, velocity and efficiency of the chemistry and the deposition. All these processes are highly variable in time and space, and their relative importance to the pollutants budgets can be quantified within a chemistry-transport models (CTM). The offline CTM CHIMERE model uses meteorological model fields and emissions fluxes and calculates deterministically their behavior in the troposphere. The calculated three-dimensional fields of chemical concentrations can be compared to measurements to analyze past periods or used to make air quality forecasts and CHIMERE has enabled a fine understanding of pollutants transport during numerous measurements campaigns. It is a part of the PREVAIR french national forecast platform, delivering pollutant concentrations up to three days in advance. The model also allows scenario studies and long term simulations for pollution trends. The modelling of photochemical air pollution has reached a good level of maturity, and the latest projects involving CHIMERE now aim at increasing our understanding of pollution impact on health at the urban scale or at the other end of the spectrum for long term air quality and climate change interlinkage studies, quantifying the emissions and transport of pollen, but also, at a larger scale, analyzing the transport of pollutants plumes emitted by volcanic eruptions and forest fires.

  10. Regional atmospheric composition modeling with CHIMERE

    Directory of Open Access Journals (Sweden)

    L. Menut

    2013-01-01

    Full Text Available Tropospheric trace gas and aerosol pollutants have adverse effects on health, environment and climate. In order to quantify and mitigate such effects, a wide range of processes leading to the formation and transport of pollutants must be considered, understood and represented in numerical models. Regional scale pollution episodes result from the combination of several factors: high emissions (from anthropogenic or natural sources, stagnant meteorological conditions, velocity and efficiency of the chemistry and the deposition. All these processes are highly variable in time and space, and their relative importance to the pollutants budgets can be quantified within a chemistry-transport models (CTM. The offline CTM CHIMERE model uses meteorological model fields and emissions fluxes and calculates deterministically their behavior in the troposphere. The calculated three-dimensional fields of chemical concentrations can be compared to measurements to analyze past periods or used to make air quality forecasts and CHIMERE has enabled a fine understanding of pollutants transport during numerous measurements campaigns. It is a part of the PREVAIR french national forecast platform, delivering pollutant concentrations up to three days in advance. The model also allows scenario studies and long term simulations for pollution trends. The modelling of photochemical air pollution has reached a good level of maturity, and the latest projects involving CHIMERE now aim at increasing our understanding of pollution impact on health at the urban scale or at the other end of the spectrum for long term air quality and climate change interlinkage studies, quantifying the emissions and transport of pollen, but also, at a larger scale, analyzing the transport of pollutants plumes emitted by volcanic eruptions and forest fires.

  11. Human Polyclonal Antibodies Produced through DNA Vaccination of Transchromosomal Cattle Provide Mice with Post-Exposure Protection against Lethal Zaire and Sudan Ebolaviruses.

    Directory of Open Access Journals (Sweden)

    Callie E Bounds

    Full Text Available DNA vaccination of transchromosomal bovines (TcBs with DNA vaccines expressing the codon-optimized (co glycoprotein (GP genes of Ebola virus (EBOV and Sudan virus (SUDV produce fully human polyclonal antibodies (pAbs that recognize both viruses and demonstrate robust neutralizing activity. Each TcB was vaccinated by intramuscular electroporation (IM-EP a total of four times and at each administration received 10 mg of the EBOV-GPco DNA vaccine and 10 mg of the SUDV-GPco DNA vaccine at two sites on the left and right sides, respectively. After two vaccinations, robust antibody responses (titers > 1000 were detected by ELISA against whole irradiated EBOV or SUDV and recombinant EBOV-GP or SUDV-GP (rGP antigens, with higher titers observed for the rGP antigens. Strong, virus neutralizing antibody responses (titers >1000 were detected after three vaccinations when measured by vesicular stomatitis virus-based pseudovirion neutralization assay (PsVNA. Maximal neutralizing antibody responses were identified by traditional plaque reduction neutralization tests (PRNT after four vaccinations. Neutralizing activity of human immunoglobulins (IgG purified from TcB plasma collected after three vaccinations and injected intraperitoneally (IP into mice at a 100 mg/kg dose was detected in the serum by PsVNA up to 14 days after administration. Passive transfer by IP injection of the purified IgG (100 mg/kg to groups of BALB/c mice one day after IP challenge with mouse adapted (ma EBOV resulted in 80% protection while all mice treated with non-specific pAbs succumbed. Similarly, interferon receptor 1 knockout (IFNAR(-/- mice receiving the purified IgG (100 mg/kg by IP injection one day after IP challenge with wild type SUDV resulted in 89% survival. These results are the first to demonstrate that filovirus GP DNA vaccines administered to TcBs by IM-EP can elicit neutralizing antibodies that provide post-exposure protection. Additionally, these data describe

  12. Evaluation of genetic melanoma vaccines in cdk4-mutant mice provides evidence for immunological tolerance against authochthonous melanomas in the skin.

    Science.gov (United States)

    Steitz, Julia; Büchs, Stefanie; Tormo, Damia; Ferrer, Aleix; Wenzel, Jörg; Huber, Christoph; Wölfel, Thomas; Barbacid, Mariano; Malumbres, Marcos; Tüting, Thomas

    2006-01-15

    We evaluated the efficacy of a candidate melanoma vaccine approach in mice genetically prone to develop melanoma due to the introduction of an oncogenic mutation (R24C) in the germline sequence of the cyclin-dependent kinase 4 (cdk4), a protein critically involved in cell cycle regulation. Melanomas were induced in cdk4-mutant mice by chemical carcinogenesis and UVB irradiation. A genetic prime-boost strategy targeting the clinically relevant differentiation antigen tyrosinase-related protein 2 (TRP2) was performed which was able to stimulate a melanocyte-specific cellular immune response associated with localized autoimmune vitiligo-like depigmentation. However, significant destruction of carcinogen-induced autochthonous melanocytic neoplasms in the skin was not observed following immunization. We provide evidence that autochthonous melanomas expressed TRP2 but not the MHC molecule H2-Kb and are immunologically tolerated in the skin. Our results highlight the importance of assessing melanoma vaccines in genetic mouse models that more adequately represent the expected clinical situation in order to identify strategies, which eventually may be of benefit for melanoma patients.

  13. Pregnancy, chimerism and lupus nephritis : a multi-centre study

    NARCIS (Netherlands)

    Hovinga, I. C. L. Kremer; Koopmans, M.; Grootscholten, C.; van der Wal, A. M.; Bijl, M.; Derksen, R. H. W. M.; Voslcuyl, A. E.; de Heer, E.; Bruijn, J. A.; Berden, J. H. M.; Rajema, I. M.

    2008-01-01

    Chimerism occurs twice as often in the kidneys of women with lupus nephritis as in normal kidneys and may he involved in the pathogenesis of systemic lupus erythematosus. Pregnancy is considered the most important source of chimerism, but the exact relationship between pregnancy, the persistence of

  14. Pregnancy, chimerism and lupus nephritis: a multi-centre study.

    NARCIS (Netherlands)

    Hovinga, I.C. Kremer; Koopmans, M.; Grootscholten, C.; Wal, A.M. van der; Bijl, M. van der; Derksen, R.H.; Voskuyl, A.E.; Heer, E. de; Bruijn, J.A.; Berden, J.H.M.; Bajema, I.M.

    2008-01-01

    Chimerism occurs twice as often in the kidneys of women with lupus nephritis as in normal kidneys and may be involved in the pathogenesis of systemic lupus erythematosus. Pregnancy is considered the most important source of chimerism, but the exact relationship between pregnancy, the persistence of

  15. Comparisons of native Shiga toxins (Stxs type 1 and 2 with chimeric toxins indicate that the source of the binding subunit dictates degree of toxicity.

    Directory of Open Access Journals (Sweden)

    Lisa M Russo

    Full Text Available Shiga toxin (Stx-producing E. coli (STEC cause food-borne outbreaks of hemorrhagic colitis. The main virulence factor expressed by STEC, Stx, is an AB5 toxin that has two antigenically distinct forms, Stx1a and Stx2a. Although Stx1a and Stx2a bind to the same receptor, globotriaosylceramide (Gb3, Stx2a is more potent than Stx1a in mice, whereas Stx1a is more cytotoxic than Stx2a in cell culture. In this study, we used chimeric toxins to ask what the relative contribution of individual Stx subunits is to the differential toxicity of Stx1a and Stx2a in vitro and in vivo. Chimeric stx1/stx2 operons were generated by PCR such that the coding regions for the A2 and B subunits of one toxin were combined with the coding region for the A1 subunit of the heterologous toxin. The toxicities of purified Stx1a, Stx2a, and the chimeric Stxs were determined on Vero and HCT-8 cell lines, while polarized HCT-8 cell monolayers grown on permeable supports were used to follow toxin translocation. In all in vitro assays, the activity of the chimeric toxin correlated with that of the parental toxin from which the B subunit originated. The origin of the native B subunit also dictated the 50% lethal dose of toxin after intraperitoneal intoxication of mice; however, the chimeric Stxs exhibited reduced oral toxicity and pH stability compared to Stx1a and Stx2a. Taken together, these data support the hypothesis that the differential toxicity of the chimeric toxins for cells and mice is determined by the origin of the B subunit.

  16. 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

  17. Angiotensin Ⅱ receptor blocker provides pancreatic β-cell protection independent of blood pressure lowering in diabetic db/db mice

    Institute of Scientific and Technical Information of China (English)

    Jia-qing SHAO; Noseki IWASHITA; Hong DU; Yang-tian WANG; Yan-yan WANG; Ming ZHAO; Jian WANG; Hirotaka WATADA; Ryuzo KAWAMORI

    2007-01-01

    Aim: Several epidemiological studies have suggested that treatment with angiotensin Ⅱ type 1 receptor blocker provided a risk reduction of developing type 2 diabetes. The aim of this study was to investigate whether and how chronic candesartan treatment can attenuate the deleterious influence of the hyperactive local intra-islet renin-angiotensin system in the diabetes state. Methods: Eight-week-old db/db mice were randomized to candesartan 1 mg/kg, candesartan 10mg/kg, manidipine 10 mg/kg, or placebo via gavage for 6 weeks. Their age-matched nondiabetic littermates db/m mice were treated with placebo and acted as nondia-betic controls. After 6 weeks' treatment, an intraperitoneal glucose tolerance test, immunohistochemical staining of oxidative stress markers, insulin, CD31, azan staining and an electron microscopy observation were performed. Results: Chronic candesartan treatment provided an improvement of glucose tolerance, and greatly rescued islet β-cell mass. Candesartan treatment also notably decreased staining intensity of oxidative stress markers, as well as attenuating intra-islet fibrosis and improving blood supply in the islet. In the electron microscopy observation, candesartan-treated animals exhibited improved granulation and less remarkable endoplasmic reticulum and Golgi bodies; furthermore, candesartan treatment greatly relieved the swelling of mitochondria to nearly normal. Both the benefits of reducing oxidative stress and ultra structure protection were in a dose-dependent and blood pressure-independent manner. Conclusion: After diabetes was initiated, candesartan treatment could not reverse the state of diabetes, but it effectively improved glucose tolerance and protected β-cell function by attenuating oxidative stress, islet fibrosis, sparsity of blood supply and ultrastructure disruption in a dose-dependent and blood pressure-independent manner.

  18. 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...

  19. Use of CTLA4Ig for induction of mixed chimerism and renal allograft tolerance in nonhuman primates.

    Science.gov (United States)

    Yamada, Y; Ochiai, T; Boskovic, S; Nadazdin, O; Oura, T; Schoenfeld, D; Cappetta, K; Smith, R-N; Colvin, R B; Madsen, J C; Sachs, D H; Benichou, G; Cosimi, A B; Kawai, T

    2014-12-01

    We have previously reported successful induction of renal allograft tolerance via a mixed chimerism approach in nonhuman primates. In those studies, we found that costimulatory blockade with anti-CD154 mAb was an effective adjunctive therapy for induction of renal allograft tolerance. However, since anti-CD154 mAb is not clinically available, we have evaluated CTLA4Ig as an alternative agent for effecting costimulation blockade in this treatment protocol. Two CTLA4Igs, abatacept and belatacept, were substituted for anti-CD154 mAb in the conditioning regimen (low dose total body irradiation, thymic irradiation, anti-thymocyte globulin and a 1-month posttransplant course of cyclosporine [CyA]). Three recipients treated with the abatacept regimen failed to develop comparable lymphoid chimerism to that achieved with anti-CD154 mAb treatment and these recipients rejected their kidney allografts early. With the belatacept regimen, four of five recipients developed chimerism and three of these achieved long-term renal allograft survival (>861, >796 and >378 days) without maintenance immunosuppression. Neither chimerism nor long-term allograft survival were achieved in two recipients treated with the belatacept regimen but with a lower, subtherapeutic dose of CyA. This study indicates that CD28/B7 blockade with belatacept can provide a clinically applicable alternative to anti-CD154 mAb for promoting chimerism and renal allograft tolerance.

  20. 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

  1. 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.

  2. 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...

  3. 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.

  4. Preparation and characterization of recombinant Llama VHH-human IgGFc chimeric antibody against H5N1 hemagglutinin from avian influenza virus%羊驼抗H5N1血凝素重链可变区-人IgGFc段嵌合抗体的制备和鉴定

    Institute of Scientific and Technical Information of China (English)

    夏立亮; 吴标; 程亚庭; 蔡家麟; 王颖; 赵国屏

    2012-01-01

    To prepare and characterize llama variable domain of heavy chain of heavy-chain antibody-human IgGlFc (VHH-hFc) chimeric antibody against hemagglutinin from H5N1 avian influenza virus, recombinant expression vector pET-22b-VHH23-hFc was constructed and VHH23-hFc chimeric antibody was expressed in E. coli BL2KDE3) strain by IPTG induction. As VHH23-hFc antibody was accumulated in inclusion bodies, two different refolding methods, dialysis and on-column refolding, were compared for the refolding efficacy and the optimal method was adopted for preparation of VHH23-hFc chimeric antibody. The activity and thermal stability of VHH antibodies were tested by ELISA. By using dialysis refolding procedure, VHH23-hFc chimeric antibody has been obtained with higher yield and good quality. The affinity constant of VHH23-hFc chimeric antibody was 2. 24 × 106 mol/L as determined by ELISA. VHH23-hFc chimeric antibody also displayed good thermal stability. The half-life span of VHH23-hFc chimeric antibody in mice was up to 35 hrs, which is comparable to conventional chimeric antibodies. Taken together, our results indicated that VHH23-hFc chimeric antibody against hemagglutinin derived from H5N1 avian influenza virus has been obtained with high activity, good thermal stability as well as longer half-life span, which provides basis for future functional study both in vitro and in vivo.%本研究旨在制备羊驼抗H5N1禽流感病毒的重链抗体可变区-人Fc段嵌合体抗体制备,对所得嵌合抗体进行制备和功能鉴定,为临床应用奠定基础.用pET-22b表达载体构建抗H5N1禽流感病毒羊驼重链可变区(VHH)-人IgG1Fc嵌合基因,以包涵体形式表达VHH23-hFc嵌合抗体蛋白,采用优化的方法复性后,获得高纯度VHH23-hFc嵌合抗体,用ELISA法鉴定嵌合抗体亲和力、热稳定性和小鼠体内的半衰期.结果显示,透析复性后原核表达的抗H5N1禽流感病毒VHH23-hFc嵌合抗体亲和力为2.24×106 mol/L,具有较好

  5. Chimeric alignment by dynamic programming: Algorithm and biological uses

    Energy Technology Data Exchange (ETDEWEB)

    Komatsoulis, G.A.; Waterman, M.S. [Univ. of Southern California, Los Angeles, CA (United States)

    1997-12-01

    A new nearest-neighbor method for detecting chimeric 16S rRNA artifacts generated during PCR amplification from mixed populations has been developed. The method uses dynamic programming to generate an optimal chimeric alignment, defined as the highest scoring alignment between a query and a concatenation of a 5{prime} and a 3{prime} segment from two separate entries from a database of related sequences. Chimeras are detected by studying the scores and form of the chimeric and global sequence alignments. The chimeric alignment method was found to be marginally more effective than k-tuple based nearest-neighbor methods in simulation studies, but its most effective use is in concert with k-tuple methods. 15 refs., 3 figs., 1 tab.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    A chimeric pestivirus of border disease virus Gifhorn and bovine viral diarrhea virus CP7 (Meyers et al., 1996) was constructed. Virulence, immunogenicity and vaccine properties of the chimeric virus were studied in a vaccination–challenge experiment in pigs. The chimeric virus proved...... to be 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....... In ‘challenge controls’, the viral load of CSFV coincided with the development of pronounced clinical symptoms. In contrast, the vaccinated pigs showed transient and weak clinical signs. Analysis of the viral load in these pigs showed 1000-fold lower viral RNA levels compared to ‘challenge controls...

  7. Does inversion abolish the left chimeric face processing advantage?

    Science.gov (United States)

    Butler, Stephen H; Harvey, Monika

    2005-12-19

    Experiments using chimeric stimuli have shown that the right hemisphere is more influential in processing facial information. Here, again, we found clear evidence that study participants used the information from the left side of the face to inform their gender decisions when chimeric male/female, female/male stimuli were presented. Most interestingly though, this effect was not only present for upright faces but also for inverted (flipped) faces (although the effect was significantly reduced). We propose that the chimeric bias effects found here argue against the idea that inversion destroys the right hemisphere superiority for faces. If this was indeed the case, flipping the chimeric faces should have resulted in a loss of the left face bias. This was not the case. PMID:16317340

  8. Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease.

    Science.gov (United States)

    Ellebrecht, Christoph T; Bhoj, Vijay G; Nace, Arben; Choi, Eun Jung; Mao, Xuming; Cho, Michael Jeffrey; Di Zenzo, Giovanni; Lanzavecchia, Antonio; Seykora, John T; Cotsarelis, George; Milone, Michael C; Payne, Aimee S

    2016-07-01

    Ideally, therapy for autoimmune diseases should eliminate pathogenic autoimmune cells while sparing protective immunity, but feasible strategies for such an approach have been elusive. Here, we show that in the antibody-mediated autoimmune disease pemphigus vulgaris (PV), autoantigen-based chimeric immunoreceptors can direct T cells to kill autoreactive B lymphocytes through the specificity of the B cell receptor (BCR). We engineered human T cells to express a chimeric autoantibody receptor (CAAR), consisting of the PV autoantigen, desmoglein (Dsg) 3, fused to CD137-CD3ζ signaling domains. Dsg3 CAAR-T cells exhibit specific cytotoxicity against cells expressing anti-Dsg3 BCRs in vitro and expand, persist, and specifically eliminate Dsg3-specific B cells in vivo. CAAR-T cells may provide an effective and universal strategy for specific targeting of autoreactive B cells in antibody-mediated autoimmune disease. PMID:27365313

  9. Neonatal transplantation tolerance is associated with a systemic reduction in memory cells, altered chimeric cell phenotype, and modified eicosanoid and cytokine production.

    Science.gov (United States)

    Ruiz, P; Nassiri, M; Gregorian, S; Viciana, A L; Streilein, J W

    1996-04-27

    Certain B10 background mice are resistant to tolerance induction following a neonatal inoculation of semiallogeneic class I/II MHC-disparate cells despite early thymic clonal deletion of alloreactive cells. The emergence of memory T cells and persistence of particular chimeric cells in the thymus has an association with this resistance. In these studies, we utilized a hemisplenectomy technique to examine systemic cell populations of adult Bl0.S (H2s, H2E-) mice that received (Bl0.S x B10.A)F1 cells at birth and before and following application (and rejection or acceptance) of Bl0.A (H2k/d, H2E+) skin grafts. Prior to skin graft challenge, tolerant mice had reduced splenic levels of memory (CD45hi, PgP-1hi, Mel-14neg) T cells as compared with the rejecting recipients and following B10.A graft challenge, the nontolerant mice showed a further increase in these cells. Elevated pretransplant levels of donor H2Kk+ cells coexpressing B220, CD11b, or CD3 were seen in the tolerant mice. Following skin grafting, splenic chimerism was reduced with differing chimeric cell phenotypes between the tolerant and nontolerant mice. In vitro production of PGE2 in a MLC was delayed in the tolerant mice with minimal production of IL-2 and IL-4. Nontolerant mice made high levels of TxB2 and heightened, early production of IL-2 and IL-4 during the MLC. Thus, tolerance induction is associated with increased numbers of particular chimeric cells, fewer peripheral lymphoid immunocompetant memory T cells, impaired eicosanoid secretion, and reduced alloreactivity and alloantigen-driven IL-2/IL-4 production. It appears that alloreactive cells necessary to break tolerance are generated when fewer class II+ (e.g., B220+, CD11b+) chimeric cells are present and that there is a coexistence of effector and regulatory T cell subpopulations in the nontolerant mice. By comparison, tolerance acquisition does not appear associated with the presence or generation of a predominant subtype of T cell but

  10. Development of a recombinant, chimeric tetravalent dengue vaccine candidate.

    Science.gov (United States)

    Osorio, Jorge E; Partidos, Charalambos D; Wallace, Derek; Stinchcomb, Dan T

    2015-12-10

    Dengue is a significant threat to public health worldwide. Currently, there are no licensed vaccines available for dengue. Takeda Vaccines Inc. is developing a live, attenuated tetravalent dengue vaccine candidate (TDV) that consists of an attenuated DENV-2 strain (TDV-2) and three chimeric viruses containing the prM and E protein genes of DENV-1, -3 and -4 expressed in the context of the attenuated TDV-2 genome backbone (TDV-1, TDV-3, and TDV-4, respectively). TDV has been shown to be immunogenic and efficacious in nonclinical animal models. In interferon-receptor deficient mice, the vaccine induces humoral neutralizing antibody responses and cellular immune responses that are sufficient to protect from lethal challenge with DENV-1, DENV-2 or DENV-4. In non-human primates, administration of TDV induces innate immune responses as well as long lasting antibody and cellular immunity. In Phase 1 clinical trials, the safety and immunogenicity of two different formulations were assessed after intradermal or subcutaneous administration to healthy, flavivirus-naïve adults. TDV administration was generally well-tolerated independent of dose and route. The vaccine induced neutralizing antibody responses to all four DENV serotypes: after a single administration of the higher formulation, 24-67%% of the subjects seroconverted to all four DENV and >80% seroconverted to three or more viruses. In addition, TDV induced CD8(+) T cell responses to the non-structural NS1, NS3 and NS5 proteins of DENV. TDV has been also shown to be generally well tolerated and immunogenic in a Phase 2 clinical trial in dengue endemic countries in adults and children as young as 18 months. Additional clinical studies are ongoing in preparation for a Phase 3 safety and efficacy study.

  11. Chimerism in wild adult populations of the broadcast spawning coral Acropora millepora on the Great Barrier Reef.

    Directory of Open Access Journals (Sweden)

    Eneour Puill-Stephan

    Full Text Available BACKGROUND: Chimeras are organisms containing tissues or cells of two or more genetically distinct individuals, and are known to exist in at least nine phyla of protists, plants, and animals. Although widespread and common in marine invertebrates, the extent of chimerism in wild populations of reef corals is unknown. METHODOLOGY/PRINCIPAL FINDINGS: The extent of chimerism was explored within two populations of a common coral, Acropora millepora, on the Great Barrier Reef, Australia, by using up to 12 polymorphic DNA microsatellite loci. At least 2% and 5% of Magnetic Island and Pelorus Island populations of A. millepora, respectively, were found to be chimeras (3% overall, based on conservative estimates. A slightly less conservative estimate indicated that 5% of colonies in each population were chimeras. These values are likely to be vast underestimates of the true extent of chimerism, as our sampling protocol was restricted to a maximum of eight branches per colony, while most colonies consist of hundreds of branches. Genotypes within chimeric corals showed high relatedness, indicating that genetic similarity is a prerequisite for long-term acceptance of non-self genotypes within coral colonies. CONCLUSIONS/SIGNIFICANCE: While some brooding corals have been shown to form genetic chimeras in their early life history stages under experimental conditions, this study provides the first genetic evidence of the occurrence of coral chimeras in the wild and of chimerism in a broadcast spawning species. We hypothesize that chimerism is more widespread in corals than previously thought, and suggest that this has important implications for their resilience, potentially enhancing their capacity to compete for space and respond to stressors such as pathogen infection.

  12. 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

  13. Chimeric TALE recombinases with programmable DNA sequence specificity.

    Science.gov (United States)

    Mercer, Andrew C; Gaj, Thomas; Fuller, Roberta P; Barbas, Carlos F

    2012-11-01

    Site-specific recombinases are powerful tools for genome engineering. Hyperactivated variants of the resolvase/invertase family of serine recombinases function without accessory factors, and thus can be re-targeted to sequences of interest by replacing native DNA-binding domains (DBDs) with engineered zinc-finger proteins (ZFPs). However, imperfect modularity with particular domains, lack of high-affinity binding to all DNA triplets, and difficulty in construction has hindered the widespread adoption of ZFPs in unspecialized laboratories. The discovery of a novel type of DBD in transcription activator-like effector (TALE) proteins from Xanthomonas provides an alternative to ZFPs. Here we describe chimeric TALE recombinases (TALERs): engineered fusions between a hyperactivated catalytic domain from the DNA invertase Gin and an optimized TALE architecture. We use a library of incrementally truncated TALE variants to identify TALER fusions that modify DNA with efficiency and specificity comparable to zinc-finger recombinases in bacterial cells. We also show that TALERs recombine DNA in mammalian cells. The TALER architecture described herein provides a platform for insertion of customized TALE domains, thus significantly expanding the targeting capacity of engineered recombinases and their potential applications in biotechnology and medicine.

  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. 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.

  16. Assessment of Free Radical Scavenging Activity of Dimethylglycine Sodium Salt and Its Role in Providing Protection against Lipopolysaccharide-Induced Oxidative Stress in Mice.

    Science.gov (United States)

    Bai, Kaiwen; Xu, Wen; Zhang, Jingfei; Kou, Tao; Niu, Yu; Wan, Xiaoli; Zhang, Lili; Wang, Chao; Wang, Tian

    2016-01-01

    In the present study, the free radical scavenging activities (against 1,1-diphenyl-2-pierylhydrazy (DPPH), 2,2'-Azinobis-(3-ethylbenzthiazoline-6- sulphonate) (ABTS+), Hydrogen peroxide (H2O2)) of dimethylglycine sodium salt (DMG-Na) were measured and compared with those of Trolox (6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid), a commonly used antioxidant. The radical scavenging activities of DMG-Na were found to be the highest at 40 mg/ml. In Experiment 2, gastric intubation in mice with 12 mg DMG-Na/0.3 ml sterile saline solution significantly increased (P effect of 12 mg DMG-Na/0.3 ml sterile saline solution, which showed the highest antioxidant capacity, was further studied using a mice model. In Experiment 3, the mice CL (CON+ lipopolysaccharide (LPS)) group showed a significant decrease (P stress by enhancing the free radical scavenging capacity, and increasing the activity of antioxidant defense system. PMID:27171376

  17. Temporal and spatial patterns of transgene expression in aging adult mice provide insights about the origins, organization, and differentiation of the intestinal epithelium.

    OpenAIRE

    Cohn, S. M.; Roth, K A; Birkenmeier, E H; Gordon, J I

    1991-01-01

    We have used liver fatty acid-binding protein/human growth hormone (L-FABP/hGH) fusion genes to explore the temporal and spatial differentiation of intestinal epithelial cells in 1- to 12-month-old transgenic mice. The intact, endogenous L-FABP gene (Fabpl) was not expressed in the colon at any time. Young adult transgenic mice containing nucleotides -596 to +21 of the rat L-FABP gene linked to the hGH gene (minus its 5' nontranscribed domain) demonstrated inappropriate expression of hGH in e...

  18. Challenge pools of hepatitis C virus genotypes 1-6 prototype strains: replication fitness and pathogenicity in chimpanzees and human liver-chimeric mouse models

    DEFF Research Database (Denmark)

    Bukh, Jens; Meuleman, Philip; Tellier, Raymond;

    2010-01-01

    Chimpanzees represent the only animal model for studies of the natural history of hepatitis C virus (HCV). To generate virus stocks of important HCV variants, we infected chimpanzees with HCV strains of genotypes 1-6 and determined the infectivity titer of acute-phase plasma pools in additional...... animals. The courses of first- and second-passage infections were similar, with early appearance of viremia, HCV RNA titers of >10(4.7) IU/mL, and development of acute hepatitis; the chronicity rate was 56%. The challenge pools had titers of 10(3)-10(5) chimpanzee infectious doses/mL. Human liver......-chimeric mice developed high-titer infections after inoculation with the challenge viruses of genotypes 1-6. Inoculation studies with different doses of the genotype 1b pool suggested that a relatively high virus dose is required to consistently infect chimeric mice. The challenge pools represent a unique...

  19. A live attenuated cold-adapted influenza A H7N3 virus vaccine provides protection against homologous and heterologous H7 viruses in mice and ferrets

    International Nuclear Information System (INIS)

    The appearance of human infections caused by avian influenza A H7 subtype viruses underscores their pandemic potential and the need to develop vaccines to protect humans from viruses of this subtype. A live attenuated H7N3 virus vaccine was generated by reverse genetics using the HA and NA genes of a low pathogenicity A/chicken/BC/CN-6/04 (H7N3) virus and the six internal protein genes of the cold-adapted A/Ann Arbor/6/60 ca (H2N2) virus. The reassortant H7N3 BC 04 ca vaccine virus was temperature sensitive and showed attenuation in mice and ferrets. Intranasal immunization with one dose of the vaccine protected mice and ferrets when challenged with homologous and heterologous H7 viruses. The reassortant H7N3 BC 04 ca vaccine virus showed comparable levels of attenuation, immunogenicity and efficacy in mice and ferret models. The safety, immunogenicity, and efficacy of this vaccine in mice and ferrets support the evaluation of this vaccine in clinical trials

  20. Phenotypic characterization of a novel virulence-factor deletion strain of Burkholderia mallei that provides partial protection against inhalational glanders in mice

    Directory of Open Access Journals (Sweden)

    Joel A. Bozue

    2016-02-01

    Full Text Available Burkholderia mallei (Bm is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN, and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders.

  1. Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia mallei That Provides Partial Protection against Inhalational Glanders in Mice.

    Science.gov (United States)

    Bozue, Joel A; Chaudhury, Sidhartha; Amemiya, Kei; Chua, Jennifer; Cote, Christopher K; Toothman, Ronald G; Dankmeyer, Jennifer L; Klimko, Christopher P; Wilhelmsen, Catherine L; Raymond, Jolynn W; Zavaljevski, Nela; Reifman, Jaques; Wallqvist, Anders

    2016-01-01

    Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders.

  2. Development of polyclonal antibodies for detection of aflatoxigenic molds involving culture filtrate and chimeric proteins expressed in Escherichia coli.

    Science.gov (United States)

    Shapira, R; Paster, N; Menasherov, M; Eyal, O; Mett, A; Meiron, T; Kuttin, E; Salomon, R

    1997-03-01

    Polyclonal antibodies (PAb) were raised against an aflatoxigenic strain of Aspergillus parasiticus by using two different sources for antibody elicitation: (i) filtrate of a culture on which the fungus had been grown (ii) and two chimeric proteins, expressed in Escherichia coli as separate products, of the genes ver-1 and apa-2, which are involved in aflatoxin biosynthesis. The gene products were amplified by PCR, and each was cloned into the E. coli expression vector pGEX2T. Upon induction, the bacteria overexpressed 38- and 33-kDa chimeric proteins corresponding to the N-terminal domains of the genes ver-1 and apa-2, respectively. The chimeric proteins were isolated and affinity purified for use as antigens. The specificity of the raised antibodies was examined by enzyme-linked immunosorbent assay (ELISA). The PAbs raised against the culture filtrate reacted with all the species of Aspergillus and Penicillium tested but not with Fusarium species or corn gain. However, the PAbs elicited against the chimeric proteins were highly specific, showing significantly higher ELISA absorbance values (A405) against A. parasiticus and A. flavus than against the other fungi tested and the corn grain. The approach of utilizing gene products associated with aflatoxin biosynthesis for antibody production therefore appears to be feasible. Such a multiantibody system combined with the PCR technique, could provide a useful tool for the rapid, sensitive, and accurate detection of aflatoxin producers present in grains and foods. PMID:9055416

  3. 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

  4. Developmental competence of porcine chimeric embryos produced by aggregation

    DEFF Research Database (Denmark)

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

    2015-01-01

    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...... 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......, aggregation was made with HMC embryos cloned using EGFP transgenic cells; the cell contribution in the formation of the inner cell mass or trophectoderm was random in chimeric blastocysts. Finally, two blastomeres from 2-cell stage embryos were fused to construct tetraploid embryos, and when diploid...

  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. 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

  7. Chimera: construction of chimeric sequences for phylogenetic analysis

    NARCIS (Netherlands)

    Leunissen, J.A.M.

    2003-01-01

    Chimera allows the construction of chimeric protein or nucleic acid sequence files by concatenating sequences from two or more sequence files in PHYLIP formats. It allows the user to interactively select genes and species from the input files. The concatenated result is stored to one single output f

  8. 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)

  9. 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

  10. 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.

  11. A technical application of quantitative next generation sequencing for chimerism evaluation

    Science.gov (United States)

    Aloisio, Michelangelo; Licastro, Danilo; Caenazzo, Luciana; Torboli, Valentina; D'eustacchio, Angela; Severini, Giovanni Maria; Athanasakis, Emmanouil

    2016-01-01

    At present, the most common genetic diagnostic method for chimerism evaluation following hematopoietic stem cell transplantation is microsatellite analysis by capillary electrophoresis. The main objective was to establish, through repeated analysis over time, if a complete chimerism was present, or if the mixed chimerism was stable, increasing or decreasing over time. Considering the recent introduction of next generation sequencing (NGS) in clinical diagnostics, a detailed study evaluating an NGS protocol was conducted, coupled with a custom bioinformatics pipeline, for chimerism quantification. Based on the technology of Ion AmpliSeq, a 44-amplicon custom chimerism panel was designed, and a custom bioinformatics pipeline dedicated to the genotyping and quantification of NGS data was coded. The custom chimerism panel allowed identification of an average of 16 informative recipient alleles. The limit of detection of the protocol was fixed at 1% due to the NGS background (NGS for chimerism quantification. PMID:27499173

  12. 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.

  13. 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.

  14. 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 ...

  15. Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model

    Science.gov (United States)

    Suarez, Eloah Rabello; Chang, De-Kuan; Sun, Jiusong; Sui, Jianhua; Freeman, Gordon J.; Signoretti, Sabina; Zhu, Quan; Marasco, Wayne A.

    2016-01-01

    Advances in the treatment of metastatic clear cell renal cell carcinoma (ccRCC) have led to improved progression-free survival of many patients; however the therapies are toxic, rarely achieve durable long-term complete responses and are not curative. Herein we used a single bicistronic lentiviral vector to develop a new combination immunotherapy that consists of human anti-carbonic anhydrase IX (CAIX)-targeted chimeric antigen receptor (CAR) T cells engineered to secrete human anti-programmed death ligand 1 (PD-L1) antibodies at the tumor site. The local antibody delivery led to marked immune checkpoint blockade. Tumor growth diminished 5 times and tumor weight reduced 50–80% when compared with the anti-CAIX CAR T cells alone in a humanized mice model of ccRCC. The expression of PD-L1 and Ki67 in the tumors decreased and an increase in granzyme B levels was found in CAR T cells. The anti-PD-L1 IgG1 isotype, which is capable of mediating ADCC, was also able to recruit human NK cells to the tumor site in vivo. These armed second-generation CAR T cells empowered to secrete human anti-PD-L1 antibodies in the ccRCC milieu to combat T cell exhaustion is an innovation in this field that should provide renewed potential for CAR T cell immunotherapy of solid tumors where limited efficacy is currently seen. PMID:27145284

  16. 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.

  17. Lentiviral Gag assembly analyzed through the functional characterization of chimeric simian immunodeficiency viruses expressing different domains of the feline immunodeficiency virus capsid protein.

    Directory of Open Access Journals (Sweden)

    María J Esteva

    Full Text Available To gain insight into the functional relationship between the capsid (CA domains of the Gag polyproteins of simian and feline immunodeficiency viruses (SIV and FIV, respectively, we constructed chimeric SIVs in which the CA-coding region was partially or totally replaced by the equivalent region of the FIV CA. The phenotypic characterization of the chimeras allowed us to group them into three categories: the chimeric viruses that, while being assembly-competent, exhibit a virion-associated unstable FIV CA; a second group represented only by the chimeric SIV carrying the N-terminal domain (NTD of the FIV CA which proved to be assembly-defective; and a third group constituted by the chimeric viruses that produce virions exhibiting a mature and stable FIV CA protein, and which incorporate the envelope glycoprotein and contain wild-type levels of viral genome RNA and reverse transcriptase. Further analysis of the latter group of chimeric SIVs demonstrated that they are non-infectious due to a post-entry impairment, such as uncoating of the viral core, reverse transcription or nuclear import of the preintegration complex. Furthermore, we show here that the carboxyl-terminus domain (CTD of the FIV CA has an intrinsic ability to dimerize in vitro and form high-molecular-weight oligomers, which, together with our finding that the FIV CA-CTD is sufficient to confer assembly competence to the resulting chimeric SIV Gag polyprotein, provides evidence that the CA-CTD exhibits more functional plasticity than the CA-NTD. Taken together, our results provide relevant information on the biological relationship between the CA proteins of primate and nonprimate lentiviruses.

  18. 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.

  19. Hepatic immunophenotyping for streptozotocin-induced hyperglycemia in mice

    Science.gov (United States)

    Lee, Young-Sun; Eun, Hyuk Soo; Kim, So Yeon; Jeong, Jong-Min; Seo, Wonhyo; Byun, Jin-Seok; Jeong, Won-Il; Yi, Hyon-Seung

    2016-01-01

    Emerging evidence revealed that diabetes induces abnormal immune responses that result in serious complications in organs. However, the effect of hyperglycemia on hepatic immunity remains obscure. We evaluated the population and function of hepatic immune cells in streptozotocin (STZ)-induced hyperglycemic mice. CC chemokine receptor 2 (CCR2)-knockout mice and mice with a depletion of regulatory T cells (DEREG) were used to investigate the migration and role of regulatory T cells (Tregs) in hyperglycemic mice. The inflammatory cytokines and hepatic transaminase levels were significantly increased in the hyperglycemic mice. The population and number of infiltrating monocytes, granulocytes, and Tregs were enhanced in the livers of the hyperglycemic mice. Hepatic monocytes other than macrophages showed the increased expression of inflammatory cytokines and chemokines in the hyperglycemic mice. The CCR2 knockout and DEREG chimeric mice exhibited increased populations of activated T cells and neutrophils compared to the WT chimeric mice, which promoted hepatic inflammation in the hyperglycemic mice. The migration of CCR2 knockout Tregs into the liver was significantly reduced compared to the WT Tregs. We demonstrated that hyperglycemia contributes to increase in infiltrating monocytes and Tregs, which are associated with hepatic immune dysfunction in mice. CCR2-mediated migration of Tregs regulates hyperglycemia-induced hepatic inflammation. PMID:27464894

  20. 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...... engineered specifically for this purpose. The E2-substituted Riems26_E2gif was derived by homologues recombination of the complete E2 protein encoding genome region from Border disease strain Gifhorn into a bacterial artificial chromosome (BAC) harbouring the genome of the CSFV vaccine strain C......-Riems. The virulence, immunogenicity and vaccine properties of Riems26_E2gif were tested in a vaccine-challenge experiment in pigs. Riems26_E2gif vaccinated pigs could be differentiated from infected pigs using a CSFV-E2 specific ELISA. Following challenge infection with highly virulent CSFV strain Koslov, all...

  1. 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.

  2. 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

  3. Engineering D-helix of antithrombin in alpha-1-proteinase inhibitor confers antiinflammatory properties on the chimeric serpin.

    Science.gov (United States)

    Yang, L; Dinarvand, P; Qureshi, S H; Rezaie, A R

    2014-07-01

    Antithrombin (AT) is a heparin-binding serpin in plasma which regulates the proteolytic activity of procoagulant proteases of the clotting cascade. In addition to being an anticoagulant, AT also exhibits antiinflammatory activities when it binds to cell surface heparan sulfate proteoglycans (HSPGs) on the endothelium via its basic residues of D-helix to elicit intracellular signalling responses. By contrast to AT, α1-proteinase inhibitor (α1-PI) is a non-heparin-binding serpin that exhibits very slow reactivity with coagulation proteases and possesses no HSPG-dependent antiinflammatory properties. To determine whether the antiinflammatory signaling specificity of AT can be transferred to α1-PI, we replaced the D-helix of human α1-PI with the corresponding sequence of human AT and expressed the chimeric serpin α1-PI/D-helix) in a bacterial expression system. High molecular weight heparin bound to α1-PI/D-helix and accelerated the inhibition of thrombin by the serpin mutant by a template mechanism reminiscent of the cofactor effect of heparin on inhibition of thrombin by AT. Like AT, α1-PI/D-helix exhibited antiinflammatory properties in both cellular and animal models. Thus, α1-PI/D-helix inhibited the barrier-disruptive effect of proinflammatory cytokines and inhibited the activation of nuclear factor-κB transcription factor in lipopolysaccharide-stimulated endothelial cells by a concentration-dependent manner. Furthermore, the chimeric serpin reduced lipopolysaccharide-mediated lethality, elicited a vascular protective effect and inhibited infiltration of activated leukocytes to the peritoneal cavity of mice in an HMGB1-mediated inflammatory model. These results suggest that grafting the D-helix of AT to α1-PI confers antiinflammatory properties on the serpin and that the chimeric serpin may have therapeutic utility for treating inflammatory disorders. PMID:24522239

  4. Rotavirus VP7 epitope chimeric proteins elicit cross-immunoreactivity in guinea pigs

    Institute of Scientific and Technical Information of China (English)

    Bingxin; Zhao; Xiaoxia; Pan; Yumei; Teng; Wenyue; Xia; Jing; Wang; Yuling; Wen; Yuanding; Chen

    2015-01-01

    VP7 of group A rotavirus(RVA) contains major neutralizing epitopes. Using the antigenic protein VP6 as the vector, chimeric proteins carrying foreign epitopes have been shown to possess good immunoreactivity and immunogenicity. In the present study, using modified VP6 as the vector,three chimeric proteins carrying epitopes derived from VP7 of RVA were constructed. The results showed that the chimeric proteins reacted with anti-VP6 and with SA11 and Wa virus strains.Antibodies from guinea pigs inoculated with the chimeric proteins recognized VP6 and VP7 of RVA and protected mammalian cells from SA11 and Wa infection in vitro. The neutralizing activities of the antibodies against the chimeric proteins were significantly higher than those against the vector protein VP6 F. Thus, development of chimeric vaccines carrying VP7 epitopes using VP6 as a vector could be a promising alternative to enhance immunization against RVAs.

  5. 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.

  6. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  8. 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.

  9. 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.

  10. 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.

  11. Long-term observations of autoimmune-prone mice treated for autoimmune disease by allogeneic bone marrow transplantation

    International Nuclear Information System (INIS)

    Long-term effects of allogeneic bone marrow transplantation (ABMT) across major histocompatibility complex barriers were studied in (NZB x NZW)F1 (B/W), BXSB, and MRL/Mr-lpr-lpr (MRL/lpr) mice with established autoimmune disease at the time of ABMT. In the BXSB or B/W mice, ABMT cured all aspects of autoimmune disease. Glomerular damage, revealed by histological study was dramatically improved. Serological abnormalities and immunologic functions also were normalized. Correction of autoimmune disease and advanced renal disease in BXSB and B/W mice regularly lasted greater than 5-6 mo and even 1 yr after ABMT. In the MRL/lpr mice, however, autoimmune and renal disease at first improved but then recurred after ABMT, apparently because of intolerance of mice for high doses of irradiation and a high degree of resistance of recipient stem cells to irradiation. In this model, H-2 typing revealed that by the time of relapse, immunocompetent cells of the chimeric mice had been replaced by host (MRL/lpr; H-2k) cells. B220+ Ly-1+ cells, present in increased numbers in untreated MRL/lpr mice, initially returned to normal levels after ABMT but then reappeared in the MRL/lpr mice that had received marrow from donors having few such lymphocytes. Thus, our results show that MRL/lpr mice possess abnormal radioresistant stem cells and provide impressive evidence that the origin of autoimmune diseases in this strain, as in the several other strains studied, resids in abnormalities present in stem cells

  12. 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

  13. Immunogenicity of candidate chimeric DNA vaccine against tuberculosis and leishmaniasis.

    Science.gov (United States)

    Dey, Ayan; Kumar, Umesh; Sharma, Pawan; Singh, Sarman

    2009-08-13

    Mycobacterium tuberculosis and Leishmania donovani are important intracellular pathogens, especially in Indian context. In India and other South East Asian countries, both these infections are highly endemic and in about 20% cases co-infection of these pathogens is reported. For both these pathogens cell mediated immunity plays most important role. The available treatment of these infections is either prolonged or cumbersome or it is ineffective in controlling the outbreaks and spread. Therefore, potentiation of a common host defense mechanism can be used to prevent both the infections simultaneously. In this study we have developed a novel chimeric DNA vaccine candidate comprising the esat-6 gene of M. tuberculosis and kinesin motor domain gene of L. donovani. After developing this novel chimera, its immunogenicity was studied in mouse model. The immune response was compared with individual constructs of esat-6 and kinesin motor domain. The results showed that immunization with chimeric DNA vaccine construct resulted in stronger IFN-gamma and IL-2 response against kinesin (3012+/-102 and 367.5+/-8.92pg/ml) and ESAT-6 (1334+/-46.5 and 245.1+/-7.72pg/ml) in comparison to the individual vaccine constructs. The reciprocal immune response (IFN-gamma and IL-2) against individual construct was lower (kinesin motor domain: 1788+/-36.48 and 341.8+/-9.801pg/ml and ESAT-6: 867.0+/-47.23 and 170.8+/-4.578pg/ml, respectively). The results also suggest that using the chimeric construct both proteins yielded a reciprocal adjuvant affect over each other as the IFN-gamma production against chimera vaccination is statistically significant (pleishmaniasis and tuberculosis and have important implication in future vaccine design.

  14. 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...

  15. Chimerism in cattle through microsurgical aggregation of morulae.

    Science.gov (United States)

    Brem, G; Tenhumberg, H; Kräußlich, H

    1984-11-01

    A cattle chimera was produced by combining four halves of two parent embryos of different breeds (Brown-Swiss x Braunvieh plus Holstein-Friesian x Holstein-Friesian) in one zona pellucida. Parent embryos in the 32-cell morula stage were recovered non-surgically, were bisected, and the combined four halves were transferred non-surgically to recipient heifers. Chimerism of coat colour was used as evidence. Combining of only two half embryos from different parents resulted in five pregnancies carried to term but none of the calves born was a chimera.

  16. 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.

  17. 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.

  18. 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

    . 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...

  19. 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

  20. 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, ...

  1. 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.

  2. 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

  3. Quantitative analysis of chimerism after allogeneic hematopoietic stem cell transplantation with molecular genetic methods

    Directory of Open Access Journals (Sweden)

    V. A. Lavrinenko

    2014-09-01

    Full Text Available Quantitative monitoring of chimerism after allogeneic hematopoietic stem cell transplantation (HSCT by molecular methods has becomea significant diagnostic tool in detection of engraftment / graft failure, predicting rejection and disease relapse. Despite the great utility of chimerism analysis there is not a unique standard method for its quantification. The objective of the present investigation was to compare perspective methods multiplex short tandem repeat polymerase chain reaction (STR-PCR and real-time PCR insertion / deletion polymorphisms (InDel-PCR for the quantification of chimerism after HSCT. We performed a study analyzing the chimerism status in 60 patients by STR-PCR and by InDel-PCR. Recipient / donor discrimination was possible with STR-PCR in all patient-donor pairs (100 %, whereas informative alleles for recipient were found in 88 % pairs with InDel-PCR. The sensitivity (detection limit of STR-PCR and InDel-PCR was 1–5 % and more than 0.01 % donor cells correspondingly. The accuracy of quantification was higher for STR-PCR than for InDel-PCR, when level of donor chimerism was 3–97 %. These methods can be successfully used to determine chimerism after allogeneic HSCT. Considering the higher sensitivity and quantification accuracy of InDel-PCR it should be chosen if donor chimerism level less 5 % or more 95 % and in other cases STR-PCR should be chosen.

  4. A technical application of quantitative next generation sequencing for chimerism evaluation.

    Science.gov (United States)

    Aloisio, Michelangelo; Licastro, Danilo; Caenazzo, Luciana; Torboli, Valentina; D'Eustacchio, Angela; Severini, Giovanni Maria; Athanasakis, Emmanouil

    2016-10-01

    At present, the most common genetic diagnostic method for chimerism evaluation following hematopoietic stem cell transplantation is microsatellite analysis by capillary electrophoresis. The main objective was to establish, through repeated analysis over time, if a complete chimerism was present, or if the mixed chimerism was stable, increasing or decreasing over time. Considering the recent introduction of next generation sequencing (NGS) in clinical diagnostics, a detailed study evaluating an NGS protocol was conducted, coupled with a custom bioinformatics pipeline, for chimerism quantification. Based on the technology of Ion AmpliSeq, a 44‑amplicon custom chimerism panel was designed, and a custom bioinformatics pipeline dedicated to the genotyping and quantification of NGS data was coded. The custom chimerism panel allowed identification of an average of 16 informative recipient alleles. The limit of detection of the protocol was fixed at 1% due to the NGS background (Torrent Personal Genome Machine guidelines. Overall, the present study added to the scientific literature, identifying novel technical details for a possible future application of NGS for chimerism quantification. PMID:27499173

  5. 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.

  6. 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.

  7. Chimeric Antigen Receptor T Cell Therapy in Hematology.

    Science.gov (United States)

    Ataca, Pınar; Arslan, Önder

    2015-12-01

    It is well demonstrated that the immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation. Adoptive T cell transfer has been improved to be more specific and potent and to cause less off-target toxicity. Currently, there are two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR) and chimeric antigen receptor (CAR) modified T cells. On 1 July 2014, the United States Food and Drug Administration granted 'breakthrough therapy' designation to anti-CD19 CAR T cell therapy. Many studies were conducted to evaluate the benefits of this exciting and potent new treatment modality. This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing process, preclinical and clinical studies, and the effectiveness and drawbacks of this strategy.

  8. Design and characterization of a chimeric multiepitope construct containing CfaB, heat-stable toxoid, CssA, CssB, and heat-labile toxin subunit B of enterotoxigenic Escherichia coli: a bioinformatic approach.

    Science.gov (United States)

    Zeinalzadeh, Narges; Salmanian, Ali Hatef; Ahangari, Ghasem; Sadeghi, Mahdi; Amani, Jafar; Bathaie, S Zahra; Jafari, Mahyat

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are the most common cause of bacterial diarrhea in children in developing countries and travelers to these areas. Enterotoxins and colonization factors (CFs) are two key virulence factors in ETEC pathogenesis, and the heterogeneity of the CFs is the bottleneck in reaching an effective vaccine. In this study, a candidate subunit vaccine, which is composed of CfaB, CssA and CssB, structural subunits of colonization factor antigen I and CS6 CFs, labile toxin subunit B, and the binding subunit of heat-labile and heat-stable toxoid, was designed to provide broad-spectrum protection against ETEC. The different features of chimeric gene, its mRNA stability, and chimeric protein properties were analyzed by using bioinformatic tools. The optimized chimeric gene was chemically synthesized and expressed successfully in a prokaryotic host. The purified protein was used for assessment of bioinformatic data by experimental methods.

  9. Evaluation of a chimeric multi-epitope-based DNA vaccine against subgroup J avian leukosis virus in chickens.

    Science.gov (United States)

    Xu, Qingqing; Cui, Ning; Ma, Xingjiang; Wang, Fangkun; Li, Hongmei; Shen, Zhiqiang; Zhao, Xiaomin

    2016-07-19

    The prokaryotic expressed recombinant chimeric multi-epitope protein X (rCMEPX) had been evaluated with good immunogenicity and protective efficacy against subgroup J avian leukosis virus (ALV-J) in our previous study. In the present research, we cloned the chimeric multi-epitope gene X into the eukaryotic expression vector pVAX1 to evaluate its potency as a DNA vaccine. The purified recombinant gp85 protein and rCMEPX were used as positive controls and a DNA prime-protein boost strategy was also studied. Six experimental groups of 7-day-old chickens (20 per group) were immunized intramuscularly three times at 2weeks interval with PBS, gp85, rCMEPX, pVAX1, pVAX-X and pVAX-X+rCMEPX respectively. The antibody titers and cellular immune responses were assayed after immunization. The efficacy of immunoprotection against the challenge of ALV-J NX0101 strain was also examined. The results showed that the DNA vaccine could elicit both neutralizing antibodies and cellular responses. Immune-challenge experiments showed good protection efficacy against ALV-J infection. Particularly, the regimen involving one priming pVAX-X and twice recombinant rCMEPX boosting, induced the highest antibody titers in all immunized groups. Our results suggest that the constructed chimeric multi-epitope DNA has potential for a candidate vaccine against ALV-J when used in proper prime-boost combinations. The data presented here may provide an alternative strategy for vaccine design in chicken ALV-J prevention.

  10. Mamu-A*01/Kb transgenic and MHC Class I knockout mice as a tool for HIV vaccine development

    International Nuclear Information System (INIS)

    We have developed a murine model expressing the rhesus macaque (RM) Mamu-A*01 MHC allele to characterize immune responses and vaccines based on antigens of importance to human disease processes. Towards that goal, transgenic (Tg) mice expressing chimeric RM (α1 and α2 Mamu-A*01 domains) and murine (α3, transmembrane, and cytoplasmic H-2Kb domains) MHC Class I molecules were derived by transgenesis of the H-2KbDb double MHC Class I knockout strain. After immunization of Mamu-A*01/Kb Tg mice with rVV-SIVGag-Pol, the mice generated CD8+ T-cell IFN-γ responses to several known Mamu-A*01 restricted epitopes from the SIV Gag and Pol antigen sequence. Fusion peptides of highly recognized CTL epitopes from SIV Pol and Gag and a strong T-help epitope were shown to be immunogenic and capable of limiting an rVV-SIVGag-Pol challenge. Mamu-A*01/Kb Tg mice provide a model system to study the Mamu-A*01 restricted T-cell response for various infectious diseases which are applicable to a study in RM.

  11. HDAC inhibitor reduces cytokine storm and facilitates induction of chimerism that reverses lupus in anti-CD3 conditioning regimen.

    Science.gov (United States)

    Li, Nainong; Zhao, Dongchang; Kirschbaum, Mark; Zhang, Chunyan; Lin, Chia-Lei; Todorov, Ivan; Kandeel, Fouad; Forman, Stephen; Zeng, Defu

    2008-03-25

    In allogeneic hematopoietic cell transplantation (HCT), donor T cell-mediated graft versus host leukemia (GVL) and graft versus autoimmune (GVA) activity play critical roles in treatment of hematological malignancies and refractory autoimmune diseases. However, graft versus host disease (GVHD), which sometimes can be fatal, remains a major obstacle in classical HCT, where recipients are conditioned with total body irradiation or high-dose chemotherapy. We previously reported that anti-CD3 conditioning allows donor CD8(+) T cells to facilitate engraftment and mediate GVL without causing GVHD. However, the clinical application of this radiation-free and GVHD preventative conditioning regimen is hindered by the cytokine storm syndrome triggered by anti-CD3 and the high-dose donor bone marrow (BM) cells required for induction of chimerism. Histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA) are known to induce apoptosis of cancer cells and reduce production of proinflammatory cytokines by nonmalignant cells. Here, we report that SAHA inhibits the proliferative and cytotoxic activity of anti-CD3-activated T cells. Administration of low-dose SAHA reduces cytokine production and ameliorates the cytokine storm syndrome triggered by anti-CD3. Conditioning with anti-CD3 and SAHA allows induction of chimerism with lower doses of donor BM cells in old nonautoimmune and autoimmune lupus mice. In addition, conditioning with anti-CD3 and SAHA allows donor CD8(+) T cell-mediated GVA activity to reverse lupus glomerulonephritis without causing GVHD. These results indicate that conditioning with anti-CD3 and HDAC inhibitors represent a radiation-free and GVHD-preventative regimen with clinical application potential.

  12. 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...

  13. Completely ES cell-derived mice produced by tetraploid complementation using inner cell mass (ICM deficient blastocysts.

    Directory of Open Access Journals (Sweden)

    Duancheng Wen

    Full Text Available Tetraploid complementation is often used to produce mice from embryonic stem cells (ESCs by injection of diploid (2n ESCs into tetraploid (4n blastocysts (ESC-derived mice. This method has also been adapted to mouse cloning and the derivation of mice from induced pluripotent stem (iPS cells. However, the underlying mechanism(s of the tetraploid complementation remains largely unclear. Whether this approach can give rise to completely ES cell-derived mice is an open question, and has not yet been unambiguously proven. Here, we show that mouse tetraploid blastocysts can be classified into two groups, according to the presence or absence of an inner cell mass (ICM. We designate these as type a (presence of ICM at blastocyst stage or type b (absence of ICM. ESC lines were readily derived from type a blastocysts, suggesting that these embryos retain a pluripotent epiblast compartment; whereas the type b blastocysts possessed very low potential to give rise to ESC lines, suggesting that they had lost the pluripotent epiblast. When the type a blastocysts were used for tetraploid complementation, some of the resulting mice were found to be 2n/4n chimeric; whereas when type b blastocysts were used as hosts, the resulting mice are all completely ES cell-derived, with the newborn pups displaying a high frequency of abdominal hernias. Our results demonstrate that completely ES cell-derived mice can be produced using ICM-deficient 4n blastocysts, and provide evidence that the exclusion of tetraploid cells from the fetus in 2n/4n chimeras can largely be attributed to the formation of ICM-deficient blastocysts.

  14. Completely ES cell-derived mice produced by tetraploid complementation using inner cell mass (ICM) deficient blastocysts.

    Science.gov (United States)

    Wen, Duancheng; Saiz, Nestor; Rosenwaks, Zev; Hadjantonakis, Anna-Katerina; Rafii, Shahin

    2014-01-01

    Tetraploid complementation is often used to produce mice from embryonic stem cells (ESCs) by injection of diploid (2n) ESCs into tetraploid (4n) blastocysts (ESC-derived mice). This method has also been adapted to mouse cloning and the derivation of mice from induced pluripotent stem (iPS) cells. However, the underlying mechanism(s) of the tetraploid complementation remains largely unclear. Whether this approach can give rise to completely ES cell-derived mice is an open question, and has not yet been unambiguously proven. Here, we show that mouse tetraploid blastocysts can be classified into two groups, according to the presence or absence of an inner cell mass (ICM). We designate these as type a (presence of ICM at blastocyst stage) or type b (absence of ICM). ESC lines were readily derived from type a blastocysts, suggesting that these embryos retain a pluripotent epiblast compartment; whereas the type b blastocysts possessed very low potential to give rise to ESC lines, suggesting that they had lost the pluripotent epiblast. When the type a blastocysts were used for tetraploid complementation, some of the resulting mice were found to be 2n/4n chimeric; whereas when type b blastocysts were used as hosts, the resulting mice are all completely ES cell-derived, with the newborn pups displaying a high frequency of abdominal hernias. Our results demonstrate that completely ES cell-derived mice can be produced using ICM-deficient 4n blastocysts, and provide evidence that the exclusion of tetraploid cells from the fetus in 2n/4n chimeras can largely be attributed to the formation of ICM-deficient blastocysts.

  15. 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

  16. 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 ...

  17. 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...

  18. 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...

  19. 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.

  20. 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

  1. Study the effect of F17S mutation on the chimeric Bacillus thermocatenulatus lipase

    Directory of Open Access Journals (Sweden)

    Seyed Hossein Khaleghinejad

    2016-06-01

    Full Text Available Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3 are one of the highest value commercial enzymes as they have potential applications in biotechnology for detergents, food, pharmaceuticals, leather, textiles, cosmetics, and paper industries; and are currently receiving considerable attention because of their potential applications in biotechnology. Bacillus thermocatenulatus Lipase 2 (BTL2 is one of the most important research targets, because of its potential industrial applications. In this study, the effect of substitution Phe17 with Ser in mutated BTL2 lipase, which conserved pentapeptide (112Ala-His-Ser-Gln-Gly116 was replaced with similar sequences (207Gly-Glu-Ser-Ala-Gly211 of Candida rugosa lipase (CLR at the nucleophilic elbow region. Docking results confirmed the mutated lipase to be better than the chimeric lipase. So, cloning was conducted, and the mutated and chimeric btl2 genes were expressed in Escherichia coli, and then the enzymes were purified by anion exchange chromatography. The mutation increased lipase lipolytic activity against most of the applied substrates, with the exception of tributyrin when compared with chimeric lipase. Further, the mutated lipase exhibited higher activity than the chimeric lipase at all temperatures. Optimum pH of the mutated lipase was obtained at pH 9.5, which was more than the chimeric one. Enzyme activity of the mutated lipase in the presence of organic solvents, detergents, and metal ions was also improved than the chimeric lipase.

  2. 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

  3. 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.

  4. Antistaphylococcal activity of bacteriophage derived chimeric protein P128

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    Vipra Aradhana A

    2012-03-01

    Full Text Available Abstract Background Bacterial drug resistance is one of the most significant challenges to human health today. In particular, effective antibacterial agents against methicillin-resistant Staphylococcus aureus (MRSA are urgently needed. A causal relationship between nasal commensal S. aureus and infection has been reported. Accordingly, elimination of nasal S. aureus reduces the risk of infection. Enzymes that degrade bacterial cell walls show promise as antibacterial agents. Bacteriophage-encoded bacterial cell wall-degrading enzymes exhibit intrinsic bactericidal activity. P128 is a chimeric protein that combines the lethal activity of the phage tail-associated muralytic enzyme of Phage K and the staphylococcal cell wall targeting-domain (SH3b of lysostaphin. Here we report results of in vitro studies evaluating the susceptibility of staphylococcal strains to this novel protein. Results Using the broth microdilution method adapted for lysostaphin, we found that P128 is effective against S. aureus clinical strains including MRSA, methicillin-sensitive S. aureus (MSSA, and a mupirocin-resistant S. aureus. Minimum bactericidal concentrations and minimum inhibitory concentrations of P128 (1-64 μg/mL were similar across the 32 S. aureus strains tested, demonstrating its bactericidal nature. In time-kill assays, P128 reduced colony-forming units by 99.99% within 1 h and inhibited growth up to 24 h. In an assay simulating topical application of P128 to skin or other biological surfaces, P128 hydrogel was efficacious when layered on cells seeded on solid media. P128 hydrogel was lethal to Staphylococci recovered from nares of healthy people and treated without any processing or culturing steps, indicating its in situ efficacy. This methodology used for in vitro assessment of P128 as an agent for eradicating nasal carriage is unique. Conclusions The novel chimeric protein P128 is a staphylococcal cell wall-degrading enzyme under development for

  5. Generation of a chimeric dust mite hypoallergen using DNA shuffling for application in allergen-specific immunotherapy.

    Science.gov (United States)

    Zhao, Bei-Bei; Diao, Ji-Dong; Liu, Zhi-Ming; Li, Chao-Pin; Jiang, Yu-Xin

    2014-01-01

    Specific immunotherapy (SIT) is the only treatment that provides long lasting relief of allergy symptoms. Unfortunately, SIT-based traditional remedies carry the risk of producing local and/or systemic side effects. To improve the safety and efficacy of SIT, it has been proposed that SIT must utilize allergens that are hypoallergenic but hyperimmunogenic. Therefore, we used DNA shuffling to generate mutant genes encoding hypoallergens with potent immunogenicity and screened them for their capacity to modify the allergic response. We tentatively shuffled the major group 1 allergen genes from house dust mite, Dermatophagoides farinae and Dermatophagoides pteronyssinus, and discovered a novel chimeric gene, termed C 1. The gene was expressed in Escherichia coli (E. coli) and the chimeric protein C 1 was purified. An animal model of asthma demonstrated that C 1 not only decreased the production of serum IgE and IgG1, and inhibited the production of IL-4 and IL-5 in the bronchoalveolar lavage fluid (BALF). C 1 also boosted the levels of IgG2a and IFN-γ, which may demonstrate a rebalance of TH1 and TH2 allergic response. Additionally, flow cytometry showed that the immunogenicity of C 1 was higher than that of ProDer f 1, but was not significantly different from that of ProDer p 1. Our findings suggest that the C 1 is hypoallergenic and yet highly immunogenic, which makes it potentially safe and effective for use in SIT of allergic asthma. PMID:25120738

  6. An improved protocol for efficient engraftment in NOD/LTSZ-SCIDIL-2Rγnull mice allows HIV replication and development of anti-HIV immune responses.

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    Maneesh Singh

    Full Text Available Cord blood hematopoietic progenitor cells (CB-HPCs transplanted immunodeficient NOD/LtsZ-scidIL2Rγ(null (NSG and NOD/SCID/IL2Rγ(null (NOG mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials.

  7. 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.

  8. 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.

  9. Chimerical pyrene-based [7]helicenes as twisted polycondensed aromatics.

    Science.gov (United States)

    Buchta, Michal; Rybáček, Jiří; Jančařík, Andrej; Kudale, Amit A; Buděšínský, Miloš; Chocholoušová, Jana Vacek; Vacek, Jaroslav; Bednárová, Lucie; Císařová, Ivana; Bodwell, Graham J; Starý, Ivo; Stará, Irena G

    2015-06-01

    Chimerical pyrene-based dibenzo[7]helicene rac-1 and 2H-pyran[7]helicene (M,R,R)-(-)-2, in which two pyrene subunits are fused to the [7]helicene/[7]heterohelicene scaffold, were synthesised by means of Ni(0) - or Co(I) -mediated [2+2+2] cycloisomerisation of dipyrenyl-acetylene-derived triynes. Pyrene-based dibenzo[7]helicene 1 was obtained in enantioenriched form by enantioselective cycloisomerisation under Ni(0) /QUINAP catalysis (57 % ee) or in enantiopure form by racemate resolution by liquid chromatography on a chiral column. 1,3-Allylic-type strain-controlled diastereoselective cycloisomerisation was employed in the synthesis of enantiopure (M,R,R)-(-)-2. Physicochemical properties of 1 and 2 encompassing the helicity assignment, stability to racemisation, X-ray crystal structure, UV/Vis, experimental/calculated electronic circular dichroism and fluorescence spectra were studied. Accordingly, comparison of the X-ray crystal structure of (M,R,R)-(-)-2 with calculated structures (DFT: B3LYP/cc-pVDZ, B97D/cc-pVDZ) indicated that its helical backbone is slightly over-flattened owing to intramolecular dispersion forces between tert-butylated pyrene subunits. Both 1 and 2 are fluorescent (with quantum yields in dichloromethane of ΦF =0.10 and 0.17, respectively) and are suggested to form intramolecular excimer states upon excitation, which are remarkably stabilised and exhibit large Stokes shifts (296 and 203 nm, respectively).

  10. 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

  11. 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

  12. 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

  13. 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

  14. Anti-tumor effect of adenovirus-mediated suicide gene therapy under control of tumor-specific and radio-inducible chimeric promoter in combination with γ-ray irradiation in vivo

    International Nuclear Information System (INIS)

    Objective: To detect the selective inhibitory effects of irradiation plus adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic acid (IAA) suicide gene system using tumor-specific and radio-inducible chimeric promoter on human hepatocellular carcinoma subcutaneously xenografted in nude mouse. Methods: Recombinant replicated-deficient adenovirus vector containing HRP gene and chimeric human telomerase reverse transcriptase (hTERT) promoter carrying 6 radio-inducible CArG elements was constructed. A human subcutaneous transplanting hepatocellular carcinoma (MHCC97 cell line) model was treated with γ-ray irradiation plus intra-tumor injections of adenoviral vector and intra-peritoneal injections of prodrug IAA. The change of tumor volume and tumor growth inhibiting rate, the survival time of nude mice, as well as histopathology of xenograft tumor and normal tissues were evaluated. Results: Thirty one days after the treatment, the relative tumor volumes in the negative, adenovirus therapy, irradiation, and combination groups were 49.23±4.55, 27.71±7.74, 28.53±10.48 and 11.58±3.23, respectively.There was a significantly statistical difference among them (F=16.288, P<0.01).The inhibition effect in the combination group was strongest as compared with that in other groups, and its inhibition ratio was 76.5%. The survival period extended to 43 d in the combination group, which showed a significantly difference with that in the control group (χ2=18.307, P<0.01). The area of tumors necrosis in the combination group was larger than that in the other groups, and the normal tissues showed no treatment-related toxic effect in all groups. However, multiple hepatocellular carcinoma metastases were observed in the liver in the control group, there were a few metastases in the monotherapy groups and no metastasis in the combination group. Conclusions: Adenovirus-mediated suicide gene therapy plus radiotherapy dramatically could inhibit tumor growth and prolong median

  15. Application of chimeric glucanase comprising mutanase and dextranase for prevention of dental biofilm formation.

    Science.gov (United States)

    Otsuka, Ryoko; Imai, Susumu; Murata, Takatoshi; Nomura, Yoshiaki; Okamoto, Masaaki; Tsumori, Hideaki; Kakuta, Erika; Hanada, Nobuhiro; Momoi, Yasuko

    2015-01-01

    Water-insoluble glucan (WIG) produced by mutans streptococci, an important cariogenic pathogen, plays an important role in the formation of dental biofilm and adhesion of biofilm to tooth surfaces. Glucanohydrolases, such as mutanase (α-1,3-glucanase) and dextranase (α-1,6-glucanase), are able to hydrolyze WIG. The purposes of this study were to construct bi-functional chimeric glucanase, composed of mutanase and dextranase, and to examine the effects of this chimeric glucanase on the formation and decomposition of biofilm. The mutanase gene from Paenibacillus humicus NA1123 and the dextranase gene from Streptococcus mutans ATCC 25175 were cloned and ligated into a pE-SUMOstar Amp plasmid vector. The resultant his-tagged fusion chimeric glucanase was expressed in Escherichia coli BL21 (DE3) and partially purified. The effects of chimeric glucanase on the formation and decomposition of biofilm formed on a glass surface by Streptococcus sobrinus 6715 glucosyltransferases were then examined. This biofilm was fractionated into firmly adherent, loosely adherent, and non-adherent WIG fractions. Amounts of WIG in each fraction were determined by a phenol-sulfuric acid method, and reducing sugars were quantified by the Somogyi-Nelson method. Chimeric glucanase reduced the formation of the total amount of WIG in a dose-dependent manner, and significant reductions of WIG in the adherent fraction were observed. Moreover, the chimeric glucanase was able to decompose biofilm, being 4.1 times more effective at glucan inhibition of biofilm formation than a mixture of dextranase and mutanase. These results suggest that the chimeric glucanase is useful for prevention of dental biofilm formation.

  16. 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

  17. Paucity of chimeric gene-transposable element transcripts in the Drosophila melanogaster genome

    Directory of Open Access Journals (Sweden)

    Petrov Dmitri A

    2005-11-01

    Full Text Available Abstract Background Recent analysis of the human and mouse genomes has shown that a substantial proportion of protein coding genes and cis-regulatory elements contain transposable element (TE sequences, implicating TE domestication as a mechanism for the origin of genetic novelty. To understand the general role of TE domestication in eukaryotic genome evolution, it is important to assess the acquisition of functional TE sequences by host genomes in a variety of different species, and to understand in greater depth the population dynamics of these mutational events. Results Using an in silico screen for host genes that contain TE sequences, we identified a set of 63 mature "chimeric" transcripts supported by expressed sequence tag (EST evidence in the Drosophila melanogaster genome. We found a paucity of chimeric TEs relative to expectations derived from non-chimeric TEs, indicating that the majority (~80% of TEs that generate chimeric transcripts are deleterious and are not observed in the genome sequence. Using a pooled-PCR strategy to assay the presence of gene-TE chimeras in wild strains, we found that over half of the observed chimeric TE insertions are restricted to the sequenced strain, and ~15% are found at high frequencies in North American D. melanogaster populations. Estimated population frequencies of chimeric TEs did not differ significantly from non-chimeric TEs, suggesting that the distribution of fitness effects for the observed subset of chimeric TEs is indistinguishable from the general set of TEs in the genome sequence. Conclusion In contrast to mammalian genomes, we found that fewer than 1% of Drosophila genes produce mRNAs that include bona fide TE sequences. This observation can be explained by the results of our population genomic analysis, which indicates that most potential chimeric TEs in D. melanogaster are deleterious but that a small proportion may contribute to the evolution of novel gene sequences such as nested or

  18. 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.

  19. Simulations of Mineral Dust Content With CHIMERE-Dust Model

    Science.gov (United States)

    Schmechtig, C.; Marticorena, B.; Menut, L.; Bergametti, G.

    2006-12-01

    Simulations of the mineral dust cycle have been performed whith CHIMERE-Dust model over a domain that includes North Africa, the Mediterranean basin and the North Tropical Atlantic Ocean (10S-60N and 90W-90E) with a 1°x1° resolution using the ECMWF (European Center for Medium-Range Weather Forecasts) meteorological fields for two years, 2000 and 2001. As a validation, we compare the simulated dust concentration fields with photometric data from the AERONET network. From the comparisons between the simulated and measured aerosol optical depth for several stations of the Mediterranean basin, the model appears to reproduce correctly the intensity and occurrences of the dust events. Over Western Africa, the results are not as satisfying since some of the most intense dust events observed on the continent and downwind are not captured by the model. In addition, the simulated events are generally underestimated compared to the measured ones. It appears that these differences in the model performances are connected to the origin of the dust plumes. For example, dust plumes coming from Libya are well simulated while dust plumes originating from the Bodélé depression not as frequent as intense as the observations suggest. Soil properties in these two regions are comparable and typical of very erodible surfaces. We thus focused on the comparison between the ECMWF 10m wind speed fields and 10m wind speed measured at the meteorological stations located in both areas. We noticed that over Libya, the measured and ECMWF 10m wind speed are in very good agreement, while the meteorological model does not reproduce the extrema of the measured wind speed in the Bodélé depression. We found that a crude empirical correction of the 10m wind field in the Bodélé Depression significantly improve the simulations in terms of occurrence and of intensity.

  20. 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

  1. Endothelial cell chimerism by fluorescence in situ hybridization in gender mismatched renal allograft biopsies

    Institute of Scientific and Technical Information of China (English)

    BAI Hong-wei; SHI Bing-yi; QIAN Ye-yong; NA Yan-qun; ZENG Xuan; ZHONG Ding-rong; LU Min; ZOU Wan-zhong; WU Shi-fei

    2007-01-01

    Background The blood vessels of a transplanted organ are the interface between donor and recipient. The endothelium in the blood vessels is thought to be the major target for graft rejection. Endothelial cells of a transplanted organ can be of recipient origin after transplantation. In this study, we tested whether endothelial chimerism correlated with the graft rejection and cold ischemia.Methods We studied the biopsy samples from 34 renal transplants of female recipients who received the kidney from a male donor for the presence of endothelial cells of recipient origin. We examined the tissue sections of renal biopsy samples by fluorescence in situ hybridization (FISH) for the presence of endothelial cells containing two X chromosomes using a biotinylated Y chromosome probe and digoxigenin labelled X chromosome probe, and then analyzed the relationship between the endothelial cell chimerism and the rejection and cold ischemia.Results Endothelial chimerism was common and irrespective of rejections (P>0.05). The cold ischemic time of chimerism group was longer than no chimerism group ((14.83±4.03) hours vs (11.27±3.87) hours, P<0.05).Conclusions There is no correlation between the percentage of recipient endothelial cells in vascular endothelial cells and the type of graft rejection. The endothelium damaged by ischemic injury might be repaired by the endothelial cells from the recipient.

  2. 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

  3. 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

  4. Quantitative assessment of hematopoietic chimerism by quantitative real-time polymerase chain reaction of sequence polymorphism systems after 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

    2011-01-01

    Background Analysis of changes in recipient and donor hematopoietic cell origin is extremely useful to monitor the effect of hematopoietic stem cell transplantation (HSCT) and sequential adoptive immunotherapy by donor lymphocyte infusions. We developed a sensitive, reliable and rapid real-time PCR method based on sequence polymorphism systems to quantitatively assess the hematopoietic chimerism after HSCT. Methods A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time PCR in 101 HSCT patients with leukemia and other hematological diseases. The chimerism kinetics of bone marrow samples of 8 HSCT patients in remission and relapse situations were followed longitudinally. Results Recipient genotype discrimination was possible in 97.0% (98 of 101) with a mean number of 2.5 (1-7)informative markers per recipient/donor pair. Using serial dilutions of plasmids containing specific SP markers, the linear correlation (r) of 0.99, the slope between -3.2 and -3.7 and the sensitivity of 0.1% were proved reproducible. By this method, it was possible to very accurately detect autologous signals in the range from 0.1% to 30%. The accuracy of the method in the very important range of autologous signals below 5% was extraordinarily high (standard deviation <1.85%),which might significantly improve detection accuracy of changes in autologous signals early in the post-transplantation course of follow-up. The main advantage of the real-time PCR method over short tandem repeat PCR chimerism assays is the absence of PCR competition and plateau biases, with demonstrated greater sensitivity and linearity. Finally, we prospectively analyzed bone marrow samples of 8 patients who received allografts and presented the chimerism kinetics of remission and relapse situations that illustrated the sensitivity level and the promising clinical application of this method. Conclusion This SP-based real-time PCR assay provides a rapid, sensitive, and accurate quantitative

  5. Patterns of Amino Acid Evolution in the Drosophila ananassae Chimeric Gene, siren, Parallel Those of Other Adh-Derived Chimeras

    Science.gov (United States)

    Shih, Hung-Jui; Jones, Corbin D.

    2008-01-01

    siren1 and siren2 are novel alcohol dehydrogenase (Adh)-derived chimeric genes in the Drosophila bipectinata complex. D. ananassae, however, harbors a single homolog of these genes. Like other Adh-derived chimeric genes, siren evolved adaptively shortly after it was formed. These changes likely shifted the catalytic activity of siren. PMID:18780749

  6. Design and production in Aspergillus niger of a chimeric protein associating a fungal feruloyl esterase and a clostridial dockerin domain

    NARCIS (Netherlands)

    Levasseur, A.; Pagès, S.; Fierobe, H.-P.; Navarro, D.; Punt, P.; Belaïch, J.-P.; Asther, M.; Record, E.

    2004-01-01

    A chimeric enzyme associating feruloyl esterase A (FAEA) from Aspergilhis niger and dockerin from Clostridium thermocellum was produced in A. niger. A completely truncated form was produced when the dockerin domain was located downstream of the FAEA (FAEA-Doc), whereas no chimeric protein was produc

  7. 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

  8. 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.

  9. Accelerated tumor progression in mice lacking the ATP receptor P2X7

    DEFF Research Database (Denmark)

    Adinolfi, Elena; Capece, Marina; Franceschini, Alessia;

    2015-01-01

    cells. Tumor size and metastatic dissemination were assessed by in vivo calliper and luciferase luminescence emission measurements along with postmortem examination. In P2X7R-deficient mice, tumor growth and metastatic spreading were accelerated strongly, compared with wild-type (wt) mice. Intratumoral...... IL-1β and VEGF release were drastically reduced, and inflammatory cell infiltration was abrogated nearly completely. Similarly, tumor growth was also greatly accelerated in wt chimeric mice implanted with P2X7R-deficient bone marrow cells, defining hematopoietic cells as a sufficient site of P2X7R...

  10. Synergistic and persistent effect of T-cell immunotherapy with anti-CD19 or anti-CD38 chimeric receptor in conjunction with rituximab on B-cell non-Hodgkin lymphoma.

    Science.gov (United States)

    Mihara, Keichiro; Yanagihara, Kazuyoshi; Takigahira, Misato; Kitanaka, Akira; Imai, Chihaya; Bhattacharyya, Joyeeta; Kubo, Takanori; Takei, Yoshifumi; Yasunaga, Shin'ichiro; Takihara, Yoshihiro; Kimura, Akiro

    2010-10-01

    Using artificial receptors, it is possible to redirect the specificity of immune cells to tumour-associated antigens, which is expected to provide a useful strategy for cancer immunotherapy. Given that B-cell non-Hodgkin lymphoma (B-NHL) cells invariably express CD19 and CD38, these antigens may be suitable molecular candidates for such immunotherapy. We transduced human peripheral T cells or a T-cell line with either anti-CD19-chimeric receptor (CAR) or anti-CD38-CAR, which contained an anti-CD19 or anti-CD38 antibody-derived single-chain variable domain respectively. Retroviral transduction led to anti-CD19-CAR or anti-CD38-CAR expression in T cells with high efficiency (>60%). The T cell line, Hut78, when transduced with anti-CD19-CAR or anti-CD38-CAR, exerted strong cytotoxicity against the B-NHL cell lines, HT and RL, and lymphoma cells isolated from patients. Interestingly, use of both CARs had an additive cytotoxic effect on HT cells in vitro. In conjunction with rituximab, human peripheral T cells expressing either anti-CD19-CAR or anti-CD38-CAR enhanced cytotoxicity against HT-luciferase cells in xenografted mice. Moreover, the synergistic tumour-suppressing activity was persistent in vivo for over 2 months. These results provide a powerful rationale for clinical testing of the combination of rituximab with autologous T cells carrying either CAR on aggressive or relapsed B-NHLs. PMID:20678160

  11. 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...

  12. 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. Graphical Abstract ᅟ.

  13. 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.

  14. 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

    CD45, a transmembrane protein tyrosine phosphatase (PTPase), is required for TCR signaling. Multiple CD45 isoforms, differing in the extracellular domain, are expressed in a tissue- and activation-specific manner, suggesting an important function for this domain. We report that a chimeric protein...

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

    Directory of Open Access Journals (Sweden)

    Sarah Djebali

    Full Text Available 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.

  16. 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

  17. Mixed chimerism following hematopoietic stem cell transplantation in pediatric thalassemia major patients: a single center experience

    Directory of Open Access Journals (Sweden)

    Elif Ünal İnce

    2010-03-01

    Full Text Available Objective: Stable mixed chimerism (MC may result in cure for thalassemia major patients following hematopoietic stem cell transplantation (HSCT, but rejection can occur. Twenty-eight HSCTs for thalassemia major were reviewed retrospectively to evaluate the clinical course of MC with possible risk factors and predictors of outcome, with a median follow-up of 1669 days (811-3576 days. Materials and Methods: Chimerism was detected by fluorescence in situ hybridization (FISH or multiplex polymerase chain reaction depending on the sex match between the donor and the recipient. Results: Primary rejection, stable MC and full donor chimerism was detected in 3.6%, 17.8% and 78.6% of patients, respectively. Clinically, 4/5 patients with stable MC had thalassemia trait with donor chimerism as low as 14%. One patient was started on pRBC transfusions at 2.5 years postHSCT. Conclusion: Stable MC can result in cure for thalassemia major patients. The clinical picture remains as the best guide for intervention until a more reliable predictor is available.

  18. 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...

  19. Minimal Residual Disease Diagnostics and Chimerism in the Post-Transplant Period in Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    Ulrike Bacher

    2011-01-01

    Full Text Available In acute myeloid leukemia (AML, the selection of poor-risk patients for allogeneic hematopoietic stem cell transplantation (HSCT is associated with rather high post-transplant relapse rates. As immunotherapeutic intervention is considered to be more effective before the cytomorphologic manifestation of relapse, post-transplant monitoring gains increasing attention in stem cell recipients with a previous diagnosis of AML. Different methods for detection of chimerism (e.g., microsatellite analysis or quantitative real-time PCR are available to quantify the ratio of donor and recipient cells in the post-transplant period. Various studies demonstrated the potential use of mixed chimerism kinetics to predict relapse of the AML. CD34+-specific chimerism is associated with a higher specificity of chimerism analysis. Nevertheless, a decrease of donor cells can have other causes as well. Therefore, efforts continue to introduce minimal residual disease (MRD monitoring based on molecular mutations in the post-transplant period. The NPM1 (nucleophosmin mutations can be monitored by sensitive quantitative real-time PCR in subsets of stem cell recipients with AML, but for approximately 20% of patients, suitable molecular mutations for post-transplant MRD monitoring are not available so far. This emphasizes the need for an expansion of the panel of MRD markers in the transplant setting.

  20. Enhanced antibody-dependent cellular phagocytosis by chimeric monoclonal antibodies with tandemly repeated Fc domains.

    Science.gov (United States)

    Nagashima, Hiroaki; Ootsubo, Michiko; Fukazawa, Mizuki; Motoi, Sotaro; Konakahara, Shu; Masuho, Yasuhiko

    2011-04-01

    We previously reported that chimeric monoclonal antibodies (mAbs) with tandemly repeated Fc domains, which were developed by introducing tandem repeats of Fc domains downstream of 2 Fab domains, augmented binding avidities for all Fcγ receptors, resulting in enhanced antibody (Ab)-dependent cellular cytotoxicity. Here we investigated regarding Ab-dependent cellular phagocytosis (ADCP) mediated by these chimeric mAbs, which is considered one of the most important mechanisms that kills tumor cells, using two-color flow cytometric methods. ADCP mediated by T3-Ab, a chimeric mAb with 3 tandemly repeated Fc domains, was 5 times more potent than that by native anti-CD20 M-Ab (M-Ab hereafter). Furthermore, T3-Ab-mediated ADCP was resistant to competitive inhibition by intravenous Ig (IVIG), although M-Ab-mediated ADCP decreased in the presence of IVIG. An Fcγ receptor-blocking study demonstrated that T3-Ab mediated ADCP via both FcγRIA and FcγRIIA, whereas M-Ab mediated ADCP exclusively via FcγRIA. These results suggest that chimeric mAbs with tandemly repeated Fc domains enhance ADCP as well as ADCC, and that Fc multimerization may significantly enhance the efficacy of therapeutic Abs. PMID:21215693

  1. 78 FR 16505 - Prospective Grant of Exclusive License: Chimeric West Nile/Dengue Viruses

    Science.gov (United States)

    2013-03-15

    ... business in San Diego, California. The patent rights in these inventions have been assigned to the... virus exhibits much more robust viral replication in cell cultures, compared to the slow growing DEN viruses. The chimeric WN/DEN virus can be used as a substitute for wild-type dengue virus in...

  2. Optimized total body irradiation for induction of renal allograft tolerance through mixed chimerism in cynomolgus monkeys

    Energy Technology Data Exchange (ETDEWEB)

    Kimikawa, Masaaki; Kawai, Tatsuo; Ota, Kazuo [Tokyo Women`s Medical Coll. (Japan)

    1996-12-01

    We previously demonstrated that a nonmyeloablative preparative regimen can induce mixed chimerism and renal allograft tolerance between MHC-disparate non-human primates. The basic regimen includes anti-thymocyte globulin (ATG), total body irradiation (TBI, 300 cGy), thymic irradiation (TI, 700 cGy), splenectomy, donor bone marrow (DBM) infusion, and posttransplant cyclosporine therapy (CYA, discontinued after 4 weeks). To evaluate the importance and to minimize the toxicity of irradiation, kidney allografts were transplanted with various manipulations of the irradiation protocol. Monkeys treated with the basic protocol without TBI and TI did not develop chimerism or long-term allograft survival. In monkeys treated with the full protocol, all six monkeys treated with two fractionated dose of 150 cGy developed chimerism and five monkeys appeared tolerant. In contrast, only two of the four monkeys treated with fractionated doses of 125 cGy developed chimerism and only one monkey survived long term. The degree of lymphocyte depletion in all recipients was proportional to the TBI dose. The fractionated TBI regimen of 150 cGy appears to be the most consistently effective regimen for establishing donor bone marrow cell engraftment and allograft tolerance. (author)

  3. Chimeric flaviviruses: novel vaccines against dengue fever, tick-borne encephalitis, and Japanese encephalitis.

    Science.gov (United States)

    Lai, Ching-Juh; Monath, Thomas P

    2003-01-01

    Many arthropod-borne flaviviruses are important human pathogens responsible for diverse illnesses, including YF, JE, TBE, and dengue. Live, attenuated vaccines have afforded the most effective and economical means of prevention and control, as illustrated by YF 17D and JE SA14-14-2 vaccines. Recent advances in recombinant DNA technology have made it possible to explore a novel approach for developing live attenuated flavivirus vaccines against other flaviviruses. Full-length cDNA clones allow construction of infectious virus bearing attenuating mutations or deletions incorporated in the viral genome. It is also possible to create chimeric flaviviruses in which the structural protein genes for the target antigens of a flavivirus are replaced by the corresponding genes of another flavivirus. By combining these molecular techniques, the DNA sequences of DEN4 strain 814669, DEN2 PDK-53 candidate vaccine and YF 17D vaccine have been used as the genetic backbone to construct chimeric flaviviruses with the required attenuation phenotype and expression of the target antigens. Encouraging results from preclinical and clinical studies have shown that several chimeric flavivirus vaccines have the safety profile and satisfactory immunogenicity and protective efficacy to warrant further evaluation in humans. The chimeric flavivirus strategy has led to the rapid development of novel live-attenuated vaccines against dengue, TBE, JE, and West Nile viruses. PMID:14714441

  4. 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

  5. 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.

  6. 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.

  7. Birth of a chimeric primate gene by capture of the transposase gene from a mobile element.

    Science.gov (United States)

    Cordaux, Richard; Udit, Swalpa; Batzer, Mark A; Feschotte, Cédric

    2006-05-23

    The emergence of new genes and functions is of central importance to the evolution of species. The contribution of various types of duplications to genetic innovation has been extensively investigated. Less understood is the creation of new genes by recycling of coding material from selfish mobile genetic elements. To investigate this process, we reconstructed the evolutionary history of SETMAR, a new primate chimeric gene resulting from fusion of a SET histone methyltransferase gene to the transposase gene of a mobile element. We show that the transposase gene was recruited as part of SETMAR 40-58 million years ago, after the insertion of an Hsmar1 transposon downstream of a preexisting SET gene, followed by the de novo exonization of previously noncoding sequence and the creation of a new intron. The original structure of the fusion gene is conserved in all anthropoid lineages, but only the N-terminal half of the transposase is evolving under strong purifying selection. In vitro assays show that this region contains a DNA-binding domain that has preserved its ancestral binding specificity for a 19-bp motif located within the terminal-inverted repeats of Hsmar1 transposons and their derivatives. The presence of these transposons in the human genome constitutes a potential reservoir of approximately 1,500 perfect or nearly perfect SETMAR-binding sites. Our results not only provide insight into the conditions required for a successful gene fusion, but they also suggest a mechanism by which the circuitry underlying complex regulatory networks may be rapidly established. PMID:16672366

  8. 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.

  9. Hybridization accompanying FRET event in labeled natural nucleoside-unnatural nucleoside containing chimeric DNA duplexes.

    Science.gov (United States)

    Bag, Subhendu Sekhar; Das, Suman K; Pradhan, Manoj Kumar; Jana, Subhashis

    2016-09-01

    Förster resonance energy transfer (FRET) is a highly efficient strategy in illuminating the structures, structural changes and dynamics of DNA, proteins and other biomolecules and thus is being widely utilized in studying such phenomena, in designing molecular/biomolecular probes for monitoring the hybridization event of two single stranded DNA to form duplex, in gene detection and in many other sensory applications in chemistry, biology and material sciences. Moreover, FRET can give information about the positional status of chromophores within the associated biomolecules with much more accuracy than other methods can yield. Toward this end, we want to report here the ability of fluorescent unnatural nucleoside, triazolylphenanthrene ((TPhen)BDo) to show FRET interaction upon hybridization with fluorescently labeled natural nucleosides, (Per)U or (OxoPy)U or (Per)U, forming two stable chimeric DNA duplexes. The pairing selectivity and the thermal duplex stability of the chimeric duplexes are higher than any of the duplexes with natural nucleoside formed. The hybridization results in a Förster resonance energy transfer (FRET) from donor triazolylphenanthrene of (TPhen)BDo to acceptor oxopyrene of (OxoPy)U and/or to perylene chromophore of (Per)U, respectively, in two chimeric DNA duplexes. Therefore, we have established the FRET process in two chimeric DNA duplexes wherein a fluorescently labeled natural nucleoside ((OxoPy)U or (Per)U) paired against an unnatural nucleoside ((TPhen)BDo) without sacrificing the duplex stability and B-DNA conformation. The hybridization accompanying FRET event in these classes of interacting fluorophores is new. Moreover, there is no report of such designed system of chimeric DNA duplex. Our observed phenomenon and the design can potentially be exploited in designing more of such efficient FRET pairs for useful application in the detection and analysis of biomolecular interactions and in material science application.

  10. 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

  11. 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

  12. 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.

  13. Immunogenicity of a virosomally-formulated Plasmodium falciparum GLURP-MSP3 chimeric protein-based malaria vaccine candidate in comparison to adjuvanted formulations

    Directory of Open Access Journals (Sweden)

    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

  14. 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.

  15. The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

    Science.gov (United States)

    Tsukahara, T; Iwase, N; Kawakami, K; Iwasaki, M; Yamamoto, C; Ohmine, K; Uchibori, R; Teruya, T; Ido, H; Saga, Y; Urabe, M; Mizukami, H; Kume, A; Nakamura, M; Brentjens, R; Ozawa, K

    2015-02-01

    Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

  16. A novel chimeric flagellum fused with the multi-epitope vaccine CTB-UE prevents Helicobacter pylori-induced gastric cancer in a BALB/c mouse model.

    Science.gov (United States)

    Song, Hui; Lv, Xiaobo; Yang, Jue; Liu, Wei; Yang, Huan; Xi, Tao; Xing, Yingying

    2015-11-01

    Helicobacter pylori (H. pylori) infection causes peptic ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma. The eradication of H. pylori might be an effective means of preventing gastric cancer. A dual-antigen epitope and dual-adjuvant vaccine called CTB-UE-CF (CCF) was constructed by combining a multi-epitope vaccine CTB-UE with a novel chimeric flagellum (CF) to simultaneously activate Toll-like receptor (TLR) 5-agonist activity and preserve the immunogenicity of H. pylori flagellum FlaA. The evaluation of efficacy to reduce H. pylori colonization was performed using BALB/c mice by oral immunization with a triple dose of this vaccine strain. Two weeks after the last immunization, mice were sacrificed to determine specific antibody levels and proinflammatory cytokine production. To determine the presence of H. pylori, we detected the number of H. pylori by real-time quantitative PCR (qPCR) and measured the urease activity in the gastric tissue. The results showed that the immunogenicity and mucosal immune responses of CCF performed significantly better than those of CTB-UE. This dual-antigen epitope and dual-adjuvant system might greatly contribute to the development of a safe and efficient therapeutic vaccine for humans against H. pylori infection.

  17. 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.

  18. 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.

  19. 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...... not include any CSFV components. In the present study, the DIVA vaccine properties of CP7_E2gif were evaluated in comparison to the conventional live attenuated Riemser C-strain vaccine. Sera and tonsil samples obtained from pigs immunised with these two vaccines were analysed. No viral RNA was found in serum...... after vaccination with CP7_E2gif, whereas some serum samples from C-strain vaccinated animals were positive. In both vaccinated groups, individual viral RNA-positive tonsil samples were detected in animals euthanised between 7 and 21 days post vaccination. Furthermore, serum samples from these animals...

  20. [Harvesting technique of chimeric multiple paddles fibular flap for wide oromandibular defects].

    Science.gov (United States)

    Foy, J-P; Qassemyar, Q; Assouly, N; Temam, S; Kolb, F

    2016-08-01

    Carcinological head and neck reconstruction still remains a challenge due to the volume and varied tissues needed. Large and wide oromandibular defects require, not just the bone but also soft tissues for the pelvilingual reconstruction and therefore, a second free flap may become necessary in addition to a fibular flap. The option of an unique chimeric flap based on the fibular artery and its branches is less known whereas it offers the advantage of a unique flap with bone, muscle and multiple skin paddles, independent of each other. The aim of this technical note is to present step by step the surgical procedure of this chimeric flap and share this method that avoids a second free flap.

  1. Replication-competent chimeric lenti-oncovirus with expanded host cell tropism.

    Science.gov (United States)

    Reiprich, S; Gundlach, B R; Fleckenstein, B; Uberla, K

    1997-04-01

    Baboon bone marrow was grafted into human immunodeficiency virus type 1-infected patients in the course of recent trials for AIDS treatment. Since the baboon genome harbors multiple copies of an endogenous oncovirus, chimeric lenti-oncoviruses could emerge in the xenotransplant recipient. To analyze the potential replication competence of hybrid viruses between different genera of retroviruses, we replaced most of the env gene of simian immunodeficiency virus with the env gene of an amphotropic murine leukemia virus. The hybrid virus could be propagated in human T-cell lines, in peripheral blood mononuclear cells of rhesus macaques, and in CD4- B-cell lines. Because of the expanded cell tropism, the hybrid virus might have a selective advantage in comparison to parental viruses. Therefore, emerging chimeric viruses may be considered a serious risk of xenotransplantation. A note of caution is also suggested for the use of pseudotyped lentiviral vectors for human gene therapy.

  2. Chimerism of allogeneic mesenchymal cells in bone marrow, liver, and spleen after mesenchymal stem cells infusion.

    Science.gov (United States)

    Meleshko, Alexander; Prakharenia, Irina; Kletski, Semen; Isaikina, Yanina

    2013-12-01

    Although an infusion of culture-expanded MSCs is applied in clinic to improve results of HSCs transplantation and for a treatment of musculoskeletal disorders, homing, and engraftment potential of culture-expanded MSC in humans is still obscure. We report two female patients who received allogeneic BM transplantation as a treatment of hematological diseases and a transplantation of MSCs from third-party male donors. Both patients died within one yr of infectious complications. Specimens of paraffin-embedded blocks of tissues from transplanted patients were taken. The aim of the study was to estimate possible homing and engraftment of allogeneic BM-derived MSCs in some tissues/organs of recipient. Sensitive real-time quantitative PCR analysis was applied with SRY gene as a target. MSC chimerism was found in BM, liver, and spleen of both patients. We conclude that sensitive RQ-PCR analysis is acceptable for low-level chimerism evaluation even in paraffin-embedded tissue specimens.

  3. 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

  4. 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

  5. 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...

  6. 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...

  7. 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...

  8. Generation of cloned and chimeric embryos/offspring using the new methods of animal biotechnology.

    Science.gov (United States)

    Skrzyszowska, Maria; Karasiewicz, Jolanta; Bednarczyk, Marek; Samiec, Marcin; Smorag, Zdzisław; Waś, Bogusław; Guszkiewicz, Andrzej; Korwin-Kossakowski, Maciej; Górniewska, Maria; Szablisty, Ewa; Modliński, Jacek A; Łakota, Paweł; Wawrzyńska, Magdalena; Sechman, Andrzej; Wojtysiak, Dorota; Hrabia, Anna; Mika, Maria; Lisowski, Mirosław; Czekalski, Przemysław; Rzasa, Janusz; Kapkowska, Ewa

    2006-01-01

    The article summarizes results of studies concerning: 1/ qualitative evaluation of pig nuclear donor cells to somatic cell cloning, 2/ developmental potency of sheep somatic cells to create chimera, 3/ efficient production of chicken chimera. The quality of nuclear donor cells is one of the most important factors to determine the efficiency of somatic cell cloning. Morphological criteria commonly used for qualitative evaluation of somatic cells may be insufficient for practical application in the cloning. Therefore, different types of somatic cells being the source of genomic DNA in the cloning procedure were analyzed on apoptosis with the use of live-DNA or plasma membrane fluorescent markers. It has been found that morphological criteria are a sufficient selection factor for qualitative evaluation of nuclear donor cells to somatic cell cloning. Developmental potencies of sheep somatic cells in embryos and chimeric animals were studied using blastocyst complementation test. Fetal fibroblasts stained with vital fluorescent dye and microsurgically placed in morulae or blastocysts were later identified in embryos cultured in vitro. Transfer of Polish merino blastocysts harbouring Heatherhead fibroblasts to recipient ewes brought about normal births at term. Newly-born animals were of merino appearance with dark patches on their noses, near the mouth and on their clovens. This overt chimerism shows that fetal fibroblasts introduced to sheep morulae/blastocysts revealed full developmental plasticity. To achieve the efficient production of chicken chimeras, the blastodermal cells from embryos of the donor breeds, (Green-legged Partridgelike breed or GPxAraucana) were transferred into the embryos of the recipient breed (White Leghorn), and the effect of chimerism on the selected reproductive and physiological traits of recipients was examined. Using the model which allowed identification of the chimerism at many loci, it has been found that 93.9% of the examined birds

  9. 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 ...

  10. Zygotes segregate entire parental genomes in distinct blastomere lineages causing cleavage-stage chimerism and mixoploidy.

    Science.gov (United States)

    Destouni, Aspasia; Zamani Esteki, Masoud; Catteeuw, Maaike; Tšuiko, Olga; Dimitriadou, Eftychia; Smits, Katrien; Kurg, Ants; Salumets, Andres; Van Soom, Ann; Voet, Thierry; Vermeesch, Joris R

    2016-05-01

    Dramatic genome dynamics, such as chromosome instability, contribute to the remarkable genomic heterogeneity among the blastomeres comprising a single embryo during human preimplantation development. This heterogeneity, when compatible with life, manifests as constitutional mosaicism, chimerism, and mixoploidy in live-born individuals. Chimerism and mixoploidy are defined by the presence of cell lineages with different parental genomes or different ploidy states in a single individual, respectively. Our knowledge of their mechanistic origin results from indirect observations, often when the cell lineages have been subject to rigorous selective pressure during development. Here, we applied haplarithmisis to infer the haplotypes and the copy number of parental genomes in 116 single blastomeres comprising entire preimplantation bovine embryos (n = 23) following in vitro fertilization. We not only demonstrate that chromosome instability is conserved between bovine and human cleavage embryos, but we also discovered that zygotes can spontaneously segregate entire parental genomes into different cell lineages during the first post-zygotic cleavage division. Parental genome segregation was not exclusively triggered by abnormal fertilizations leading to triploid zygotes, but also normally fertilized zygotes can spontaneously segregate entire parental genomes into different cell lineages during cleavage of the zygote. We coin the term "heterogoneic division" to indicate the events leading to noncanonical zygotic cytokinesis, segregating the parental genomes into distinct cell lineages. Persistence of those cell lines during development is a likely cause of chimerism and mixoploidy in mammals. PMID:27197242

  11. 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

  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. Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation

    Science.gov (United States)

    Stahl, Tanja; Rothe, Caroline; Böhme, Manja U.; Kohl, Aloisa; Kröger, Nicolaus; Fehse, Boris

    2016-01-01

    Accurate and sensitive determination of hematopoietic chimerism is a crucial diagnostic measure after allogeneic stem cell transplantation to monitor engraftment and potentially residual disease. Short tandem repeat (STR) amplification, the current “gold standard” for chimerism assessment facilitates reliable accuracy, but is hampered by its limited sensitivity (≥1%). Digital PCR (dPCR) has been shown to combine exact quantification and high reproducibility over a very wide measurement range with excellent sensitivity (routinely ≤0.1%) and thus represents a promising alternative to STR analysis. We here aimed at developing a whole panel of digital-PCR based assays for routine diagnostic. To this end, we tested suitability of 52 deletion/insertion polymorphisms (DIPs) for duplex analysis in combination with either a reference gene or a Y-chromosome specific PCR. Twenty-nine DIPs with high power of discrimination and good performance were identified, optimized and technically validated. We tested the newly established assays on retrospective patient samples that were in parallel also measured by STR amplification and found excellent correlation. Finally, a screening plate for initial genotyping with DIP-specific duplex dPCR assays was designed for convenient assay selection. In conclusion, we have established a comprehensive dPCR system for precise and high-sensitivity measurement of hematopoietic chimerism, which should be highly useful for clinical routine diagnostics. PMID:27618030

  15. 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.

  16. Modulating p56Lck in T-Cells by a Chimeric Peptide Comprising Two Functionally Different Motifs of Tip from Herpesvirus saimiri

    Directory of Open Access Journals (Sweden)

    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. Analysis of the atmospheric composition during the summer 2013 over the Mediterranean area using the CHARMEX measurements and the CHIMERE model

    Directory of Open Access Journals (Sweden)

    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.

  18. 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

  19. 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.

  20. 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...

  1. 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...

  2. Mutant SOD1 in cell types other than motor neurons and oligodendrocytes accelerates onset of disease in ALS mice

    OpenAIRE

    Yamanaka, Koji; Boillee, Severine; Roberts, Elizabeth A.; Garcia, Michael L.; McAlonis-Downes, Melissa; Mikse, Oliver R.; Cleveland, Don W.; Lawrence S B Goldstein

    2008-01-01

    Dominant mutations in ubiquitously expressed superoxide dismutase (SOD1) cause familial ALS by provoking premature death of adult motor neurons. To test whether mutant damage to cell types beyond motor neurons is required for the onset of motor neuron disease, we generated chimeric mice in which all motor neurons and oligodendrocytes expressed mutant SOD1 at a level sufficient to cause fatal, early-onset motor neuron disease when expressed ubiquitously, but did so in a cellular environment co...

  3. 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

  4. Polyethylene glycol (PEG) linked to near infrared (NIR) dyes conjugated to chimeric anti-carcinoembryonic antigen (CEA) antibody enhances imaging of liver metastases in a nude-mouse model of human colon cancer.

    Science.gov (United States)

    Maawy, Ali A; Hiroshima, Yukihiko; Zhang, Yong; Luiken, George A; Hoffman, Robert M; Bouvet, Michael

    2014-01-01

    We report here that polyethylene glycol (PEG) linked to near infrared dyes conjugated to chimeric mouse-human anti-carcinoembryonic antigen (CEA) antibody greatly improves imaging of liver metastases in a nude mouse model of colon-cancer experimental metastases. PEGylated and non-PEGylated DyLight 650 and 750 dyes were conjugated to the chimeric anti-CEA antibody. The dyes were initially injected intravenously into nude mice without tumors. Tissue biodistribution was determined by tissue sonication and analyzing tissue dye concentration profiles over time. PEGylated dyes had significantly lower accumulation in the liver (p = 0.03 for the 650 dyes; p = 0.002 for the 750 dyes) compared to non-PEGylated dyes. In an experimental liver metastasis model of HT-29 colon cancer, PEGylated dyes conjugated to the anti-CEA antibody showed good labeling of metastatic tumors with high contrast between normal and malignant tissue which was not possible with the non-PEGylated dyes since there was so much non-specific accumulation in the liver. PEGylation of the DyLight 650 and 750 NIR dyes significantly altered tissue biodistribution, allowing brighter tissue labeling, decreased accumulation in normal organs, particularly the liver. This enabled high fidelity and high contrast imaging of liver metastases.

  5. Polyethylene glycol (PEG linked to near infrared (NIR dyes conjugated to chimeric anti-carcinoembryonic antigen (CEA antibody enhances imaging of liver metastases in a nude-mouse model of human colon cancer.

    Directory of Open Access Journals (Sweden)

    Ali A Maawy

    Full Text Available We report here that polyethylene glycol (PEG linked to near infrared dyes conjugated to chimeric mouse-human anti-carcinoembryonic antigen (CEA antibody greatly improves imaging of liver metastases in a nude mouse model of colon-cancer experimental metastases. PEGylated and non-PEGylated DyLight 650 and 750 dyes were conjugated to the chimeric anti-CEA antibody. The dyes were initially injected intravenously into nude mice without tumors. Tissue biodistribution was determined by tissue sonication and analyzing tissue dye concentration profiles over time. PEGylated dyes had significantly lower accumulation in the liver (p = 0.03 for the 650 dyes; p = 0.002 for the 750 dyes compared to non-PEGylated dyes. In an experimental liver metastasis model of HT-29 colon cancer, PEGylated dyes conjugated to the anti-CEA antibody showed good labeling of metastatic tumors with high contrast between normal and malignant tissue which was not possible with the non-PEGylated dyes since there was so much non-specific accumulation in the liver. PEGylation of the DyLight 650 and 750 NIR dyes significantly altered tissue biodistribution, allowing brighter tissue labeling, decreased accumulation in normal organs, particularly the liver. This enabled high fidelity and high contrast imaging of liver metastases.

  6. The nature of tolerance in adult recipient mice made tolerant of alloantigens with supralethal irradiation followed by syngeneic bone marrow cell transplantation plus injection of F1 spleen cells

    International Nuclear Information System (INIS)

    The length of time after syngeneic bone marrow reconstitution when tolerance to alloantigens can be induced in adult mice during T cell differentiation from bone marrow cells was studied by exposing those T cells to (recipient x donor)F1 spleen cells. Supralethally irradiated C3H/He Slc(C3H; H-2k) mice were reconstituted with 1 x 10(7) syngeneic T cell-depleted bone marrow cells and then injected intravenously with 5 x 10(7) (C3H x C57BL/6[B6])F1 (B6C3F1; H-2bxk) or (C3H x AKR/J[AKR])F1 (AKC3F1; H-2kxk) spleen cells at various intervals. In the fully allogeneic combination of B6C3F1----C3H, EL-4 tumor originating from B6 was accepted, and survival of grafted B6 skin was significantly prolonged in the tolerant C3H mice treated with irradiation on day -1 followed by injection of syngeneic bone marrow cells on day 0 plus B6C3F1 spleen cells on days 0, 5, or 10, in a tolerogen-specific manner. In the multiminor histocompatibility antigen-disparate combination of AKC3F1----C3H, AKR skin grafts were permanently accepted in the tolerant C3H mice treated with AKC3F1 spleen cells on days 0, 5, 10, or 15. Immunological parameters, including cytotoxic T lymphocyte activity and delayed foot-pad reaction (DFR), were almost completely suppressed in C3H mice made tolerant of B6 or AKR antigens. A chimeric assay using a direct immunofluorescence method revealed that the tolerant C3H mice given B6C3F1 spleen cells on day 0 were mixed-chimeric for at least 8 weeks after syngeneic bone marrow reconstitution, but not definitely chimeric thereafter. The C3H mice given AKC3F1 spleen cells on day 0 were chimeric even 43 weeks after syngeneic bone marrow reconstitution, but the C3H mice given AKC3F1 spleen cells on day 15 showed temporal chimerism that disappeared within 43 weeks. The untolerant mice were never detectably chimeric

  7. Evaluating Human T-Cell Therapy of Cytomegalovirus Organ Disease in HLA-Transgenic Mice.

    Directory of Open Access Journals (Sweden)

    Simone Thomas

    2015-07-01

    Full Text Available Reactivation of human cytomegalovirus (HCMV can cause severe disease in recipients of hematopoietic stem cell transplantation. Although preclinical research in murine models as well as clinical trials have provided 'proof of concept' for infection control by pre-emptive CD8 T-cell immunotherapy, there exists no predictive model to experimentally evaluate parameters that determine antiviral efficacy of human T cells in terms of virus control in functional organs, prevention of organ disease, and host survival benefit. We here introduce a novel mouse model for testing HCMV epitope-specific human T cells. The HCMV UL83/pp65-derived NLV-peptide was presented by transgenic HLA-A2.1 in the context of a lethal infection of NOD/SCID/IL-2rg-/- mice with a chimeric murine CMV, mCMV-NLV. Scenarios of HCMV-seropositive and -seronegative human T-cell donors were modeled by testing peptide-restimulated and T-cell receptor-transduced human T cells, respectively. Upon transfer, the T cells infiltrated host tissues in an epitope-specific manner, confining the infection to nodular inflammatory foci. This resulted in a significant reduction of viral load, diminished organ pathology, and prolonged survival. The model has thus proven its potential for a preclinical testing of the protective antiviral efficacy of HCMV epitope-specific human T cells in the evaluation of new approaches to an immunotherapy of CMV disease.

  8. HVR1-mediated antibody evasion of highly infectious in vivo adapted HCV in humanised mice

    DEFF Research Database (Denmark)

    Prentoe, Jannick; Verhoye, Lieven; Velázquez Moctezuma, Rodrigo;

    2016-01-01

    OBJECTIVE: HCV is a major cause of chronic liver disease worldwide, but the role of neutralising antibodies (nAbs) in its natural history remains poorly defined. We analysed the in vivo role of hypervariable region 1 (HVR1) for HCV virion properties, including nAb susceptibility. DESIGN: Analysis...... of HCV from human liver chimeric mice infected with cell-culture-derived prototype genotype 2a recombinant J6/JFH1 or HVR1-deleted variant J6/JFH1ΔHVR1 identified adaptive mutations, which were analysed by reverse genetics in Huh7.5 and CD81-deficient S29 cells. The increased in vivo genomic stability......06 IgG in vitro. CONCLUSIONS: The A876P-substitution bridges in vitro and in vivo studies using J6/JFH1-based recombinants. We provide the first in vivo evidence that HVR1 protects cross-genotype conserved HCV neutralisation epitopes, which advocates the possibility of using HVR1-deleted viruses...

  9. Influence of cell physiological state on gene delivery to T lymphocytes by chimeric adenovirus Ad5F35.

    Science.gov (United States)

    Zhang, Wen-feng; Shao, Hong-wei; Wu, Feng-lin; Xie, Xin; Li, Zhu-ming; Bo, Hua-ben; Shen, Han; Wang, Teng; Huang, Shu-lin

    2016-01-01

    Adoptive transfer of genetically-modified T cells is a promising approach for treatment of both human malignancies and viral infections. Due to its ability to efficiently infect lymphocytes, the chimeric adenovirus Ad5F35 is potentially useful as an immunotherapeutic for the genetic modification of T cells. In previous studies, it was found that the infection efficiency of Ad5F35 was significantly increased without enhanced expression of the viral receptor after T cell stimulation; however, little is known about the underlying mechanism. Nonetheless, cell physiology has long been thought to affect viral infection. Therefore, we aimed to uncover the physiologic changes responsible for the increased infection efficiency of Ad5F35 following T cell stimulation. Given the complexity of intracellular transport we analyzed viral binding, entry, and escape using a Jurkat T cell model and found that both cell membrane fluidity and endosomal escape of Ad5F35 were altered under different physiological states. This, in turn, resulted in differences in the amount of virus entering cells and reaching the cytoplasm. These results provide additional insight into the molecular mechanisms underlying Ad5F35 infection of T cells and consequently, will help further the clinical application of genetically-modified T cells for immunotherapy. PMID:26972139

  10. 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.

  11. 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.

  12. Cytotoxic T cell response against the chimeric ETV6-AML1 protein in childhood acute lymphoblastic leukemia.

    Science.gov (United States)

    Yotnda, P; Garcia, F; Peuchmaur, M; Grandchamp, B; Duval, M; Lemonnier, F; Vilmer, E; Langlade-Demoyen, P

    1998-07-15

    Cytotoxic T lymphocytes (CTL) are potent effector cells that could provide long term antitumor immunity if induced by appropriate vaccines. CTL recognize 8-14 amino acid-long peptides processed intracellularly and presented by MHC class I molecules. A well-characterized example of a potential tumor antigen in childhood pre-B Acute Lymphoblastic Leukemia (ALL) results from the chromosomal translocation 12;21 leading to the fusion of the ETV6 and AML1 genes. This translocation is observed in > 25% of ALL-patients. In this study, we have examined whether the chimeric ETV6-AML1 protein could serve as a tumor specific antigen for CTL in HLA-A2.1 individuals. We have identified a nonapeptide (RIAECILGM), encoded by the fusion region of the ETV6-AML1 protein, that binds to HLA-A2.1 molecules and induces specific primary CTL in peripheral blood lymphocytes from healthy donors. These CTL specifically lysed HLA-A2.1 tumor cells endogeneously expressing the ETV6-AML fusion protein. CTL with similar functional capacities were found with high frequencies and cloned from one patient's bone marrow indicating that ETV6-AML1-specific anti-ALL CTL are, at least in some patients, spontaneously stimulated and might participate to host antileukemia defense.

  13. 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

  14. Marrow transplantation from tolerant donors to treat and prevent autoimmune diseases in BXSB mice

    International Nuclear Information System (INIS)

    Autoimmune-prone BXSB male mice were supralethally irradiated and transplanted with CBA/H bone marrow cells. A complete and long-term chimerism was established when donor mice had been induced to develop tolerance of BXSB male antigens by combined treatment with BXSB male spleen cells and cyclophosphamide. Such chimeras did not express autoimmune phenomena or develop lethal autoimmune manifestations. Nor did the recipient mice develop the wasting syndrome or evidence of persistent immunodeficiencies that have been seen in other strains of autoimmune-resistant mice that had been transplanted with bone marrow cells across major histocompatibility complex barriers following an initial purging of the bone marrow of Thy-1+ cells using anti-Thy-1+C

  15. 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.

  16. Isolation and chimerization of a highly neutralizing antibody conferring passive protection against lethal Bacillus anthracis infection.

    Directory of Open Access Journals (Sweden)

    Ronit Rosenfeld

    Full Text Available Several studies have demonstrated that the passive transfer of protective antigen (PA-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29. Guinea pigs were fully protected against infection by 40LD(50B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD(50 of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.

  17. 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

  18. 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.

  19. Pretransplant mobilization with granulocyte colony-stimulating factor improves B-cell reconstitution by lentiviral vector gene therapy in SCID-X1 mice.

    Science.gov (United States)

    Huston, Marshall W; Riegman, Adriaan R A; Yadak, Rana; van Helsdingen, Yvette; de Boer, Helen; van Til, Niek P; Wagemaker, Gerard

    2014-10-01

    Hematopoietic stem cell (HSC) gene therapy is a demonstrated effective treatment for X-linked severe combined immunodeficiency (SCID-X1), but B-cell reconstitution and function has been deficient in many of the gene therapy treated patients. Cytoreductive preconditioning is known to improve HSC engraftment, but in general it is not considered for SCID-X1 since the poor health of most of these patients at diagnosis and the risk of toxicity preclude the conditioning used in standard bone marrow stem cell transplantation. We hypothesized that mobilization of HSC by granulocyte colony-stimulating factor (G-CSF) should create temporary space in bone marrow niches to improve engraftment and thereby B-cell reconstitution. In the present pilot study supplementing our earlier preclinical evaluation (Huston et al., 2011), Il2rg(-/-) mice pretreated with G-CSF were transplanted with wild-type lineage negative (Lin(-)) cells or Il2rg(-/-) Lin(-) cells transduced with therapeutic IL2RG lentiviral vectors. Mice were monitored for reconstitution of lymphocyte populations, level of donor cell chimerism, and antibody responses as compared to 2 Gy total body irradiation (TBI), previously found effective in promoting B-cell reconstitution. The results demonstrate that G-CSF promotes B-cell reconstitution similar to low-dose TBI and provides proof of principle for an alternative approach to improve efficacy of gene therapy in SCID patients without adverse effects associated with cytoreductive conditioning. PMID:25222508

  20. 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...

  1. Construction of chimeric enzymes out of maize endosperm branching enzymes I and II: activity and properties.

    Science.gov (United States)

    Kuriki, T; Stewart, D C; Preiss, J

    1997-11-14

    Branching enzyme I and II isoforms from maize endosperm (mBE I and mBE II, respectively) have quite different properties, and to elucidate the domain(s) that determines the differences, chimeric genes consisting of part mBE I and part mBE II were constructed. When expressed under the control of the T7 promoter in Escherichia coli, several of the chimeric enzymes were inactive. The only fully active chimeric enzyme was mBE II-I BspHI, in which the carboxyl-terminal part of mBE II was exchanged for that of mBE I at a BspHI restriction site and was purified to homogeneity and characterized. Another chimeric enzyme, mBE I-II HindIII, in which the amino-terminal end of mBE II was replaced with that of mBE I, had very little activity and was only partially characterized. The purified mBE II-I BspHI exhibited higher activity than wild-type mBE I and mBE II when assayed by the phosphorylase a stimulation assay. mBE II-I BspHI had substrate specificity (preference for amylose rather than amylopectin) and catalytic capacity similar to mBE I, despite the fact that only the carboxyl terminus was from mBE I, suggesting that the carboxyl terminus may be involved in determining substrate specificity and catalytic capacity. In chain transfer experiments, mBE II-I BspHI transferred more short chains (with a degree of polymerization of around 6) in a fashion similar to mBE II. In contrast, mBE I-II HindIII transferred more long chains (with a degree of polymerization of around 11-12), similar to mBE I, suggesting that the amino terminus of mBEs may play a role in the size of oligosaccharide chain transferred. This study challenges the notion that the catalytic centers for branching enzymes are exclusively located in the central portion of the enzyme; it suggests instead that the amino and carboxyl termini may also be involved in determining substrate preference, catalytic capacity, and chain length transfer.

  2. 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...

  3. Chimerism in a child with severe combined immunodeficiency: a case report.

    Science.gov (United States)

    Aureli, Anna; Piancatelli, Daniela; Monaco, Palmina I; Ozzella, Giuseppina; Canossi, Angelica; Piazza, Antonina; Isacchi, Giancarlo; Caniglia, Maurizio; Adorno, Domenico

    2006-09-01

    Severe combined immunodeficiency (SCID) represents a group of rare, sometimes fatal, congenital disorders in which there is a combined absence of T-lymphocyte and B-lymphocyte function. Children with SCID die within two years of age, if untreated. The effective treatment for SCID is a hematopoietic stem cell transplantation (HSCT). It has been repeatedly described that in peripheral blood of infants with SCID maternal T cells can be found. Here we report a case of blood chimerism in a one-year-old boy with SCID.

  4. 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...

  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. Allogeneic Bone Marrow Transplant from MRL/MpJ Super-Healer Mice Does Not Improve Articular Cartilage Repair in the C57Bl/6 Strain.

    Directory of Open Access Journals (Sweden)

    Catherine A Leonard

    Full Text Available Articular cartilage has been the focus of multiple strategies to improve its regenerative/ repair capacity. The Murphy Roths Large (MRL/MpJ "super-healer" mouse demonstrates an unusual enhanced regenerative capacity in many tissues and provides an opportunity to further study endogenous cartilage repair. The objective of this study was to test whether the super-healer phenotype could be transferred from MRL/MpJ to non-healer C57Bl/6 mice by allogeneic bone marrow transplant.The healing of 2mm ear punches and full thickness cartilage defects was measured 4 and 8 weeks after injury in control C57Bl/6 and MRL/MpJ "super-healer" mice, and in radiation chimeras reconstituted with bone marrow from the other mouse strain. Healing was assessed using ear hole diameter measurement, a 14 point histological scoring scale for the cartilage defect and an adapted version of the Osteoarthritis Research Society International scale for assessment of osteoarthritis in mouse knee joints.Normal and chimeric MRL mice showed significantly better healing of articular cartilage and ear wounds along with less severe signs of osteoarthritis after cartilage injury than the control strain. Contrary to our hypothesis, however, bone marrow transplant from MRL mice did not confer improved healing on the C57Bl/6 chimeras, either in regards to ear wound healing or cartilage repair.The elusive cellular basis for the MRL regenerative phenotype still requires additional study and may possibly be dependent on additional cell types external to the bone marrow.

  7. Vaccine efficacy in senescent mice challenged with recombinant SARS-CoV bearing epidemic and zoonotic spike variants.

    Directory of Open Access Journals (Sweden)

    Damon Deming

    2006-12-01

    Full Text Available BACKGROUND: In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV was identified as the etiological agent of severe acute respiratory syndrome, a disease characterized by severe pneumonia that sometimes results in death. SARS-CoV is a zoonotic virus that crossed the species barrier, most likely originating from bats or from other species including civets, raccoon dogs, domestic cats, swine, and rodents. A SARS-CoV vaccine should confer long-term protection, especially in vulnerable senescent populations, against both the 2003 epidemic strains and zoonotic strains that may yet emerge from animal reservoirs. We report the comprehensive investigation of SARS vaccine efficacy in young and senescent mice following homologous and heterologous challenge. METHODS AND FINDINGS: Using Venezuelan equine encephalitis virus replicon particles (VRP expressing the 2003 epidemic Urbani SARS-CoV strain spike (S glycoprotein (VRP-S or the nucleocapsid (N protein from the same strain (VRP-N, we demonstrate that VRP-S, but not VRP-N vaccines provide complete short- and long-term protection against homologous strain challenge in young and senescent mice. To test VRP vaccine efficacy against a heterologous SARS-CoV, we used phylogenetic analyses, synthetic biology, and reverse genetics to construct a chimeric virus (icGDO3-S encoding a synthetic S glycoprotein gene of the most genetically divergent human strain, GDO3, which clusters among the zoonotic SARS-CoV. icGD03-S replicated efficiently in human airway epithelial cells and in the lungs of young and senescent mice, and was highly resistant to neutralization with antisera directed against the Urbani strain. Although VRP-S vaccines provided complete short-term protection against heterologous icGD03-S challenge in young mice, only limited protection was seen in vaccinated senescent animals. VRP-N vaccines not only failed to protect from homologous or heterologous challenge, but resulted in enhanced

  8. Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae: effects of insert position and host background

    DEFF Research Database (Denmark)

    Stentebjerg-Olesen, B; Pallesen, L; Jensen, LB;

    1997-01-01

    with respect to host background in three different Escherichia coli strains, i.e. an isogenic set of K-12 strains, differing in the presence of an indigenous fim gene cluster, as well as a wild-type isolate. Immunization of rabbits with purified chimeric fimbriae resulted in serum which specifically recognized......The potential of the major structural protein of type 1 fimbriae as a display system for heterologous sequences was tested. As a reporter-epitope, a heterologous sequence mimicking a neutralizing epitope of the cholera toxin B chain was inserted, in one or two copies, into four different positions....... 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...

  9. Characteristics of macrophages in irradiation chimeras in mice reconstituted with allogeneic bone marrow cells

    International Nuclear Information System (INIS)

    Biological and immunological characteristics of the reticuloendothelial system of irradiation bone marrow chimeric mice and macrophages collected from various tissue sources of the mice were studied. The chimeras showed comparable activities in carbon clearance to those of normal donor or recipient mice. The macrophages from spleen, lymph node, bone marrow, peripheral blood, liver, peritoneal cavity, and lung were demonstrated to be of donor marrow origin. They showed almost the same enzyme activities and phagocytic capability of sheep erythrocytes (SRBC, E), SRBC sensitized with anti-SRBC IgG (EA), and SRBC sensitized with anti-SRBC IgM and coated with complement (EAC) as those of normal mice. Proportions of Fc receptor and complement receptor-positive cells are also in normal range. In addition, the antigen-presenting capability of the chimeric macrophages for in vitro primary antibody response to SRBC was intact. These observations suggest that the reticuloendothelial system and macrophages of allogeneic bone marrow chimeras where donor and recipient differ at the major histocompatibility complex have no defect so far as could be ascertained by the present study

  10. Pathogenesis of Lassa Fever Virus Infection: I. Susceptibility of Mice to Recombinant Lassa Gp/LCMV Chimeric Virus

    OpenAIRE

    Lee, Andrew M.; Cruite, Justin; Welch, Megan J.; Sullivan, Brian; Oldstone, Michael B. A.

    2013-01-01

    Lassa virus (LASV) is a BSL-4 restricted agent. To allow study of infection by LASV under BSL-2 conditions, we generated a recombinant virus in which the LASV glycoprotein (Gp) was placed on the backbone of lymphocytic choriomeningitis virus (LCMV) Cl13 nucleoprotein, Z and polymerase genes (rLCMV Cl13/LASV Gp). The recombinant virus displayed high tropism for dendritic cells following in vitro or in vivo infection. Inoculation of immunocompetent adults resulted in an acute infection, generat...

  11. Hepatitis E virus genotype three infection of human liver chimeric mice as a model for chronic HEV infection

    NARCIS (Netherlands)

    M.D.B. van de Garde (Martijn); S.D. Pas (Suzan); G. van der Net (Guido); R.A. de Man (Robert); A.D.M.E. Osterhaus (Albert); B.L. Haagmans (Bart); A. Boonstra (Andre); T. Vanwolleghem (Thomas)

    2016-01-01

    textabstractGenotype (gt) 3 hepatitis E virus (HEV) infections are emerging in Western countries. Immunosuppressed patients are at risk of chronic HEV infection and progressive liver damage, but no adequate model system currently mimics this disease course. Here we explore the possibilities of in vi

  12. Evaluation of cellular responses for a chimeric HBsAg-HCV core DNA vaccine in BALB/c mice

    Directory of Open Access Journals (Sweden)

    Maryam Yazdanian

    2015-01-01

    Conclusion: Fusion of HBsAg to HCVcp in the context of a DNA vaccine modality could augment Th1-oriented cellular and CTL responses toward a protective epitope, comparable to that of HCVcp (subunit HCV vaccine immunization.

  13. 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

  14. Application of Humanized Mice in Drug Metabolism and Toxicity%人源化小鼠模型在药物代谢和毒性研究中的应用

    Institute of Scientific and Technical Information of China (English)

    武之涛; 任进; 潘国宇

    2013-01-01

    OBJECTIVE To discuss the application of humanized mice model in drug metabolism and toxicity.METHODS By sorting foreign scientific literature,the history of the development of humanized mice model and its application in drug metabolizing enzymes and toxicity studies were reviewed and summarized to provide support for early drug development.RESULTS AND CONCLUSION The humanized mice model could reduce interspecies differences and has been increasingly used in drug metabolism and safety evaluation in foreign countries.Transgenic mice can be applied to studying the metabolism of drugs metabolized by certain enzyme.In the chimeric mice body,the disposal of drugs could be more systematic and comprehensive mirrored,predicting this model has enormous potential in drug discovery and development.%目的 阐述人源化小鼠模型在药物代谢和毒性研究中的应用.方法 通过整理国外相关科技文献,对人源化小鼠模型的发展历史和在药物代谢酶与药物毒性研究上的应用进行回顾、总结,为早期药物的研发提供支持.结果与结论 人源化小鼠模型可以克服种属差异,在国外已被逐渐用于药物代谢研究和药物安全性评价.转基因小鼠可以专一的用于研究某个药物代谢酶对药物的代谢.嵌合小鼠可以更系统和全面地反映药物在体内的处置,在药物的发现和开发中具有较大应用潜能.

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

    Institute of Scientific and Technical Information of China (English)

    Huang Qiman; Yang Qunying; Elisabeth Martinez; Guo Sandui

    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.

  16. Inter-specific coral chimerism: genetically distinct multicellular structures associated with tissue loss in Montipora capitata.

    Directory of Open Access Journals (Sweden)

    Thierry M Work

    Full Text Available Montipora white syndrome (MWS results in tissue-loss that is often lethal to Montipora capitata, a major reef building coral that is abundant and dominant in the Hawai'ian Archipelago. Within some MWS-affected colonies in Kane'ohe Bay, Oahu, Hawai'i, we saw unusual motile multicellular structures within gastrovascular canals (hereafter referred to as invasive gastrovascular multicellular structure-IGMS that were associated with thinning and fragmentation of the basal body wall. IGMS were in significantly greater densities in coral fragments manifesting tissue-loss compared to paired normal fragments. Mesenterial filaments from these colonies yielded typical M. capitata mitochondrial haplotypes (CO1, CR, while IGMS from the same colony consistently yielded distinct haplotypes previously only found in a different Montipora species (Montipora flabellata. Protein profiles showed consistent differences between paired mesenterial filaments and IGMS from the same colonies as did seven microsatellite loci that also exhibited an excess of alleles per locus inconsistent with a single diploid organism. We hypothesize that IGMS are a parasitic cellular lineage resulting from the chimeric fusion between M. capitata and M. flabellata larvae followed by morphological reabsorption of M. flabellata and subsequent formation of cell-lineage parasites. We term this disease Montiporaiasis. Although intra-specific chimerism is common in colonial animals, this is the first suspected inter-specific example and the first associated with tissue loss.

  17. Report of a chimeric origin of transposable elements in a bovine-coding gene.

    Science.gov (United States)

    Almeida, L M; Amaral, M E J; Silva, I T; Silva, W A; Riggs, P K; Carareto, C M

    2008-02-01

    Despite the wide distribution of transposable elements (TEs) in mammalian genomes, part of their evolutionary significance remains to be discovered. Today there is a substantial amount of evidence showing that TEs are involved in the generation of new exons in different species. In the present study, we searched 22,805 genes and reported the occurrence of TE-cassettes in coding sequences of 542 cow genes using the RepeatMasker program. Despite the significant number (542) of genes with TE insertions in exons only 14 (2.6%) of them were translated into protein, which we characterized as chimeric genes. From these chimeric genes, only the FAST kinase domains 3 (FASTKD3) gene, present on chromosome BTA 20, is a functional gene and showed evidence of the exaptation event. The genome sequence analysis showed that the last exon coding sequence of bovine FASTKD3 is approximately 85% similar to the ART2A retrotransposon sequence. In addition, comparison among FASTKD3 proteins shows that the last exon is very divergent from those of Homo sapiens, Pan troglodytes and Canis familiares. We suggest that the gene structure of bovine FASTKD3 gene could have originated by several ectopic recombinations between TE copies. Additionally, the absence of TE sequences in all other species analyzed suggests that the TE insertion is clade-specific, mainly in the ruminant lineage.

  18. 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.

  19. 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

  20. 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.

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

    Science.gov (United States)

    Zhao, T S; Xia, Y H

    2016-06-01

    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 E(rns) 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

  2. 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.

  3. The chimeric eukaryote: origin of the nucleus from the karyomastigont in amitochondriate protists

    Science.gov (United States)

    Margulis, L.; Dolan, M. F.; Guerrero, R.

    2000-01-01

    We present a testable model for the origin of the nucleus, the membrane-bounded organelle that defines eukaryotes. A chimeric cell evolved via symbiogenesis by syntrophic merger between an archaebacterium and a eubacterium. The archaebacterium, a thermoacidophil resembling extant Thermoplasma, generated hydrogen sulfide to protect the eubacterium, a heterotrophic swimmer comparable to Spirochaeta or Hollandina that oxidized sulfide to sulfur. Selection pressure for speed swimming and oxygen avoidance led to an ancient analogue of the extant cosmopolitan bacterial consortium "Thiodendron latens." By eubacterial-archaebacterial genetic integration, the chimera, an amitochondriate heterotroph, evolved. This "earliest branching protist" that formed by permanent DNA recombination generated the nucleus as a component of the karyomastigont, an intracellular complex that assured genetic continuity of the former symbionts. The karyomastigont organellar system, common in extant amitochondriate protists as well as in presumed mitochondriate ancestors, minimally consists of a single nucleus, a single kinetosome and their protein connector. As predecessor of standard mitosis, the karyomastigont preceded free (unattached) nuclei. The nucleus evolved in karyomastigont ancestors by detachment at least five times (archamoebae, calonymphids, chlorophyte green algae, ciliates, foraminifera). This specific model of syntrophic chimeric fusion can be proved by sequence comparison of functional domains of motility proteins isolated from candidate taxa.

  4. Engraftment of bone marrow-derived cells after nonlethal radiation in syngeneic C57BL/6mice%Engraftment of bone marrow-derived cells after nonlethal radiation in syngeneic C57BL/6 mice

    Institute of Scientific and Technical Information of China (English)

    Wu Liao; Tan Li; Wang Yu; Liu Dengqun; Shi Chunmeng

    2015-01-01

    Objective To study the characteristics of cell engraftment in mice at a lower dose under nonlethal radiated condition.Methods A syngeneic C57BL/6 mouse model,transplanted with 1 × 107 bone marrow cells and exposed to 2.5 Gy whole body irradiation (WBI),was selected to study the chimerism of cells from green fluorescent protein positive (GFP +) transgenic mice.The control group was injected with GFP + cells without receiving irradiation.In addition,an allogenic transplantation model of BALB/c mice was also investigated which was infused by GFP + cells from C57BL/6 mice.The engraftment of bone marrow-derived cells (BMDCs) was detected by immunohistochemistry in bone marrow,liver,lung,small intestine and spleen.Results The transplanted bone marrow cells successfully grafted in the haematopoietic tissues from syngeneic GFP transgenic mice.The transplanted GFP+ cells were also detected in the non-haematopoietic tissues,such as the small intestine,liver,spleen and lung,after irradiation.However,a lethal dose irradiation of 8 Gy was required to establish successful chimerism in allogeneic transplantation model by infusing the bone marrow cells from C57BL/6 mice to BALB/c mice.Conclusions Bone marrow-derived cells can be successfully grafted into various recipient tissues receiving a 2.5 Gy dose of radiation in syngeneic mice,but not in allogeneic mice.This nonlethal model may help to further study the plasticity and mechanism of bone marrow-derived cells in tissue repair and regeneration after radiation injury.

  5. 78 FR 70955 - Prospective Grant of Exclusive Patent License: GMCSF-BclxL-Derived Chimeric Therapeutics for Use...

    Science.gov (United States)

    2013-11-27

    ...- Derived Chimeric Therapeutics for Use in Treatment of Cancer, Neutropenia, CNS Injury and Parkinson's.... Receptors for GMCSF are found on a ] variety of normal tissues, including hematopoietic stem cells, neurons... also be used in patients receiving stem cell transplantation or in ex vivo expansion of...

  6. 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...

  7. 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...

  8. Fiber-chimeric adenoviruses expressing fibers from serotype 16 and 50 improve gene transfer to human pancreatic adenocarcinoma

    NARCIS (Netherlands)

    Kuhlmann, K.F.D.; Geer, M.A. van; Bakker, C.T.; Dekker, J.E.M.; Havenga, M.J.E.; Oude Elferink, R.P.J.; Gouma, D.J.; Bosma, P.J.; Wesseling, J.G.

    2009-01-01

    Survival of patients with pancreatic cancer is poor. Adenoviral (Ad) gene therapy employing the commonly used serotype 5 reveals limited transduction efficiency due to the low amount of coxsackie-adenovirus receptor on pancreatic cancer cells. To identify fiber-chimeric adenoviruses with improved ge

  9. 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

  10. 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.

  11. Hypothesis: Artifacts, Including Spurious Chimeric RNAs with a Short Homologous Sequence, Caused by Consecutive Reverse Transcriptions and Endogenous Random Primers.

    Science.gov (United States)

    Peng, Zhiyu; Yuan, Chengfu; Zellmer, Lucas; Liu, Siqi; Xu, Ningzhi; Liao, D Joshua

    2015-01-01

    Recent RNA-sequencing technology and associated bioinformatics have led to identification of tens of thousands of putative human chimeric RNAs, i.e. RNAs containing sequences from two different genes, most of which are derived from neighboring genes on the same chromosome. In this essay, we redefine "two neighboring genes" as those producing individual transcripts, and point out two known mechanisms for chimeric RNA formation, i.e. transcription from a fusion gene or trans-splicing of two RNAs. By our definition, most putative RNA chimeras derived from canonically-defined neighboring genes may either be technical artifacts or be cis-splicing products of 5'- or 3'-extended RNA of either partner that is redefined herein as an unannotated gene, whereas trans-splicing events are rare in human cells. Therefore, most authentic chimeric RNAs result from fusion genes, about 1,000 of which have been identified hitherto. We propose a hypothesis of "consecutive reverse transcriptions (RTs)", i.e. another RT reaction following the previous one, for how most spurious chimeric RNAs, especially those containing a short homologous sequence, may be generated during RT, especially in RNA-sequencing wherein RNAs are fragmented. We also point out that RNA samples contain numerous RNA and DNA shreds that can serve as endogenous random primers for RT and ensuing polymerase chain reactions (PCR), creating artifacts in RT-PCR.

  12. 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

  13. Immunization of neonatal mice with LAMP/p55 HIV gag DNA elicits robust immune responses that last to adulthood

    International Nuclear Information System (INIS)

    Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses, as measured by the breadth of the Gag peptide-specific IFN-γ, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric LAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory.

  14. Cassava is not a goitrogen in mice

    International Nuclear Information System (INIS)

    To examine the effect of cassava on the thyroid function of mice, the authors fed fresh cassava root to mice and compared this diet with low iodine diet and Purina. Cassava provided a low iodine intake and increased urine thiocyanate excretion and serum thiocyanate levels. Mice on cassava lost weight. The thyroid glands of mice on cassava were not enlarged, even when normalized for body weight. The 4- and 24-hr thyroid uptakes of mice on cassava were similar to those of mice on low iodine diets. Protein-bound [125I]iodine at 24 hr was high in mice on either the cassava or low iodine diets. The thyroid iodide trap (T/M) was similar in mice on cassava and low iodine diets. When thiocyanate was added in vitro to the incubation medium, T/M was reduced in all groups of mice; under these conditions, thiocyanate caused a dose-related inhibition of T/M. The serum thyroxine (T4) and triiodothyronine (T3) concentrations of mice on cassava were reduced compared with mice on Purina diet. Thyroid T4 and T3 contents of mice on cassava were relatively low compared with mice on Purina diet. Hepatic T3 content and T4 5'-monodeiodination in liver homogenates were reduced in mice on cassava compared with other groups. The data show that cassava does not cause goiter in mice. The thiocyanate formed from ingestation of cassava is insufficient to inhibit thyroid iodide transport or organification of iodide. The cassava diet leads to rapid turnover of hormonal iodine because it is a low iodine diet. It also impairs 5'-monodeiodination of T4 which may be related to nutritional deficiency. These data in mice do not support the concept that cassava per se has goitrogenic action in man

  15. Germ line transcription in mice bearing neor gene downstream of Igamma3 exon in the Ig heavy chain locus.

    Science.gov (United States)

    Samara, Maha; Oruc, Zeliha; Dougier, Hei-Lanne; Essawi, Tamer; Cogné, Michel; Khamlichi, Ahmed Amine

    2006-04-01

    Class switch recombination (CSR) is preceded by germ line transcription that initiates from promoters upstream of switch (S) sequences and terminates downstream of associated constant genes. Previous work showed that germ line transcripts and their processing are required for CSR and that germ line transcription is regulated in a major part by a regulatory region located downstream of the Ig heavy chain locus. This long-range, polarized effect can be disturbed by inserting an expressed neomycine resistance (neo(r)) gene. To contribute to a better understanding of the mechanism of such a long-distance regulation, we generated knock-in mice in which a neo(r) gene was inserted downstream of Igamma3 exon leaving intact all the necessary elements for germ line transcription and splicing. We show that the expressed neo(r) gene interferes with transcription initiation from Igamma3, and that it impairs but does not block S recombination to Cgamma3. Moreover, we show for the first time that the neo(r) gene provides through chimeric neo(r)-Cgamma3 transcripts the necessary elements for splicing of germ line transcripts by activating two novel cryptic splice sites, one in the coding region of the intronless neo(r) gene and the other in the Igamma3-Cgamma3 intron.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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'

  1. 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.

  2. 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.

  3. 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.``

  4. Self-assembling chimeric polypeptide-doxorubicin conjugate nanoparticles that abolish tumours after a single injection

    Science.gov (United States)

    Andrew Mackay, J.; Chen, Mingnan; McDaniel, Jonathan R.; Liu, Wenge; Simnick, Andrew J.; Chilkoti, Ashutosh

    2009-12-01

    New strategies to self-assemble biocompatible materials into nanoscale, drug-loaded packages with improved therapeutic efficacy are needed for nanomedicine. To address this need, we developed artificial recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into sub-100-nm-sized, near-monodisperse nanoparticles on conjugation of diverse hydrophobic molecules, including chemotherapeutics. These CPs consist of a biodegradable polypeptide that is attached to a short Cys-rich segment. Covalent modification of the Cys residues with a structurally diverse set of hydrophobic small molecules, including chemotherapeutics, leads to spontaneous formation of nanoparticles over a range of CP compositions and molecular weights. When used to deliver chemotherapeutics to a murine cancer model, CP nanoparticles have a fourfold higher maximum tolerated dose than free drug, and induce nearly complete tumour regression after a single dose. This simple strategy can promote co-assembly of drugs, imaging agents and targeting moieties into multifunctional nanomedicines.

  5. 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...... of rituximab. The large majority of patients also received Pred and, in some cases, other concomitant immunosuppressive treatment during part of their rituximab treatment. A complete response (CR) was defined as a rise in the platelet count > 100 x 10(9)/L, a partial response (PR) as a rise in the platelet...

  6. Chimeric Antigen Receptor T Cell (Car T Cell Therapy In Hematology

    Directory of Open Access Journals (Sweden)

    Pinar Ataca

    2015-12-01

    Full Text Available It is well demonstrated that immune system can control and eliminate cancer cells. Immune-mediated elimination of tumor cells has been discovered and is the basis of both cancer vaccines and cellular therapies including hematopoietic stem cell transplantation (HSCT. Adoptive T cell transfer has been improved to be more specific and potent and cause less off-target toxicities. Currently, there are two forms of engineered T cells being tested in clinical trials: T cell receptor (TCR and chimeric antigen receptor (CAR modified T cells. On July 1, 2014, the United States Food and Drug Administration granted ‘breakthrough therapy’ designation to anti-CD19 CAR T cell therapy. Many studies were conducted to evaluate the beneficiaries of this exciting and potent new treatment modality. This review summarizes the history of adoptive immunotherapy, adoptive immunotherapy using CARs, the CAR manufacturing process, preclinical-clinical studies, effectiveness and drawbacks of this strategy.

  7. [Research of Human-mouse Chimeric Antibodies Against Ebola Virus Nucleoprotein].

    Science.gov (United States)

    Zhou, Rongping; Sun, Lina; Liu, Yang; Wu, Wei; Li, Chuan; Liang, Mifang; Qiu, Peihong

    2016-01-01

    The Ebola virus is highly infectious and can result in death in ≤ 90% of infected subjects. Detection of the Ebola virus and diagnosis of infection are extremely important for epidemic control. Presently, Chinese laboratories detect the nucleic acids of the Ebola virus by real-time reverse transcription-polymerase chain reaction (RT-PCR). However, such detection takes a relatively long time and necessitates skilled personnel and expensive equipment. Enzyme-linked immunosorbent assay (ELISA) of serum is simple, easy to operate, and can be used to ascertain if a patient is infected with the Ebola virus as well as the degree of infection. Hence, ELISA can be used in epidemiological investigations and is a strong complement to detection of nucleic acids. Cases of Ebola hemorrhagic fever have not been documented in China, so quality-control material for positive serology is needed. Construction and expression of human-mouse chimeric antibodies against the nucleoprotein of the Ebola virus was carried out. Genes encoding variable heavy (VH) and variable light (VL) chains were extracted and amplified from murine hybridoma cells. Genes encoding the VH and VL chains of monoclonal antibodies were amplified by RT-PCR. According to sequence analyses, a primer was designed to amplify functional sequences relative to VH and VL chain. The eukaryotic expression vector HL51-14 carrying some human antibody heavy chain- and light chain-constant regions was used. IgG antibodies were obtained by transient transfection of 293T cells. Subsequently, immunological detection and immunological identification were identified by ELISA, immunofluorescence assay, and western blotting. These results showed that we constructed and purified two human- mouse chimeric antibodies.

  8. 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

  9. Solitary fibrous tumors: loss of chimeric protein expression and genomic instability mark dedifferentiation.

    Science.gov (United States)

    Dagrada, Gian P; Spagnuolo, Rosalin D; Mauro, Valentina; Tamborini, Elena; Cesana, Luca; Gronchi, Alessandro; Stacchiotti, Silvia; Pierotti, Marco A; Negri, Tiziana; Pilotti, Silvana

    2015-08-01

    Solitary fibrous tumors, which are characterized by their broad morphological spectrum and unpredictable behavior, are rare mesenchymal neoplasias that are currently divided into three main variants that have the NAB2-STAT6 gene fusion as their unifying molecular lesion: usual, malignant and dedifferentiated solitary fibrous tumors. The aims of this study were to validate molecular and immunohistochemical/biochemical approaches to diagnose the range of solitary fibrous tumors by focusing on the dedifferentiated variant, and to reveal the genetic events associated with dedifferentiation by integrating the findings of array comparative genomic hybridization. We studied 29 usual, malignant and dedifferentiated solitary fibrous tumors from 24 patients (including paired samples from five patients whose tumors progressed to the dedifferentiated form) by means of STAT6 immunohistochemistry and (when frozen material was available) reverse-transcriptase polymerase chain reaction and biochemistry. In addition, the array comparative genomic hybridization findings were used to profile 12 tumors from nine patients. The NAB2/STAT6 fusion was detected in all of the tumors, but immunohistochemistry and western blotting indicated that chimeric protein expression was atypical or absent in 9 out of 11 dedifferentiated tumors. The comparative genomic hybridization results revealed that the usual and malignant solitary fibrous tumors had a simple profile, whereas the genome of the dedifferentiated tumors was complex and unstable, and suggested that 13q and 17p deletions and TP53 mutations may be present in malignant lesions before the full expression of a dedifferentiated phenotype. Solitary fibrous tumor dedifferentiation is associated with the loss of chimeric oncoprotein expression, genomic instability, and cell decommitment and reprogramming. The assessment of dedifferentiated solitary fibrous tumors is based on the presence of the fusion transcripts and, in principle, negative

  10. 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.

  11. 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.

  12. Therapy Provider Phase Information

    Data.gov (United States)

    U.S. Department of Health & Human Services — The Therapy Provider Phase Information dataset is a tool for providers to search by their National Provider Identifier (NPI) number to determine their phase for...

  13. Psychopharmacological Studies in Mice.

    Science.gov (United States)

    Matsuda, Toshio

    2016-01-01

    Since 1998, when the laboratory of Medicinal Pharmacology was established in the Graduate School of Pharmaceutical Sciences, Osaka University, I have been interested in psychopharmacological research topics. During this period, we identified a number of novel regulatory mechanisms that control the prefrontal dopamine system through functional interaction between serotonin1A and dopamine D2 receptors or between serotonin1A and σ1 receptors. Our findings suggest that strategies that enhance the prefrontal dopamine system may have therapeutic potential in the treatment of psychiatric disorders. We also found that environmental factors during development strongly impact the psychological state in adulthood. Furthermore, we clarified the pharmacological profiles of the acetylcholinesterase inhibitors donepezil, galantamine, and rivastigmine, providing novel insights into their mechanisms of action. Finally, we developed the female encounter test, a novel method for evaluating motivation in mice. This simple method should help advance future psychopharmacological research. In this review, we summarize the major findings obtained from our recent studies in mice.

  14. Linkage disequilibrium in wild mice.

    Directory of Open Access Journals (Sweden)

    Cathy C Laurie

    2007-08-01

    Full Text Available Crosses between laboratory strains of mice provide a powerful way of detecting quantitative trait loci for complex traits related to human disease. Hundreds of these loci have been detected, but only a small number of the underlying causative genes have been identified. The main difficulty is the extensive linkage disequilibrium (LD in intercross progeny and the slow process of fine-scale mapping by traditional methods. Recently, new approaches have been introduced, such as association studies with inbred lines and multigenerational crosses. These approaches are very useful for interval reduction, but generally do not provide single-gene resolution because of strong LD extending over one to several megabases. Here, we investigate the genetic structure of a natural population of mice in Arizona to determine its suitability for fine-scale LD mapping and association studies. There are three main findings: (1 Arizona mice have a high level of genetic variation, which includes a large fraction of the sequence variation present in classical strains of laboratory mice; (2 they show clear evidence of local inbreeding but appear to lack stable population structure across the study area; and (3 LD decays with distance at a rate similar to human populations, which is considerably more rapid than in laboratory populations of mice. Strong associations in Arizona mice are limited primarily to markers less than 100 kb apart, which provides the possibility of fine-scale association mapping at the level of one or a few genes. Although other considerations, such as sample size requirements and marker discovery, are serious issues in the implementation of association studies, the genetic variation and LD results indicate that wild mice could provide a useful tool for identifying genes that cause variation in complex traits.

  15. Identification of Immunodominant Responses to the Plasmodium falciparum Antigens PfUIS3, PfLSA1 and PfLSAP2 in Multiple Strains of Mice.

    Directory of Open Access Journals (Sweden)

    Rhea J Longley

    Full Text Available Malaria, caused by the Plasmodium parasite, remains a serious global public health concern. A vaccine could have a substantial impact on eliminating this disease, alongside other preventative measures. We recently described the development of three novel, viral vectored vaccines expressing either of the antigens PfUIS3, PfLSA1 and PfLSAP2. Each vaccination regimen provided high levels of protection against chimeric parasite challenge in a mouse model, largely dependent on CD8+ T cells. In this study we aimed to further characterize the induced cellular immune response to these vaccines. We utilized both the IFNγ enzyme-linked immunosorbent spot assay and intracellular cytokine staining to achieve this aim. We identified immunodominant peptide responses for CD4+ and CD8+ T cells for each of the antigens in BALB/c, C57BL/6 and HLA-A2 transgenic mice, creating a useful tool for researchers for subsequent study of these antigens. We also compared these immunodominant peptides with those generated from epitope prediction software, and found that only a small proportion of the large number of epitopes predicted by the software were identifiable experimentally. Furthermore, we characterized the polyfunctionality of the induced CD8+ T cell responses. These findings contribute to our understanding of the immunological mechanisms underlying these protective vaccines, and provide a useful basis for the assessment of these and related vaccines as clinical constructs.

  16. Provide, provide: the economics of aging.

    Science.gov (United States)

    1998-06-26

    Most older persons face two potentially serious economic problems: (a) declining earning power and (b) declining health that can be partly offset by increased utilization of health care. The decline in earning power is largely attributable to physiological changes and to obsolescence of skills and knowledge. These adverse effects are exacerbated by public and private policies that reduce the incentives of older persons to continue work and increase the cost to employers of employing older workers. The problems of earnings replacement and health care payment are usually discussed separately, but there are several reasons why they should be considered together. First, there are often tradeoffs between the two. Money is money, and for most people there is never enough to go around. This is self-evident where private funds are concerned. Low-income elderly, for instance, frequently must choose between expensive prescription drugs and an adequate diet. For middle-income elderly, the choice may be between saving on medigap insurance or forgoing an airplane trip to a grandchild's graduation. Difficult choices are also inherent in the allocation of public funds. The same tax receipts that could be used to maintain or increase retirement benefits could be used to fund additional care, and vice versa. In discussing these tradeoffs, some analysts assert that people will gladly give up other goods and services for medical care that cures illness, relieves pain, or restores function. Others believe that some people would forgo some health insurance in order to maintain access to other goods and services. A second reason for looking at the two problems together is that they pose similar questions for public policy. How much should each generation provide for its own needs in old age, and how much should be provided by the generations that follow? How much provision should be voluntary, how much compulsory? How much intra-generational redistribution is appropriate after age 65

  17. Maternal Microchimerism Leads to the Presence of Interleukin-2 in Interleukin-2 Knock Out Mice: Implications for the Role of Interleukin-2 in Thymic Function

    OpenAIRE

    Wrenshall, Lucile E.; Stevens, Elliot T.; Smith, Deandra R.; Miller, John D.

    2007-01-01

    The role of interleukin-2 (IL-2) in thymic development is uncertain. Not surprisingly, IL-2 knockout (KO) mice have been used to address this question. However, as we report here, such mice are chimeric, containing both IL-2 KO cells and IL-2-expressing cells transferred in utero from their heterozygous mothers. These cells produce IL-2 in amounts detectable by conventional means, and their presence in lymphoid tissues confounds efforts to define the true IL-2 KO phenotype. To minimize the am...

  18. The neurovirulence and neuroinvasiveness of chimeric tick-borne encephalitis/dengue virus can be attenuated by introducing defined mutations into the envelope and NS5 protein genes and the 3' non-coding region of the genome

    International Nuclear Information System (INIS)

    Tick-borne encephalitis (TBE) is a severe disease affecting thousands of people throughout Eurasia. Despite the use of formalin-inactivated vaccines in endemic areas, an increasing incidence of TBE emphasizes the need for an alternative vaccine that will induce a more durable immunity against TBE virus (TBEV). The chimeric attenuated virus vaccine candidate containing the structural protein genes of TBEV on a dengue virus genetic background (TBEV/DEN4) retains a high level of neurovirulence in both mice and monkeys. Therefore, attenuating mutations were introduced into the envelope (E315) and NS5 (NS5654,655) proteins, and into the 3' non-coding region (Δ30) of TBEV/DEN4. The variant that contained all three mutations (vΔ30/E315/NS5654,655) was significantly attenuated for neuroinvasiveness and neurovirulence and displayed a reduced level of replication and virus-induced histopathology in the brains of mice. The high level of safety in the central nervous system indicates that vΔ30/E315/NS5654,655 should be further evaluated as a TBEV vaccine.

  19. Specific tumor labeling enhanced by polyethylene glycol linkage of near infrared dyes conjugated to a chimeric anti-carcinoembryonic antigen antibody in a nude mouse model of human pancreatic cancer

    Science.gov (United States)

    Maawy, Ali A.; Hiroshima, Yukihiko; Zhang, Yong; Luiken, George A.; Hoffman, Robert M.; Bouvet, Michael

    2014-10-01

    Labeling of metastatic tumors can aid in their staging and resection of cancer. Near infrared (NIR) dyes have been used in the clinic for tumor labeling. However, there can be a nonspecific uptake of dye by the liver, lungs, and lymph nodes, which hinders detection of metastasis. In order to overcome these problems, we have used two NIR dyes (DyLight 650 and 750) conjugated to a chimeric anti-carcinoembryonic antigen antibody to evaluate how polyethylene glycol linkage (PEGylation) can improve specific tumor labeling in a nude mouse model of human pancreatic cancer. The conjugated PEGylated and non-PEGylated DyLight 650 and 750 dyes were injected intravenously into non-tumor-bearing nude mice. Serum samples were collected at various time points in order to determine serum concentrations and elimination kinetics. Conjugated PEGylated dyes had significantly higher serum dye concentrations than non-PEGylated dyes (p=0.005 for the 650 dyes and pdyes). Human pancreatic tumors subcutaneously implanted into nude mice were labeled with antibody-dye conjugates and serially imaged. Labeling with conjugated PEGylated dyes resulted in significantly brighter tumors compared to the non-PEGylated dyes (pdyes; p=0.01 for 750 dyes). PEGylation of the NIR dyes also decreased their accumulation in lymph nodes, liver, and lung. These results demonstrate enhanced selective tumor labeling by PEGylation of dyes conjugated to a tumor-specific antibody, suggesting their future clinical use in fluorescence-guided surgery.

  20. A new Toxoplasma gondii chimeric antigen containing fragments of SAG2, GRA1, and ROP1 proteins-impact of immunodominant sequences size on its diagnostic usefulness.

    Science.gov (United States)

    Ferra, Bartłomiej; Holec-Gąsior, Lucyna; Kur, Józef

    2015-09-01

    This study presents the first evaluation of new Toxoplasma gondii recombinant chimeric antigens containing three immunodominant regions of SAG2, GRA1, and one of two ROP1 fragments differing in length for the serodiagnosis of human toxoplasmosis. The recombinant chimeric antigens SAG2-GRA1-ROP1L (with large fragment of ROP1, 85-396 amino acid residues) and SAG2-GRA1-ROP1S (with a small fragment of ROP1, 85-250 amino acid residues) were obtained as fusion proteins containing His6-tags at both ends using an Escherichia coli expression system. The diagnostic utility of these chimeric antigens was determined using the enzyme-linked immunosorbent assay (ELISA) for the detection of specific anti-T. gondii immunoglobulin G (IgG). The IgG ELISA results obtained for the chimeric antigens were compared to those obtained for the use of Toxoplasma lysate antigen (TLA) and for a mixture of recombinant antigens containing rSAG2, rGRA1, and rROP1. The sensitivity of the IgG ELISA was similar for the SAG2-GRA1-ROP1L chimeric antigen (100 %), the mixture of three proteins (99.4 %) and the TLA (97.1 %), whereas the sensitivity of IgG ELISA with the SAG2-GRA1-ROP1S chimeric antigen was definitely lower, reaching 88.4 %. In conclusion, this study shows that SAG2-GRA1-ROP1L chimeric antigen can be useful for serodiagnosis of human toxoplasmosis with the use of the IgG ELISA assay. Therefore, the importance of proper selection of protein fragments for the construction of chimeric antigen with the highest reactivity in ELISA test is demonstrated.

  1. Characterization and Functional Analysis of scFv-based Chimeric Antigen Receptors to Redirect T Cells to IL13Rα2-positive Glioma.

    Science.gov (United States)

    Krenciute, Giedre; Krebs, Simone; Torres, David; Wu, Meng-Fen; Liu, Hao; Dotti, Gianpietro; Li, Xiao-Nan; Lesniak, Maciej S; Balyasnikova, Irina V; Gottschalk, Stephen

    2016-02-01

    Immunotherapy with T cells expressing chimeric antigen receptors (CARs) is an attractive approach to improve outcomes for patients with glioblastoma (GBM). IL13Rα2 is expressed at a high frequency in GBM but not in normal brain, making it a promising CAR T-cell therapy target. IL13Rα2-specific CARs generated up to date contain mutated forms of IL13 as an antigen-binding domain. While these CARs target IL13Rα2, they also recognize IL13Rα1, which is broadly expressed. To overcome this limitation, we constructed a panel of IL13Rα2-specific CARs that contain the IL13Rα2-specific single-chain variable fragment (scFv) 47 as an antigen binding domain, short or long spacer regions, a transmembrane domain, and endodomains derived from costimulatory molecules and CD3.ζ (IL13Rα2-CARs). IL13Rα2-CAR T cells recognized IL13Rα2-positive target cells in coculture and cytotoxicity assays with no cross-reactivity to IL13Rα1. However, only IL13Rα2-CAR T cells with a short spacer region produced IL2 in an antigen-dependent fashion. In vivo, T cells expressing IL13Rα2-CARs with short spacer regions and CD28.ζ, 41BB.ζ, and CD28.OX40.ζ endodomains had potent anti-glioma activity conferring a significant survival advantage in comparison to mice that received control T cells. Thus, IL13Rα2-CAR T cells hold the promise to improve current IL13Rα2-targeted immunotherapy approaches for GBM and other IL13Rα2-positive malignancies. PMID:26514825

  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. Human Prolactin Improves Engraftment and Reconstitution of Human Peripheral Blood Lymphocytes in SCID Mice

    Institute of Scientific and Technical Information of China (English)

    Rui Sun; Jian Zhang; Cai Zhang; Jianhua Zhang; Shujuan Liang; Anyuan Sun; Junfu Wang; Zhigang Tian

    2004-01-01

    Recombinant human prolactin (rhPRL) was administered to huPBL-SCID mice to determine its effects on human immunologic reconsfitution and function. The huPBL-SCID mice were given 10 μg I.p. Injection of rhPRL every other day for a total of 10 injections after huPBL were transferred. The results demonstrated that rhPRL improved the engraftment of lymphocytes into thymus, lymph nodes and spleens, showing that the cellularities of these organs increased although the cellularities tended to vary depending on the donor. The amounts of human T cells (HLA-ABC+/CD3+) increased greatly in thymus (14.2 folds), spleen (4.16 folds) and lymph nodes (40.18 folds) after rhPRL injections. The amounts of human B cells (HLA-ABC+/CD19+) also increased greatly in lymph nodes (42.5 folds) and spleen (5.78 folds). The lymph node cells from the rhPRL-treated huPBL-SCID mice were more sensitive to PHA stimulation ([3H] thymidine incorporation). The supernatant of PHA-stimulated PBL from rhPRL-treated huPBL/SCID chimerism contained more cytokines (IFN-γ and IL-2). The natural cytotoxicity against human sensitive target cells, K562 cells, from spleen and bone marrow of hPBL/SCID chimerism was significantly enhanced by rhPRL administration. The lymph node cells were stimulated with LPS in vitro for 3 days and the lymphocytes from the rhPRL-treated huPBL-SCID mice were more sensitive to mitogen stimulation. Both serum total IgG level and IgM level of rhPRL-treated huPBL/SCID chimerism were increased, and even without DT-rechallenge the base line of DT-specific IgG was elevated after rhPRL treatment in huPBL-SCID mice. Thus, rhPRL stimulation promotes reconstitution of human immune system in huPBL-SCID mice.

  4. Human Prolactin Improves Engraftment and Reconstitution of Human Peripheral Blood Lymphocytes in SCID Mice

    Institute of Scientific and Technical Information of China (English)

    RuiSun; JianZhang; CaiZhang; JianhuaZhang; ShujuanLiang; AnyuanSun; JunfuWang; ZhigangTian

    2004-01-01

    Recombinant human prolactin (rhPRL) was administered to huPBL-SCID mice to determine its effects on human immunologic reconstitution and function. The huPBL-SCID mice were given 10 μg i.p. injection of rhPRL every other day for a total of 10 injections after huPBL were transfered. The results demonstrated that rhPRL improved the engraftment of lymphocytes into thymus, lymph nodes and spleens, showing that the cellularities of these organs increased although the cellularities tended to vary depending on the donor. The amounts of human T cells (HLA-ABC+/CD3+) increased greatly in thymus (14.2 folds), spleen (4.16 folds) and lymph nodes (40.18 folds) after rhPRL injections. The amounts of human B cells (HLA-ABC+/CD19+) also increased greatly in lymph nodes (42.5 folds) and spleen (5.78 folds). The lymph node cells from the rhPRL-treated huPBL-SCID mice were more sensitive to PHA stimulation (〔3H〕thymidine incorporation). The supernatant of PHA-stimulated PBL from rhPRL-treated huPBL/SCID chimerism contained more cytokines (IFN-γ and IL-2). The natural cytotoxicity against human sensitive target cells, K562 cells, from spleen and bone marrow of hPBL/SCID chimerism was significantly enhanced by rhPRL administration. The lymph node cells were stimulated with LPS in vitro for 3 days and the lymphocytes from the rhPRL-treated huPBL-SCID mice were more sensitive to mitogen stimulation. Both serum total IgG level and IgM level of rhPRL-treated huPBL/SCID chimerism were increased, and even without DT-rechallenge the base line of DT-specific IgG was elevated after rhPRL treatment in huPBL-SCID mice. Thus, rhPRL stimulation promotes reconstitution of human immune system in huPBL-SCID mice. Cellular & Molecular Immunology. 2004;1(2):129-136.

  5. Detection of Salmonella invA by isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) in Zambia.

    Science.gov (United States)

    Isogai, Emiko; Makungu, Chitwambi; Yabe, John; Sinkala, Patson; Nambota, Andrew; Isogai, Hiroshi; Fukushi, Hideto; Silungwe, Manda; Mubita, Charles; Syakalima, Michelo; Hang'ombe, Bernard Mudenda; Kozaki, Shunji; Yasuda, Jun

    2005-01-01

    The isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) is a new isothermal DNA amplification method composed of exo Bca DNA polymerase, RNaseH and DNA-RNA chimeric primers. We detected invA of Salmonella from chicken carcasses, egg yolk and cattle fecal samples. Fifty-three of 59 isolates were invA-positive in ICAN-chromatostrip detection. The result was consistent with those obtained by standard PCR. Salmonella invA was detected in 12 of 14 carcass rinses by ICAN, while in 7 of 14 rinses by standard PCR. These results indicate that ICAN is an efficient, sensitive and simple system to detect invA of Salmonella species in developing countries such as Zambia.

  6. Characterization of oligosaccharide structures on a chimeric respiratory syncytial virus protein expressed in insect cell line Sf9

    Energy Technology Data Exchange (ETDEWEB)

    Wathen, M.W.; Aeed, P.A.; Elhammer, A.P. (Upjohn Co., Kalamazoo, MI (United States))

    1991-03-19

    The oligosaccharide structures added to a chimeric protein (FG) composed of the extracellular domains of respiratory syncytial virus F and G proteins, expressed in the insect cell line Sf9, were investigated. Cells were labeled in vivo with ({sup 3}H)glucosamine and infected wit a recombinant baculovirus containing the FG gene. The secreted chimeric protein was isolated by immunoprecipitation and subjected to oligosaccharide analysis. The FG protein contains two types of O-linked oligosaccharides: GalNAc and Gal{beta}1-3GalNAc constituting 17 and 66% of the total number of structures respectively. Only one type of N-linked oligosaccharide, constituting the remaining 17% of the structures on FG, was detected: a trimannosyl core structure with a fucose residue linked {alpha}1-6 to the asparagine-linked N-acetylglucosamine.

  7. Recruitment of SHP-1 protein tyrosine phosphatase and signalling by a chimeric T-cell receptor-killer inhibitory receptor

    DEFF Research Database (Denmark)

    Christensen, M D; Geisler, C

    2000-01-01

    Receptors expressing the immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic tail play an important role in the negative regulation of natural killer and B-cell activation. A subpopulation of T cells expresses the ITIM containing killer cell inhibitory receptor (KIR), which...... recognize MHC class I molecules. Following coligation of KIR with an activating receptor, the tyrosine in the ITIM is phosphorylated and the cytoplasmic protein tyrosine phosphatase SHP-1 is recruited to the ITIM via its SH2 domains. It is still not clear how SHP-1 affects T-cell receptor (TCR) signalling....... In this study, we constructed a chimeric TCR-KIR receptor. We demonstrated that SHP-1 is recruited to the chimeric TCR-KIR receptor following T-cell stimulation with either anti-TCR monoclonal antibody (MoAb) or superantigen. However, in spite of this we could not detect any effect of SHP-1 on TCR signalling...

  8. 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

    2016-01-26

    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.

  9. 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)...

  10. 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

  11. 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...

  12. 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...

  13. 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.

  14. 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.

  15. Useful oriented immobilization of antibodies on chimeric magnetic particles: direct correlation of biomacromolecule orientation with biological activity by AFM studies.

    Science.gov (United States)

    Marciello, Marzia; Filice, Marco; Olea, David; Velez, Marisela; Guisan, José M; Mateo, Cesar

    2014-12-16

    The preparation and performance of a suitable chimeric biosensor based on antibodies (Abs) immobilized on lipase-coated magnetic particles by means of a standing orienting strategy are presented. This novel system is based on hydrophobic magnetic particles coated with modified lipase molecules able to orient and further immobilize different Abs in a covalent way without any previous site-selective chemical modification of biomacromolecules. Different key parameters attending the process were studied and optimized. The optimal preparation was performed using a controlled loading (1 nmol Ab g(-1) chimeric support) at pH 9 and a short reaction time to recover a biological activity of about 80%. AFM microscopy was used to study and confirm the Abs-oriented immobilization on lipase-coated magnetic particles and the final achievement of a highly active and recyclable chimeric immune sensor. This direct technique was demonstrated to be a powerful alternative to the indirect immunoactivity assay methods for the study of biomacromolecule-oriented immobilizations.

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. Enhanced human immunodeficiency virus Type 1 expression and neuropathogenesis in knockout mice lacking Type I interferon responses.

    Science.gov (United States)

    He, Hongxia; Sharer, Leroy R; Chao, Wei; Gu, Chao-Jiang; Borjabad, Alejandra; Hadas, Eran; Kelschenbach, Jennifer; Ichiyama, Koji; Do, Meilan; Potash, Mary Jane; Volsky, David J

    2014-01-01

    The roles of Type I interferon (IFN) in human immunodeficiency virus Type 1 (HIV-1) neuropathogenesis are poorly understood; both protective and deleterious effects of IFN signaling have been described. We used genetically modified mice deficient in the Type I IFN receptor (IFNRKO) to analyze the progress of HIV-1 brain infection and neuropathogenesis in the absence of IFN signaling. IFNRKO and wild-type (WT) mice on the 129xSv/Ev or C57BL/6 strain backgrounds were infected systemically with EcoHIV, a chimeric HIV-1 that productively infects mice. IFNRKO mice showed higher HIV-1 expression in spleen and peritoneal macrophages and greater virus infiltration into the brain compared to WT mice. Neuropathogenesis was studied by histopathological, immunohistochemical, immunofluorescence, and polymerase chain reaction analyses of brain tissues after the virus was inoculated into the brain by stereotaxic intracerebral injection. Both IFNRKO and WT mice showed readily detectable HIV-1 and brain lesions, including microglial activation, astrocytosis, and increased expression of genes coding for inflammatory cytokines and chemokines typical of human HIV-1 brain disease. Parameters of HIV-1 neuropathogenesis, including HIV-1 expression in microglia/macrophages, were significantly greater in IFNRKO than in WT mice. Our results show unequivocally that Type I IFN signaling and responses limit HIV-1 infection and pathogenesis in the brains of mice.

  4. Prospects for adoptive immunotherapy of pancreatic cancer using chimeric antigen receptor-engineered T-cells.

    Science.gov (United States)

    Alrifai, Doraid; Sarker, Debashis; Maher, John

    2016-01-01

    Adoptive immunotherapy using chimeric antigen receptor (CAR) engineered T-cells is emerging as a powerful new approach to cancer immunotherapy. CARs are fusion molecules that couple the antibody-like binding of a native cell surface target to the delivery of a bespoke T-cell activating signal. Recent studies undertaken by several centers have demonstrated highly compelling efficacy in patients with acute and chronic B-cell malignancies. However, comparable therapeutic activity has not been achieved in solid tumors. Modern management of pancreatic ductal adenocarcinoma (PDAC) remains ineffective, reflected in the virtual equivalence of annual incidence and mortality statistics for this tumor type. Increasing evidence indicates that these tumors are recognized by the immune system, but deploy powerful evasion strategies that limit natural immune surveillance and render efforts at immunotherapy challenging. Here, we review preclinical and clinical studies that have been initiated or completed in an effort to develop CAR-based immunotherapy for PDAC. We also consider the hurdles to the effective clinical development of this exciting new therapeutic modality.

  5. 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

  6. Optimizing RNA/ENA chimeric antisense oligonucleotides using in vitro splicing.

    Science.gov (United States)

    Takeshima, Yasuhiro; Yagi, Mariko; Matsuo, Masafumi

    2012-01-01

    A molecular therapy for Duchenne muscular dystrophy (DMD) that converts dystrophin mRNA from out-of-frame to in-frame transcripts by inducing exon skipping with antisense oligonucleotides (AOs) is now approaching clinical application. To exploit the broad therapeutic applicability of exon skipping therapy, it is necessary to identify AOs that are able to induce efficient and specific exon skipping. To optimize AOs, we have established an in vitro splicing system using cultured DMD myocytes. Here, we describe the process of identifying the best AO.Cultured DMD myocytes are established from a biopsy sample and the target exon is chosen. A series of AOs are designed to cover the whole target exon sequence. As AOs, we use 15-20-mer chimeric oligonucleotides consisting of 2'-O-methyl RNA and modified nucleic acid (2'-O, 4'-C-ethylene-bridged nucleic acid). Each AO is transfected individually into cultured DMD myocytes, and the resulting mRNA is analyzed by reverse transcription-PCR. The ability of each AO to induce exon skipping is examined by comparing the amount of cDNA with and without exon skipping. If necessary, having roughly localized the target region, another set of AOs are designed and the exon skipping abilities of the new AOs are examined. Finally, one AO is determined as the best for the molecular therapy.Our simple and reliable methods using an in vitro splicing system have enabled us to identify optimized AOs against many exons of the DMD gene.

  7. 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)

  8. Chimeric adaptor proteins translocate diverse type VI secretion system effectors in Vibrio cholerae.

    Science.gov (United States)

    Unterweger, Daniel; Kostiuk, Benjamin; Ötjengerdes, Rina; Wilton, Ashley; Diaz-Satizabal, Laura; Pukatzki, Stefan

    2015-08-13

    Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines.

  9. Horizontal transfer of an adaptive chimeric photoreceptor from bryophytes to ferns.

    Science.gov (United States)

    Li, Fay-Wei; Villarreal, Juan Carlos; Kelly, Steven; Rothfels, Carl J; Melkonian, Michael; Frangedakis, Eftychios; Ruhsam, Markus; Sigel, Erin M; Der, Joshua P; Pittermann, Jarmila; Burge, Dylan O; Pokorny, Lisa; Larsson, Anders; Chen, Tao; Weststrand, Stina; Thomas, Philip; Carpenter, Eric; Zhang, Yong; Tian, Zhijian; Chen, Li; Yan, Zhixiang; Zhu, Ying; Sun, Xiao; Wang, Jun; Stevenson, Dennis W; Crandall-Stotler, Barbara J; Shaw, A Jonathan; Deyholos, Michael K; Soltis, Douglas E; Graham, Sean W; Windham, Michael D; Langdale, Jane A; Wong, Gane Ka-Shu; Mathews, Sarah; Pryer, Kathleen M

    2014-05-01

    Ferns are well known for their shade-dwelling habits. Their ability to thrive under low-light conditions has been linked to the evolution of a novel chimeric photoreceptor--neochrome--that fuses red-sensing phytochrome and blue-sensing phototropin modules into a single gene, thereby optimizing phototropic responses. Despite being implicated in facilitating the diversification of modern ferns, the origin of neochrome has remained a mystery. We present evidence for neochrome in hornworts (a bryophyte lineage) and demonstrate that ferns acquired neochrome from hornworts via horizontal gene transfer (HGT). Fern neochromes are nested within hornwort neochromes in our large-scale phylogenetic reconstructions of phototropin and phytochrome gene families. Divergence date estimates further support the HGT hypothesis, with fern and hornwort neochromes diverging 179 Mya, long after the split between the two plant lineages (at least 400 Mya). By analyzing the draft genome of the hornwort Anthoceros punctatus, we also discovered a previously unidentified phototropin gene that likely represents the ancestral lineage of the neochrome phototropin module. Thus, a neochrome originating in hornworts was transferred horizontally to ferns, where it may have played a significant role in the diversification of modern ferns.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. Cancer T cell immunotherapy with bispecific antibodies and chimeric antigen receptors.

    Science.gov (United States)

    Lacher, Markus D; Provenzano, Maurizio

    2013-09-01

    Solid tumors contain several different types of malignant cells. This cellular heterogeneity complicates therapy for at least two reasons. First, each subpopulation may respond differently to a given treatment. Second, cancer cells are plastic, and thus may convert from a therapy-sensitive to a therapy-resistant cell type represented by another subpopulation. Therefore, successful therapies will have to target numerous malignant cell types, not just the rapidly proliferating cells as most standard treatments do. Immunotherapies with T cells engineered to recognize cancer cells via bispecific antibodies (bsAbs) or chimeric antigen receptors (CARs) are particularly promising approaches with potential to ablate both dividing and non/slow-dividing subpopulations of cancer cells. Here, we discuss several patents associated with exceptionally effective bsAbs of the tandem single-chain variable fragment (taFv) class and untangle a part of the complex network of patents directly or indirectly related to CARs. Furthermore, we speculate on the future of bsAbs and CARs for both treatment and prevention of solid tumors such as prostate cancer. PMID:23688207

  16. 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.

  17. Elimination of progressive mammary cancer by repeated administrations of chimeric antigen receptor-modified T cells.

    Science.gov (United States)

    Globerson-Levin, Anat; Waks, Tova; Eshhar, Zelig

    2014-05-01

    Continuous oncogenic processes that generate cancer require an on-going treatment approach to eliminate the transformed cells, and prevent their further development. Here, we studied the ability of T cells expressing a chimeric antibody-based receptor (CAR) to offer a therapeutic benefit for breast cancer induced by erbB-2. We tested CAR-modified T cells (T-bodies) specific to erbB-2 for their antitumor potential in a mouse model overexpressing a human erbB-2 transgene that develops mammary tumors. Comparing the antitumor reactivity of CAR-modified T cells under various therapeutic settings, either prophylactic, prior to tumor development, or therapeutically. We found that repeated administration of CAR-modified T cells is required to eliminate spontaneously developing mammary cancer. Systemic, as well as intratumoral administered CAR-modified T cells accumulated at tumor sites and eventually eliminated the malignant cells. Interestingly, within a few weeks after a single CAR T cells' administration, and rejection of primary lesion, tumors usually relapsed both in treated mammary gland and at remote sites; however, repeated injections of CAR-modified T cells were able to control the secondary tumors. Since spontaneous tumors can arise repeatedly, especially in the case of syndromes characterized by specific susceptibility to cancer, multiple administrations of CAR-modified T cells can serve to control relapsing disease.

  18. 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

  19. Construction of a genetically engineered chimeric apoprotein consisting of sequences derived from lidamycin and neocarzinostatin.

    Science.gov (United States)

    Jiang, Wenguo; Shang, Boyang; Li, Liang; Zhang, Shenghua; Zhen, Yongsu

    2016-01-01

    Neocarzinostatin (NCS) consists of an enediyne chromophore and an apoprotein (NCP). Lidamycin (LDM) is composed of another active enediyne chromophore (AE) and an acidic protein (LDP). Although the structures of NCP and LDP are very similar, LDM has been shown to have an increased tumor-suppressive activity than that of NCS. The aim of this study was to construct a chimeric protein (CMP) that consists of both the terminus residue of NCP and an LDP pocket-forming residue that can bind AE. This CMP will have a structure similar to NCS and an antitumor activity similar to LDM. The assembling efficiency of LDP, CMP, and NCP was 73.9, 1.5, and 1.1%, respectively. The cytotoxicity was consistent with their assembling efficiency of AE in proteins. When CMP-AE and NCP-AE were administered at equivalent AE doses of LDM, the inhibition rate of CMP-AE was the same as LDM and significantly higher than that of NCP-AE. Our study implied that the binding activity between LDP and AE was very specific. The terminus residue of LDP could affect the specifically binding activity. The pocket-forming residue could confer a protective function to the chromophore. Further investigation of its bioactivity might serve as a new drug design strategy and drug-delivery carrier in targeted cancer therapy.

  20. 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.

  1. 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.

  2. 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.

  3. Enhanced cytotoxicity of natural killer cells following the acquisition of chimeric antigen receptors through trogocytosis.

    Directory of Open Access Journals (Sweden)

    Fu-Nan Cho

    Full Text Available Natural killer (NK cells have the capacity to target tumors and are ideal candidates for immunotherapy. Viral vectors have been used to genetically modify in vitro expanded NK cells to express chimeric antigen receptors (CARs, which confer cytotoxicity against tumors. However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome. In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy. A K562 cell line expressing high levels of anti-CD19 CARs was generated as a donor cell to transfer the anti-CD19 CARs onto NK cells via trogocytosis. Anti-CD19 CAR expression was observed in expanded NK cells after these cells were co-cultured for one hour with freeze/thaw-treated donor cells expressing anti-CD19 CARs. Immunofluorescence analysis confirmed the localization of the anti-CD19 CARs on the NK cell surface. Acquisition of anti-CD19 CARs via trogocytosis enhanced NK cell-mediated cytotoxicity against the B-cell acute lymphoblastic leukemia (B-ALL cell lines and primary B-ALL cells derived from patients. To our knowledge, this is the first report that describes the increased cytotoxicity of NK cells following the acquisition of CARs via trogocytosis. This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.

  4. Myeloblastic leukemia cells conditionally blocked by myc-estrogen receptor chimeric transgenes for terminal differentiation coupled to growth arrest and apoptosis.

    Science.gov (United States)

    Selvakumaran, M; Liebermann, D; Hoffman-Liebermann, B

    1993-05-01

    Conditional mutants of the myeloblastic leukemic M1 cell line, expressing the chimeric mycer transgene, have been established. It is shown that M1 mycer cells, like M1, undergo terminal differentiation coupled to growth arrest and programmed cell death (apoptosis) after treatment with the physiologic differentiation inducer interleukin-6. However, when beta-estradiol is included in the culture medium, M1 mycer cells respond to differentiation inducers like M1 myc cell lines, where the differentiation program is blocked at an intermediate stage. By manipulating the function of the mycer transgene product, it is shown that there is a 10-hour window during myeloid differentiation, from 30 to 40 hours after the addition of the differentiation inducer, when the terminal differentiation program switches from being dependent on c-myc suppression to becoming c-myc suppression independent, where activation of c-myc has no apparent effect on mature macrophages. M1 mycer cell lines provide a powerful tool to increase our understanding of the role of c-myc in normal myelopoiesis and in leukemogenesis, also providing a strategy to clone c-myc target genes.

  5. Induction of Antibodies and T Cell Responses by a Recombinant Influenza Virus Carrying an HIV-1 TatΔ51–59 Protein in Mice

    Directory of Open Access Journals (Sweden)

    B. Garulli

    2014-01-01

    Full Text Available Recombinant influenza viruses hold promise as vectors for vaccines to prevent transmission of mucosal pathogens. In this study, we generated a recombinant WSN/TatΔ51–59 virus in which Tat protein lacking residues 51 to 59 of the basic domain was inserted into the N-terminus of the hemagglutinin (HA of A/WSN/33 virus. The TatΔ51–59 insertion into the viral HA caused a 2-log reduction in viral titers in cell culture, compared with the parental A/WSN/33 virus, and severely affected virus replication in vivo. Nevertheless, Tat-specific antibodies and T cell responses were elicited upon a single intranasal immunization of BALB/c mice with WSN/TatΔ51–59 virus. Moreover, Tat-specific immune responses were also detected following vaccine administration via the vaginal route. These data provide further evidence that moderately large HIV antigens can be delivered by chimeric HA constructs and elicit specific immune responses, thus increasing the options for the potential use of recombinant influenza viruses, and their derivatives, for prophylactic and therapeutic vaccines.

  6. A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice

    International Nuclear Information System (INIS)

    Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-γ secretion and protection experiments, mediated by CD4+ and CD8+ cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

  7. In vivo expansion of co-transplanted T cells impacts on tumor re-initiating activity of human acute myeloid leukemia in NSG mice.

    Directory of Open Access Journals (Sweden)

    Malte von Bonin

    Full Text Available Human cells from acute myeloid leukemia (AML patients are frequently transplanted into immune-compromised mouse strains to provide an in vivo environment for studies on the biology of the disease. Since frequencies of leukemia re-initiating cells are low and a unique cell surface phenotype that includes all tumor re-initiating activity remains unknown, the underlying mechanisms leading to limitations in the xenotransplantation assay need to be understood and overcome to obtain robust engraftment of AML-containing samples. We report here that in the NSG xenotransplantation assay, the large majority of mononucleated cells from patients with AML fail to establish a reproducible myeloid engraftment despite high donor chimerism. Instead, donor-derived cells mainly consist of polyclonal disease-unrelated expanded co-transplanted human T lymphocytes that induce xenogeneic graft versus host disease and mask the engraftment of human AML in mice. Engraftment of mainly myeloid cell types can be enforced by the prevention of T cell expansion through the depletion of lymphocytes from the graft prior transplantation.

  8. Expression of MPB83 from Mycobacterium bovis in Brucella abortus S19 induces specific cellular immune response against the recombinant antigen in BALB/c mice.

    Science.gov (United States)

    Sabio y García, Julia V; Bigi, Fabiana; Rossetti, Osvaldo; Campos, Eleonora

    2010-12-01

    Immunodominant MPB83 antigen from Mycobacterium bovis was expressed as a chimeric protein fused to either β-galactosidase, outer membrane lipoprotein OMP19 or periplasmic protein BP26 in gram-negative Brucella abortus S19, in all cases driven by each gene's own promoter. All fusion proteins were successfully expressed and localized in the expected subcellular fraction. Moreover, OMP19-MPB83 was processed as a lipoprotein when expressed in B. abortus. Splenocytes from BALB/c mice immunized with the recombinant S19 strains carrying the genes coding for the heterologous antigens in replicative plasmids, showed equally specific INF-γ production in response to MPB83 stimulation. Association to the lipid moiety of OMP19 presented no advantage in terms of immunogenicity for MPB83. In contrast, fusion to BP26, which was encoded by an integrative plasmid, resulted in a weaker immune response. None of the constructions affected the survival rate or the infection pattern of Brucella. We concluded that B. abortus S19 is an appropriate candidate for the expression of M. bovis antigens both associated to the membrane or cytosolic fraction and may provide the basis for a future combined vaccine for bovine brucellosis and tuberculosis. PMID:20888425

  9. MAX Provider Characteristics

    Data.gov (United States)

    U.S. Department of Health & Human Services — The MAX Provider Characteristics (PC) File Implementation Report describes the design, implementation, and results of the MAXPC prototype, which was based on three...

  10. 人乳头瘤病毒11型L1/E7嵌合DNA疫苗的构建及其诱导的免疫效应%Construction and immunogenicity evaluation of chimerical DNA vaccine of human papillomavirus type 11

    Institute of Scientific and Technical Information of China (English)

    黄朝晖; 李莉华; 郭子健; 刘志辉; 任金冬; 宋明旭; 周希科; 王飞; 毕志刚

    2009-01-01

    目的 构建人乳头瘤病毒11型L1/E7嵌合DNA疫苗pcDNA3 L1-E7并研究其在小鼠中诱导的免疫效应.方法 用分子克隆技术构建重组真核表达质粒peDNA3 L1-E7.重组质粒DNA股四头肌注射免疫小鼠,用ELISA方法 检测L1、E7特异性抗体和脾细胞分泌的IL-2、γ-INF;MTT法检测脾淋巴细胞增殖反应.结果 成功构建pcDNA3 L1-E7.免疫小鼠后,重组质粒可诱导机体产生特异性脾淋巴细胞增殖及IL-2、γ-INF分泌增加,并诱导机体产生HPV 11-E7 IgG和HPV 11-L1 IgG抗体.结论 嵌合DNA疫苗pcDNA3 L1-E7能诱导小鼠产生特异性的细胞免疫和体液免疫反应.%Objective To construct chimerical DNA vaccine plasmid of human papiUomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity. Methods Molecular cloning techniques were used to construct recombinant plasmid PeDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection. IL-2 and γ-INF secreted by immunized spleens lymphocyte and HPV 11 LI or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay. Results The chimerical DNA plasmid of pcDNA3 LI-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and γ-INF were detected in vaccinated mice. Conclusion Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.

  11. Palatable meal anticipation in mice.

    Directory of Open Access Journals (Sweden)

    Cynthia T Hsu

    Full Text Available The ability to sense time and anticipate events is a critical skill in nature. Most efforts to understand the neural and molecular mechanisms of anticipatory behavior in rodents rely on daily restricted food access, which induces a robust increase of locomotor activity in anticipation of daily meal time. Interestingly, rats also show increased activity in anticipation of a daily palatable meal even when they have an ample food supply, suggesting a role for brain reward systems in anticipatory behavior, and providing an alternate model by which to study the neurobiology of anticipation in species, such as mice, that are less well adapted to "stuff and starve" feeding schedules. To extend this model to mice, and exploit molecular genetic resources available for that species, we tested the ability of wild-type mice to anticipate a daily palatable meal. We observed that mice with free access to regular chow and limited access to highly palatable snacks of chocolate or "Fruit Crunchies" avidly consumed the snack but did not show anticipatory locomotor activity as measured by running wheels or video-based behavioral analysis. However, male mice receiving a snack of high fat chow did show increased food bin entry prior to access time and a modest increase in activity in the two hours preceding the scheduled meal. Interestingly, female mice did not show anticipation of a daily high fat meal but did show increased activity at scheduled mealtime when that meal was withdrawn. These results indicate that anticipation of a scheduled food reward in mice is behavior, diet, and gender specific.

  12. Primary Human Hepatocytes Repopulate Livers of Mice After In Vitro Culturing and Lentiviral-Mediated Gene Transfer.

    Science.gov (United States)

    Bierwolf, Jeanette; Volz, Tassilo; Lütgehetmann, Marc; Allweiss, Lena; Riecken, Kristoffer; Warlich, Michael; Fehse, Boris; Kalff, Joerg C; Dandri, Maura; Pollok, Joerg-Matthias

    2016-05-01

    Cell-based therapies represent a promising alternative to orthotopic liver transplantation. However, therapeutic effects are limited by low cell engraftment rates. We recently introduced a technique creating human hepatocyte spheroids for potential therapeutic application. The aim of this study was to evaluate whether these spheroids are suitable for engraftment in diseased liver tissues. Intrasplenic spheroid transplantation into immunodeficient uPA/SCID/beige mice was performed. Hepatocyte transduction ability prior to transplantation was tested by lentiviral labeling using red-green-blue (RGB) marking. Eight weeks after transplantation, animals were sacrificed and livers were analyzed by immunohistochemistry and immunofluorescence. To investigate human hepatocyte-specific gene expression profiles in mice, quantitative real-time-PCR was applied. Human albumin and alpha-1-antitrypsin concentrations in mouse serum were quantified to assess the levels of human chimerism. Precultured human hepatocytes reestablished their physiological liver tissue architecture and function upon transplantation in mice. Positive immunohistochemical labeling of the proliferating cell nuclear antigen revealed that human hepatocytes retained their in vivo proliferation capacity. Expression profiles of human genes analyzed in chimeric mouse livers resembled levels determined in native human tissue. Extensive vascularization of human cell clusters was detected by demonstration of von Willebrand factor activity. To model gene therapy approaches, lentiviral transduction was performed ex vivo and fluorescent microscopic imaging revealed maintenance of RGB marking in vivo. Altogether, this is the first report demonstrating that cultured and retroviral transduced human hepatocyte spheroids are able to engraft and maintain their regenerative potential in vivo. PMID:27068494

  13. Experimental study of MSCs promoting haploid hematopoietic stem cell transplantation in treatment for mice with acute radiation syndrome

    International Nuclear Information System (INIS)

    Objective: To investigate the mechanism of mesenchymal stem cells in enhancing the effects of haploid matched bone marrow cells transplantation in mice with acute radiation syndrome (ARS). Methods: The survival of mice infused with different levels of MSCs and bone marrow cells after 8 Gy TBI were examined. BALB/c female mice irradiated with 8 Gy of 60Co γ-rays were randomly divided into two groups, MSCs group, infused with MSCs of female CB6F1 mice labeled with cm-DiI and bone marrow monocytes of male CB6F1, Control group, only infused with bone marrow monocytes. Peripheral blood counts, T-lymphocyte subpopulation of peripheral, blood cells, the sty-gene chimerism of bone marrow of the receiptors, the distribution of MSCs in the receiptors, the occurrence time of cGVHD, pathologic variety of medulla were observed. Results: MSCs improved the survival of mice after 8 TBI, but 1.5 x 108/kg of MSCs increased the mortality of irradiated mice. In comparison with the control group, leukocytes and plastocytes recovered rapidly in MSCs group. Megacaryocytes in sternum marrows grew lastly in MSC group. The percent of CD3 and CD4 positive cells in the MSCs group were higher than those in control post-transplantation. The sty-gene chimerism of bone marrow of the receiptors was higher in the MSCs group than that in the control at 30 d. The MSCs were distributed in intestine, thymus, bone marrow, liver, heart of the receiptors at 30 d. The cGVHD occurrence was 30 d later in MSCs group than that of the control. Conclusions: MSCs could improve stem cell engraftment, enhance T-lymphocyte and plastocytes recovery, delay occurrence of cGVHD, repair injured organs and increase survivals. It is indicated that MSCs can enhance the treatment effects of haploid hematopoietic stem cells transplant for ARS. (authors)

  14. Provider of Services File

    Data.gov (United States)

    U.S. Department of Health & Human Services — The POS file consists of two data files, one for CLIA labs and one for 18 other provider types. The file names are CLIA and OTHER. If downloading the file, note it...

  15. What HERA may provide?

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Hannes [DESY, Hamburg (Germany); De Roeck, Albert [CERN, Genf (Switzerland); Bartles, Jochen [Univ. Hamburg (DE). Institut fuer Theoretische Physik II] (and others)

    2008-09-15

    More than 100 people participated in a discussion session at the DIS08 workshop on the topic What HERA may provide. A summary of the discussion with a structured outlook and list of desirable measurements and theory calculations is given. (orig.)

  16. The Provident Principal.

    Science.gov (United States)

    McCall, John R.

    This monograph offers leadership approaches for school principals. Discussion applies the business leadership theory of Warren Bennis and Burt Nanus to the role of the principal. Each of the booklet's three parts concludes with discussion questions. Part 1, "Visions and Values for the Provident Principal," demonstrates the importance of…

  17. Carotid chemoreceptor development in mice.

    Science.gov (United States)

    Shirahata, Machiko; Kostuk, Eric W; Pichard, Luis E

    2013-01-01

    Mice are the most suitable species for understanding genetic aspects of postnatal developments of the carotid body due to the availability of many inbred strains and knockout mice. Our study has shown that the carotid body grows differentially in different mouse strains, indicating the involvement of genes. However, the small size hampers investigating functional development of the carotid body. Hypoxic and/or hyperoxic ventilatory responses have been investigated in newborn mice, but these responses are indirect assessment of the carotid body function. Therefore, we need to develop techniques of measuring carotid chemoreceptor neural activity from young mice. Many studies have taken advantage of the knockout mice to understand chemoreceptor function of the carotid body, but they are not always suitable for addressing postnatal development of the carotid body due to lethality during perinatal periods. Various inbred strains with well-designed experiments will provide useful information regarding genetic mechanisms of the postnatal carotid chemoreceptor development. Also, targeted gene deletion is a critical approach.

  18. 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

  19. 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

  20. Achieving Provider Engagement

    Science.gov (United States)

    Greenfield, Geva; Pappas, Yannis; Car, Josip; Majeed, Azeem; Harris, Matthew

    2014-01-01

    The literature on integrated care is limited with respect to practical learning and experience. Although some attention has been paid to organizational processes and structures, not enough is paid to people, relationships, and the importance of these in bringing about integration. Little is known, for example, about provider engagement in the organizational change process, how to obtain and maintain it, and how it is demonstrated in the delivery of integrated care. Based on qualitative data from the evaluation of a large-scale integrated care initiative in London, United Kingdom, we explored the role of provider engagement in effective integration of services. Using thematic analysis, we identified an evolving engagement narrative with three distinct phases: enthusiasm, antipathy, and ambivalence, and argue that health care managers need to be aware of the impact of professional engagement to succeed in advancing the integrated care agenda. PMID:25212855

  1. Providence Their Guide

    Directory of Open Access Journals (Sweden)

    A.D. Naudé

    2012-02-01

    Full Text Available In his preface to this book the author quotes from Milton's Paradise lost, from which the title of this book is derived: 'Som natural tears they drop'd, but wip'd them soon; The World was all before them, where to choose Thir place of rest, and Providence thir guide; They hand in hand, with wand ring steps and slow, Through Eden took thir solitarie way.

  2. Why healthcare providers merge.

    Science.gov (United States)

    Postma, Jeroen; Roos, Anne-Fleur

    2016-04-01

    In many OECD countries, healthcare sectors have become increasingly concentrated as a result of mergers. However, detailed empirical insight into why healthcare providers merge is lacking. Also, we know little about the influence of national healthcare policies on mergers. We fill this gap in the literature by conducting a survey study on mergers among 848 Dutch healthcare executives, of which 35% responded (resulting in a study sample of 239 executives). A total of 65% of the respondents was involved in at least one merger between 2005 and 2012. During this period, Dutch healthcare providers faced a number of policy changes, including increasing competition, more pressure from purchasers, growing financial risks, de-institutionalisation of long-term care and decentralisation of healthcare services to municipalities. Our empirical study shows that healthcare providers predominantly merge to improve the provision of healthcare services and to strengthen their market position. Also efficiency and financial reasons are important drivers of merger activity in healthcare. We find that motives for merger are related to changes in health policies, in particular to the increasing pressure from competitors, insurers and municipalities. PMID:26055501

  3. 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

  4. 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

  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.

  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. 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.

  8. 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.

  9. 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.

  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. Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy.

    Science.gov (United States)

    Mock, Ulrike; Nickolay, Lauren; Philip, Brian; Cheung, Gordon Weng-Kit; Zhan, Hong; Johnston, Ian C D; Kaiser, Andrew D; Peggs, Karl; Pule, Martin; Thrasher, Adrian J; Qasim, Waseem

    2016-08-01

    Novel cell therapies derived from human T lymphocytes are exhibiting enormous potential in early-phase clinical trials in patients with hematologic malignancies. Ex vivo modification of T cells is currently limited to a small number of centers with the required infrastructure and expertise. The process requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, automation of these procedures is being developed. The CliniMACS Prodigy (Miltenyi Biotec) has recently been adapted for lentiviral transduction of T cells and here we analyse the feasibility of a clinically compliant T-cell engineering process for the manufacture of T cells encoding chimeric antigen receptors (CAR) for CD19 (CAR19), a widely targeted antigen in B-cell malignancies. Using a closed, single-use tubing set we processed mononuclear cells from fresh or frozen leukapheresis harvests collected from healthy volunteer donors. Cells were phenotyped and subjected to automated processing and activation using TransAct, a polymeric nanomatrix activation reagent incorporating CD3/CD28-specific antibodies. Cells were then transduced and expanded in the CentriCult-Unit of the tubing set, under stabilized culture conditions with automated feeding and media exchange. The process was continuously monitored to determine kinetics of expansion, transduction efficiency and phenotype of the engineered cells in comparison with small-scale transductions run in parallel. We found that transduction efficiencies, phenotype and function of CAR19 T cells were comparable with existing procedures and overall T-cell yields sufficient for anticipated therapeutic dosing. The automation of closed-system T-cell engineering should improve dissemination of emerging immunotherapies and greatly widen applicability.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system.

    Directory of Open Access Journals (Sweden)

    Mark A Little

    Full Text Available Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3 antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener's granulomatosis. Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ⁻/⁻ mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17% more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.

  17. Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system.

    LENUS (Irish Health Repository)

    Little, Mark A

    2012-01-01

    Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3) antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener\\'s granulomatosis). Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ⁻\\/⁻ mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17%) more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.

  18. Providing Compassion through Flow

    Directory of Open Access Journals (Sweden)

    Lydia Royeen

    2015-07-01

    Full Text Available Meg Kral, MS, OTR/L, CLT, is the cover artist for the Summer 2015 issue of The Open Journal of Occupational Therapy. Her untitled piece of art is an oil painting and is a re-creation of a photograph taken while on vacation. Meg is currently supervisor of outpatient services at Rush University Medical Center. She is lymphedema certified and has a specific interest in breast cancer lymphedema. Art and occupational therapy serve similar purposes for Meg: both provide a sense of flow. She values the outcomes, whether it is a piece of art or improved functional status

  19. The Provident Sea

    Science.gov (United States)

    Cushing, David H.

    1988-09-01

    The Provident Sea describes the history of fish stock management (including whales and seals). The book traces, on the basis of the original scientific material, the history of the management of "the provident sea" up to recent times when problems of over-exploitation have had dramatic effects upon stocks. The need for management arose mainly from the increasing industrialization of capture. Hence the preindustrial fisheries are covered, in particular the old cod fishery on the Grand Bank and the herring fishery in the North Sea, as an essential background to current problems. The origins of fisheries and whaling science are described, as is the development up to 1965 of the science and institution in fisheries, whaling, and sealing. In the sixties and seventies, certain major fishing nations took a heavy harvest of fish stocks using sophisticated and efficient gathering methods. This in turn led to conflict and one consequence was the "Law of the Sea" conference set up to try and resolve these issues.

  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. Individually Ventilated Cages Impose Cold Stress on Laboratory Mice: A Source of Systemic Experimental Variability

    OpenAIRE

    David, John M.; Knowles, Scott; Lamkin, Donald M.; Stout, David B.

    2013-01-01

    Individual ventilated cages (IVC) are increasing in popularity. Although mice avoid IVC in preference testing, they show no aversion when provided additional nesting material or the cage is not ventilated. Given the high ventilation rate in IVC, we developed 3 hypotheses: that mice housed in IVC experience more cold stress than do mice housed in static cages; that IVC-induced cold stress affects the results of experiments using mice; and that, when provided shelters, mice behaviorally thermor...

  2. THE HUMORAL AND CELLULAR IMMUNE RESPONSES INDUCED BY HPV18L1-E6/E7 DNA VACCINES IN MICE

    Institute of Scientific and Technical Information of China (English)

    Yang Jin; Li Xu; Li Ang; Wang Yili; Si Lüsheng

    2006-01-01

    Objective To construct eukaryotic expression vector of HPV18 L1- E6, E7 chimeric gene and examine the humoral and cellular immune responses induced by this DNA vaccines in mice. Methods The C-terminal of major capsid protein L1 gene and mutant zinc finger domains of early E6/7 oncogenes in HPV18 were integrated and inserted into eukaryotic expression vector pVAX1 to generate vaccines pVAX1-L1E6Mxx, E7Mxx. CHO cells were transiently transfected with the individual construct. Target protein expressions in the lysate of the transfected cells were measured by ELISA and immunocytochemistry. After BALB/c mice were vaccinated with various recombinant plasmids(pVAX1-L1-E6M3 or pVAX1-L1-E7M3) and immunie adjuvants (pLXHDmB7-2 or LTB) through different administration routes (intramuscular or intranasal) , the great cellular immune responses were produced as revealed by delayed-type hypersensitivity (DTH) and lymphocyte proliferation, and the expression of IL-4 and IFN- γ cells in CD4+ and CD8+subpopulations. Results The highly efficient expression of pVAX1-L1E6Mxx, E7Mxx vector in host eukaryotic cells were demonstrated both by ELISA and immunocytochemistry. The level of specific serum IgG against HPV in experiment groups mice was much higher than that of control group, and intranuscular immunization group had the highest antibody level. Intramuscular immunization groups were superior to intranasal immunization groups in DTH response, splenocyte proliferation and CD8+ IFN-γ + cells number, but CD4+ IL4+ cell number was higher in intranasal immunization groups. The immunization groups using pLXHDmB7-2 as adjuvant were superior to other groups in immunoresponse. Conclusion These DNA vaccines produce remarkable cellular and humoral immuneresponses in the mouse and may provide as prophylatic and therapeutic candidates for HPV induced cancer treatment.

  3. Minor Contribution of Chimeric Host-HIV Readthrough Transcripts to the Level of HIV Cell-Associated gag RNA.

    Science.gov (United States)

    Pasternak, Alexander O; DeMaster, Laura K; Kootstra, Neeltje A; Reiss, Peter; O'Doherty, Una; Berkhout, Ben

    2015-11-11

    Cell-associated HIV unspliced RNA is an important marker of the viral reservoir. HIV gag RNA-specific assays are frequently used to monitor reservoir activation. Because HIV preferentially integrates into actively transcribed genes, some of the transcripts detected by these assays may not represent genuine HIV RNA but rather chimeric host-HIV readthrough transcripts. Here, we demonstrate that in HIV-infected patients on suppressive combination antiretroviral therapy, such host-derived transcripts do not significantly contribute to the HIV gag RNA level.

  4. 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...

  5. 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...

  6. 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...

  7. 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

  8. 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...

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

    Energy Technology Data Exchange (ETDEWEB)

    Sun, L.K.; Curtis, P.; Rakowicz-Szulczynska, E.; Ghrayeb, J.; Chang, N.; Morrison, S.L.; Koprowski, H.

    1987-01-01

    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 ..gamma..3 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.

  10. 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...

  11. Database of Physiological Parameters for Early Life Rats and Mice

    Data.gov (United States)

    U.S. Environmental Protection Agency — The Database of Physiological Parameters for Early Life Rats and Mice provides information based on scientific literature about physiological parameters. Modelers...

  12. 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

  13. Chimerization at the AQP2–AQP3 locus is the genetic basis of melarsoprol–pentamidine cross-resistance in clinical Trypanosoma brucei gambiense isolates

    Directory of Open Access Journals (Sweden)

    Fabrice E. Graf

    2015-08-01

    Full Text Available Aquaglyceroporin-2 is a known determinant of melarsoprol–pentamidine cross-resistance in Trypanosoma brucei brucei laboratory strains. Recently, chimerization at the AQP2–AQP3 tandem locus was described from melarsoprol–pentamidine cross-resistant Trypanosoma brucei gambiense isolates from sleeping sickness patients in the Democratic Republic of the Congo. Here, we demonstrate that reintroduction of wild-type AQP2 into one of these isolates fully restores drug susceptibility while expression of the chimeric AQP2/3 gene in aqp2–aqp3 null T. b. brucei does not. This proves that AQP2–AQP3 chimerization is the cause of melarsoprol–pentamidine cross-resistance in the T. b. gambiense isolates.

  14. What HERA May Provide?

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Hannes; /DESY; De Roeck, Albert; /CERN; Bartels, Jochen; /Hamburg U., Inst. Theor. Phys. II; Behnke, Olaf; Blumlein, Johannes; /DESY; Brodsky, Stanley; /SLAC /Durham U., IPPP; Cooper-Sarkar, Amanda; /Oxford U.; Deak, Michal; /DESY; Devenish, Robin; /Oxford U.; Diehl, Markus; /DESY; Gehrmann, Thomas; /Zurich U.; Grindhammer, Guenter; /Munich, Max Planck Inst.; Gustafson, Gosta; /CERN /Lund U., Dept. Theor. Phys.; Khoze, Valery; /Durham U., IPPP; Knutsson, Albert; /DESY; Klein, Max; /Liverpool U.; Krauss, Frank; /Durham U., IPPP; Kutak, Krzysztof; /DESY; Laenen, Eric; /NIKHEF, Amsterdam; Lonnblad, Leif; /Lund U., Dept. Theor. Phys.; Motyka, Leszek; /Hamburg U., Inst. Theor. Phys. II /Birmingham U. /Southern Methodist U. /DESY /Piemonte Orientale U., Novara /CERN /Paris, LPTHE /Hamburg U. /Penn State U.

    2011-11-10

    More than 100 people participated in a discussion session at the DIS08 workshop on the topic What HERA may provide. A summary of the discussion with a structured outlook and list of desirable measurements and theory calculations is given. The HERA accelerator and the HERA experiments H1, HERMES and ZEUS stopped running in the end of June 2007. This was after 15 years of very successful operation since the first collisions in 1992. A total luminosity of {approx} 500 pb{sup -1} has been accumulated by each of the collider experiments H1 and ZEUS. During the years the increasingly better understood and upgraded detectors and HERA accelerator have contributed significantly to this success. The physics program remains in full swing and plenty of new results were presented at DIS08 which are approaching the anticipated final precision, fulfilling and exceeding the physics plans and the previsions of the upgrade program. Most of the analyses presented at DIS08 were still based on the so called HERA I data sample, i.e. data taken until 2000, before the shutdown for the luminosity upgrade. This sample has an integrated luminosity of {approx} 100 pb{sup -1}, and the four times larger statistics sample from HERA II is still in the process of being analyzed.

  15. What HERA May Provide?

    International Nuclear Information System (INIS)

    More than 100 people participated in a discussion session at the DIS08 workshop on the topic What HERA may provide. A summary of the discussion with a structured outlook and list of desirable measurements and theory calculations is given. The HERA accelerator and the HERA experiments H1, HERMES and ZEUS stopped running in the end of June 2007. This was after 15 years of very successful operation since the first collisions in 1992. A total luminosity of ∼ 500 pb-1 has been accumulated by each of the collider experiments H1 and ZEUS. During the years the increasingly better understood and upgraded detectors and HERA accelerator have contributed significantly to this success. The physics program remains in full swing and plenty of new results were presented at DIS08 which are approaching the anticipated final precision, fulfilling and exceeding the physics plans and the previsions of the upgrade program. Most of the analyses presented at DIS08 were still based on the so called HERA I data sample, i.e. data taken until 2000, before the shutdown for the luminosity upgrade. This sample has an integrated luminosity of ∼ 100 pb-1, and the four times larger statistics sample from HERA II is still in the process of being analyzed.

  16. Mixed T Lymphocyte Chimerism after Allogeneic Hematopoietic Transplantation Is Predictive for Relapse of Acute Myeloid Leukemia and Myelodysplastic Syndromes.

    Science.gov (United States)

    Lee, Hans C; Saliba, Rima M; Rondon, Gabriela; Chen, Julianne; Charafeddine, Yasmeen; Medeiros, L Jeffrey; Alatrash, Gheath; Andersson, Borje S; Popat, Uday; Kebriaei, Partow; Ciurea, Stefan; Oran, Betul; Shpall, Elizabeth; Champlin, Richard

    2015-11-01

    Chimerism testing after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) represents a promising tool for predicting disease relapse, although its precise role in this setting remains unclear. We investigated the predictive value of T lymphocyte chimerism analysis at 90 to 120 days after allo-HSCT in 378 patients with AML/MDS who underwent busulfan/fludarabine-based myeloablative preparative regimens. Of 265 (70%) patients with available T lymphocyte chimerism data, 43% of patients in first or second complete remission (CR1/CR2) at the time of transplantation had complete (100%) donor T lymphocytes at day +90 to +120 compared with 60% of patients in the non-CR1/CR2 cohort (P = .005). In CR1/CR2 patients, donor T lymphocyte chimerism ≤ 85% at day +90 to +120 was associated with a higher frequency of 3-year disease progression (29%; 95% confidence interval [CI], 18% to 46% versus 15%; 95% CI, 9% to 23%; hazard ratio [HR], 2.1; P = .04). However, in the more advanced, non-CR1/CR2 cohort, mixed T lymphocyte chimerism was not associated with relapse (37%; 95% CI, 20% to 66% versus 34%; 95% CI, 25% to 47%; HR, 1.3; P = .60). These findings demonstrate that early T lymphocyte chimerism testing at day +90 to +120 is a useful approach for predicting AML/MDS disease recurrence in patients in CR1/CR2 at the time of transplantation. PMID:26183077

  17. Graft-versus-leukemia effects from donor lymphocyte infusion after nonmyeloablative allogeneic bone marrow transplantation in mice

    Institute of Scientific and Technical Information of China (English)

    DU Bing; LI De-peng; XU Kai-lin; PAN Xiu-ying

    2005-01-01

    Background Nonmyeloablative allogeneic bone marrow transplantation has been used since the 1990s as a new hematological stem cell transplantation strategy for treating hematological diseases. The purpose of this study was to explore the graft-versus-leukemia (GVL) effects of donor lymphocyte infusions (DLIs) after nonmyeloablative allogeneic bone marrow transplantations, while assessing the declines in treatment-associated morbidity, mortality, and graft-versus-host disease (GVHD).Methods A total of 615 (H-2k) mice were injected with L615 tumor cells and received 500 cGy (60Coγ-ray) irradiation three days later, followed by an allogeneic bone marrow transplantation (allo-BMT). The allo-grafts consisted of 3×107 bone marrow cells and 1×107 spleen cells from BALB/C (H-2d) donor mice. Two days after the allo-BMT, the recipient mice were given 200 mg/kg of cyclophosphamide. Subsequently, recipient mice were infused with either donor spleen cells (2×107) on day 14 or 21, or donor spleen cells (5×107) pretreated with hydrocortisone and cyclosporin A (CsA) in vitro on day 14 post-BMT.Results The median survival time of mice that received DLI on day 21 and pretreated DLI on day 14 post-BMT was longer than that of controls and the day 14 DLI group (P<0.01). No evidence of severe GVHD was observed in the day 21 DLI group nor in the day 14 treated DLI group. Mixed chimerism was confirmed in the day 14 DLI group, the day 14 treated DLI group, and the day 21 DLI group on the thirteenth day post-transplantation; full donor chimerism was observed two weeks after DLI.Conclusion Donor lymphocyte infusion after nonmyeloablative bone marrow transplantation may reduce transplantation-associated morbidity and mortality while strengthening graft-versus-leukemia effects.

  18. 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

  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. Cytoplasmic male sterility-associated chimeric open reading frames identified by mitochondrial genome sequencing of four Cajanus genotypes.

    Science.gov (United States)

    Tuteja, Reetu; Saxena, Rachit K; Davila, Jaime; Shah, Trushar; Chen, Wenbin; Xiao, Yong-Li; Fan, Guangyi; Saxena, K B; Alverson, Andrew J; Spillane, Charles; Town, Christopher; Varshney, Rajeev K

    2013-10-01

    The hybrid pigeonpea (Cajanus cajan) breeding technology based on cytoplasmic male sterility (CMS) is currently unique among legumes and displays major potential for yield increase. CMS is defined as a condition in which a plant is unable to produce functional pollen grains. The novel chimeric open reading frames (ORFs) produced as a results of mitochondrial genome rearrangements are considered to be the main cause of CMS. To identify these CMS-related ORFs in pigeonpea, we sequenced the mitochondrial genomes of three C. cajan lines (the male-sterile line ICPA 2039, the maintainer line ICPB 2039, and the hybrid line ICPH 2433) and of the wild relative (Cajanus cajanifolius ICPW 29). A single, circular-mapping molecule of length 545.7 kb was assembled and annotated for the ICPA 2039 line. Sequence annotation predicted 51 genes, including 34 protein-coding and 17 RNA genes. Comparison of the mitochondrial genomes from different Cajanus genotypes identified 31 ORFs, which differ between lines within which CMS is present or absent. Among these chimeric ORFs, 13 were identified by comparison of the related male-sterile and maintainer lines. These ORFs display features that are known to trigger CMS in other plant species and to represent the most promising candidates for CMS-related mitochondrial rearrangements in pigeonpea. PMID:23792890

  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. 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

  4. Bioactivity and structural properties of chimeric analogs of the starfish SALMFamide neuropeptides S1 and S2.

    Science.gov (United States)

    Jones, Christopher E; Otara, Claire B; Younan, Nadine D; Viles, John H; Elphick, Maurice R

    2014-10-01

    The starfish SALMFamide neuropeptides S1 (GFNSALMFamide) and S2 (SGPYSFNSGLTFamide) are the prototypical members of a family of neuropeptides that act as muscle relaxants in echinoderms. Comparison of the bioactivity of S1 and S2 as muscle relaxants has revealed that S2 is ten times more potent than S1. Here we investigated a structural basis for this difference in potency by comparing the bioactivity and solution conformations (using NMR and CD spectroscopy) of S1 and S2 with three chimeric analogs of these peptides. A peptide comprising S1 with the addition of S2's N-terminal tetrapeptide (Long S1 or LS1; SGPYGFNSALMFamide) was not significantly different to S1 in its bioactivity and did not exhibit concentration-dependent structuring seen with S2. An analog of S1 with its penultimate residue substituted from S2 (S1(T); GFNSALTFamide) exhibited S1-like bioactivity and structure. However, an analog of S2 with its penultimate residue substituted from S1 (S2(M); SGPYSFNSGLMFamide) exhibited loss of S2-type bioactivity and structural properties. Collectively, our data indicate that the C-terminal regions of S1 and S2 are the key determinants of their differing bioactivity. However, the N-terminal region of S2 may influence its bioactivity by conferring structural stability in solution. Thus, analysis of chimeric SALMFamides has revealed how neuropeptide bioactivity is determined by a complex interplay of sequence and conformation.

  5. In silico analysis and modeling of ACP-MIP–PilQ chimeric antigen from Neisseria meningitidis serogroup B

    Science.gov (United States)

    Gholami, Mehrdad; Salimi Chirani, Alireza; Moshiri, Mona; Sedighi, Mansour; Pournajaf, Abazar; Tohidfar, Masoud; Irajian, Gholamreza

    2015-01-01

    Background: Neisseria meningitidis, a life-threatening human pathogen with the potential to cause large epidemics, can be isolated from the nasopharynx of 5–15% of adults. The aim of the current study was to evaluate biophysical and biochemical properties and immunological aspects of chimeric acyl-carrier protein-macrophage infectivity potentiator protein-type IV pilus biogenesis protein antigen (ACP-MIP-PilQ) from N. meningitidis serogroup B strain. Methods: Biochemical properties and multiple alignments were predicted by appropriate web servers. Secondary molecular structures were predicted based on Chou and Fasman, Garnier-Osguthorpe-Robson, and Neural Network methods. Tertiary modeling elucidated conformational properties of the chimeric protein. Proteasome cleavage and transporter associated with antigen processing (TAP) binding sites, and T- and B-cell antigenic epitopes, were predicted using bioinformatic web servers. Results: Based on our in silico and immunoinformatics analyses, the ACP-MIP-PilQ protein (AMP) can induce high-level cross-strain bactericidal activity. In addition, several immune proteasomal cleavage sites were detected. The 22 epitopes associated with MHC class I and class II (DR) alleles were confirmed in the AMP. Thirty linear B-cell epitopes as antigenic regions were predicted from the full-length protein. Conclusion: All predicted properties of the AMP indicate it could be a good candidate for further immunological in vitro and in vivo studies. PMID:26989750

  6. 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)

  7. [Detection of mixed lymphoid chimerism after allogeneic bone marrow transplantation: demonstration by interphase cytogenetics in paraffin-embedded tissue].

    Science.gov (United States)

    Friedrich, T; Ott, G; Kalla, J; Helbig, W; Schwenke, H; Kubel, M; Pönisch, W; Feyer, P; Friedrich, A

    1994-01-01

    In bone marrow transplantation (BMT) the detection of residual host lymphoid or haematopoietic cells surviving conditioning therapy is because of its association to graft-versus-host disease, graft-versus-leukemia reaction, and relapse of leukemia a matter of great interest. We studied the occurrence of this mixed lymphoid chimerism (MC) in the formol-fixed lymphatic tissue of lymph nodes and spleen from 21 autopsies after allogeneic sex-mismatched BMT (5 females, 16 males, survival 5 to 1140 days after BMT). In situ hybridisation with biotinylated centromer-specific anti-X- and anti-Y-chromosome probes was performed on pepsin-digested paraffin sections. The number of double X-, single X-, and Y-chromosome bearing cells was analysed microscopically. Because of artefacts only 14 cases remained for valid investigation. MC was detected in 6 cases (5 out of 11 males 5 days to 840 days and 1 out of 3 females 76 days after BMT). MC occurred after whole body irradiation with 10 Gy (n = 5) and 7 Gy (n = 1). In 1 autopsy relapse of leukemia caused host cell infiltration. Cases with MC did not express histological signs of acute or chronic graft-versus-host disease, but 5 out of 8 with complete lymphoid chimerism did. The sensitivity of interphase cytogenetics on paraffin embedded tissue is low.

  8. Cytoplasmic male sterility-associated chimeric open reading frames identified by mitochondrial genome sequencing of four Cajanus genotypes.

    Science.gov (United States)

    Tuteja, Reetu; Saxena, Rachit K; Davila, Jaime; Shah, Trushar; Chen, Wenbin; Xiao, Yong-Li; Fan, Guangyi; Saxena, K B; Alverson, Andrew J; Spillane, Charles; Town, Christopher; Varshney, Rajeev K

    2013-10-01

    The hybrid pigeonpea (Cajanus cajan) breeding technology based on cytoplasmic male sterility (CMS) is currently unique among legumes and displays major potential for yield increase. CMS is defined as a condition in which a plant is unable to produce functional pollen grains. The novel chimeric open reading frames (ORFs) produced as a results of mitochondrial genome rearrangements are considered to be the main cause of CMS. To identify these CMS-related ORFs in pigeonpea, we sequenced the mitochondrial genomes of three C. cajan lines (the male-sterile line ICPA 2039, the maintainer line ICPB 2039, and the hybrid line ICPH 2433) and of the wild relative (Cajanus cajanifolius ICPW 29). A single, circular-mapping molecule of length 545.7 kb was assembled and annotated for the ICPA 2039 line. Sequence annotation predicted 51 genes, including 34 protein-coding and 17 RNA genes. Comparison of the mitochondrial genomes from different Cajanus genotypes identified 31 ORFs, which differ between lines within which CMS is present or absent. Among these chimeric ORFs, 13 were identified by comparison of the related male-sterile and maintainer lines. These ORFs display features that are known to trigger CMS in other plant species and to represent the most promising candidates for CMS-related mitochondrial rearrangements in pigeonpea.

  9. 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.

  10. 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

  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. Responses of Male C57BL/6N Mice to Observing the Euthanasia of Other Mice.

    Science.gov (United States)

    Boivin, Gregory P; Bottomley, Michael A; Grobe, Nadja

    2016-01-01

    The AVMA Panel on Euthanasia recommends that sensitive animals should not be present during the euthanasia of others, especially of their own species, but does not provide guidelines on how to identify a sensitive species. To determine if mice are a sensitive species we reviewed literature on empathy in mice, and measured the cardiovascular and activity response of mice observing euthanasia of conspecifics. We studied male 16-wk-old C57BL/6N mice and found no increase in cardiovascular parameters or activity in the response of the mice to observing CO2 euthanasia. Mice observing decapitation had an increase in all values, but this was paralleled by a similar increase during mock decapitations in which no animals were handled or euthanized. We conclude that CO2 euthanasia of mice does not have an impact on other mice in the room, and that euthanasia by decapitation likely only has an effect due to the noise of the guillotine. We support the conceptual idea that mice are both a sensitive species and display empathy, but under the controlled circumstances of the euthanasia procedures used in this study there was no signaling of stress to witnessing inhabitants in the room. PMID:27423146

  13. Targeted disruption of the mouse adenine phosphoribosyltransferase (aprt) gene and the production of APRT-deficient mice

    Energy Technology Data Exchange (ETDEWEB)

    Engle, S.J.; Chen, J.; Tischfield, J.A. [Indiana Univ., School of Medicine, Indianapolis, IN (United States)] [and others

    1994-09-01

    Adenine phosphoribosyltransferase (APRT: EC 2.4.2.7), a ubiquitously expressed purine salvage enzyme, catalyzes the synthesis of AMP and inorganic pyrophosphate from existing adenine and 5-phosphoribosyl-1-pyrophosphate. Deficiency of this enzyme in humans results in the accumulation of 2,8-dihydroxyadenine leading to crystalluria and nephrolithiasis. In order to facilitate our study of this rare, autosomal recessive disorder, we applied the advances in gene targeting technology and mouse embryonic stem (ES) cell culture to the production of APRT-deficient mice. A positive-negative targeting strategy was used. The tageting vector contain 5.6 kb of the mouse APRT gene, a neomycin resistance gene in exon 3 as a positive selection marker, and a HSV thymidine ki