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Sample records for chimeric human parainfluenza

  1. Human Parainfluenza Viruses

    Science.gov (United States)

    ... Search Form Controls Cancel Submit Search The CDC Human Parainfluenza Viruses (HPIVs) Note: Javascript is disabled or ... CDC.gov . Recommend on Facebook Tweet Share Compartir Human parainfluenza viruses (HPIVs) commonly cause respiratory illnesses in ...

  2. About Human Parainfluenza Viruses (HPIVs)

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    ... Healthcare Professionals Clinical Overview Laboratory Diagnosis HPIV Seasons Resources & References About Human Parainfluenza Viruses (HPIVs) Recommend on Facebook Tweet Share Compartir Symptoms & Illnesses Lists symptoms and ...

  3. Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge

    International Nuclear Information System (INIS)

    Bukreyev, Alexander; Marzi, Andrea; Feldmann, Friederike; Zhang Liqun; Yang Lijuan; Ward, Jerrold M.; Dorward, David W.; Pickles, Raymond J.; Murphy, Brian R.; Feldmann, Heinz; Collins, Peter L.

    2009-01-01

    We generated a new live-attenuated vaccine against Ebola virus (EBOV) based on a chimeric virus HPIV3/ΔF-HN/EboGP that contains the EBOV glycoprotein (GP) as the sole transmembrane envelope protein combined with the internal proteins of human parainfluenza virus type 3 (HPIV3). Electron microscopy analysis of the virus particles showed that they have an envelope and surface spikes resembling those of EBOV and a particle size and shape resembling those of HPIV3. When HPIV3/ΔF-HN/EboGP was inoculated via apical surface of an in vitro model of human ciliated airway epithelium, the virus was released from the apical surface; when applied to basolateral surface, the virus infected basolateral cells but did not spread through the tissue. Following intranasal (IN) inoculation of guinea pigs, scattered infected cells were detected in the lungs by immunohistochemistry, but infectious HPIV3/ΔF-HN/EboGP could not be recovered from the lungs, blood, or other tissues. Despite the attenuation, the virus was highly immunogenic, and a single IN dose completely protected the animals against a highly lethal intraperitoneal challenge of guinea pig-adapted EBOV

  4. Human and Mouse Eosinophils Have Antiviral Activity against Parainfluenza Virus.

    Science.gov (United States)

    Drake, Matthew G; Bivins-Smith, Elizabeth R; Proskocil, Becky J; Nie, Zhenying; Scott, Gregory D; Lee, James J; Lee, Nancy A; Fryer, Allison D; Jacoby, David B

    2016-09-01

    Respiratory viruses cause asthma exacerbations. Because eosinophils are the prominent leukocytes in the airways of 60-70% of patients with asthma, we evaluated the effects of eosinophils on a common respiratory virus, parainfluenza 1, in the lung. Eosinophils recruited to the airways of wild-type mice after ovalbumin sensitization and challenge significantly decreased parainfluenza virus RNA in the lungs 4 days after infection compared with nonsensitized animals. This antiviral effect was also seen in IL-5 transgenic mice with an abundance of airway eosinophils (NJ.1726) but was lost in transgenic eosinophil-deficient mice (PHIL) and in IL-5 transgenic mice crossed with eosinophil-deficient mice (NJ.1726-PHIL). Loss of the eosinophil granule protein eosinophil peroxidase, using eosinophil peroxidase-deficient transgenic mice, did not reduce eosinophils' antiviral effect. Eosinophil antiviral mechanisms were also explored in vitro. Isolated human eosinophils significantly reduced parainfluenza virus titers. This effect did not involve degradation of viral RNA by eosinophil granule RNases. However, eosinophils treated with a nitric oxide synthase inhibitor lost their antiviral activity, suggesting eosinophils attenuate viral infectivity through production of nitric oxide. Consequently, eosinophil nitric oxide production was measured with an intracellular fluorescent probe. Eosinophils produced nitric oxide in response to virus and to a synthetic agonist of the virus-sensing innate immune receptor, Toll-like receptor (TLR) 7. IFNγ increased expression of eosinophil TLR7 and potentiated TLR7-induced nitric oxide production. These results suggest that eosinophils promote viral clearance in the lung and contribute to innate immune responses against respiratory virus infections in humans.

  5. Identification of a natural human serotype 3 parainfluenza virus

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    Wang Xiao-Jing

    2011-02-01

    Full Text Available Abstract Parainfluenza virus is an important pathogen threatening the health of animals and human, which brings human many kinds of disease, especially lower respiratory tract infection involving infants and young children. In order to control the virus, it is necessary to fully understand the molecular basis resulting in the genetic diversity of the virus. Homologous recombination is one of mechanisms for the rapid change of genetic diversity. However, as a negative-strand virus, it is unknown whether the recombination can naturally take place in human PIV. In this study, we isolated and identified a mosaic serotype 3 human PIV (HPIV3 from in China, and also provided several putative PIV mosaics from previous reports to reveal that the recombination can naturally occur in the virus. In addition, two swine PIV3 isolates transferred from cattle to pigs were found to have mosaic genomes. These results suggest that homologous recombination can promote the genetic diversity and potentially bring some novel biologic characteristics of HPIV.

  6. Attenuated Human Parainfluenza Virus Type 1 Expressing Ebola Virus Glycoprotein GP Administered Intranasally Is Immunogenic in African Green Monkeys.

    Science.gov (United States)

    Lingemann, Matthias; Liu, Xueqiao; Surman, Sonja; Liang, Bo; Herbert, Richard; Hackenberg, Ashley D; Buchholz, Ursula J; Collins, Peter L; Munir, Shirin

    2017-05-15

    The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to develop vaccines against EBOV disease. We describe the development and preclinical evaluation of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the membrane-anchored form of EBOV glycoprotein GP, as an intranasal (i.n.) EBOV vaccine. GP was codon optimized and expressed either as a full-length protein or as an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were replaced with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (C Δ170 ). The constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, replicating at low titers over several days, in the respiratory tract of African green monkeys (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and the TMCT modification did not increase packaging or immunogenicity but rather reduced the stability of GP expression during in vivo replication. In conclusion, we identified an attenuated and immunogenic i.n. vaccine candidate expressing GP from the pre-N position. It is expected to be well tolerated in humans and is available for clinical evaluation. IMPORTANCE EBOV hemorrhagic fever is one of the most lethal viral infections and lacks a licensed vaccine. Contact of fluids from infected individuals, including droplets or aerosols, with mucosal surfaces is an important route of EBOV spread during a natural outbreak, and aerosols also might be exploited for intentional virus spread. Therefore, vaccines that protect

  7. Expression of the Surface Glycoproteins of Human Parainfluenza Virus Type 3 by Bovine Parainfluenza Virus Type 3, a Novel Attenuated Virus Vaccine Vector

    OpenAIRE

    Haller, Aurelia A.; Miller, Tessa; Mitiku, Misrach; Coelingh, Kathleen

    2000-01-01

    Bovine parainfluenza virus type 3 (bPIV3) is being evaluated as an intranasal vaccine for protection against human PIV3 (hPIV3). In young infants, the bPIV3 vaccine appears to be infectious, attenuated, immunogenic, and genetically stable, which are desirable characteristics for an RNA virus vector. To test the potential of the bPIV3 vaccine strain as a vector, an infectious DNA clone of bPIV3 was assembled and recombinant bPIV3 (r-bPIV3) was rescued. r-bPIV3 displayed a temperature-sensitive...

  8. Epidemiology and clinical presentation of the four human parainfluenza virus types

    Directory of Open Access Journals (Sweden)

    Liu Wen-Kuan

    2013-01-01

    Full Text Available Abstract Background Human parainfluenza viruses (HPIVs are important causes of upper respiratory tract illness (URTI and lower respiratory tract illness (LRTI. To analyse epidemiologic and clinical characteristics of the four types of human parainfluenza viruses (HPIVs, patients with acute respiratory tract illness (ARTI were studied in Guangzhou, southern China. Methods Throat swabs (n=4755 were collected and tested from children and adults with ARTI over a 26-month period, and 4447 of 4755 (93.5% patients’ clinical presentations were recorded for further analysis. Results Of 4755 patients tested, 178 (3.7% were positive for HPIV. Ninety-nine (2.1% samples were positive for HPIV-3, 58 (1.2% for HPIV-1, 19 (0.4% for HPIV-2 and 8 (0.2% for HPIV-4. 160/178 (88.9% HPIV-positive samples were from paediatric patients younger than 5 years old, but no infant under one month of age was HPIV positive. Seasonal peaks of HPIV-3 and HPIV-1 occurred as autumn turned to winter and summer turned to autumn. HPIV-2 and HPIV-4 were detected less frequently, and their frequency of isolation increased when the frequency of HPIV-3 and HPIV-1 declined. HPIV infection led to a wide spectrum of symptoms, and more “hoarseness” (p=0.015, “abnormal pulmonary breathing sound” (p Conclusions HPIV infection led to a wide spectrum of symptoms, and similar clinical manifestations were found in the patients with four different types of HPIVs. The study suggested pathogenic activity of HPIV in gastrointestinal illness. The clinical presentation of HPIV infection may differ by patient age.

  9. Identification of Interferon-Stimulated Gene Proteins That Inhibit Human Parainfluenza Virus Type 3.

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    Rabbani, M A G; Ribaudo, Michael; Guo, Ju-Tao; Barik, Sailen

    2016-12-15

    A major arm of cellular innate immunity is type I interferon (IFN), represented by IFN-α and IFN-β. Type I IFN transcriptionally induces a large number of cellular genes, collectively known as IFN-stimulated gene (ISG) proteins, which act as antivirals. The IFIT (interferon-induced proteins with tetratricopeptide repeats) family proteins constitute a major subclass of ISG proteins and are characterized by multiple tetratricopeptide repeats (TPRs). In this study, we have interrogated IFIT proteins for the ability to inhibit the growth of human parainfluenza virus type 3 (PIV3), a nonsegmented negative-strand RNA virus of the Paramyxoviridae family and a major cause of respiratory disease in children. We found that IFIT1 significantly inhibited PIV3, whereas IFIT2, IFIT3, and IFIT5 were less effective or not at all. In further screening a set of ISG proteins we discovered that several other such proteins also inhibited PIV3, including IFITM1, IDO (indoleamine 2,3-dioxygenase), PKR (protein kinase, RNA activated), and viperin (virus inhibitory protein, endoplasmic reticulum associated, interferon inducible)/Cig5. The antiviral effect of IDO, the enzyme that catalyzes the first step of tryptophan degradation, could be counteracted by tryptophan. These results advance our knowledge of diverse ISG proteins functioning as antivirals and may provide novel approaches against PIV3. The innate immunity of the host, typified by interferon (IFN), is a major antiviral defense. IFN inhibits virus growth by inducing a large number of IFN-stimulated gene (ISG) proteins, several of which have been shown to have specific antiviral functions. Parainfluenza virus type 3 (PIV3) is major pathogen of children, and no reliable vaccine or specific antiviral against it currently exists. In this article, we report several ISG proteins that strongly inhibit PIV3 growth, the use of which may allow a better antiviral regimen targeting PIV3. Copyright © 2016, American Society for Microbiology

  10. Modeling cognition and disease using human glial chimeric mice

    DEFF Research Database (Denmark)

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

    2015-01-01

    , oligodendrocytes as well. As a result, the recipient brains may become inexorably humanized with regards to their resident glial populations, yielding human glial chimeric mouse brains. These brains provide us a fundamentally new tool by which to assess the species-specific attributes of glia in modulating human...... for studying the human-specific contributions of glia to psychopathology, as well as to higher cognition. As such, the assessment of human glial chimeric mice may provide us new insight into the species-specific contributions of glia to human cognitive evolution, as well as to the pathogenesis of human...

  11. Prevalence of Human Parainfluenza Viruses and Noroviruses Genomes on Office Fomites.

    Science.gov (United States)

    Stobnicka, Agata; Gołofit-Szymczak, Małgorzata; Wójcik-Fatla, Angelina; Zając, Violetta; Korczyńska-Smolec, Joanna; Górny, Rafał L

    2018-06-01

    The aim of this study was to evaluate the potential role of office fomites in respiratory (human parainfluenza virus 1-HPIV1, human parainfluenza virus 3-HPIV3) and enteric (norovirus GI-NoV GI, norovirus GII-NoV GII) viruses transmission by assessing the occurrence of these viruses on surfaces in office buildings. Between 2016 and 2017, a total of 130 surfaces from open-space and non-open-space rooms in office buildings located in one city were evaluated for HPIV1, HPIV3, NoV GI, and NoV GII viral RNA presence. Detection of viruses was performed by RT-qPCR method. Study revealed 27 positive samples, among them 59.3% were HPIV3-positive, 25.9% HPIV1-positive, and 14.8% NoV GII-positive. All tested surfaces were NoV GI-negative. Statistical analysis of obtained data showed that the surfaces of office equipment including computer keyboards and mice, telephones, and desktops were significantly more contaminated with respiratory viruses than the surfaces of building equipment elements such as door handles, light switches, or ventilation tracts (χ 2 p = 0.006; Fisher's Exact p = 0.004). All examined surfaces were significantly more contaminated with HPIVs than NoVs (χ 2 p = 0.002; Fisher's Exact p = 0.003). Office fomites in open-space rooms were more often contaminated with HPIVs than with NoVs (χ 2 p = 0.016; Fisher's Exact p = 0.013). The highest average concentration of HPIVs RNA copies was observed on telephones (1.66 × 10 2 copies/100 cm 2 ), while NoVs on the light switches (1.40 × 10 2 copies/100 cm 2 ). However, the Kruskal-Wallis test did not show statistically significant differences in concentration levels of viral RNA copies on surfaces between the all tested samples. This study unequivocally showed that individuals in office environment may have contact with both respiratory and enteric viral particles present on frequently touched surfaces.

  12. A study of genetic variability of human parainfluenza virus type 1 in Croatia, 2011-2014.

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    Košutić-Gulija, Tanja; Slovic, Anamarija; Ljubin-Sternak, Sunčanica; Mlinarić-Galinović, Gordana; Forčić, Dubravko

    2016-08-01

    Molecular epidemiology of human parainfluenza viruses type 1 (HPIV1) was investigated. Samples were collected from patients hospitalized in Croatia during the three consecutive epidemic seasons (2011-2014). Results indicated co-circulation of two major genetic clusters of HPIV1. Samples from the current study refer to clades II and III in a phylogenetic tree of haemagglutinin-neuraminidase (HN) gene. Additional phylogenetic trees of fusion (F) and phosphoprotein (P) genes confirmed the topology. Analysis of nucleotide diversity of entire P, F and HN genes demonstrated similar values: 0.0255, 0.0236 and 0.0237, respectively. However, amino acid diversity showed F protein to be the most conserved, while P protein was the most tolerant to mutations. Potential N- and O-glycosylation sites suggested that HPIV1 HN protein is abundantly glycosylated, and a specific N-glycosylation pattern could distinguish between clades II and III. Analysis of potential O-glycosylation sites in F protein indicated that samples from this study have two potential O-glycosylation sites, while publicly available sequences have five potential sites. This study provides data on the molecular characterization and epidemic pattern of HPIV1 in Croatia.

  13. [Detection and Analysis of Human Parainfluenza Virus Infection in Hospitalized Adults with Acute Respiratory Tract Infections].

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    Li, Xing-Qiao; Liu, Xue-Wei; Zhou, Tao; Pei, Xiao-Fang

    2017-11-01

    To investigate the prevalence and gene characteristics of different groups of human parainfluenza virus (HPIV) infection in hospitalized adults with acute respiratory tract infections (ARI). RT-PCR was used to detect HPIV hemagglutinin (HA) DNA,which was extracted from sputum samples of 1 039 adult patients with ARI from March,2014 to June,2016. The HA gene amplified from randomly selected positive samples were sequenced to analyze the homology and variation. 10.6% (110/1 039) of these samples were positive for HPIV,including 8 cases of HPIV-1,22 cases of HPIV-2,46 cases of HPIV-3 and 34 cases of HPIV-4. Detectable rate varied among different groups of HPIV according to seasons of the year and ages of patients. No significant differences were found between the positive samples and the reference sequences. Compared with different reference strains of different regions,the genetic distance of nucleotide is the smallest between the strains tested in this study and the reference strains of other provinces and cities in China. In Chengdu region,HPIV virus is highly detected in ARI,all subtypes were detected with HPIV-3 being the main subtype.

  14. Haemophilus parainfluenzae urethritis among homosexual men.

    Science.gov (United States)

    Hsu, Meng-Shiuan; Wu, Mei-Yu; Lin, Tsui-Hsien; Liao, Chun-Hsing

    2015-08-01

    Haemophilus parainfluenzae is a common inhabitant of the human upper respiratory tract of the normal oral microflora. We report three men who had been having unprotected sex with men (MSM) and subsequently acquired H. parainfluenzae urethritis, which was confirmed by 16S rRNA gene sequencing analysis. Two men were treated with ceftriaxone and doxycycline, and the third man was treated with clarithromycin. All three patients responded to treatment. This case series highlights the potential role of H. parainfluenzae as a sexually transmitted genitourinary pathogen. Copyright © 2012. Published by Elsevier B.V.

  15. 5-Hydroxytryptophan, a major product of tryptophan degradation, is essential for optimal replication of human parainfluenza virus.

    Science.gov (United States)

    Rabbani, M A G; Barik, Sailen

    2017-03-01

    Interferon (IFN) exerts its antiviral effect by inducing a large family of cellular genes, named interferon (IFN)-stimulated genes (ISGs). An intriguing member of this family is indoleamine 2,3-dioxygenase (IDO), which catalyzes the first and rate-limiting step of the main branch of tryptophan (Trp) degradation, the kynurenine pathway. We recently showed that IDO strongly inhibits human parainfluenza virus type 3 (PIV3), a significant respiratory pathogen. Here, we show that 5-hydoxytryptophan (5-HTP), the first product of an alternative branch of Trp degradation and a serotonin precursor, is essential to protect virus growth against IDO in cell culture. We also show that the apparent antiviral effect of IDO on PIV3 is not due to the generation of the kynurenine pathway metabolites, but rather due to the depletion of intracellular Trp by IDO, as a result of which this rare amino acid becomes unavailable for the alternative, proviral 5-HTP pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. In vitro and in vivo properties of human/mouse chimeric monoclonal antibody specific for common acute lymphocytic leukemia antigen

    International Nuclear Information System (INIS)

    Saga, T.; Endo, K.; Koizumi, M.; Kawamura, Y.; Watanabe, Y.; Konishi, J.; Ueda, R.; Nishimura, Y.; Yokoyama, M.; Watanabe, T.

    1990-01-01

    A human/mouse chimeric monoclonal antibody specific for a common acute lymphocytic leukemia antigen was efficiently obtained by ligating human heavy-chain enhancer element to the chimeric heavy- and light-chain genes. Cell binding and competitive inhibition assays of both radioiodine and indium-111- (111In) labeled chimeric antibodies demonstrated in vitro immunoreactivity identical with that of the parental murine monoclonal antibodies. The biodistribution of the radiolabeled chimeric antibody in tumor-bearing nude mice was similar to that of the parental murine antibody. Tumor accumulation of radioiodinated parental and chimeric antibodies was lower than that of 111 In-labeled antibodies, probably because of dehalogenation of the radioiodinated antibodies. Indium-111-labeled chimeric antibody clearly visualized xenografted tumor. These results suggest that a human/mouse chimeric antibody can be labeled with 111 In and radioiodine without the loss of its immunoreactivity, and that chimeric antibody localizes in vivo in the same way as the parental murine antibody

  17. Chimeric animal models in human stem cell biology.

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    Glover, Joel C; Boulland, Jean-Luc; Halasi, Gabor; Kasumacic, Nedim

    2009-01-01

    The clinical use of stem cells for regenerative medicine is critically dependent on preclinical studies in animal models. In this review we examine some of the key issues and challenges in the use of animal models to study human stem cell biology-experimental standardization, body size, immunological barriers, cell survival factors, fusion of host and donor cells, and in vivo imaging and tracking. We focus particular attention on the various imaging modalities that can be used to track cells in living animals, comparing their strengths and weaknesses and describing technical developments that are likely to lead to new opportunities for the dynamic assessment of stem cell behavior in vivo. We then provide an overview of some of the most commonly used animal models, their advantages and disadvantages, and examples of their use for xenotypic transplantation of human stem cells, with separate reviews of models involving rodents, ungulates, nonhuman primates, and the chicken embryo. As the use of human somatic, embryonic, and induced pluripotent stem cells increases, so too will the range of applications for these animal models. It is likely that increasingly sophisticated uses of human/animal chimeric models will be developed through advances in genetic manipulation, cell delivery, and in vivo imaging.

  18. An Amino Acid of Human Parainfluenza Virus Type 3 Nucleoprotein Is Critical for Template Function and Cytoplasmic Inclusion Body Formation

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    Zhang, Shengwei; Chen, Longyun; Zhang, Guangyuan; Yan, Qin; Yang, Xiaodan; Ding, Binbin; Tang, Qiaopeng; Sun, Shengjun; Hu, Zhulong

    2013-01-01

    The nucleoprotein (N) and phosphoprotein (P) interaction of nonsegmented negative-strand RNA viruses is essential for viral replication; this includes N0-P (N0, free of RNA) interaction and the interaction of N-RNA with P. The precise site(s) within N that mediates the N-P interaction and the detailed regulating mechanism, however, are less clear. Using a human parainfluenza virus type 3 (HPIV3) minigenome assay, we found that an N mutant (NL478A) did not support reporter gene expression. Using in vivo and in vitro coimmunoprecipitation, we found that NL478A maintains the ability to form NL478A0-P, to self-assemble, and to form NL478A-RNA but that NL478A-RNA does not interact with P. Using an immunofluorescence assay, we found that N-P interaction provides the minimal requirement for the formation of cytoplasmic inclusion bodies, which contain viral RNA, N, P, and polymerase in HPIV3-infected cells. NL478A was unable to form inclusion bodies when coexpressed with P, but the presence of N rescued the ability of NL478A to form inclusion bodies and the transcriptional function of NL478A, thereby suggesting that hetero-oligomers formed by N and NL478A are functional and competent to form inclusion bodies. Furthermore, we found that NL478A is also defective in virus growth. To our knowledge, we are the first to use a paramyxovirus to identify a precise amino acid within N that is critical for N-RNA and P interaction but not for N0-P interaction for the formation of inclusion bodies, which appear to be bona fide sites of RNA synthesis. PMID:24027324

  19. Application of chimeric mice with humanized liver for study of human-specific drug metabolism.

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    Bateman, Thomas J; Reddy, Vijay G B; Kakuni, Masakazu; Morikawa, Yoshio; Kumar, Sanjeev

    2014-06-01

    Human-specific or disproportionately abundant human metabolites of drug candidates that are not adequately formed and qualified in preclinical safety assessment species pose an important drug development challenge. Furthermore, the overall metabolic profile of drug candidates in humans is an important determinant of their drug-drug interaction susceptibility. These risks can be effectively assessed and/or mitigated if human metabolic profile of the drug candidate could reliably be determined in early development. However, currently available in vitro human models (e.g., liver microsomes, hepatocytes) are often inadequate in this regard. Furthermore, the conduct of definitive radiolabeled human ADME studies is an expensive and time-consuming endeavor that is more suited for later in development when the risk of failure has been reduced. We evaluated a recently developed chimeric mouse model with humanized liver on uPA/SCID background for its ability to predict human disposition of four model drugs (lamotrigine, diclofenac, MRK-A, and propafenone) that are known to exhibit human-specific metabolism. The results from these studies demonstrate that chimeric mice were able to reproduce the human-specific metabolite profile for lamotrigine, diclofenac, and MRK-A. In the case of propafenone, however, the human-specific metabolism was not detected as a predominant pathway, and the metabolite profiles in native and humanized mice were similar; this was attributed to the presence of residual highly active propafenone-metabolizing mouse enzymes in chimeric mice. Overall, the data indicate that the chimeric mice with humanized liver have the potential to be a useful tool for the prediction of human-specific metabolism of xenobiotics and warrant further investigation.

  20. Recurrent chimeric RNAs enriched in human prostate cancer identified by deep sequencing

    Science.gov (United States)

    Kannan, Kalpana; Wang, Liguo; Wang, Jianghua; Ittmann, Michael M.; Li, Wei; Yen, Laising

    2011-01-01

    Transcription-induced chimeric RNAs, possessing sequences from different genes, are expected to increase the proteomic diversity through chimeric proteins or altered regulation. Despite their importance, few studies have focused on chimeric RNAs especially regarding their presence/roles in human cancers. By deep sequencing the transcriptome of 20 human prostate cancer and 10 matched benign prostate tissues, we obtained 1.3 billion sequence reads, which led to the identification of 2,369 chimeric RNA candidates. Chimeric RNAs occurred in significantly higher frequency in cancer than in matched benign samples. Experimental investigation of a selected 46 set led to the confirmation of 32 chimeric RNAs, of which 27 were highly recurrent and previously undescribed in prostate cancer. Importantly, a subset of these chimeras was present in prostate cancer cell lines, but not detectable in primary human prostate epithelium cells, implying their associations with cancer. These chimeras contain discernable 5′ and 3′ splice sites at the RNA junction, indicating that their formation is mediated by splicing. Their presence is also largely independent of the expression of parental genes, suggesting that other factors are involved in their production and regulation. One chimera, TMEM79-SMG5, is highly differentially expressed in human cancer samples and therefore a potential biomarker. The prevalence of chimeric RNAs may allow the limited number of human genes to encode a substantially larger number of RNAs and proteins, forming an additional layer of cellular complexity. Together, our results suggest that chimeric RNAs are widespread, and increased chimeric RNA events could represent a unique class of molecular alteration in cancer. PMID:21571633

  1. Inhibition of Primary Clinical Isolates of Human Parainfluenza Virus by DAS181 in Cell Culture and in a Cotton Rat Model

    OpenAIRE

    Jones, B. G.; Hayden, R.T.; Hurwitz, J. L.

    2013-01-01

    DAS181 is a novel drug in development for the treatment of influenza as well as human parainfluenza viruses (hPIV). Previous studies demonstrated that DAS181 inhibited laboratory strains of hPIV, but no tests were conducted with primary clinical isolates of hPIV. To fill this gap, we studied six primary isolates including hPIV-2 and hPIV-3. First tests showed that the amplification of all viruses in vitro was reproducibly inhibited with DAS181 drug concentrations ranging between 0.1 and 1 nM....

  2. Current status of prediction of drug disposition and toxicity in humans using chimeric mice with humanized liver.

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    Kitamura, Shigeyuki; Sugihara, Kazumi

    2014-01-01

    1. Human-chimeric mice with humanized liver have been constructed by transplantation of human hepatocytes into several types of mice having genetic modifications that injure endogenous liver cells. Here, we focus on liver urokinase-type plasminogen activator-transgenic severe combined immunodeficiency (uPA/SCID) mice, which are the most widely used human-chimeric mice. Studies so far indicate that drug metabolism, drug transport, pharmacological effects and toxicological action in these mice are broadly similar to those in humans. 2. Expression of various drug-metabolizing enzymes is known to be different between humans and rodents. However, the expression pattern of cytochrome P450, aldehyde oxidase and phase II enzymes in the liver of human-chimeric mice resembles that in humans, not that in the host mice. 3. Metabolism of various drugs, including S-warfarin, zaleplon, ibuprofen, naproxen, coumarin, troglitazone and midazolam, in human-chimeric mice is mediated by human drug-metabolizing enzymes, not by host mouse enzymes, and thus resembles that in humans. 4. Pharmacological and toxicological effects of various drugs in human-chimeric mice are also similar to those in humans. 5. The current consensus is that chimeric mice with humanized liver are useful to predict drug metabolism catalyzed by cytochrome P450, aldehyde oxidase and phase II enzymes in humans in vivo and in vitro. Some remaining issues are discussed in this review.

  3. Protein chimerism: novel source of protein diversity in humans adds complexity to bottom-up proteomics.

    Science.gov (United States)

    Casado-Vela, Juan; Lacal, Juan Carlos; Elortza, Felix

    2013-01-01

    Three main molecular mechanisms are considered to contribute expanding the repertoire and diversity of proteins present in living organisms: first, at DNA level (gene polymorphisms and single nucleotide polymorphisms); second, at messenger RNA (pre-mRNA and mRNA) level including alternative splicing (also termed differential splicing or cis-splicing); finally, at the protein level mainly driven through PTM and specific proteolytic cleavages. Chimeric mRNAs constitute an alternative source of protein diversity, which can be generated either by chromosomal translocations or by trans-splicing events. The occurrence of chimeric mRNAs and proteins is a frequent event in cells from the immune system and cancer cells, mainly as a consequence of gene rearrangements. Recent reports support that chimeric proteins may also be expressed at low levels under normal physiological circumstances, thus, representing a novel source of protein diversity. Notably, recent publications demonstrate that chimeric protein products can be successfully identified through bottom-up proteomic analyses. Several questions remain unsolved, such as the physiological role and impact of such chimeric proteins or the potential occurrence of chimeric proteins in higher eukaryotic organisms different from humans. The occurrence of chimeric proteins certainly seems to be another unforeseen source of complexity for the proteome. It may be a process to take in mind not only when performing bottom-up proteomic analyses in cancer studies but also in general bottom-up proteomics experiments. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Alix serves as an adaptor that allows human parainfluenza virus type 1 to interact with the host cell ESCRT system.

    Directory of Open Access Journals (Sweden)

    Jim Boonyaratanakornkit

    Full Text Available The cellular ESCRT (endosomal sorting complex required for transport system functions in cargo-sorting, in the formation of intraluminal vesicles that comprise multivesicular bodies (MVB, and in cytokinesis, and this system can be hijacked by a number of enveloped viruses to promote budding. The respiratory pathogen human parainfluenza virus type I (HPIV1 encodes a nested set of accessory C proteins that play important roles in down-regulating viral transcription and replication, in suppressing the type I interferon (IFN response, and in suppressing apoptosis. Deletion or mutation of the C proteins attenuates HPIV1 in vivo, and such mutants are being evaluated preclinically and clinically as vaccines. We show here that the C proteins interact and co-localize with the cellular protein Alix, which is a member of the class E vacuolar protein sorting (Vps proteins that assemble at endosomal membranes into ESCRT complexes. The HPIV1 C proteins interact with the Bro1 domain of Alix at a site that is also required for the interaction between Alix and Chmp4b, a subunit of ESCRT-III. The C proteins are ubiquitinated and subjected to proteasome-mediated degradation, but the interaction with AlixBro1 protects the C proteins from degradation. Neither over-expression nor knock-down of Alix expression had an effect on HPIV1 replication, although this might be due to the large redundancy of Alix-like proteins. In contrast, knocking down the expression of Chmp4 led to an approximately 100-fold reduction in viral titer during infection with wild-type (WT HPIV1. This level of reduction was similar to that observed for the viral mutant, P(C- HPIV1, in which expression of the C proteins were knocked out. Chmp4 is capable of out-competing the HPIV1 C proteins for binding Alix. Together, this suggests a possible model in which Chmp4, through Alix, recruits the C proteins to a common site on intracellular membranes and facilitates budding.

  5. The biological characteristics of anti-CD71 mouse/human chimeric antibody

    International Nuclear Information System (INIS)

    Wang Shuo; Jiang Lin; Lei Ping; Zhu Huifen; Shen Guanxin; Cui Wuren; Wang Yanggong

    2002-01-01

    Objective: To study the biological characteristics of an anti-CD71 mouse/human chimeric antibody (D2C). Methods: Analysis of the chimeric Ab production in culture supernatant was made by the standard concentration curve method with ELISA. The antibody was purified by DEAE-Sephredax-A50 ion-exchange chromatography and was confirmed by SDS-PAGE. The competition inhibition studies for binding to the same epitope on CD71 were performed between the chimeric Ab(D2C) in the culture supernatant was about 0.5-5 μg/ml in 5-day cultures when seeded at 1 x 10 5 cells/5ml compared with 12.5-25 μg/ml in the supernatant from their parental monoclonal Ab(7579). The purified chimeric Ab(D2C) from mouse ascetics was 1-2 mg/ml. The SDS-PAGE analysis of purified chimeric Ab(D2C) with discontinuous system confirmed two protein bands of 55 kDa and 25 kDa. It was clear that both chimeric Ab(D2C) and murine monoclonal Ab (7579) compete effectively to join the same epitope of CD71 each other. The chimeric antibody's affinity constant (Ka), quantitated by Scatchard analysis, is about 9.34-9.62 x 10 9 L/mol. Conclusion: The chimeric Ab(D2C) produced from the transfectomas is stable. The binding capacity of the chimeric Ab(D2C) to the antigen (CD71) was retained

  6. Identification of Metabolism and Excretion Differences of Procymidone between Rats and Humans Using Chimeric Mice: Implications for Differential Developmental Toxicity.

    Science.gov (United States)

    Abe, Jun; Tomigahara, Yoshitaka; Tarui, Hirokazu; Omori, Rie; Kawamura, Satoshi

    2018-02-28

    A metabolite of procymidone, hydroxylated-PCM, causes rat-specific developmental toxicity due to higher exposure to it in rats than in rabbits or monkeys. When procymidone was administered to chimeric mice with rat or human hepatocytes, the plasma level of hydroxylated-PCM was higher than that of procymidone in rat chimeric mice, and the metabolic profile of procymidone in intact rats was well reproduced in rat chimeric mice. In human chimeric mice, the plasma level of hydroxylated-PCM was less, resulting in a much lower exposure. The main excretion route of hydroxylated-PCM-glucuronide was bile (the point that hydroxylated-PCM enters the enterohepatic circulation) in rat chimeric mice, and urine in human chimeric mice. These data suggest that humans, in contrast to rats, extensively form the glucuronide and excrete it in urine, as do rabbits and monkeys. Overall, procymidone's potential for causing teratogenicity in humans must be low compared to that in rats.

  7. 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...... 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...

  8. Rats and mice immunised with chimeric human/mouse proteinase 3 produce autoantibodies to mouse Pr3 and rat granulocytes

    NARCIS (Netherlands)

    van der Geld, Ymke M.; Hellmark, Thomas; Selga, Daina; Heeringa, Peter; Huitema, Minke G.; Limburg, Pieter C.; Kallenberg, Cees G. M.

    2007-01-01

    Aim: In this study, we employed chimeric human/ mouse Proteinase 3 ( PR3) proteins as tools to induce an autoantibody response to PR3 in rats and mice. Method: Rats and mice were immunised with recombinant human PR3 ( HPR3), recombinant murine PR3 ( mPR3), single chimeric human/ mouse PR3 ( HHm,

  9. Chimeric mice transplanted with human hepatocytes as a model for prediction of human drug metabolism and pharmacokinetics.

    Science.gov (United States)

    Sanoh, Seigo; Ohta, Shigeru

    2014-03-01

    Preclinical studies in animal models are used routinely during drug development, but species differences of pharmacokinetics (PK) between animals and humans have to be taken into account in interpreting the results. Human hepatocytes are also widely used to examine metabolic activities mediated by cytochrome P450 (P450) and other enzymes, but such in vitro metabolic studies also have limitations. Recently, chimeric mice with humanized liver (h-chimeric mice), generated by transplantation of human donor hepatocytes, have been developed as a model for the prediction of metabolism and PK in humans, using both in vitro and in vivo approaches. The expression of human-specific metabolic enzymes and metabolic activities was confirmed in humanized liver of h-chimeric mice with high replacement ratios, and several reports indicate that the profiles of P450 and non-P450 metabolism in these mice adequately reflect those in humans. Further, the combined use of h-chimeric mice and r-chimeric mice, in which endogenous hepatocytes are replaced with rat hepatocytes, is a promising approach for evaluation of species differences in drug metabolism. Recent work has shown that data obtained in h-chimeric mice enable the semi-quantitative prediction of not only metabolites, but also PK parameters, such as hepatic clearance, of drug candidates in humans, although some limitations remain because of differences in the metabolic activities, hepatic blood flow and liver structure between humans and mice. In addition, fresh h-hepatocytes can be isolated reproducibly from h-chimeric mice for metabolic studies. Copyright © 2013 John Wiley & Sons, Ltd.

  10. Rhabdomyolysis Associated with Parainfluenza Virus

    Directory of Open Access Journals (Sweden)

    Miltiadis Douvoyiannis

    2013-01-01

    Full Text Available Influenza virus is the most frequently reported viral cause of rhabdomyolysis. A 7-year-old child is presented with rhabdomyolysis associated with parainfluenza type 2 virus. Nine cases of rhabdomyolysis associated with parainfluenza virus have been reported. Complications may include electrolyte disturbances, acute renal failure, and compartment syndrome.

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

    Science.gov (United States)

    Wu, Jun; Izpisua Belmonte, Juan Carlos

    2016-06-01

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

  12. Development of Murine Cyp3a Knockout Chimeric Mice with Humanized Liver.

    Science.gov (United States)

    Kato, Kota; Ohbuchi, Masato; Hamamura, Satoko; Ohshita, Hiroki; Kazuki, Yasuhiro; Oshimura, Mitsuo; Sato, Koya; Nakada, Naoyuki; Kawamura, Akio; Usui, Takashi; Kamimura, Hidetaka; Tateno, Chise

    2015-08-01

    We developed murine CYP3A knockout ko chimeric mice with humanized liver expressing human P450S similar to those in humans and whose livers and small intestines do not express murine CYP3A this: approach may overcome effects of residual mouse metabolic enzymes like Cyp3a in conventional chimeric mice with humanized liver, such as PXB-mice [urokinase plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice repopulated with over 70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes were transplanted into Cyp3a KO/uPA/SCID host mice, human albumin levels logarithmically increased until approximately 60 days after transplantation, findings similar to those in PXB-mice. Quantitative real-time-polymerase chain reaction analyses showed that hepatic human P450s, UGTs, SULTs, and transporters mRNA expression levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression in mouse liver and intestine. Findings for midazolam and triazolam metabolic activities in liver microsomes were comparable between Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a, hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplants) were 8.4- and 61-fold upregulated compared with PXB-mice, respectively. However, human hepatocyte transplantation successfully restored Cyp2b10 level nearly fully and Cyp2c55 level partly (still 13-fold upregulated) compared with those in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by human hepatocyte transplantation in Cyp3a KO chimeric mice. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  13. Targeted induction of interferon-λ in humanized chimeric mouse liver abrogates hepatotropic virus infection.

    Science.gov (United States)

    Nakagawa, Shin-ichiro; Hirata, Yuichi; Kameyama, Takeshi; Tokunaga, Yuko; Nishito, Yasumasa; Hirabayashi, Kazuko; Yano, Junichi; Ochiya, Takahiro; Tateno, Chise; Tanaka, Yasuhito; Mizokami, Masashi; Tsukiyama-Kohara, Kyoko; Inoue, Kazuaki; Yoshiba, Makoto; Takaoka, Akinori; Kohara, Michinori

    2013-01-01

    The interferon (IFN) system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV) and hepatitis B virus (HBV). This study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC). Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs) in the livers and sera of these humanized chimeric mice. Following treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level) of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-β in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic) tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1), suggesting dual recognition of LIC-pIC by both sensor adaptor pathways. These findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.

  14. Targeted induction of interferon-λ in humanized chimeric mouse liver abrogates hepatotropic virus infection.

    Directory of Open Access Journals (Sweden)

    Shin-ichiro Nakagawa

    Full Text Available BACKGROUND & AIMS: The interferon (IFN system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV and hepatitis B virus (HBV. METHODS: This study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC. Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs in the livers and sera of these humanized chimeric mice. RESULTS: Following treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-β in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1, suggesting dual recognition of LIC-pIC by both sensor adaptor pathways. CONCLUSIONS: These findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.

  15. Chimeric Human Skin Substitute Tissue: A Novel Treatment Option for the Delivery of Autologous Keratinocytes.

    Science.gov (United States)

    Rasmussen, Cathy A; Allen-Hoffmann, B Lynn

    2012-04-01

    For patients suffering from catastrophic burns, few treatment options are available. Chimeric coculture of patient-derived autologous cells with a "carrier" cell source of allogeneic keratinocytes has been proposed as a means to address the complex clinical problem of severe skin loss. Currently, autologous keratinocytes are harvested, cultured, and expanded to form graftable epidermal sheets. However, epidermal sheets are thin, are extremely fragile, and do not possess barrier function, which only develops as skin stratifies and matures. Grafting is typically delayed for up to 4 weeks to propagate a sufficient quantity of the patient's cells for application to wound sites. Fully stratified chimeric bioengineered skin substitutes could not only provide immediate wound coverage and restore barrier function, but would simultaneously deliver autologous keratinocytes to wounds. The ideal allogeneic cell source for this application would be an abundant supply of clinically evaluated, nontumorigenic, pathogen-free, human keratinocytes. To evaluate this potential cell-based therapy, mixed populations of a green fluorescent protein-labeled neonatal human keratinocyte cell line (NIKS) and unlabeled primary keratinocytes were used to model the allogeneic and autologous components of chimeric monolayer and organotypic cultures. Relatively few autologous keratinocytes may be required to produce fully stratified chimeric skin substitute tissue substantially composed of autologous keratinocyte-derived regions. The need for few autologous cells interspersed within an allogeneic "carrier" cell population may decrease cell expansion time, reducing the time to patient application. This study provides proof of concept for utilizing NIKS keratinocytes as the allogeneic carrier for the generation of bioengineered chimeric skin substitute tissues capable of providing immediate wound coverage while simultaneously supplying autologous human cells for tissue regeneration.

  16. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Mice with chimeric livers are an improved model for human lipoprotein metabolism.

    Science.gov (United States)

    Ellis, Ewa C S; Naugler, Willscott Edward; Nauglers, Scott; Parini, Paolo; Mörk, Lisa-Mari; Jorns, Carl; Zemack, Helen; Sandblom, Anita Lövgren; Björkhem, Ingemar; Ericzon, Bo-Göran; Wilson, Elizabeth M; Strom, Stephen C; Grompe, Markus

    2013-01-01

    Rodents are poor model for human hyperlipidemias because total cholesterol and low density lipoprotein levels are very low on a normal diet. Lipoprotein metabolism is primarily regulated by hepatocytes and we therefore assessed whether chimeric mice extensively repopulated with human cells can model human lipid and bile acid metabolism. FRG [ F ah(-/-) R ag2(-/-)Il2r g (-/-)]) mice were repopulated with primary human hepatocytes. Serum lipoprotein lipid composition and distribution (VLDL, LDL, and HDL) was analyzed by size exclusion chromatography. Bile was analyzed by LC-MS or by GC-MS. RNA expression levels were measured by quantitative RT-PCR. Chimeric mice displayed increased LDL and VLDL fractions and a lower HDL fraction compared to wild type, thus significantly shifting the ratio of LDL/HDL towards a human profile. Bile acid analysis revealed a human-like pattern with high amounts of cholic acid and deoxycholic acid (DCA). Control mice had only taurine-conjugated bile acids as expcted, but highly repopulated mice had glycine-conjugated cholic acid as found in human bile. RNA levels of human genes involved in bile acid synthesis including CYP7A1, and CYP27A1 were significantly upregulated as compared to human control liver. However, administration of recombinant hFGF19 restored human CYP7A1 levels to normal. Humanized-liver mice showed a typical human lipoprotein profile with LDL as the predominant lipoprotein fraction even on a normal diet. The bile acid profile confirmed presence of an intact enterohepatic circulation. Although bile acid synthesis was deregulated in this model, this could be fully normalized by FGF19 administration. Taken together these data indicate that chimeric FRG-mice are a useful new model for human lipoprotein and bile-acid metabolism.

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

    International Nuclear Information System (INIS)

    Yamamura, Miyuki; Hinoda, Yuji; Sasaki, Shigeru; Tsujisaki, Masayuki; Imai, Kohzoh; Oriuchi, Noboru; Endo, Keigo.

    1996-01-01

    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,κ). 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 γ1 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 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)

  19. Vaccinia virus recombinants expressing chimeric proteins of human immunodeficiency virus and gamma interferon are attenuated for nude mice.

    OpenAIRE

    Giavedoni, L D; Jones, L; Gardner, M B; Gibson, H L; Ng, C T; Barr, P J; Yilma, T

    1992-01-01

    We have developed a method for attenuating vaccinia virus recombinants by expressing a fusion protein of a lymphokine and an immunogen. Chimeric genes were constructed that coded for gamma interferon (IFN-gamma) and structural proteins of the human immunodeficiency virus type 1 (HIV-1). In this study, we describe the biological and immunological properties of vaccinia virus recombinants expressing chimeric genes of murine or human IFN-gamma with glycoprotein gp120, gag, and a fragment of gp41...

  20. Safety and immunogenicity of an intranasal Sendai virus-based human parainfluenza virus type 1 vaccine in 3- to 6-year-old children.

    Science.gov (United States)

    Adderson, Elisabeth; Branum, Kristen; Sealy, Robert E; Jones, Bart G; Surman, Sherri L; Penkert, Rhiannon; Freiden, Pamela; Slobod, Karen S; Gaur, Aditya H; Hayden, Randall T; Allison, Kim; Howlett, Nanna; Utech, Jill; Allay, Jim; Knight, James; Sleep, Susan; Meagher, Michael M; Russell, Charles J; Portner, Allen; Hurwitz, Julia L

    2015-03-01

    Human parainfluenza virus type 1 (hPIV-1) is the most common cause of laryngotracheobronchitis (croup), resulting in tens of thousands of hospitalizations each year in the United States alone. No licensed vaccine is yet available. We have developed murine PIV-1 (Sendai virus [SeV]) as a live Jennerian vaccine for hPIV-1. Here, we describe vaccine testing in healthy 3- to 6-year-old hPIV-1-seropositive children in a dose escalation study. One dose of the vaccine (5 × 10(5), 5 × 10(6), or 5 × 10(7) 50% egg infectious doses) was delivered by the intranasal route to each study participant. The vaccine was well tolerated by all the study participants. There was no sign of vaccine virus replication in the airway in any participant. Most children exhibited an increase in antibody binding and neutralizing responses toward hPIV-1 within 4 weeks from the time of vaccination. In several children, antibody responses remained above incoming levels for at least 6 months after vaccination. Data suggest that SeV may provide a benefit to 3- to 6-year-old children, even when vaccine recipients have preexisting cross-reactive antibodies due to previous exposures to hPIV-1. Results encourage the testing of SeV administration in young seronegative children to protect against the serious respiratory tract diseases caused by hPIV-1 infections. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. Human parainfluenza virus type 2 hemagglutinin-neuramindase gene: sequence and phylogenetic analysis of the Saudi strain Riyadh 105/2009

    Directory of Open Access Journals (Sweden)

    Almajhdi Fahad N

    2012-12-01

    Full Text Available Abstract Background Although human parainfluenza type 2 (HPIV-2 virus is an important respiratory pathogen, a little is known about strains circulating in Saudi Arabia. Findings Among 180 nasopharyngeal aspirates collected from suspected cases in Riyadh, only one sample (0.56% was confirmed HPIV-2 positive by nested RT-PCR. The sample that was designated Riyadh 105/2009 was used for sequencing and phylogenetic analysis of the most variable virus gene; the haemagglutinin-neuramindase (HN. Comparison of HN gene of Riyadh 105/2009 strain and the relevant sequences available in GenBank revealed a strong relationship with Oklahoma-94-2009 strain. Phylogenetic analysis indicated four different clusters of HPIV-2 strains (G1-4. Twenty-three amino acid substitutions were recorded for Riyadh 105/2009, from which four are unique. The majority of substitutions (n=18 had changed their amino acids characteristics. By analyzing the effect of the recorded substitutions on the protein function using SIFT program, only two located at positions 360 and 571 were predicted to be deleterious. Conclusions The presented changes of Riyadh 105/2009 strain may possess potential effect on the protein structure and/or function level. This is the first report that describes partial characterization of Saudi HPIV-2 strain.

  2. Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.

    Science.gov (United States)

    Ikeda, Koki; Koga, Tomoaki; Sasaki, Fumiyuki; Ueno, Ayumi; Saeki, Kazuko; Okuno, Toshiaki; Yokomizo, Takehiko

    2017-05-13

    DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Chimeric mice with humanized liver: Application in drug metabolism and pharmacokinetics studies for drug discovery.

    Science.gov (United States)

    Naritomi, Yoichi; Sanoh, Seigo; Ohta, Shigeru

    2018-02-01

    Predicting human drug metabolism and pharmacokinetics (PK) is key to drug discovery. In particular, it is important to predict human PK, metabolite profiles and drug-drug interactions (DDIs). Various methods have been used for such predictions, including in vitro metabolic studies using human biological samples, such as hepatic microsomes and hepatocytes, and in vivo studies using experimental animals. However, prediction studies using these methods are often inconclusive due to discrepancies between in vitro and in vivo results, and interspecies differences in drug metabolism. Further, the prediction methods have changed from qualitative to quantitative to solve these issues. Chimeric mice with humanized liver have been developed, in which mouse liver cells are mostly replaced with human hepatocytes. Since human drug metabolizing enzymes are expressed in the liver of these mice, they are regarded as suitable models for mimicking the drug metabolism and PK observed in humans; therefore, these mice are useful for predicting human drug metabolism and PK. In this review, we discuss the current state, issues, and future directions of predicting human drug metabolism and PK using chimeric mice with humanized liver in drug discovery. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  4. The pharmacokinetics and metabolism of lumiracoxib in chimeric humanized and murinized FRG mice.

    Science.gov (United States)

    Dickie, A P; Wilson, C E; Schreiter, K; Wehr, R; Wilson, E M; Bial, J; Scheer, N; Wilson, I D; Riley, R J

    2017-07-01

    The pharmacokinetics and metabolism of lumiracoxib were studied, after administration of single 10mg/kg oral doses to chimeric liver-humanized and murinized FRG mice. In the chimeric humanized mice, lumiracoxib reached peak observed concentrations in the blood of 1.10±0.08μg/mL at 0.25-0.5h post-dose with an AUC inf of 1.74±0.52μgh/mL and an effective half-life for the drug of 1.42±0.72h (n=3). In the case of the murinized animals peak observed concentrations in the blood were determined as 1.15±0.08μg/mL at 0.25h post-dose with an AUC inf of 1.94±0.22μgh/mL and an effective half-life of 1.28±0.02h (n=3). Analysis of blood indicated only the presence of unchanged lumiracoxib. Metabolic profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated metabolites detected. The profiles obtained in humanized mice were different compared to murinized animals with e.g., a higher proportion of the dose detected in the form of acyl glucuronide metabolites and much reduced amounts of taurine conjugates. Comparison of the metabolic profiles obtained from the present study with previously published data from C57bl/6J mice and humans, revealed a greater though not complete match between chimeric humanized mice and humans, such that the liver-humanized FRG model may represent a useful approach to assessing the biotransformation of such compounds in humans. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. A chimeric human-mouse model of Sjögren's syndrome.

    Science.gov (United States)

    Young, Nicholas A; Wu, Lai-Chu; Bruss, Michael; Kaffenberger, Benjamin H; Hampton, Jeffrey; Bolon, Brad; Jarjour, Wael N

    2015-01-01

    Despite recent advances in the understanding of Sjögren's Syndrome (SjS), the pathogenic mechanisms remain elusive and an ideal model for early drug discovery is not yet available. To establish a humanized mouse model of SjS, peripheral blood mononuclear cells (PBMCs) from healthy volunteers or patients with SjS were transferred into immunodeficient NOD-scid IL-2rγ(null) mouse recipients to produce chimeric mice. While no difference was observed in the distribution of cells, chimeric mice transferred with PBMCs from SjS patients produced enhanced cytokine levels, most significantly IFN-γ and IL-10. Histological examination revealed enhanced inflammatory responses in the lacrimal and salivary glands of SjS chimeras, as measured by digital image analysis and blinded histopathological scoring. Infiltrates were primarily CD4+, with minimal detection of CD8+ T-cells and B-cells. These results demonstrate a novel chimeric mouse model of human SjS that provides a unique in vivo environment to test experimental therapeutics and investigate T-cell disease pathology. Copyright © 2014. Published by Elsevier Inc.

  6. Ethical acceptability of research on human-animal chimeric embryos: summary of opinions by the Japanese Expert Panel on Bioethics.

    Science.gov (United States)

    Mizuno, Hiroshi; Akutsu, Hidenori; Kato, Kazuto

    2015-01-01

    Human-animal chimeric embryos are embryos obtained by introducing human cells into a non-human animal embryo. It is envisaged that the application of human-animal chimeric embryos may make possible many useful research projects including producing three-dimensional human organs in animals and verification of the pluripotency of human ES cells or iPS cells in vivo. The use of human-animal chimeric embryos, however, raises several ethical and moral concerns. The most fundamental one is that human-animal chimeric embryos possess the potential to develop into organisms containing human-derived tissue, which may lead to infringing upon the identity of the human species, and thus impairing human dignity. The Japanese Expert Panel on Bioethics in the Cabinet Office carefully considered the scientific significance and ethical acceptability of the issue and released its "Opinions regarding the handling of research using human-animal chimeric embryos". The Panel proposed a framework of case-by-case review, and suggested that the following points must be carefully reviewed from the perspective of ethical acceptability: (a) Types of animal embryos and types of animals receiving embryo transfers, particularly in dealing with non-human primates; (b) Types of human cells and organs intended for production, particularly in dealing with human nerve or germ cells; and (c) Extent of the period required for post-transfer studies. The scientific knowledge that can be gained from transfer into an animal uterus and from the production of an individual must be clarified to avoid unnecessary generation of chimeric animals. The time is ripe for the scientific community and governments to start discussing the ethical issues for establishing a global consensus.

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

    International Nuclear Information System (INIS)

    Yi, Ka Hee; Kim, Chang Min

    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

  8. 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.

  9. The C proteins of human parainfluenza virus type 1 block IFN signaling by binding and retaining Stat1 in perinuclear aggregates at the late endosome.

    Directory of Open Access Journals (Sweden)

    Henrick Schomacker

    Full Text Available Interferons (IFNs play a crucial role in the antiviral immune response. Whereas the C proteins of wild-type human parainfluenza virus type 1 (WT HPIV1 inhibit both IFN-β induction and signaling, a HPIV1 mutant encoding a single amino acid substitution (F170S in the C proteins is unable to block either host response. Here, signaling downstream of the type 1 IFN receptor was examined in Vero cells to define at what stage WT HPIV1 can block, and F170S HPIV1 fails to block, IFN signaling. WT HPIV1 inhibited phosphorylation of both Stat1 and Stat2, and this inhibition was only slightly reduced for F170S HPIV1. Degradation of Stat1 or Stat2 was not observed. The HPIV1 C proteins were found to accumulate in the perinuclear space, often forming large granules, and co-localized with Stat1 and the cation-independent mannose 6-phosphate receptor (M6PR that is a marker for late endosomes. Upon stimulation with IFN-β, both the WT and F170S C proteins remained in the perinuclear space, but only the WT C proteins prevented Stat1 translocation to the nucleus. In addition, WT HPIV1 C proteins, but not F170S C proteins, co-immunoprecipitated both phosphorylated and unphosphorylated Stat1. Our findings suggest that the WT HPIV1 C proteins form a stable complex with Stat1 in perinuclear granules that co-localize with M6PR, and that this direct interaction between the WT HPIV1 C proteins and Stat1 is the basis for the ability of HPIV1 to inhibit IFN signaling. The F170S mutation in HPIV1 C did not prevent perinuclear co-localization with Stat1, but apparently weakened this interaction such that, upon IFN stimulation, Stat1 was translocated to the nucleus to induce an antiviral response.

  10. A Tryptophan-Rich Motif in the Human Parainfluenza Virus Type 2 V Protein Is Critical for the Blockade of Toll-Like Receptor 7 (TLR7)- and TLR9-Dependent Signaling▿

    OpenAIRE

    Kitagawa, Yoshinori; Yamaguchi, Mayu; Zhou, Min; Komatsu, Takayuki; Nishio, Machiko; Sugiyama, Tsuyoshi; Takeuchi, Kenji; Itoh, Masae; Gotoh, Bin

    2011-01-01

    Plasmacytoid dendritic cells (pDCs) do not produce alpha interferon (IFN-α) unless viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs and macrophages. We show here that even paramyxoviruses, whose infections are restricted to the respiratory tract, have a V protein able to prevent Toll-like receptor 7 (TLR7)- and TLR9-dependent IFN-α induction specific to pDCs. Mutational analysis of human parainfluenza virus type 2 demonstrates that the second ...

  11. Attenuation and efficacy of human parainfluenza virus type 1 (HPIV1 vaccine candidates containing stabilized mutations in the P/C and L genes

    Directory of Open Access Journals (Sweden)

    Skiadopoulos Mario H

    2007-07-01

    Full Text Available Abstract Background Two recombinant, live attenuated human parainfluenza virus type 1 (rHPIV1 mutant viruses have been developed, using a reverse genetics system, for evaluation as potential intranasal vaccine candidates. These rHPIV1 vaccine candidates have two non-temperature sensitive (non-ts attenuating (att mutations primarily in the P/C gene, namely CR84GHNT553A (two point mutations used together as a set and CΔ170 (a short deletion mutation, and two ts att mutations in the L gene, namely LY942A (a point mutation, and LΔ1710–11 (a short deletion, the last of which has not been previously described. The latter three mutations were specifically designed for increased genetic and phenotypic stability. These mutations were evaluated on the HPIV1 backbone, both individually and in combination, for attenuation, immunogenicity, and protective efficacy in African green monkeys (AGMs. Results The rHPIV1 mutant bearing the novel LΔ1710–11 mutation was highly ts and attenuated in AGMs and was immunogenic and efficacious against HPIV1 wt challenge. The rHPIV1-CR84G/Δ170HNT553ALY942A and rHPIV1-CR84G/Δ170HNT553ALΔ1710–11 vaccine candidates were highly ts, with shut-off temperatures of 38°C and 35°C, respectively, and were highly attenuated in AGMs. Immunization with rHPIV1-CR84G/Δ170HNT553ALY942A protected against HPIV1 wt challenge in both the upper and lower respiratory tracts. In contrast, rHPIV1-CR84G/Δ170HNT553ALΔ1710–11 was not protective in AGMs due to over-attenuation, but it is expected to replicate more efficiently and be more immunogenic in the natural human host. Conclusion The rHPIV1-CR84G/Δ170HNT553ALY942A and rHPIV1-CR84G/Δ170HNT553ALΔ1710–11 vaccine candidates are clearly highly attenuated in AGMs and clinical trials are planned to address safety and immunogenicity in humans.

  12. [Construction of the lentiviral expression vector for anti-p185(erbB2) mouse/human chimeric antibody].

    Science.gov (United States)

    Liu, Fang; Li, Li; Zhang, Wei; Wang, Qi

    2013-04-01

    This research was to construct the lentiviral expression vector for anti- p185(erbB2) mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-p185(erbB2) mAb and the constant regions of human IgG1 (kappa and gamma1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody expression was detected by RT-PCR and direct ELISA. The results showed that after 293T cells were transfected with recombination plasmid, both light and heavy chains of the chimeric antibody genes could express together. The chimeric antibody expressed could bind to p185(erbB2) specifically. This research may lay a sound foundation for further study of anti-p185(erbB2) engineered antibody.

  13. Biomimetic chimeric peptide-tethered hydrogels for human mesenchymal stem cell delivery.

    Science.gov (United States)

    Shim, Gayong; Kim, Gunwoo; Choi, Junhyeok; Yi, TacGhee; Cho, Yun Kyoung; Song, Sun Uk; Byun, Youngro; Oh, Yu-Kyoung

    2015-12-01

    Here, we report a chimeric peptide-tethered fibrin hydrogel scaffold for delivery of human mesenchymal stem cells (hMSC). Osteopontin-derived peptide (OP) was used as an hMSC-tethering moiety. OP showed hMSC adhesion properties and enhanced hMSC proliferation. A natural fibrin-binding protein-derived peptide (FBP) was tested for its ability to tether hMSC to the fibrin gel matrix. FBP loading on fibrin gels was 8.2-fold higher than that of a scrambled peptide (scFBP). FBP-loaded fibrin gels were retained at injection sites longer than scFBP-loaded fibrin gels, showing a 15.9-fold higher photon intensity of fluorescent FBP-grafted fibrin gels than fluorescent scFBP-loaded fibrin gels 48 h after injection. On the basis of the fibrin gel-binding properties of FBP and the hMSC-binding and proliferation-supporting properties of OP, we constructed chimeric peptides containing FBP and OP linked with a spacer (FBPsOP). Four days after transplantation, the survival of hMSC in FBPsOP-grafted fibrin gels was 3.9-fold higher than hMSC in fibrin gels alone. Our results suggest the potential of FBPsOP-grafted fibrin gels as a bioactive delivery system for enhanced survival of stem cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Comparison of predictability for human pharmacokinetics parameters among monkeys, rats, and chimeric mice with humanised liver.

    Science.gov (United States)

    Miyamoto, Maki; Iwasaki, Shinji; Chisaki, Ikumi; Nakagawa, Sayaka; Amano, Nobuyuki; Hirabayashi, Hideki

    2017-12-01

    1. The aim of the present study was to evaluate the usefulness of chimeric mice with humanised liver (PXB mice) for the prediction of clearance (CL t ) and volume of distribution at steady state (Vd ss ), in comparison with monkeys, which have been reported as a reliable model for human pharmacokinetics (PK) prediction, and with rats, as a conventional PK model. 2. CL t and Vd ss values in PXB mice, monkeys and rats were determined following intravenous administration of 30 compounds known to be mainly eliminated in humans via the hepatic metabolism by various drug-metabolising enzymes. Using single-species allometric scaling, human CL t and Vd ss values were predicted from the three animal models. 3. Predicted CL t values from PXB mice exhibited the highest predictability: 25 for PXB mice, 21 for monkeys and 14 for rats were predicted within a three-fold range of actual values among 30 compounds. For predicted human Vd ss values, the number of compounds falling within a three-fold range was 23 for PXB mice, 24 for monkeys, and 16 for rats among 29 compounds. PXB mice indicated a higher predictability for CL t and Vd ss values than the other animal models. 4. These results demonstrate the utility of PXB mice in predicting human PK parameters.

  15. A tryptophan-rich motif in the human parainfluenza virus type 2 V protein is critical for the blockade of toll-like receptor 7 (TLR7)- and TLR9-dependent signaling.

    Science.gov (United States)

    Kitagawa, Yoshinori; Yamaguchi, Mayu; Zhou, Min; Komatsu, Takayuki; Nishio, Machiko; Sugiyama, Tsuyoshi; Takeuchi, Kenji; Itoh, Masae; Gotoh, Bin

    2011-05-01

    Plasmacytoid dendritic cells (pDCs) do not produce alpha interferon (IFN-α) unless viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs and macrophages. We show here that even paramyxoviruses, whose infections are restricted to the respiratory tract, have a V protein able to prevent Toll-like receptor 7 (TLR7)- and TLR9-dependent IFN-α induction specific to pDCs. Mutational analysis of human parainfluenza virus type 2 demonstrates that the second Trp residue of the Trp-rich motif (Trp-X(3)-Trp-X(9)-Trp) in the C-terminal domain unique to V, a determinant for IRF7 binding, is critical for the blockade of TLR7/9-dependent signaling.

  16. 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.

  17. Fluorescently labeled chimeric anti-CEA antibody improves detection and resection of human colon cancer in a patient-derived orthotopic xenograft (PDOX) nude mouse model.

    Science.gov (United States)

    Metildi, Cristina A; Kaushal, Sharmeela; Luiken, George A; Talamini, Mark A; Hoffman, Robert M; Bouvet, Michael

    2014-04-01

    The aim of this study was to evaluate a new fluorescently labeled chimeric anti-CEA antibody for improved detection and resection of colon cancer. Frozen tumor and normal human tissue samples were stained with chimeric and mouse antibody-fluorophore conjugates for comparison. Mice with patient-derived orthotopic xenografts (PDOX) of colon cancer underwent fluorescence-guided surgery (FGS) or bright-light surgery (BLS) 24 hr after tail vein injection of fluorophore-conjugated chimeric anti-CEA antibody. Resection completeness was assessed using postoperative images. Mice were followed for 6 months for recurrence. The fluorophore conjugation efficiency (dye/mole ratio) improved from 3-4 to >5.5 with the chimeric CEA antibody compared to mouse anti-CEA antibody. CEA-expressing tumors labeled with chimeric CEA antibody provided a brighter fluorescence signal on frozen human tumor tissues (P = 0.046) and demonstrated consistently lower fluorescence signals in normal human tissues compared to mouse antibody. Chimeric CEA antibody accurately labeled PDOX colon cancer in nude mice, enabling improved detection of tumor margins for more effective FGS. The R0 resection rate increased from 86% to 96% with FGS compared to BLS. Improved conjugating efficiency and labeling with chimeric fluorophore-conjugated antibody resulted in better detection and resection of human colon cancer in an orthotopic mouse model. © 2013 Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

    2016-01-01

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

  19. Acute HBV infection in humanized chimeric mice has multiphasic viral kinetics.

    Science.gov (United States)

    Ishida, Yuji; Chung, Tje Lin; Imamura, Michio; Hiraga, Nobuhiko; Sen, Suranjana; Yokomichi, Hiroshi; Tateno, Chise; Canini, Laetitia; Perelson, Alan S; Uprichard, Susan L; Dahari, Harel; Chayama, Kazuaki

    2018-03-23

    Chimeric uPA/SCID mice reconstituted with humanized livers are useful for studying HBV infection in the absence of an adaptive immune response. However, the detailed characterization of HBV infection kinetics necessary to enable in-depth mechanistic studies in this novel in vivo HBV infection model is lacking. To characterize HBV kinetics post-inoculation (p.i.) to steady state, 42 mice were inoculated with HBV. Serum HBV DNA was frequently measured from 1 minute to 63 days p.i. Total intrahepatic HBV DNA, HBV cccDNA, and HBV RNA was measured in a subset of mice at 2, 4, 6, 10, and 13 weeks p.i. HBV half-life (t 1/2 ) was estimated using a linear mixed-effects model. During the first 6 h p.i. serum HBV declined in repopulated uPA/SCID mice with a t 1/2 =62 min [95%CI=59-67min]. Thereafter, viral decline slowed followed by a 2 day lower plateau. Subsequent viral amplification was multiphasic with an initial mean doubling time of t 2 =8±3 h followed by an interim plateau before prolonged amplification (t 2 =2±0.5 days) to a final HBV steady state of 9.3±0.3 log copies/ml. Serum HBV and intrahepatic HBV DNA were positively correlated (R 2 =0.98). HBV infection in uPA/SCID chimeric mice is highly dynamic despite the absence of an adaptive immune response. The serum HBV t 1/2 in humanized uPA/SCID mice was estimated to be ∼1 h regardless of inoculum size. The HBV acute infection kinetics presented here is an important step in characterizing this experimental model system so that it can be effectively used to elucidate the dynamics of the HBV lifecycle and thus possibly reveal effective antiviral drug targets. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

  20. Endothelial cell chimerism associated with graft rejection after human lung transplantation.

    OpenAIRE

    Ratajczak , Philippe; Murata , Hideyuki; Meignin , Véronique; Groussard , Odile; Fournier , Michel; Socié , Gérard; Mal , Hervé; Janin , Anne

    2008-01-01

    International audience; Endotheliitis is a major sign of graft rejection. Recipient-derived endothelial cells found in two series of liver and kidney transplants were related to graft rejection. Here, we assessed the presence and the number of chimeric endothelial cells in lung transplants, and their relation with graft rejection. In six males grafted with female lungs out of 193 lung transplantations, endothelial chimerism was studied by combined XY-fluorescent in situ hybridization with CD3...

  1. Current management of parainfluenza pneumonitis in immunocompromised patients: a review

    Directory of Open Access Journals (Sweden)

    Falsey AR

    2012-08-01

    Full Text Available Ann R FalseyUniversity of Rochester, Rochester General Hospital, Rochester, NY, USAAbstract: Parainfluenza viruses (PIV are common respiratory viruses that belong to the Paramyxoviridae family. PIV infection can lead to a wide variety of clinical syndromes ranging from mild upper respiratory illness to severe pneumonia. Severe disease can be seen in elderly or chronically ill persons and may be fatal in persons with compromised immune systems, particularly children with severe combined immunodeficiency disease syndrome and hematopathic stem cell transplant recipients. At present, there are no licensed antiviral agents for the treatment of PIV infection. Aerosolized or systemic ribavirin in combination with intravenous gamma globulin has been reported in small, uncontrolled series and case reports of immunocompromised patients. A number of agents show antiviral activity in vitro and in animals, but none are currently approved for human use.Keywords: parainfluenza virus, antiviral agents, immunocompromised host

  2. Chimeric L2-Based Virus-Like Particle (VLP Vaccines Targeting Cutaneous Human Papillomaviruses (HPV.

    Directory of Open Access Journals (Sweden)

    Bettina Huber

    Full Text Available Common cutaneous human papillomavirus (HPV types induce skin warts, whereas species beta HPV are implicated, together with UV-radiation, in the development of non-melanoma skin cancer (NMSC in immunosuppressed patients. Licensed HPV vaccines contain virus-like particles (VLP self-assembled from L1 major capsid proteins that provide type-restricted protection against mucosal HPV infections causing cervical and other ano-genital and oro-pharyngeal carcinomas and warts (condylomas, but do not target heterologous HPV. Experimental papillomavirus vaccines have been designed based on L2 minor capsid proteins that contain type-common neutralization epitopes, to broaden protection to heterologous mucosal and cutaneous HPV types. Repetitive display of the HPV16 L2 cross-neutralization epitope RG1 (amino acids (aa 17-36 on the surface of HPV16 L1 VLP has greatly enhanced immunogenicity of the L2 peptide. To more directly target cutaneous HPV, L1 fusion proteins were designed that incorporate the RG1 homolog of beta HPV17, the beta HPV5 L2 peptide aa53-72, or the common cutaneous HPV4 RG1 homolog, inserted into DE surface loops of HPV1, 5, 16 or 18 L1 VLP scaffolds. Baculovirus expressed chimeric proteins self-assembled into VLP and VLP-raised NZW rabbit immune sera were evaluated by ELISA and L1- and L2-based pseudovirion (PsV neutralizing assays, including 12 novel beta PsV types. Chimeric VLP displaying the HPV17 RG1 epitope, but not the HPV5L2 aa53-72 epitope, induced cross-neutralizing humoral immune responses to beta HPV. In vivo cross-protection was evaluated by passive serum transfer in a murine PsV challenge model. Immune sera to HPV16L1-17RG1 VLP (cross- protected against beta HPV5/20/24/38/96/16 (but not type 76, while antisera to HPV5L1-17RG1 VLP cross-protected against HPV20/24/96 only, and sera to HPV1L1-4RG1 VLP cross-protected against HPV4 challenge. In conclusion, RG1-based VLP are promising next generation vaccine candidates to target

  3. Parainfluenza

    Science.gov (United States)

    ... SL, Steinbach WJ, Hotez PJ, eds. Feigin and Cherry's Textbook of Pediatric Infectious Diseases . 7th ed. Philadelphia, PA: Elsevier Saunders; 2014:chap 178. Review Date 8/21/2016 Updated by: Denis Hadjiliadis, MD, MHS, Paul F. Harron, Jr. Associate Professor of Medicine, Pulmonary, ...

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

    Science.gov (United States)

    Windrem, Martha S; Schanz, Steven J; Morrow, Carolyn; Munir, Jared; Chandler-Militello, Devin; Wang, Su; Goldman, Steven A

    2014-11-26

    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 proportion of mouse GPCs fell as a function of time, concomitant with the mitotic expansion and spread of donor hGPCs. By a year after neonatal xenograft, the forebrain GPC populations of implanted mice were largely, and often entirely, of human origin. Thus, neonatally implanted hGPCs outcompeted 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. Copyright © 2014 the authors 0270-6474/14/3416153-09$15.00/0.

  5. Identification and characterization of metabolites of ASP015K, a novel oral Janus kinase inhibitor, in rats, chimeric mice with humanized liver, and humans.

    Science.gov (United States)

    Nakada, Naoyuki; Oda, Kazuo

    2015-01-01

    1. Here, we elucidated the structure of metabolites of novel oral Janus kinase inhibitor ASP015K in rats and humans and evaluated the predictability of human metabolites using chimeric mice with humanized liver (PXB mice). 2. Rat biological samples collected after oral dosing of (14)C-labelled ASP015K were examined using a liquid chromatography-radiometric detector and mass spectrometer (LC-RAD/MS). The molecular weight of metabolites in human and the liver chimeric mouse biological samples collected after oral dosing of non-labelled ASP015K was also investigated via LC-MS. Metabolites were also isolated from rat bile samples and analyzed using nuclear magnetic resonance. 3. Metabolic pathways of ASP015K in rats and humans were found to be glucuronide conjugation, methyl conjugation, sulfate conjugation, glutathione conjugation, hydroxylation of the adamantane ring and N-oxidation of the 1H-pyrrolo[2,3-b]pyridine ring. The main metabolite of ASP015K in rats was the glucuronide conjugate, while the main metabolite in humans was the sulfate conjugate. Given that human metabolites were produced by human hepatocytes in chimeric mice with humanized liver, this human model mouse was believed to be useful in predicting the human metabolic profile of various drug candidates.

  6. Dystrophin Expressing Chimeric (DEC) Human Cells Provide a Potential Therapy for Duchenne Muscular Dystrophy.

    Science.gov (United States)

    Siemionow, Maria; Cwykiel, Joanna; Heydemann, Ahlke; Garcia, Jesus; Marchese, Enza; Siemionow, Krzysztof; Szilagyi, Erzsebet

    2018-06-01

    Duchenne Muscular Dystrophy (DMD) is a progressive and lethal disease caused by mutations of the dystrophin gene. Currently no cure exists. Stem cell therapies targeting DMD are challenged by limited engraftment and rejection despite the use of immunosuppression. There is an urgent need to introduce new stem cell-based therapies that exhibit low allogenic profiles and improved cell engraftment. In this proof-of-concept study, we develop and test a new human stem cell-based approach to increase engraftment, limit rejection, and restore dystrophin expression in the mdx/scid mouse model of DMD. We introduce two Dystrophin Expressing Chimeric (DEC) cell lines created by ex vivo fusion of human myoblasts (MB) derived from two normal donors (MB N1 /MB N2 ), and normal and DMD donors (MB N /MB DMD ). The efficacy of fusion was confirmed by flow cytometry and confocal microscopy based on donor cell fluorescent labeling (PKH26/PKH67). In vitro, DEC displayed phenotype and genotype of donor parent cells, expressed dystrophin, and maintained proliferation and myogenic differentiation. In vivo, local delivery of both DEC lines (0.5 × 10 6 ) restored dystrophin expression (17.27%±8.05-MB N1 /MB N2 and 23.79%±3.82-MB N /MB DMD ) which correlated with significant improvement of muscle force, contraction and tolerance to fatigue at 90 days after DEC transplant to the gastrocnemius muscles (GM) of dystrophin-deficient mdx/scid mice. This study establishes DEC as a potential therapy for DMD and other types of muscular dystrophies.

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

    Directory of Open Access Journals (Sweden)

    Chise Tateno

    Full Text Available 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

  8. 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

  9. Clinical usefulness of human-mouse chimeric Fab monoclonal antibody A7 for radioimmunoguided surgery

    International Nuclear Information System (INIS)

    Yamamoto, Kazuhito

    1999-01-01

    This study was designed to determine the clinical usefulness of radioimmunoguided surgery (RIGS) using the human-mouse chimeric Fab monoclonal antibody A7 (chA7Fab) for colorectal cancer patients. Whole murine monoclonal antibody A7 (whole A7) and chA7Fab were labelled with 125 I and 131 I, and their biodistributions were investigated experimentally and clinically. Radioactivities of the antibodies in the tissues were measured by a portable gamma detecting probe (GDP) purchased from Neoprobe Corp.. Of the four labelled antibodies used in a mouse model, 125 I-chA7Fab revealed the highest tumor/surrounding tissue ratio and all values were greater than 2.0. All tumor/surrounding tissue ratios of 131 I-chA7Fab were greater than 1.5, but the values were lower than those of 125 I-chA7Fab. Due to the limited clinical use of 125 I in Japan, 131 I was used as a radio-tracer for chA7Fab in the clinical trial. RIGS using 131 I-chA7Fab was performed on ten colorectal cancer patients. Tumor localization was intraoperatively determined in four of ten patients using the GDP. Liver metastasis and lymph node metastasis were identified in two patients and one patient, respectively. The GDP revealed tumor/surrounding tissue ratios of 1.5 or greater in eight of the ten resected tumors. Although radioimmunoguided surgery using chA7Fab is a promising tool to intraoperatively determine the tumor localization of colorectal cancer, 125 I and not 131 I should be used as a tracer for radioimmunoguided surgery to increase the accuracy of chA7Fab. (author)

  10. 21 CFR 866.3400 - Parainfluenza virus serological reagents.

    Science.gov (United States)

    2010-04-01

    ... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3400 Parainfluenza... that consist of antigens and antisera used in serological tests to identify antibodies to parainfluenza...

  11. Recombinant human parainfluenza virus type 2 with mutations in V that permit cellular interferon signaling are not attenuated in non-human primates

    Science.gov (United States)

    Schaap-Nutt, Anne; D’Angelo, Christopher; Amaro-Carambot, Emerito; Nolan, Sheila M.; Davis, Stephanie; Wise, Shenelle-Marie; Higgins, Caraline; Bradley, Konrad; Kim, Olivia; Mayor, Reina; Skiadopoulos, Mario H.; Collins, Peter L.; Murphy, Brian R.; Schmidt, Alexander C.

    2010-01-01

    The HPIV2 V protein inhibits type I interferon (IFN) induction and signaling. To manipulate the V protein, whose coding sequence overlaps that of the polymerase-associated phosphoprotein (P), without altering the P protein, we generated an HPIV2 virus in which P and V are expressed from separate genes (rHPIV2-P+V). rHPIV2-P+V replicated like HPIV2-WT in vitro and in non-human primates. HPIV2-P+V was modified by introducing two separate mutations into the V protein to create rHPIV2-L101E/L102E and rHPIV2-Δ122–127. In contrast to HPIV2-WT, both mutant viruses were unable to degrade STAT2, leaving virus-infected cells susceptible to IFN. Neither mutant, nor HPIV2-WT, induced significant amounts of IFN-β in infected cells. Surprisingly, neither rHPIV2-L101E/L102E nor rHPIV2-Δ122–127 was attenuated in two species of non-human primates. This indicates that loss of HPIV2's ability to inhibit IFN signaling is insufficient to attenuate virus replication in vivo as long as IFN induction is still inhibited. PMID:20667570

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

    Directory of Open Access Journals (Sweden)

    Dan Xu

    2014-04-01

    Full Text Available 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.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.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 of chimeric mice in preclinical toxicology

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

    Science.gov (United States)

    Xu, Dan; Nishimura, Toshi; Nishimura, Sachiko; Zhang, Haili; Zheng, Ming; Guo, Ying-Ying; Masek, Marylin; Michie, Sara A; Glenn, Jeffrey; Peltz, Gary

    2014-04-01

    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. 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. 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 of chimeric mice in preclinical toxicology studies could improve

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

    Science.gov (United States)

    Kamimura, Hidetaka; Ito, Satoshi

    2016-01-01

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

  15. Metabolism of methylstenbolone studied with human liver microsomes and the uPA⁺/⁺-SCID chimeric mouse model.

    Science.gov (United States)

    Geldof, Lore; Lootens, Leen; Polet, Michael; Eichner, Daniel; Campbell, Thane; Nair, Vinod; Botrè, Francesco; Meuleman, Philip; Leroux-Roels, Geert; Deventer, Koen; Eenoo, Peter Van

    2014-07-01

    Anti-doping laboratories need to be aware of evolutions on the steroid market and elucidate steroid metabolism to identify markers of misuse. Owing to ethical considerations, in vivo and in vitro models are preferred to human excretion for nonpharmaceutical grade substances. In this study the chimeric mouse model and human liver microsomes (HLM) were used to elucidate the phase I metabolism of a new steroid product containing, according to the label, methylstenbolone. Analysis revealed the presence of both methylstenbolone and methasterone, a structurally closely related steroid. Via HPLC fraction collection, methylstenbolone was isolated and studied with both models. Using HLM, 10 mono-hydroxylated derivatives (U1-U10) and a still unidentified derivative of methylstenbolone (U13) were detected. In chimeric mouse urine only di-hydroxylated metabolites (U11-U12) were identified. Although closely related, neither methasterone nor its metabolites were detected after administration of isolated methylstenbolone. Administration of the steroid product resulted mainly in the detection of methasterone metabolites, which were similar to those already described in the literature. Methylstenbolone metabolites previously described were not detected. A GC-MS/MS multiple reaction monitoring method was developed to detect methylstenbolone misuse. In one out of three samples, previously tested positive for methasterone, methylstenbolone and U13 were additionally detected, indicating the applicability of the method. Copyright © 2014 John Wiley & Sons, Ltd.

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

    International Nuclear Information System (INIS)

    Lu Pingping; Meng Zhiyun; Dou Guifang; Wu Yingliang; Wang Minwei

    2008-01-01

    To investigate the bioactivity and application of 125 I labeled human mouse chimeric monoclonal SM03, SM03 was labeled with 125 I 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. 125 I-SM03 was applied to screen cell lines which express the most abundant CD22 antigen. The purity and recovery of 125 I-SM03 were >99% and >47%, respectively. The bioactivity of 125 I- SM03 and SM03 hasn't significant difference in statistics. Ramos cell line had the strongest special radioactivity when 125 I-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 125 I-SM03 not only can be used for detect agent, but also may be put into market for NHL therapy. (authors)

  17. Formation of the accumulative human metabolite and human-specific glutathione conjugate of diclofenac in TK-NOG chimeric mice with humanized livers.

    Science.gov (United States)

    Kamimura, Hidetaka; Ito, Satoshi; Nozawa, Kohei; Nakamura, Shota; Chijiwa, Hiroyuki; Nagatsuka, Shin-ichiro; Kuronuma, Miyuki; Ohnishi, Yasuyuki; Suemizu, Hiroshi; Ninomiya, Shin-ichi

    2015-03-01

    3'-Hydroxy-4'-methoxydiclofenac (VI) is a human-specific metabolite known to accumulate in the plasma of patients after repeated administration of diclofenac sodium. Diclofenac also produces glutathione-conjugated metabolites, some of which are human-specific. In the present study, we investigated whether these metabolites could be generated in humanized chimeric mice produced from TK-NOG mice. After a single oral administration of diclofenac to humanized mice, the unchanged drug in plasma peaked at 0.25 hour and then declined with a half-life (t1/2) of 2.4 hours. 4'-Hydroxydiclofenac (II) and 3'-hydroxydiclofenac also peaked at 0.25 hour and were undetectable within 24 hours. However, VI peaked at 8 hours and declined with a t1/2 of 13 hours. When diclofenac was given once per day, peak and trough levels of VI reached plateau within 3 days. Studies with administration of II suggested VI was generated via II as an intermediate. Among six reported glutathione-conjugated metabolites of diclofenac, M1 (5-hydroxy-4-(glutathion-S-yl)diclofenac) to M6 (2'-(glutathion-S-yl)monoclofenac), we found three dichlorinated conjugates [M1, M2 (4'-hydroxy-3'-(glutathion-S-yl)diclofenac), and M3 (5-hydroxy-6-(glutathion-S-yl)diclofenac)], and a single monochlorinated conjugate [M4 (2'-hydroxy-3'-(glutathion-S-yl)monoclofenac) or M5 (4'-hydroxy-2'-(glutathion-S-yl)monoclofenac)], in the bile of humanized chimeric mice. M4 and M5 are positional isomers and have been previously reported as human-specific in vitro metabolites likely generated via arene oxide and quinone imine-type intermediates, respectively. The biliary monochlorinated metabolite exhibited the same mass spectrum as those of M4 and M5, and we discuss whether this conjugate corresponded to M4 or M5. Overall, humanized TK-NOG chimeric mice were considered to be a functional tool for the study of drug metabolism of diclofenac in humans. Copyright © 2015 by The American Society for Pharmacology and Experimental

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

    International Nuclear Information System (INIS)

    Murata, Yoko.

    1995-01-01

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

  19. HBsAg-redirected T cells exhibit antiviral activity in HBV-infected human liver chimeric mice.

    Science.gov (United States)

    Kruse, Robert L; Shum, Thomas; Tashiro, Haruko; Barzi, Mercedes; Yi, Zhongzhen; Whitten-Bauer, Christina; Legras, Xavier; Bissig-Choisat, Beatrice; Garaigorta, Urtzi; Gottschalk, Stephen; Bissig, Karl-Dimiter

    2018-04-06

    Chronic hepatitis B virus (HBV) infection remains incurable. Although HBsAg-specific chimeric antigen receptor (HBsAg-CAR) T cells have been generated, they have not been tested in animal models with authentic HBV infection. We generated a novel CAR targeting HBsAg and evaluated its ability to recognize HBV+ cell lines and HBsAg particles in vitro. In vivo, we tested whether human HBsAg-CAR T cells would have efficacy against HBV-infected hepatocytes in human liver chimeric mice. HBsAg-CAR T cells recognized HBV-positive cell lines and HBsAg particles in vitro as judged by cytokine production. However, HBsAg-CAR T cells did not kill HBV-positive cell lines in cytotoxicity assays. Adoptive transfer of HBsAg-CAR T cells into HBV-infected humanized mice resulted in accumulation within the liver and a significant decrease in plasma HBsAg and HBV-DNA levels compared with control mice. Notably, the fraction of HBV core-positive hepatocytes among total human hepatocytes was greatly reduced after HBsAg-CAR T cell treatment, pointing to noncytopathic viral clearance. In agreement, changes in surrogate human plasma albumin levels were not significantly different between treatment and control groups. HBsAg-CAR T cells have anti-HBV activity in an authentic preclinical HBV infection model. Our results warrant further preclinical exploration of HBsAg-CAR T cells as immunotherapy for HBV. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  20. High Resistance of Human Parainfluenza Type 2 Virus Protein-Expressing Cells to the Antiviral and Anti-Cell Proliferative Activities of Alpha/Beta Interferons: Cysteine-Rich V-Specific Domain Is Required for High Resistance to the Interferons

    OpenAIRE

    Nishio, Machiko; Tsurudome, Masato; Ito, Morihiro; Kawano, Mitsuo; Komada, Hiroshi; Ito, Yasuhiko

    2001-01-01

    Human parainfluenza type 2 virus (hPIV-2)-infected HeLa (HeLa-CA) cells and hPIV-2 V-expressing HeLa (HeLa-V) cells show high resistance to alpha/beta interferons (IFN-α/β) irrespective of whether vesicular stomatitis virus or Sindbis virus is used as a challenge virus. When Sindbis virus is used, these cells show high susceptibility to human IFN-γ. Furthermore, the multiplication of HeLa-V cells is not inhibited by IFN-α/β. HeLa cells expressing the N-terminally truncated V protein show resi...

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

    DEFF Research Database (Denmark)

    Moisini, Ioana; Nguyen, Phuong; Fugger, Lars

    2008-01-01

    Therapies that Ag-specifically target pathologic T lymphocytes responsible for multiple sclerosis (MS) and other autoimmune diseases would be expected to have improved therapeutic indices compared with Ag-nonspecific therapies. We have developed a cellular immunotherapy that uses chimeric receptors...... mouse model system. Finally, the chimeric receptor-modified CTL ameliorated or blocked experimental allergic encephalomyelitis (EAE) disease mediated by MBP(84-102)/DR2-specific T lymphocytes. These results provide support for the further development of redirected therapeutic T cells able to counteract...... pathologic, self-specific T lymphocytes, and specifically validate humanized MBP-DR2-zeta chimeric receptors as a potential therapeutic in MS. Udgivelsesdato: 2008-Mar-1...

  2. Identification of amino acids essential for the human parainfluenza type 2 virus V protein to lower the intracellular levels of the STAT2

    International Nuclear Information System (INIS)

    Kozuka, Yuji; Yamashita, Yasufumi; Kawano, Mitsuo; Tsurudome, Masato; Ito, Morihiro; Nishio, Machiko; Komada, Hiroshi; Ito, Yasuhiko

    2003-01-01

    The V protein of SV41 targets STAT1, while a specific loss of STAT2 is induced by the hPIV2 V protein. We established HeLa cells constitutively expressing various chimeric proteins between the hPIV2 and SV41 V proteins, and which STAT (STAT1 or 2) was expressed in these cells was analyzed. Both the P-V common domain and the V specific domain of hPIV2 V protein are necessary for STAT2 lowering. The internal domain (aa145-173) containing a large number of nonidentical amino acids between hPIV2 and SV41 does not direct STAT tropism, and the regions necessary for STAT2 lowering are discontinuous. The N-terminal domain (aa1-104) and the internal domain (aa126-196) of the hPIV2 V protein do not determine STAT tropism. HeLa cells expressing A105E or H108P show distinct expression of STAT2, but do show low expression or a loss of STAT1, indicating that the amino acid residues 105 and 108 of the hPIV2 V protein are essential for STAT2 lowering. Interestingly, there is an important amino acid(s) in the region (aa121-125) for STAT2 lowering, and the presence of either amino acid residue 123 or 125 of the hPIV2 V protein is necessary for lowering of STAT2. In addition, HeLa cells expressing S216D or 1217R expressed STAT2, but no STAT1, indicating that the amino acid residues 216 and 217 of the hPIV2 V protein are indispensable for STAT2 lowering. HeLa/hPIV2V cells and HeLa/S104/P are resistant to IFN-β, while they are sensitive to IFN-γ. On the other hand, HeLa/SV41V, HeLa/S216D, and HeLa1217R cells are resistant to both IFNs. Intriguingly, HeLa/A105E and HeLa/H108P cells were found to be sensitive to IFN-γ

  3. Radiotoxicity of systemically administered 211At-labeled human/mouse chimeric monoclonal antibody: a long-term survival study with histologic analysis

    International Nuclear Information System (INIS)

    McLendon, Roger E.; Archer, Gary E.; Larsen, Roy H.; Akabani, Gamal; Bigner, Darell D.; Zalutsky, Michael R.

    1999-01-01

    Purpose: The antitenascin human/mouse chimeric monoclonal antibody labeled with the α-particle-emitting radionuclide 211 At is of interest as an endo radiotherapeutic agent for the treatment of brain tumors. To facilitate the investigation of 211 At-labeled chimeric 81C6 in patients, the long-term radiotoxicity of this radiopharmaceutical has been evaluated. Methods and Materials: Antibody labeling was performed using N-succinimidyl 3-[ 211 At]astato-benzoate. After an initial dose-finding experiment, a second toxicity study was carried out at 4 dose levels in groups of 30 non thyroid blocked B6C3F 1 mice per group (15 males, 15 females). Male mice received either saline or 15-81 kBq/g and females received either saline or 16-83 kBq/g of 211 At-labeled antibody. Ten animals (5 males, 5 females) were followed for 6 months and the remainder for 1 year. Results: The lethal dose in 10% of animals (LD 10 ) for 211 At-labeled chimeric 81C6 was 46 kBq/g in females and 102 kBq/g in males. Toxic effects--perivascular fibrosis of the intraventricular septum of the heart, bone marrow suppression, splenic white pulp atrophy, and spermatic maturational delay--generally were confined to a few animals receiving the highest doses of labeled antibody. Conclusions: The LD 10 of 211 At-labeled chimeric 81C6 in this mouse strain was about half that of [ 211 At]astatide. These results establish the preclinical maximum tolerated dose of 211 At-labeled chimeric 81C6 and define in the mouse the target organs for toxicity. These studies will be useful for determining starting doses for clinical studies with 211 At-labeled chimeric 81C6

  4. Epiglottitis with an abscess caused by Haemophilus parainfluenzae

    DEFF Research Database (Denmark)

    Juul, Marie Louise; Johansen, Helle Krogh; Homøe, Preben

    2014-01-01

    A healthy 23-year-old man was admitted under the diagnosis of acute epiglottitis. Flexible fiber laryngoscopic examination showed a swollen epiglottis with an abscess. Microbiologic swab showed Haemophilus parainfluenzae, non-haemolytic Streptococcus and non-haemolytic Streptococcus salivarius. O....... Only in 1984 a case of acute epiglottitis due to H. parainfluenzae has been described in the literature. Still, in this case we think that H. parainfluenzae was the most likely pathogen causing the abscess....

  5. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    Directory of Open Access Journals (Sweden)

    Vanesa Alonso-Camino

    2013-01-01

    Full Text Available A human single-chain variable fragment (scFv antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs. The repertoire was fused to a first-generation T cell receptor ζ (TCRζ-based chimeric antigen receptor (CAR. We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2 bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR and the selection context (cell synapse, which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.

  6. MINOR HUMAN-ANTIBODY RESPONSE TO A MOUSE AND CHIMERIC MONOCLONAL-ANTIBODY AFTER A SINGLE IV INFUSION IN OVARIAN-CARCINOMA PATIENTS - A COMPARISON OF 5 ASSAYS

    NARCIS (Netherlands)

    BUIST, MR; KENEMANS, P; VANKAMP, GJ; Haisma, Hidde

    The human anti-(mouse Ig) antibody (HAMA) response was measured in serum of 52 patients suspected of having ovarian carcinoma who had received an i.v. injection of either the murine monoclonal antibody (mAb) OV-TL 3 F(ab')(2) (n = 28, 1 mg) or the chimeric mouse/human mAb MOv18 (cMOv18; n = 24, 3

  7. Minor human antibody response to a mouse and chimeric monoclonal antibody after a single i.v. infusion in ovarian carcinoma patients: a comparison of five assays

    NARCIS (Netherlands)

    Buist, M. R.; Kenemans, P.; van Kamp, G. J.; Haisma, H. J.

    1995-01-01

    The human anti-(mouse Ig) antibody (HAMA) response was measured in serum of 52 patients suspected of having ovarian carcinoma who had received an i.v. injection of either the murine monoclonal antibody (mAb) OV-TL 3 F(ab')2 (n = 28, 1 mg) or the chimeric mouse/human mAb MOv18 (cMOv18; n = 24, 3 mg).

  8. Safety and infectivity of two doses of live-attenuated recombinant cold-passaged human parainfluenza type 3 virus vaccine rHPIV3cp45 in HPIV3-seronegative young children.

    Science.gov (United States)

    Englund, Janet A; Karron, Ruth A; Cunningham, Coleen K; Larussa, Philip; Melvin, Ann; Yogev, Ram; Handelsman, Ed; Siberry, George K; Thumar, Bhavanji; Schappell, Elizabeth; Bull, Catherine V; Chu, Helen Y; Schaap-Nutt, Anne; Buchholz, Ursula; Collins, Peter L; Schmidt, Alexander C

    2013-11-19

    Human parainfluenza virus type 3 (HPIV3) is a common cause of upper and lower respiratory tract illness in infants and young children. Live-attenuated cold-adapted HPIV3 vaccines have been evaluated in infants but a suitable interval for administration of a second dose of vaccine has not been defined. HPIV3-seronegative children between the ages of 6 and 36 months were randomized 2:1 in a blinded study to receive two doses of 10⁵ TCID₅₀ (50% tissue culture infectious dose) of live-attenuated, recombinant cold-passaged human PIV3 vaccine (rHPIV3cp45) or placebo 6 months apart. Serum antibody levels were assessed prior to and approximately 4-6 weeks after each dose. Vaccine virus infectivity, defined as detection of vaccine-HPIV3 in nasal wash and/or a≥4-fold rise in serum antibody titer, and reactogenicity were assessed on days 3, 7, and 14 following immunization. Forty HPIV3-seronegative children (median age 13 months; range 6-35 months) were enrolled; 27 (68%) received vaccine and 13 (32%) received placebo. Infectivity was detected in 25 (96%) of 26 evaluable vaccinees following doses 1 and 9 of 26 subject (35%) following dose 2. Among those who shed virus, the median duration of viral shedding was 12 days (range 6-15 days) after dose 1 and 6 days (range 3-8 days) after dose 2, with a mean peak log₁₀ viral titer of 3.4 PFU/mL (SD: 1.0) after dose 1 compared to 1.5 PFU/mL (SD: 0.92) after dose 2. Overall, reactogenicity was mild, with no difference in rates of fever and upper respiratory infection symptoms between vaccine and placebo groups. rHPIV3cp45 was immunogenic and well-tolerated in seronegative young children. A second dose administered 6 months after the initial dose was restricted in those previously infected with vaccine virus; however, the second dose boosted antibody responses and induced antibody responses in two previously uninfected children. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Engineering chimeric human and mouse major histocompatibility complex (MHC) class I tetramers for the production of T-cell receptor (TCR) mimic antibodies

    Science.gov (United States)

    Bentley, Carol; Yates, Jenna; Salimi, Maryam; Greig, Jenny; Wiblin, Sarah; Hassanali, Tasneem; Banham, Alison H.

    2017-01-01

    Therapeutic monoclonal antibodies targeting cell surface or secreted antigens are among the most effective classes of novel immunotherapies. However, the majority of human proteins and established cancer biomarkers are intracellular. Peptides derived from these intracellular proteins are presented on the cell surface by major histocompatibility complex class I (MHC-I) and can be targeted by a novel class of T-cell receptor mimic (TCRm) antibodies that recognise similar epitopes to T-cell receptors. Humoural immune responses to MHC-I tetramers rarely generate TCRm antibodies and many antibodies recognise the α3 domain of MHC-I and β2 microglobulin (β2m) that are not directly involved in presenting the target peptide. Here we describe the production of functional chimeric human-murine HLA-A2-H2Dd tetramers and modifications that increase their bacterial expression and refolding efficiency. These chimeric tetramers were successfully used to generate TCRm antibodies against two epitopes derived from wild type tumour suppressor p53 (RMPEAAPPV and GLAPPQHLIRV) that have been used in vaccination studies. Immunisation with chimeric tetramers yielded no antibodies recognising the human α3 domain and β2m and generated TCRm antibodies capable of specifically recognising the target peptide/MHC-I complex in fully human tetramers and on the cell surface of peptide pulsed T2 cells. Chimeric tetramers represent novel immunogens for TCRm antibody production and may also improve the yield of tetramers for groups using these reagents to monitor CD8 T-cell immune responses in HLA-A2 transgenic mouse models of immunotherapy. PMID:28448627

  10. The remarkable stability of chimeric, sialic acid-derived alpha/delta-peptides in human blood plasma.

    Science.gov (United States)

    Saludes, Jonel P; Natarajan, Arutselvan; DeNardo, Sally J; Gervay-Hague, Jacquelyn

    2010-05-01

    Peptides are labile toward proteolytic enzymes, and structural modifications are often required to prolong their metabolic half-life and increase resistance. One modification is the incorporation of non-alpha-amino acids into the peptide to deter recognition by hydrolytic enzymes. We previously reported the synthesis of chimeric alpha/delta-peptides from glutamic acids (Glu) and the sialic acid derivative Neu2en. Conformational analyses revealed these constructs adopt secondary structures in water and may serve as conformational surrogates of polysialic acid. Polysialic acid is a tumor-associated polysaccharide and is correlated with cancer metastasis. Soluble polysialic acid is rapidly cleared from the blood limiting its potential for vaccine development. One motivation in developing structural surrogates of polysialic acid was to create constructs with increased bioavailability. Here, we report plasma stability profiles of Glu/Neu2en alpha/delta-peptides. DOTA was conjugated at the peptide N-termini by solid phase peptide synthesis, radiolabeled with (111)In, incubated in human blood plasma at 37 degrees C, and their degradation patterns monitored by cellulose acetate electrophoresis and radioactivity counting. Results indicate that these peptides exhibit a long half-life that is two- to three-orders of magnitude higher than natural alpha-peptides. These findings provide a viable platform for the synthesis of plasma stable, sialic acid-derived peptides that may find pharmaceutical application.

  11. Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice

    Science.gov (United States)

    Shih, Yao-Ming; Sun, Cheng-Pu; Chou, Hui-Hsien; Wu, Tzu-Hui; Chen, Chun-Chi; Wu, Ping-Yi; Enya Chen, Yu-Chen; Bissig, Karl-Dimiter; Tao, Mi-Hua

    2015-01-01

    Selection of escape mutants with mutations within the target sequence could abolish the antiviral RNA interference activity. Here, we investigated the impact of a pre-existing shRNA-resistant HBV variant on the efficacy of shRNA therapy. We previously identified a highly potent shRNA, S1, which, when delivered by an adeno-associated viral vector, effectively inhibits HBV replication in HBV transgenic mice. We applied the “PICKY” software to systemically screen the HBV genome, then used hydrodynamic transfection and HBV transgenic mice to identify additional six highly potent shRNAs. Human liver chimeric mice were infected with a mixture of wild-type and T472C HBV, a S1-resistant HBV variant, and then treated with a single or combined shRNAs. The presence of T472C mutant compromised the therapeutic efficacy of S1 and resulted in replacement of serum wild-type HBV by T472C HBV. In contrast, combinatorial therapy using S1 and P28, one of six potent shRNAs, markedly reduced titers for both wild-type and T472C HBV. Interestingly, treatment with P28 alone led to the emergence of escape mutants with mutations in the P28 target region. Our results demonstrate that combinatorial RNAi therapy can minimize the escape of resistant viral mutants in chronic HBV patients. PMID:26482836

  12. Tumor-Targeted Human T Cells Expressing CD28-Based Chimeric Antigen Receptors Circumvent CTLA-4 Inhibition.

    Directory of Open Access Journals (Sweden)

    Maud Condomines

    Full Text Available Adoptive T cell therapy represents a promising treatment for cancer. Human T cells engineered to express a chimeric antigen receptor (CAR recognize and kill tumor cells in a MHC-unrestricted manner and persist in vivo when the CAR includes a CD28 costimulatory domain. However, the intensity of the CAR-mediated CD28 activation signal and its regulation by the CTLA-4 checkpoint are unknown. We investigated whether T cells expressing an anti-CD19, CD3 zeta and CD28-based CAR (19-28z displayed the same proliferation and anti-tumor abilities than T cells expressing a CD3 zeta-based CAR (19z1 costimulated through the CD80/CD28, ligand/receptor pathway. Repeated in vitro antigen-specific stimulations indicated that 19-28z+ T cells secreted higher levels of Th1 cytokines and showed enhanced proliferation compared to those of 19z1+ or 19z1-CD80+ T cells. In an aggressive pre-B cell leukemia model, mice treated with 19-28z+ T cells had 10-fold reduced tumor progression compared to those treated with 19z1+ or 19z1-CD80+ T cells. shRNA-mediated CTLA-4 down-regulation in 19z1-CD80+ T cells significantly increased their in vivo expansion and anti-tumor properties, but had no effect in 19-28z+ T cells. Our results establish that CTLA-4 down-regulation may benefit human adoptive T cell therapy and demonstrate that CAR design can elude negative checkpoints to better sustain T cell function.

  13. Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor.

    Science.gov (United States)

    Zhao, Yangbing; Moon, Edmund; Carpenito, Carmine; Paulos, Chrystal M; Liu, Xiaojun; Brennan, Andrea L; Chew, Anne; Carroll, Richard G; Scholler, John; Levine, Bruce L; Albelda, Steven M; June, Carl H

    2010-11-15

    Redirecting T lymphocyte antigen specificity by gene transfer can provide large numbers of tumor-reactive T lymphocytes for adoptive immunotherapy. However, safety concerns associated with viral vector production have limited clinical application of T cells expressing chimeric antigen receptors (CAR). T lymphocytes can be gene modified by RNA electroporation without integration-associated safety concerns. To establish a safe platform for adoptive immunotherapy, we first optimized the vector backbone for RNA in vitro transcription to achieve high-level transgene expression. CAR expression and function of RNA-electroporated T cells could be detected up to a week after electroporation. Multiple injections of RNA CAR-electroporated T cells mediated regression of large vascularized flank mesothelioma tumors in NOD/scid/γc(-/-) mice. Dramatic tumor reduction also occurred when the preexisting intraperitoneal human-derived tumors, which had been growing in vivo for >50 days, were treated by multiple injections of autologous human T cells electroporated with anti-mesothelin CAR mRNA. This is the first report using matched patient tumor and lymphocytes showing that autologous T cells from cancer patients can be engineered to provide an effective therapy for a disseminated tumor in a robust preclinical model. Multiple injections of RNA-engineered T cells are a novel approach for adoptive cell transfer, providing flexible platform for the treatment of cancer that may complement the use of retroviral and lentiviral engineered T cells. This approach may increase the therapeutic index of T cells engineered to express powerful activation domains without the associated safety concerns of integrating viral vectors. Copyright © 2010 AACR.

  14. Parainfluenza virus as a cause of acute respiratory infection in hospitalized children.

    Science.gov (United States)

    Pecchini, Rogério; Berezin, Eitan Naaman; Souza, Maria Cândida; Vaz-de-Lima, Lourdes de Andrade; Sato, Neuza; Salgado, Maristela; Ueda, Mirthes; Passos, Saulo Duarte; Rangel, Raphael; Catebelota, Ana

    2015-01-01

    Human parainfluenza viruses account for a significant proportion of lower respiratory tract infections in children. To assess the prevalence of Human parainfluenza viruses as a cause of acute respiratory infection and to compare clinical data for this infection against those of the human respiratory syncytial virus. A prospective study in children younger than five years with acute respiratory infection was conducted. Detection of respiratory viruses in nasopharyngeal aspirate samples was performed using the indirect immunofluorescence reaction. Length of hospital stay, age, clinical history and physical exam, clinical diagnoses, and evolution (admission to Intensive Care Unit or general ward, discharge or death) were assessed. Past personal (premature birth and cardiopathy) as well as family (smoking and atopy) medical factors were also assessed. A total of 585 patients were included with a median age of 7.9 months and median hospital stay of six days. No difference between the HRSV+ and HPIV+ groups was found in terms of age, gender or length of hospital stay. The HRSV+ group had more fever and cough. Need for admission to the Intensive Care Unit was similar for both groups but more deaths were recorded in the HPIV+ group. The occurrence of parainfluenza peaked during the autumn in the first two years of the study. Parainfluenza was responsible for significant morbidity, proving to be the second-most prevalent viral agent in this population after respiratory syncytial virus. No difference in clinical presentation was found between the two groups, but mortality was higher in the HPIV+ group. Copyright © 2015. Published by Elsevier Editora Ltda.

  15. Novel Atlantic bottlenose dolphin parainfluenza virus TtPIV-1 clusters with bovine PIV-3 genotype B strains

    Science.gov (United States)

    Parainfluenza virus 3 (PIV-3) is a common viral infection not only in humans, but many other species. Serological evidence suggests that nearly 100% of children in the United States have been infected with PIV-3 by five years of age. Similarly, in cattle PIV-3 is commonly associated with bovine re...

  16. Tears from children with chronic hepatitis B virus (HBV) infection are infectious vehicles of HBV transmission: experimental transmission of HBV by tears, using mice with chimeric human livers.

    Science.gov (United States)

    Komatsu, Haruki; Inui, Ayano; Sogo, Tsuyoshi; Tateno, Akihiko; Shimokawa, Reiko; Fujisawa, Tomoo

    2012-08-15

    Body fluids such as saliva, urine, sweat, and tears from hepatitis B virus (HBV) carriers are potential sources of HBV transmission. Thirty-nine children and 8 adults who were chronically infected with HBV were enrolled. Real-time polymerase chain reaction was used for the quantification of HBV DNA. HBV DNA was detected in 73.7% of urine samples (14 of 19), 86.8% of saliva samples (33 of 38), 100% of tear samples (11 of 11), and 100% of sweat samples (9 of 9). Mean HBV DNA levels (±SD) in urine, saliva, tears, and sweat were 4.3 ± 1.1 log copies/mL, 5.9 ± 1.2 log copies/mL, 6.2 ± 0.7 log copies/mL, and 5.2 ± 0.6 log copies/mL, respectively. A statistically significant correlation was observed between the HBV DNA level in serum specimens and HBV DNA levels in saliva and tear specimens (r = 0.88; P Tear specimens from a child were injected intravenously into 2 human hepatocyte-transplanted chimeric mice. One week after inoculation, both chimeric mice had serum positive for HBV DNA. The levels of HBV DNA in tear specimens from young children were high. Tears were confirmed to be infectious, using chimeric mice. Strict precautions should be taken against direct contact with body fluids from HBV carriers with high-level viremia.

  17. Analytical characterization of ch14.18: a mouse-human chimeric disialoganglioside-specific therapeutic antibody.

    Science.gov (United States)

    Soman, Gopalan; Kallarakal, Abraham T; Michiel, Dennis; Yang, Xiaoyi; Saptharish, Nirmala; Jiang, Hengguang; Giardina, Steve; Gilly, John; Mitra, George

    2012-01-01

    Ch14.18 is a mouse-human chimeric monoclonal antibody to the disialoganglioside (GD2) glycolipid. In the clinic, this antibody has been shown to be effective in the treatment of children with high-risk neuroblastoma, either alone or in combination therapy. Extensive product characterization is a prerequisite to addressing the potential issues of product variability associated with process changes and manufacturing scale-up. Charge heterogeneity, glycosylation profile, molecular state and aggregation, interaction (affinity) with Fcγ receptors and functional or biological activities are a few of the critical characterization assays for assessing product comparability for this antibody. In this article, we describe the in-house development and qualification of imaged capillary isoelectric focusing to assess charge heterogeneity, analytical size exclusion chromatography with online static and dynamic light scattering (DLS), batch mode DLS for aggregate detection, biosensor (surface plasmon resonance)-based Fcγ receptor antibody interaction kinetics, N-glycoprofiling with PNGase F digestion, 2-aminobenzoic acid labeling and high performance liquid chromatography and N-glycan analysis using capillary electrophoresis. In addition, we studied selected biological activity assays, such as complement-dependent cytotoxicity. The consistency and reproducibility of the assays are established by comparing the intra-day and inter-day assay results. Applications of the methodologies to address stability or changes in product characteristics are also reported. The study results reveal that the ch14.18 clinical product formulated in phosphate-buffered saline at a concentration of 5 mg/ml and stored at 2-8°C is stable for more than five years.

  18. Regression of established renal cell carcinoma in nude mice using lentivirus-transduced human T cells expressing a human anti-CAIX chimeric antigen receptor

    Directory of Open Access Journals (Sweden)

    Agnes Shuk-Yee Lo

    2014-01-01

    Full Text Available Carbonic anhydrase IX (CAIX is a tumor-associated antigen and marker of hypoxia that is overexpressed on > 90% of clear-cell type renal cell carcinoma (RCC but not on neighboring normal kidney tissue. Here, we report on the construction of two chimeric antigen receptors (CARs that utilize a carbonic anhydrase (CA domain mapped, human single chain antibody (scFv G36 as a targeting moiety but differ in their capacity to provide costimulatory signaling for optimal T cell proliferation and tumor cell killing. The resulting anti-CAIX CARs were expressed on human primary T cells via lentivirus transduction. CAR-transduced T cells (CART cells expressing second-generation G36-CD28-TCRζ exhibited more potent in vitro antitumor effects on CAIX+ RCC cells than first-generation G36-CD8-TCRζ including cytotoxicity, cytokine secretion, proliferation, and clonal expansion. Adoptive G36-CD28-TCRζ CART cell therapy combined with high-dose interleukin (IL-2 injection also lead to superior regression of established RCC in nude mice with evidence of tumor cell apoptosis and tissue necrosis. These results suggest that the fully human G36-CD28-TCRζ CARs should provide substantial improvements over first-generation mouse anti-CAIX CARs in clinical use through reduced human anti-mouse antibody responses against the targeting scFv and administration of lower doses of T cells during CART cell therapy of CAIX+ RCC.

  19. 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

  20. Preclinical Safety Studies of Enadenotucirev, a Chimeric Group B Human-Specific Oncolytic Adenovirus

    Directory of Open Access Journals (Sweden)

    Sam Illingworth

    2017-06-01

    Full Text Available Enadenotucirev is an oncolytic group B adenovirus identified by a process of bio-selection for the ability to selectively propagate in and rapidly kill carcinoma cells. It is resistant to inactivation by human blood components, potentially enabling intravenous dosing in patients with metastatic cancer. However, there are no known permissive animal models described for group B adenoviruses that could facilitate a conventional approach to preclinical safety studies. In this manuscript, we describe our tailored preclinical strategy designed to evaluate the key biological properties of enadenotucirev. As enadenotucirev does not replicate in animal cells, a panel of primary human cells was used to evaluate enadenotucirev replication selectivity in vitro, demonstrating that virus genome levels were >100-fold lower in normal cells relative to tumor cells. Acute intravenous tolerability in mice was used to assess virus particle-mediated toxicology and effects on innate immunity. These studies showed that particle toxicity could be ameliorated by dose fractionation, using an initial dose of virus to condition the host such that cytokine responses to subsequent doses were significantly attenuated. This, in turn, supported the initiation of a phase I intravenous clinical trial with a starting dose of 1 × 1010 virus particles given on days 1, 3, and 5.

  1. Chimeric antigen receptors with human scFvs preferentially induce T cell anti-tumor activity against tumors with high B7H6 expression.

    Science.gov (United States)

    Gacerez, Albert T; Hua, Casey K; Ackerman, Margaret E; Sentman, Charles L

    2018-05-01

    B7H6 is emerging as a promising tumor antigen that is known to be expressed on a wide array of tumors and is reported to stimulate anti-tumor responses from the immune system. As such, B7H6 presents a good target for tumor-specific immunotherapies. B7H6-specific chimeric antigen receptors (CAR) based on a murine antibody showed successful targeting and elimination of tumors expressing B7H6. However, mouse single chain variable fragments (scFvs) have the potential to induce host anti-CAR responses that may limit efficacy, so human scFvs specific for B7H6 were selected by yeast surface display. In this study, we validate the functionality of these human scFvs when formatted into chimeric antigen receptors. The data indicate that T cells expressing these B7H6-specific human scFvs as CARs induced potent anti-tumor activity in vitro and in vivo against tumors expressing high amounts of B7H6. Importantly, these human scFv-based CARs are sensitive to changes in B7H6 expression which may potentially spare non-tumor cells that express B7H6 and provides the foundation for future clinical development.

  2. HUMAN PAPILLOMA VIRUS IMMUNOGEN CREATION ON THE BASE OF CHIMERIC RECOMBINANT PROTEIN L2E7

    Directory of Open Access Journals (Sweden)

    I. S. Malakhov

    2016-01-01

    Full Text Available The cervical cancer is one of the most common diseases in world. This malignancy is the seventh highest prevalence oncological disease worldwide and the second highest prevalence oncological disease of women in the world. Meanwhile women need to be infected by human papilloma virus (HPV is absolutely necessary for it further evolution, HPV DNA was found in 99.97% cases of disease. Except cervical cancer, HPV cause 85% of rectal cancer, 50% of the vulva, vagina and penis cancers, 20% of oropharyngeal cancer and 10% of larynx and esophagus cancers. In 2009, 14 000 women were diagnosed with cervical cancer in Russia. The growth in morbidity was 19% (in comparison with 1999. The most effective recognised measure for almost each infection prophylaxis is a vaccination. Two human papilloma virus vaccines are available in Russia nowadays — Gardasil and Cervarix, produced in Belgium and the Netherlands respectively. Cervarix is a bivalent vaccine based on virus-like particles (VLP of two types. Recombinant major capsid proteins L1 HPV 16 and HPV 18 express in baculovirus expression system and self-assembled into virus-like particles (about 70 percent of cervical cancers are caused by HPV 16 and HPV 18. VLP of each strain produced in different baculovirus vectors and then combined in single drug. Gardasil is like Cervarix with few exceptions. Producing organisms are fungi S. cerevisiae in this case, and this vaccine contains low-risk HPV 6 and HPV 11 VLP. Thus, Gardasil is quadrivalent HPV-6/11/16/18 vaccine. These vaccines are very effective in averting infection of disease and don’t have significant side-effects, however they have some disadvantages. Firstly, they have a high price because of necessity of their expression in eukaryotic cells. Secondly, they are strain-specific, so vaccines are completely effective only for virus’s strains which are represented in the vaccine. Thirdly, it`s the absence of therapeutic (treatment of established

  3. Human hepatocytes support the hypertrophic but not the hyperplastic response to the murine nongenotoxic hepatocarcinogen sodium phenobarbital in an in vivo study using a chimeric mouse with humanized liver.

    Science.gov (United States)

    Yamada, Tomoya; Okuda, Yu; Kushida, Masahiko; Sumida, Kayo; Takeuchi, Hayato; Nagahori, Hirohisa; Fukuda, Takako; Lake, Brian G; Cohen, Samuel M; Kawamura, Satoshi

    2014-11-01

    High doses of sodium phenobarbital (NaPB), a constitutive androstane receptor (CAR) activator, have been shown to produce hepatocellular tumors in rodents by a mitogenic mode of action (MOA) involving CAR activation. The effect of 1-week dietary treatment with NaPB on liver weight and histopathology, hepatic CYP2B enzyme activity and CYP2B/3A mRNA expression, replicative DNA synthesis and selected genes related to cell proliferation, and functional transcriptomic and metabolomic analyses was studied in male CD-1 mice, Wistar Hannover (WH) rats, and chimeric mice with human hepatocytes. The treatment of chimeric mice with 1000-1500-ppm NaPB resulted in plasma levels around 3-5-fold higher than those observed in human subjects given therapeutic doses of NaPB. NaPB produced dose-dependent increases in hepatic CYP2B activity and CYP2B/3A mRNA levels in all animal models. Integrated functional metabolomic and transcriptomic analyses demonstrated that the responses to NaPB in the human liver were clearly different from those in rodents. Although NaPB produced a dose-dependent increase in hepatocyte replicative DNA synthesis in CD-1 mice and WH rats, no increase in replicative DNA synthesis was observed in human hepatocyte-originated areas of chimeric mice. In addition, treatment with NaPB had no effect on Ki-67, PCNA, GADD45β, and MDM2 mRNA expression in chimeric mice, whereas significant increases were observed in CD-1 mice and/or WH rats. However, increases in hepatocyte replicative DNA synthesis were observed in chimeric mice both in vivo and in vitro after treatment epidermal growth factor. Thus, although NaPB could activate CAR in both rodent and human hepatocytes, NaPB did not increase replicative DNA synthesis in human hepatocytes of chimeric mice, whereas it was mitogenic to rat and mouse hepatocytes. As human hepatocytes are refractory to the mitogenic effects of NaPB, the MOA for NaPB-induced rodent liver tumor formation is thus not relevant for humans. © The

  4. Identification of structural traits that increase the antimicrobial activity of a chimeric peptide of human β-defensins 2 and 3.

    Science.gov (United States)

    Spudy, Björn; Sönnichsen, Frank D; Waetzig, Georg H; Grötzinger, Joachim; Jung, Sascha

    2012-10-12

    Antimicrobial peptides participate in the first line of defence of many organisms against pathogens. In humans, the family of β-defensins plays a pivotal role in innate immunity. Two human β-defensins, β-defensin-2 and -3 (HBD2 and HBD3), show substantial sequence identity and structural similarity. However, HBD3 kills Staphylococcus (S.) aureus with a 4- to 8-fold higher efficiency compared to HBD2, whereas their activities against Escherichia (E.) coli are very similar. The generation of six HBD2/HBD3-chimeric molecules led to the identification of distinct molecular regions which mediate their divergent killing properties. One of the chimeras (chimera C3) killed both E. coli and S. aureus with an even higher efficacy compared to the wild-type molecules. Due to the broad spectrum of its antimicrobial activity against many human multidrug-resistant pathogens, this HBD2/HBD3-chimeric peptide represents a promising candidate for a new class of antibiotics. In order to investigate the structural basis of its exceptional antimicrobial activity, the peptide's tertiary structure was determined by NMR spectroscopy, which allowed its direct comparison to the published structures of HBD2 and HBD3 and the identification of the activity-increasing molecular features. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Immunological circumvention of multiple organ metastases of multidrug resistant human small cell lung cancer cells by mouse-human chimeric anti-ganglioside GM2 antibody KM966.

    Science.gov (United States)

    Hanibuchi, M; Yano, S; Nishioka, Y; Yanagawa, H; Miki, T; Sone, S

    2000-01-01

    serum against SBC-3/DOX cells to a similar extent compared with parental SBC-3 cells. Pretreatment of human effector cells with various cytokines induced further enhancement of the KM966-dependent ADCC against SBC-3/DOX cells. Intravenous injection of SBC-3 or SBC-3/DOX cells into natural killer (NK) cell-depleted severe combined immunodeficient (SCID) mice developed metastases in multiple organs (liver, kidneys and lymph nodes). Interestingly, SBC-3/DOX cells produced metastases more rapidly than SBC-3 cells, suggesting more aggressive phenotype of SBC-3/DOX cells than their parental cells in vivo. Systemic treatment with KM966, given on days 2 and 7, drastically inhibited the formation of multiple-organ metastases produced by both SBC-3 and SBC-3/DOX cells, indicating that KM966 can eradicate metastasis by SCLC cells irrespective of MDR phenotype. These findings suggest that the mouse-human chimeric KM966 targets the GM2 antigen, and might be useful for the immunological circumvention of multiple-organ metastases of refractory SCLC.

  6. Expression of a Chimeric Antigen Receptor Specific for Donor HLA Class I Enhances the Potency of Human Regulatory T Cells in Preventing Human Skin Transplant Rejection.

    Science.gov (United States)

    Boardman, D A; Philippeos, C; Fruhwirth, G O; Ibrahim, M A A; Hannen, R F; Cooper, D; Marelli-Berg, F M; Watt, F M; Lechler, R I; Maher, J; Smyth, L A; Lombardi, G

    2017-04-01

    Regulatory T cell (Treg) therapy using recipient-derived Tregs expanded ex vivo is currently being investigated clinically by us and others as a means of reducing allograft rejection following organ transplantation. Data from animal models has demonstrated that adoptive transfer of allospecific Tregs offers greater protection from graft rejection compared to polyclonal Tregs. Chimeric antigen receptors (CAR) are clinically translatable synthetic fusion proteins that can redirect the specificity of T cells toward designated antigens. We used CAR technology to redirect human polyclonal Tregs toward donor-MHC class I molecules, which are ubiquitously expressed in allografts. Two novel HLA-A2-specific CARs were engineered: one comprising a CD28-CD3ζ signaling domain (CAR) and one lacking an intracellular signaling domain (ΔCAR). CAR Tregs were specifically activated and significantly more suppressive than polyclonal or ΔCAR Tregs in the presence of HLA-A2, without eliciting cytotoxic activity. Furthermore, CAR and ΔCAR Tregs preferentially transmigrated across HLA-A2-expressing endothelial cell monolayers. In a human skin xenograft transplant model, adoptive transfer of CAR Tregs alleviated the alloimmune-mediated skin injury caused by transferring allogeneic peripheral blood mononuclear cells more effectively than polyclonal Tregs. Our results demonstrated that the use of CAR technology is a clinically applicable refinement of Treg therapy for organ transplantation. © 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

  7. 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...

  8. L1 Cell Adhesion Molecule-Specific Chimeric Antigen Receptor-Redirected Human T Cells Exhibit Specific and Efficient Antitumor Activity against Human Ovarian Cancer in Mice.

    Directory of Open Access Journals (Sweden)

    Hao Hong

    Full Text Available New therapeutic modalities are needed for ovarian cancer, the most lethal gynecologic malignancy. Recent clinical trials have demonstrated the impressive therapeutic potential of adoptive therapy using chimeric antigen receptor (CAR-redirected T cells to target hematological cancers, and emerging studies suggest a similar impact may be achieved for solid cancers. We sought determine whether genetically-modified T cells targeting the CE7-epitope of L1-CAM, a cell adhesion molecule aberrantly expressed in several cancers, have promise as an immunotherapy for ovarian cancer, first demonstrating that L1-CAM was highly over-expressed on a panel of ovarian cancer cell lines, primary ovarian tumor tissue specimens, and ascites-derived primary cancer cells. Human central memory derived T cells (TCM were then genetically modified to express an anti-L1-CAM CAR (CE7R, which directed effector function upon tumor antigen stimulation as assessed by in vitro cytokine secretion and cytotoxicity assays. We also found that CE7R+ T cells were able to target primary ovarian cancer cells. Intraperitoneal (i.p. administration of CE7R+ TCM induced a significant regression of i.p. established SK-OV-3 xenograft tumors in mice, inhibited ascites formation, and conferred a significant survival advantage compared with control-treated animals. Taken together, these studies indicate that adoptive transfer of L1-CAM-specific CE7R+ T cells may offer a novel and effective immunotherapy strategy for advanced ovarian cancer.

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

    Science.gov (United States)

    Kamenz, Thomas; Caca, Karel; Blüthner, Thilo; Tannapfel, Andrea; Mössner, Joachim; Wiedmann, Marcus

    2006-01-01

    AIM: To investigate the c-kit expression in biliary tract cancer cell lines and histological sections from patients with extrahepatic cholangiocarcinoma (CC) and to evaluate the efficacy of in vitro and in vitro treatment with imatinib mesilate. METHODS: The protein expression of c-kit in the human biliary tract cancer cell lines Mz-ChA-2 and EGI-1 and histological sections from 19 patients with extrahepatic CC was assessed by immunoblotting, immunocytochemistry, and immunohistochemistry. The anti-proliferative effect of imatinib mesilate on biliary tract cancer cell lines Mz-ChA-2 and EGI-1 was studied in vitro by automated cell counting. In addition, immunodeficient NMRI mice (TaconicTM) were subcutaneously injected with 5 x 106 cells of cell lines MzChA-2 and EGI-1. After having reached a tumour volume of 200 mm3, daily treatment was started intraperitoneally with imatinib mesilate at a dose of 50 mg/kg or normal saline (NS). Tumor volume was calculated with a Vernier caliper. After 14 d, mice were sacrificed with tumors excised and tumor mass determined. RESULTS: Immunoblotting revealed presence of c-kit in Mz-ChA-2 and absence in EGI-1 cells. Immunocytochemistry with c-kit antibodies displayed a cytoplasmatic and membraneous localization of receptor protein in Mz-ChA-2 cells and absence of c-kit in EGI-1 cells. c-kit was expressed in 7 of 19 (37%) extrahepatic human CC tissue samples, 2 showed a moderate and 5 a rather weak immunostaining. Imatinib mesilate at a low concentration of 5 µmol/L caused a significant growth inhibition in the c-kit positive cell line Mz-ChA-2 (31%), but not in the c-kit negative cell line EGI-1 (0%) (P < 0.05). Imatinib mesilate at an intermediate concentration of 10 µmol/L inhibited cellular growth of both cell lines (51% vs 57%). Imatinib mesilate at a higher concentration of 20 µmol/L seemed to have a general toxic effect on both cell lines. The IC50 values were 9.7 µmol/L and 11 µmol/L, respectively. After 14 d of in vitro

  10. CXCR3 Directs Antigen-Specific Effector CD4+ T Cell Migration to the Lung During Parainfluenza Virus Infection

    DEFF Research Database (Denmark)

    Kohlmeier, Jacob E; Cookenham, Tres; Miller, Shannon C

    2009-01-01

    effector CD4(+) T cell migration to the lungs. To assess the role of CCR5 and CXCR3 in vivo, we directly compared the migration of Ag-specific wild-type and chemokine receptor-deficient effector T cells in mixed bone marrow chimeric mice during a parainfluenza virus infection. CXCR3-deficient effector CD4......(+) T cells were 5- to 10-fold less efficient at migrating to the lung compared with wild-type cells, whereas CCR5-deficient effector T cells were not impaired in their migration to the lung. In contrast to its role in trafficking, CXCR3 had no impact on effector CD4(+) T cell proliferation, phenotype......, or function in any of the tissues examined. These findings demonstrate that CXCR3 controls virus-specific effector CD4(+) T cell migration in vivo, and suggest that blocking CXCR3-mediated recruitment may limit T cell-induced immunopathology during respiratory virus infections....

  11. Challenge Pools of Hepatitis C Virus Genotypes 1–6 Prototype Strains: Replication Fitness and Pathogenicity in Chimpanzees and Human Liver–Chimeric Mouse Models

    Science.gov (United States)

    Bukh, Jens; Meuleman, Philip; Tellier, Raymond; Engle, Ronald E.; Feinstone, Stephen M.; Eder, Gerald; Satterfield, William C.; Govindarajan, Sugantha; Krawczynski, Krzysztof; Miller, Roger H.; Leroux-Roels, Geert; Purcell, Robert H.

    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 >104.7 IU/mL, and development of acute hepatitis; the chronicity rate was 56%. The challenge pools had titers of 103–105 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 resource for studies of HCV molecular virology and for studies of pathogenesis, protective immunity, and vaccine efficacy in vivo. PMID:20353362

  12. Close-up of the alpha-1,3-Gal epitope as defined by a monoclonal chimeric IgE and human serum using saturation transfer difference (STD) NMR

    DEFF Research Database (Denmark)

    Plum, Melanie; Michel, Yvonne; Wallach, Katharina

    2011-01-01

    of an alpha-Gal-specific murine IgM antibody was employed to construct chimeric IgE and IgG antibodies. Reactivity and specificity of the resulting antibodies were assessed by means of ELISA and receptor binding studies. Using defined carbohydrates, interaction of the IgE and human serum was assessed...... by mediator release assays, surface plasmon resonance (SPR) and STD NMR analyses. The alpha-Gal-specific chimeric IgE and IgG antibodies were proven functional regarding interaction with antigen and Fc receptors. SPR measurements demonstrated affinities in the micromolar range. In contrast to a reference...

  13. 9 CFR 113.316 - Canine Parainfluenza Vaccine.

    Science.gov (United States)

    2010-01-01

    ... furnished or approved by Animal and Plant Health Inspection Service. (4) The rectal temperature of each dog...: (1) Twenty-five canine parainfluenza susceptible dogs (20 vaccinates and 5 controls) shall be used as test animals. Nasal swabs shall be collected from each dog on the day the first dose of vaccine is...

  14. 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

    2016-06-07

    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.

  15. Chimeric Recombinant Human Metapneumoviruses with the Nucleoprotein or Phosphoprotein Open Reading Frame Replaced by That of Avian Metapneumovirus Exhibit Improved Growth In Vitro and Attenuation In Vivo

    Science.gov (United States)

    Pham, Quynh N.; Biacchesi, Stéphane; Skiadopoulos, Mario H.; Murphy, Brian R.; Collins, Peter L.; Buchholz, Ursula J.

    2005-01-01

    Chimeric versions of recombinant human metapneumovirus (HMPV) were generated by replacing the nucleoprotein (N) or phosphoprotein (P) open reading frame with its counterpart from the closely related avian metapneumovirus (AMPV) subgroup C. In Vero cells, AMPV replicated to an approximately 100-fold-higher titer than HMPV. Surprisingly, the N and P chimeric viruses replicated to a peak titer that was 11- and 25-fold higher, respectively, than that of parental HMPV. The basis for this effect is not known but was not due to obvious changes in the efficiency of gene expression. AMPV and the N and P chimeras were evaluated for replication, immunogenicity, and protective efficacy in hamsters. AMPV was attenuated compared to HMPV in this mammalian host on day 5 postinfection, but not on day 3, and only in the nasal turbinates. In contrast, the N and P chimeras were reduced approximately 100-fold in both the upper and lower respiratory tract on day 3 postinfection, although there was little difference by day 5. The N and P chimeras induced a high level of neutralizing serum antibodies and protective efficacy against HMPV; AMPV was only weakly immunogenic and protective against HMPV challenge, reflecting antigenic differences. In African green monkeys immunized intranasally and intratracheally, the mean peak titer of the P chimera was reduced 100- and 1,000-fold in the upper and lower respiratory tracts, whereas the N chimera was reduced only 10-fold in the lower respiratory tract. Both chimeras were comparable to wild-type HMPV in immunogenicity and protective efficacy. Thus, the P chimera is a promising live HMPV vaccine candidate that paradoxically combines improved growth in vitro with attenuation in vivo. PMID:16306583

  16. Parainfluenza virus infections in a tropical city: clinical and epidemiological aspects

    Directory of Open Access Journals (Sweden)

    Mariana Mota Moura Fé

    Full Text Available Little information on the epidemiology and clinical characteristics of human parainfluenza virus (HPIV infections, especially in children from tropical countries, has been published. The aim of this study was to determine the frequency of HPIV infections in children attended at a large hospital in Fortaleza in Northeast Brazil, and describe seasonal patterns, clinical and epidemiological characteristics of these infections. From January 2001 to December 2006, a total of 3070 nasopharyngeal aspirates collected from children were screened by indirect immunofluorescence for human parainfluenza viruses 1, 2, and 3 (HPIV-1, 2 and 3 and other respiratory viruses. Viral antigens were identified in 933 samples and HPIV in 117. The frequency of HPIV-3, HPIV-1 and HPIV-2 was of 83.76%, 11.96% and 4.27%, respectively. Only HPIV-3 showed a seasonal occurrence, with most cases observed from September to November, and with an inverse relationship to the rainy season. Most HPIV-3 infections seen in outpatients were diagnosed as upper respiratory tract infections.

  17. Development of A Chimeric Antigen Receptor Targeting C-Type Lectin-Like Molecule-1 for Human Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    Eduardo Laborda

    2017-10-01

    Full Text Available The treatment of patients with acute myeloid leukemia (AML with targeted immunotherapy is challenged by the heterogeneity of the disease and a lack of tumor-exclusive antigens. Conventional immunotherapy targets for AML such as CD33 and CD123 have been proposed as targets for chimeric antigen receptor (CAR-engineered T-cells (CAR-T-cells, a therapy that has been highly successful in the treatment of B-cell leukemia and lymphoma. However, CD33 and CD123 are present on hematopoietic stem cells, and targeting with CAR-T-cells has the potential to elicit long-term myelosuppression. C-type lectin-like molecule-1 (CLL1 or CLEC12A is a myeloid lineage antigen that is expressed by malignant cells in more than 90% of AML patients. CLL1 is not expressed by healthy Hematopoietic Stem Cells (HSCs, and is therefore a promising target for CAR-T-cell therapy. Here, we describe the development and optimization of an anti-CLL1 CAR-T-cell with potent activity on both AML cell lines and primary patient-derived AML blasts in vitro while sparing healthy HSCs. Furthermore, in a disseminated mouse xenograft model using the CLL1-positive HL60 cell line, these CAR-T-cells completely eradicated tumor, thus supporting CLL1 as a promising target for CAR-T-cells to treat AML while limiting myelosuppressive toxicity.

  18. 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.

  19. Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells

    Science.gov (United States)

    Munisvaradass, Rusheni; Kumar, Suresh; Govindasamy, Chandramohan; Alnumair, Khalid S.; Mok, Pooi Ling

    2017-01-01

    Breast cancer is a common malignancy among women. The innate and adaptive immune responses failed to be activated owing to immune modulation in the tumour microenvironment. Decades of scientific study links the overexpression of human epidermal growth factor receptor 2 (ERBB2) antigen with aggressive tumours. The Chimeric Antigen Receptor (CAR) coding for specific tumour-associated antigens could initiate intrinsic T-cell signalling, inducing T-cell activation, and cytotoxic activity without the need for major histocompatibility complex recognition. This renders CAR as a potentially universal immunotherapeutic option. Herein, we aimed to establish CAR in CD3+ T-cells, isolated from human peripheral blood mononucleated cells that could subsequently target and induce apoptosis in the ERBB2 overexpressing human breast cancer cell line, SKBR3. Constructed CAR was inserted into a lentiviral plasmid containing a green fluorescent protein tag and produced as lentiviral particles that were used to transduce activated T-cells. Transduced CAR-T cells were then primed with SKBR3 cells to evaluate their functionality. Results showed increased apoptosis in SKBR3 cells co-cultured with CAR-T cells compared to the control (non–transduced T-cells). This study demonstrates that CAR introduction helps overcome the innate limitations of native T-cells leading to cancer cell apoptosis. We recommend future studies should focus on in vivo cytotoxicity of CAR-T cells against ERBB2 expressing tumours. PMID:28885562

  20. Development of β-lactoglobulin-specific chimeric human IgEκ monoclonal antibodies for in vitro safety assessment of whey hydrolysates.

    Science.gov (United States)

    Knipping, Karen; Simons, Peter J; Buelens-Sleumer, Laura S; Cox, Linda; den Hartog, Marcel; de Jong, Niels; Teshima, Reiko; Garssen, Johan; Boon, Louis; Knippels, Léon M J

    2014-01-01

    Cow's milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow's milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays. An oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays. Mice were immunized with bovine β-lactoglobulin, and subsequently the variable domains of dissimilar anti-β-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains. After sensitization with this pool of anti-β-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa β-lactoglobulin, and 5-10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow's milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-β-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on β-lactoglobulin, which were also recognized by serum IgEs from cow's milk allergic children. Usage of our 'unlimited' source and well-defined pool of β-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow's milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula.

  1. Human/murine chimeric 81C6 F(ab')2 fragment: preclinical evaluation of a potential construct for the targeted radiotherapy of malignant glioma

    International Nuclear Information System (INIS)

    Boskovitz, Abraham; Akabani, Gamal H.; Pegram, Charles N.; Bigner, Darrell D.; Zalutsky, Michael R.

    2004-01-01

    We have obtained encouraging responses in recent Phase I studies evaluating 131 I-labeled human/murine chimeric 81C6 anti-tenascin monoclonal antibody (ch81C6) administered into surgically-created tumor resection cavities in brain tumor patients. However, because the blood clearance is slow, hematologic toxicity has been higher than seen with murine 81C6 (mu81C6). In the current study, a series of paired-label experiments were performed in athymic mice bearing subcutaneous D-245 MG human glioma xenografts to compare the biodistribution of the fragment ch81C6 F(ab') 2 labeled using Iodogen to a) intact ch81C6, b) mu81C6, and c) ch81C6 F(ab') 2 labeled using N-succinimidyl 3-[ 131 I]iodobenzoate. Tumor retention of radioiodine activity for the F(ab') 2 fragment was comparable to that for intact ch81C6 for the first 24 h and to that for mu81C6 for the first 48 h; as expected, blood and other normal tissue levels declined faster for ch81C6 F(ab') 2. Radiation dosimetry calculations suggest that 131 I-labeled ch81C6 F(ab') 2 may warrant further evaluation as a targeted radiotherapeutic for the treatment of brain tumors

  2. Comparative analysis of chimeric ZFP-, TALE- and Cas9-piggyBac transposases for integration into a single locus in human cells.

    Science.gov (United States)

    Luo, Wentian; Galvan, Daniel L; Woodard, Lauren E; Dorset, Dan; Levy, Shawn; Wilson, Matthew H

    2017-08-21

    Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of 'targeted-only' cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

  3. Redirected Primary Human Chimeric Antigen Receptor Natural Killer Cells As an “Off-the-Shelf Immunotherapy” for Improvement in Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Olaf Oberschmidt

    2017-06-01

    Full Text Available Primary human natural killer (NK cells recognize and subsequently eliminate virus infected cells, tumor cells, or other aberrant cells. However, cancer cells are able to develop tumor immune escape mechanisms to undermine this immune control. To overcome this obstacle, NK cells can be genetically modified to express chimeric antigen receptors (CARs in order to improve specific recognition of cancer surface markers (e.g., CD19, CD20, and ErbB2. After target recognition, intracellular CAR domain signaling (CD3ζ, CD28, 4-1BB, and 2B4 leads to activation of PI3K or DNAX proteins (DAP10, DAP12 and finally to enhanced cytotoxicity, proliferation, and/or interferon γ release. This mini-review summarizes both the first preclinical trials with CAR-engineered primary human NK cells and the translational implications for “off-the-shelf immunotherapy” in cancer treatment. Signal transduction in NK cells as well as optimization of CAR signaling will be described, becoming more and more a focal point of interest in addition to redirected T cells. Finally, strategies to overcome off-target effects will be discussed in order to improve future clinical trials and to avoid attacking healthy tissues.

  4. [Cloning of VH and VL Gene of Human anti-IL1RAP McAb and Construction of Recombinant Chimeric Receptor].

    Science.gov (United States)

    Yin, Ling-Ling; Ruan, Su-Hong; Tian, Yu; Zhao, Kai; Xu, Kai Lin

    2015-10-01

    To clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs). The VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells. The VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired. Human anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.

  5. A tetravalent vaccine comprising hexon-chimeric adenoviruses elicits balanced protective immunity against human adenovirus types 3, 7, 14 and 55.

    Science.gov (United States)

    Tian, Xingui; Jiang, Zaixue; Fan, Ye; Qiu, Shuyan; Zhang, Ling; Li, Xiao; Zhou, Zhichao; Liu, Tiantian; Ma, Qiang; Lu, Xiaomei; Zhong, Baimao; Zhou, Rong

    2018-04-04

    Human adenovirus (Ad) species B contains several of the most important types associated with acute respiratory diseases, Ad3, -7, -14 and -55, which often lead to severe lower respiratory tract diseases and epidemic outbreaks. However, there is currently no Ad vaccine approved for general use. The major capsid protein, hexon, is the primary determinant recognized by neutralizing antibodies (NAbs). In this study, four recombinant Ads that have the same genome sequence as Ad3 with the exception of the hexon genes, rAd3EGFP, rAd3H7, rAd3H14 and rAd3H55, were combined as a tetravalent Ad candidate vaccine against Ad3, -7, -14 and -55. The replication efficiencies of chimeric rAd3H14, rAd3H7 and rAd3H55 were similar to that of rAd3EGFP. Recombinant rAd3EGFP, rAd3H7, rAd3H14 and rAd3H55 induced high titers of NAbs against Ad3, -7, -14 and -55, respectively, which were comparable to those induced by wild-type Ads. The mixture of the four recombinant Ads in equal proportions, rAdMix, or rAdMix inactivated by β-propiolactone, induced balanced NAb responses against Ad3, -7, -14 and -55 in mice without reciprocal immunological interference. In co-culture the four recombinant Ads replicated with a similar efficiency without reciprocal inhibition, and the progeny virions may be chimeric. Purified co-culture, rAdMix-C, also elicited balanced immune responses, suggesting a simple method for multivalent vaccine production. These results indicate the possible advantage of the four Ads as a live combined vaccine. Importantly, pre-immunization with rAdMix conferred protection against Ad3, -7, -14 or -55 challenge in mice in vivo. Thus, this research provides a novel tetravalent Ad vaccine candidate against Ad3, -7, -14 and -55. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. 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.

  7. Silencing of BCR/ABL Chimeric Gene in Human Chronic Myelogenous Leukemia Cell Line K562 by siRNA-Nuclear Export Signal Peptide Conjugates.

    Science.gov (United States)

    Shinkai, Yasuhiro; Kashihara, Shinichi; Minematsu, Go; Fujii, Hirofumi; Naemura, Madoka; Kotake, Yojiro; Morita, Yasutaka; Ohnuki, Koichiro; Fokina, Alesya A; Stetsenko, Dmitry A; Filichev, Vyacheslav V; Fujii, Masayuki

    2017-06-01

    Herein we described the synthesis of siRNA-NES (nuclear export signal) peptide conjugates by solid phase fragment coupling and the application of them to silencing of bcr/abl chimeric gene in human chronic myelogenous leukemia cell line K562. Two types of siRNA-NES conjugates were prepared, and both sense strands at 5' ends were covalently linked to a NES peptide derived from TFIIIA and HIV-1 REV, respectively. Significant enhancement of silencing efficiency was observed for both of them. siRNA-TFIIIA NES conjugate suppressed the expression of BCR/ABL gene to 8.3% at 200 nM and 11.6% at 50 nM, and siRNA-HIV-1REV NES conjugate suppressed to 4.0% at 200 nM and 6.3% at 50 nM, whereas native siRNA suppressed to 36.3% at 200 nM and 30.2% at 50 nM. We could also show complex of siRNA-NES conjugate and designed amphiphilic peptide peptideβ7 could be taken up into cells with no cytotoxicity and showed excellent silencing efficiency. We believe that the complex siRNA-NES conjugate and peptideβ7 is a promising candidate for in vivo use and therapeutic applications.

  8. Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway.

    Directory of Open Access Journals (Sweden)

    Nan-Sun Kim

    Full Text Available A chimeric protein vaccine composed of the cholera toxin B subunit fused to proinsulin (CTB-INS was shown to suppress type 1 diabetes onset in NOD mice and upregulate biosynthesis of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1 in human dendritic cells (DCs. Here we demonstrate siRNA inhibition of the NF-κB-inducing kinase (NIK suppresses vaccine-induced IDO1 biosynthesis as well as IKKα phosphorylation. Chromatin immunoprecipitation (ChIP analysis of CTB-INS inoculated DCs showed that RelB bound to NF-κB consensus sequences in the IDO1 promoter, suggesting vaccine stimulation of the non-canonical NF-κB pathway activates IDO1 expression in vivo. The addition of Tumor Necrosis Factor Associated Factors (TRAF TRAF 2, 3 and TRAF6 blocking peptides to vaccine inoculated DCs was shown to inhibit IDO1 biosynthesis. This experimental outcome suggests vaccine activation of the TNFR super-family receptor pathway leads to upregulation of IDO1 biosynthesis in CTB-INS inoculated dendritic cells. Together, our experimental data suggest the CTB-INS vaccine uses a TNFR-dependent signaling pathway of the non-canonical NF-κB signaling pathway resulting in suppression of dendritic cell mediated type 1 diabetes autoimmunity.

  9. ChLpMab-23: Cancer-Specific Human-Mouse Chimeric Anti-Podoplanin Antibody Exhibits Antitumor Activity via Antibody-Dependent Cellular Cytotoxicity.

    Science.gov (United States)

    Kaneko, Mika K; Nakamura, Takuro; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Yamada, Shinji; Yanaka, Miyuki; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

    2017-06-01

    Podoplanin is expressed in many cancers, including oral cancers and brain tumors. The interaction between podoplanin and its receptor C-type lectin-like receptor 2 (CLEC-2) has been reported to be involved in cancer metastasis and tumor malignancy. We previously established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-23 (IgG 1 , kappa), one of the mouse anti-podoplanin mAbs, was shown to be a CasMab. However, we have not shown the usefulness of LpMab-23 for antibody therapy against podoplanin-expressing cancers. In this study, we first determined the minimum epitope of LpMab-23 and revealed that Gly54-Leu64 peptide, especially Gly54, Thr55, Ser56, Glu57, Asp58, Arg59, Tyr60, and Leu64 of podoplanin, is a critical epitope of LpMab-23. We further produced human-mouse chimeric LpMab-23 (chLpMab-23) and investigated whether chLpMab-23 exerts antibody-dependent cellular cytotoxicity (ADCC) and antitumor activity. In flow cytometry, chLpMab-23 showed high sensitivity against a podoplanin-expressing glioblastoma cell line, LN319, and an oral cancer cell line, HSC-2. chLpMab-23 also showed ADCC activity against podoplanin-expressing CHO cells (CHO/podoplanin). In xenograft models with HSC-2 and CHO/podoplanin, chLpMab-23 exerts antitumor activity using human natural killer cells, indicating that chLpMab-23 could be useful for antibody therapy against podoplanin-expressing cancers.

  10. Demonstration of Hepatitis C Virus RNA with In Situ Hybridization Employing a Locked Nucleic Acid Probe in Humanized Liver of Infected Chimeric Mice and in Needle-Biopsied Human Liver

    Directory of Open Access Journals (Sweden)

    Kazuya Shiogama

    2013-01-01

    Full Text Available Background. In situ hybridization (ISH with high sensitivity has been requested to demonstrate hepatitis C virus (HCV RNA in formalin-fixed, paraffin-embedded (FFPE sections of the liver. Methods. ISH employing a locked-nucleic-acid- (LNA-modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected and of needle-biopsied livers from HCV-infected patients. Results. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82% of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73% of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions. HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma. Conclusion. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.

  11. Parainfluenza Virus Infection Sensitizes Cancer Cells to DNA-Damaging Agents: Implications for Oncolytic Virus Therapy.

    Science.gov (United States)

    Fox, Candace R; Parks, Griffith D

    2018-04-01

    A parainfluenza virus 5 (PIV5) with mutations in the P/V gene (P/V-CPI - ) is restricted for spread in normal cells but not in cancer cells in vitro and is effective at reducing tumor burdens in mouse model systems. Here we show that P/V-CPI - infection of HEp-2 human laryngeal cancer cells results in the majority of the cells dying, but unexpectedly, over time, there is an emergence of a population of cells that survive as P/V-CPI - persistently infected (PI) cells. P/V-CPI - PI cells had elevated levels of basal caspase activation, and viability was highly dependent on the activity of cellular inhibitor-of-apoptosis proteins (IAPs) such as Survivin and XIAP. In challenge experiments with external inducers of apoptosis, PI cells were more sensitive to cisplatin-induced DNA damage and cell death. This increased cisplatin sensitivity correlated with defects in DNA damage signaling pathways such as phosphorylation of Chk1 and translocation of damage-specific DNA binding protein 1 (DDB1) to the nucleus. Cisplatin-induced killing of PI cells was sensitive to the inhibition of wild-type (WT) p53-inducible protein 1 (WIP1), a phosphatase which acts to terminate DNA damage signaling pathways. A similar sensitivity to cisplatin was seen with cells during acute infection with P/V-CPI - as well as during acute infections with WT PIV5 and the related virus human parainfluenza virus type 2 (hPIV2). Our results have general implications for the design of safer paramyxovirus-based vectors that cannot establish PI as well as the potential for combining chemotherapy with oncolytic RNA virus vectors. IMPORTANCE There is intense interest in developing oncolytic viral vectors with increased potency against cancer cells, particularly those cancer cells that have gained resistance to chemotherapies. We have found that infection with cytoplasmically replicating parainfluenza virus can result in increases in the killing of cancer cells by agents that induce DNA damage, and this is linked

  12. Haemophilus parainfluenzae Strain ATCC 33392 Forms Biofilms In Vitro and during Experimental Otitis Media Infections.

    Science.gov (United States)

    Pang, Bing; Swords, W Edward

    2017-09-01

    Haemophilus parainfluenzae is a nutritionally fastidious, Gram-negative bacterium with an oropharyngeal/nasopharyngeal carriage niche that is associated with a range of opportunistic infections, including infectious endocarditis and otitis media (OM). These infections are often chronic/recurrent in nature and typically involve bacterial persistence within biofilm communities that are highly resistant to host clearance. This study addresses the primary hypothesis that H. parainfluenzae forms biofilm communities that are important determinants of persistence in vivo The results from in vitro biofilm studies confirmed that H. parainfluenzae formed biofilm communities within which the polymeric matrix was mainly composed of extracellular DNA and proteins. Using a chinchilla OM infection model, we demonstrated that H. parainfluenzae formed surface-associated biofilm communities containing bacterial and host components that included neutrophil extracellular trap (NET) structures and that the bacteria mainly persisted in these biofilm communities. We also used this model to examine the possible interaction between H. parainfluenzae and its close relative Haemophilus influenzae , which is also commonly carried within the same host environments and can cause OM. The results showed that coinfection with H. influenzae promoted clearance of H. parainfluenzae from biofilm communities during OM infection. The underlying mechanisms for bacterial persistence and biofilm formation by H. parainfluenzae and knowledge about the survival defects of H. parainfluenzae during coinfection with H. influenzae are topics for future work. Copyright © 2017 American Society for Microbiology.

  13. Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy.

    Science.gov (United States)

    Zhuang, Xiaolei; Watts, Norman R; Palmer, Ira W; Kaufman, Joshua D; Dearborn, Altaira D; Trenbeath, Joni L; Eren, Elif; Steven, Alasdair C; Rader, Christoph; Wingfield, Paul T

    2017-10-06

    Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in Escherichia coli , had unprecedentedly high binding affinities ( K d ∼10 -12 m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.

  14. Antitumor activity of chLpMab-2, a human-mouse chimeric cancer-specific antihuman podoplanin antibody, via antibody-dependent cellular cytotoxicity.

    Science.gov (United States)

    Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Abe, Shinji; Nishioka, Yasuhiko; Kunita, Akiko; Fukayama, Masashi; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

    2017-04-01

    Human podoplanin (hPDPN), a platelet aggregation-inducing transmembrane glycoprotein, is expressed in different types of tumors, and it binds to C-type lectin-like receptor 2 (CLEC-2). The overexpression of hPDPN is involved in invasion and metastasis. Anti-hPDPN monoclonal antibodies (mAbs) such as NZ-1 have shown antitumor and antimetastatic activities by binding to the platelet aggregation-stimulating (PLAG) domain of hPDPN. Recently, we developed a novel mouse anti-hPDPN mAb, LpMab-2, using the cancer-specific mAb (CasMab) technology. In this study we developed chLpMab-2, a human-mouse chimeric anti-hPDPN antibody, derived from LpMab-2. chLpMab-2 was produced using fucosyltransferase 8-knockout (KO) Chinese hamster ovary (CHO)-S cell lines. By flow cytometry, chLpMab-2 reacted with hPDPN-expressing cancer cell lines including glioblastomas, mesotheliomas, and lung cancers. However, it showed low reaction with normal cell lines such as lymphatic endothelial and renal epithelial cells. Moreover, chLpMab-2 exhibited high antibody-dependent cellular cytotoxicity (ADCC) against PDPN-expressing cells, despite its low complement-dependent cytotoxicity. Furthermore, treatment with chLpMab-2 abolished tumor growth in xenograft models of CHO/hPDPN, indicating that chLpMab-2 suppressed tumor development via ADCC. In conclusion, chLpMab-2 could be useful as a novel antibody-based therapy against hPDPN-expressing tumors. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  15. Use of retroviral-mediated gene transfer to deliver and test function of chimeric antigen receptors in human T-cells

    Directory of Open Access Journals (Sweden)

    Ana C. Parente-Pereira

    2014-07-01

    Full Text Available Chimeric antigen receptors (CARs are genetically delivered fusion molecules that elicit T-cell activation upon binding of a native cell surface molecule. These molecules can be used to generate a large number of memory and effector T-cells that are capable of recognizing and attacking tumor cells. Most commonly, stable CAR expression is achieved in T-cells using retroviral vectors. In the method described here, retroviral vectors are packaged in a two-step procedure. First, H29D human retroviral packaging cells (a derivative of 293 cells are transfected with the vector of interest, which is packaged transiently in vesicular stomatitis virus (VSV G pseudotyped particles. These particles are used to deliver the vector to PG13 cells, which achieve stable packaging of gibbon ape leukaemia virus (GALV-pseudotyped particles that are suitable for infection of human T-cells. The key advantage of the method reported here is that it robustly generates polyclonal PG13 cells that are 100% positive for the vector of interest. This means that efficient gene transfer may be repeatedly achieved without the need to clone individual PG13 cells for experimental pre-clinical testing. To achieve T-cell transduction, cells must first be activated using a non-specific mitogen. Phytohemagglutinin (PHA provides an economic and robust stimulus to achieve this. After 48-72 h, activated T-cells and virus-conditioned medium are mixed in RetroNectin-coated plasticware, which enhances transduction efficiency. Transduced cells are analyzed for gene transfer efficiency by flow cytometry 48 h following transduction and may then be tested in several assays to evaluate CAR function, including target-dependent cytotoxicity, cytokine production and proliferation.

  16. First complete genome sequence of parainfluenza virus 5 isolated from lesser panda.

    Science.gov (United States)

    Zhai, Jun-Qiong; Zhai, Shao-Lun; Lin, Tao; Liu, Jian-Kui; Wang, He-Xing; Li, Bing; Zhang, He; Zou, Shu-Zhan; Zhou, Xia; Wu, Meng-Fan; Chen, Wu; Luo, Man-Lin

    2017-05-01

    Parainfluenza virus 5 (PIV5) is widespread in mammals and humans. Up to now, there is little information about PIV5 infection in lesser pandas. In this study, a PIV5 variant (named ZJQ-221) was isolated from a lesser panda with respiratory disease in Guangzhou zoo in Guangdong province, southern China. The full-length genome of ZJQ-221 was found to be 15,246 nucleotides and consisted of seven non-overlapping genes encoding eight proteins (i.e., NP, V, P, M, F, SH, HN and L). Sequence alignment and genetic analysis revealed that ZJQ-221 shared a close relationship with a PIV5 strain of canine-origin (1168-1) from South Korea. The findings of this study confirm the presence of PIV5 in lesser panda and indicate this mammal as a possible natural reservoir. Furthermore they highlight the urgent need to strengthen viral surveillance and control of PIV5 in zoo animals.

  17. Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof

    OpenAIRE

    2011-01-01

    The present invention relates to infectious DNA clones, infectious chimeric DNA clones of porcine circovirus (PCV), vaccines and means of protecting pigs against viral infection or postweaning multisystemic wasting syndrome (PMWS) caused by PCV2. The new chimeric infectious DNA clone and its derived, avirulent chimeric virus are constructed from the nonpathogenic PCV1 in which the immunogenic ORF gene of the pathogenic PCV2 replaces a gene of the nonpathogenic PCV1, preferably in the same pos...

  18. Polymicrobial infective endocarditis caused by Neisseria sicca and Haemophilus parainfluenzae

    Directory of Open Access Journals (Sweden)

    Nikoloz Koshkelashvili

    2016-01-01

    Full Text Available Infective endocarditis is a common clinical problem in industrialized countries. Risk factors include abnormal cardiac valves, a history of endocarditis, intracardiac devices, prosthetic valves and intravenous drug use. We report a case of polymicrobial infective endocarditis in a 33 year-old female with a history chronic heroin use caused by Neisseria sicca and Haemophilus parainfluenzae. We believe the patient was exposed to these microbes by cleansing her skin with saliva prior to injection. Pairing a detailed history with the consideration of atypical agents is crucial in the proper diagnosis and management of endocarditis in patients with high-risk injection behaviors.

  19. Rational and efficient preparation of a chimeric protein containing a tandem dimer of thrombopoietin mimetic peptide fused to human growth hormone in Escherichia coli.

    Science.gov (United States)

    Wang, Song; Shen, Mingqiang; Xu, Yang; Chen, Fang; Chen, Mo; Chen, Shilei; Wang, Aiping; Zhang, Zhou; Ran, Xinze; Cheng, Tianmin; Su, Yongping; Wang, Junping

    2013-04-01

    The 14-mer thrombopoietin mimetic peptide (TMP), especially in the form of dimer, displayed potent megakaryocytopoiesis activity in vitro. However, it is difficult to prepare such short peptide with high bioactivity through gene-engineering approaches. In this study, a chimeric protein containing a tandem dimer of TMP (dTMP) fused to human growth hormone (hGH), a kind of hematopoietic growth factor that activates the same signal pathways as thrombopoietin, was produced in Escherichia coli by soluble expression. By rational utilization of the XmnI and EcoRV restriction sites, a PCR fragment encoding dTMP-GH was inserted into the plasmid vector pMAL-p2X at the position right after Xa factor cleavage site, in frame with maltose-binding protein (MBP) gene. Under optimized conditions, a high-level expression of soluble MBP-dTMP-GH fusion protein was obtained. By application of amylose resin chromatography, Xa factor digestion, hydrophobic chromatography followed by gel filtration, the dTMP-GH fusion protein was separated. Finally, a relatively high yield of dTMP-GH fusion protein with high purity (>98%) and without redundant amino acid was achieved, as identified by high-performance liquid chromatography, mass spectrometry, and amino acid sequencing. The functional assays showed that dTMP-GH could promote the proliferation of megakaryoblast cells and maturation of murine megakaryocytes derived from bone marrow, in a dose-dependent manner. Moreover, an enhanced effect of dTMP-GH on megakaryocytopoiesis was found as compared with equimolar concentration of dTMP and rhGH. This work provides a new avenue to generate thrombopoietic agents based on TMP.

  20. Expression, purification and characterization of the recombinant chimeric IgE Fc-fragment opossum-human-opossum (OSO), an active immunotherapeutic vaccine component.

    Science.gov (United States)

    Xu, Bingze; Lundgren, Mats; Magnusson, Ann-Christine; Fuentes, Alexis

    2010-11-01

    The active vaccine component recombinant chimeric IgE Fc-fragment opossum-human-opossum (OSO) has been expressed in CHO-K1 cells. It contains two identical polypeptide chains with 338 amino acid residues in each chain connected by two disulfide bridges. The cell lines were adapted to suspension culture in a serum-free medium. An expression level of 60 mg/L was obtained after 8 days in a shaking flask at a temperature of 31.5 degrees C. The OSO protein has been purified to homogeneity by a combination of three chromatographic steps. Virus inactivation and reduction by solvent detergent treatment and nano-filtration were included in the process. The residual host cell protein content was less than 50 ng/mg OSO as analyzed by ELISA. Purity was analyzed by SDS-PAGE under reducing and non-reducing conditions and was estimated by densitometry to be above 99.0%. The dimer content was less than 0.1% as estimated by analytical size exclusion chromatography. The molecular mass, as estimated by SDS-PAGE, is 90 kDa. A value of around 74 kDa was calculated from its amino acid composition. This indicates that the protein is heavily glycosylated containing around 18% carbohydrate. Isoelectric focusing in polyacrylamide gel disclosed a ladder type band pattern with pI values in the pH-range 7.0-8.3, indicating a variation in the sialic acid content. The OSO protein is not stable at temperatures above 40 degrees C and at pH values below 4 indicating that virus inactivation by incubating the protein solution at higher temperature or at lower pH is not possible. Copyright 2010 Elsevier Inc. All rights reserved.

  1. Labelling of anti-human bladder tumor chimeric antibody with 99Tcm and radioimmunoimaging of bladder carcinoma xenograft in nude mice

    International Nuclear Information System (INIS)

    Zhang Chunli; Wang Rongfu; Fu Zhanli; Bai Yin; Ding Yi; Yu Lizhang

    2003-01-01

    Objective: To study the in vitro immunoreactivity and in vivo tissue distribution, tumor targeting property of anti-human bladder tumor human-murine chimeric antibody (ch-BDI) labeled with 99 Tc m and to investigate its possibility for being used in guiding diagnosis and guiding therapy of bladder cancer. Methods: The ch-BDI was labeled with 99 Tc m by improved Schwarz method and the labeled antibody was purified by Sephadex G-50. Labeling yield and radiochemical purity were measured by paper chromatography. The immunoreactive fraction and association constant (K a ) were measured by Lindmo method and Scatchard analysis, respectively. 11.1 MBq (30 μg) 99 Tc m -ch-BDI was intravenously injected into nude mice bearing human bladder cancer xenografts in the right thigh and radioimmunoimaging (RII) was performed 2, 6, 20 and 24 h postinjection. The images were processed by region of interest (ROI) method to acquire the counts of whole body and the tumor and the counts ratios of tumor to contralateral normal tissue or to tissues of other non-tumor bearing organs. The mice were killed after 24 h postinjection imaging and tissue distribution was measured. %ID/g and target to nontarget (T/NT) ratios were calculated. Results: The labeling yield and radiochemical purity of 99 Tc m -ch-BDI were (66.5±7.3)% and >90%, respectively. The immunoreactive fraction was 76% and K a was 3.56 x 10 9 L/mol. RII showed that the tumor was clearly visualized 6 h postinjection and becoming clearer along with time prolonging. The radioactivity of whole body decreased rapidly with time, whereas the radioactivity of the tumor decreased slowly. The T/NT ratios was increased with time. Biodistribution results showed that tumor uptake was 17.4%ID/g 24 h postinjection. T/NT ratios were very high except for the kidney. T/NT ratios for brain, muscle, intestinal wall, bone and heart wall were 136.0, 55.1, 39.3, 29.7 and 27.9, respectively. Conclusion: 99 Tc m -ch-BDI exhibits excellent

  2. Aberrant chimeric RNA GOLM1-MAK10 encoding a secreted fusion protein as a molecular signature for human esophageal squamous cell carcinoma

    Science.gov (United States)

    Zhang, Hao; Lin, Wan; Kannan, Kalpana; Luo, Liming; Li, Jing; Chao, Pei-Wen; Wang, Yan; Chen, Yu-Ping; Gu, Jiang; Yen, Laising

    2013-01-01

    It is increasingly recognized that chimeric RNAs may exert a novel layer of cellular complexity that contributes to oncogenesis and cancer progression, and could be utilized as molecular biomarkers and therapeutic targets. To date yet no fusion chimeric RNAs have been identified in esophageal cancer, the 6th most frequent cause of cancer death in the world. While analyzing the expression of 32 recurrent cancer chimeric RNAs in esophageal squamous cell carcinoma (ESCC) from patients and cancer cell lines, we identified GOLM1-MAK10, as a highly cancer-enriched chimeric RNA in ESCC. In situ hybridization revealed that the expression of the chimera is largely restricted to cancer cells in patient tumors, and nearly undetectable in non-neoplastic esophageal tissue from normal subjects. The aberrant chimera closely correlated with histologic differentiation and lymph node metastasis. Furthermore, we demonstrate that chimera GOLM1-MAK10 encodes a secreted fusion protein. Mechanistic studies reveal that GOLM1-MAK10 is likely derived from transcription read-through/splicing rather than being generated from a fusion gene. Collectively, these findings provide novel insights into the molecular mechanism involved in ESCC and provide a novel potential target for future therapies. The secreted fusion protein translated from GOLM1-MAK10 could also serve as a unique protein signature detectable by standard non-invasive assays. These observations are critical as there is no clinically useful molecular signature available for detecting this deadly disease or monitoring the treatment response. PMID:24243830

  3. Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

    International Nuclear Information System (INIS)

    Zambetti, G.; Stein, J.; Stein, G.

    1987-01-01

    The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 β-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression

  4. Development of Anti-Human Mesothelin-Targeted Chimeric Antigen Receptor Messenger RNA-transfected Peripheral Blood Lymphocytes for Ovarian Cancer Therapy.

    Science.gov (United States)

    Hung, Chien-Fu; Xu, Xuequn; Li, Linhong; Ma, Ying; Jin, Qiu; Viley, Angelia; Allen, Cornell; Natarajan, Pachai; Shivakumar, Rama; Peshwa, Madhusudan V; Emens, Leisha A

    2018-04-02

    CD19-targeted chimeric antigen receptor (CAR) engineered T/natural killer (NK)-cell therapies can result in durable clinical responses in B-cell malignancies. However, CAR-based immunotherapies have been much less successful in solid cancers, in part due to "on-target off-tumor" toxicity related to expression of target tumor antigens on normal tissue. Based on preliminary observations of safety and clinical activity in proof-of-concept clinical trials, tumor antigen-specific messenger RNA (mRNA) CAR transfection into selected, activated, and expanded T/NK cells may permit prospective control of "on-target off-tumor" toxicity. To develop a commercial product for solid tumors, mesothelin was selected as an antigen target based on its association with poor prognosis and overexpression in multiple solid cancers. It was hypothesized that selecting, activating, and expanding cells ex vivo prior to mRNA CAR transfection would not be necessary, thus simplifying the complexity and cost of manufacturing. Now, the development of anti-human mesothelin mRNA CAR transfected peripheral blood lymphocytes (CARMA-hMeso) is reported, demonstrating the manufacture and cryopreservation of multiple cell aliquots for repeat administrations from a single human leukapheresis. A rapid, automated, closed system for cGMP-compliant transfection of mRNA CAR in up to 20 × 10 9 peripheral blood lymphocytes was developed. Here we show that CARMA-hMeso cells recognize and lyse tumor cells in a mesothelin-specific manner. Expression of CAR was detectable over approximately 7 days in vitro, with a progressive decline of CAR expression that appears to correlate with in vitro cell expansion. In a murine ovarian cancer model, a single intraperitoneal injection of CARMA-hMeso resulted in the dose-dependent inhibition of tumor growth and improved survival of mice. Furthermore, repeat weekly intraperitoneal administrations of the optimal CARMA-hMeso dose further prolonged disease control and survival

  5. Chimeric mitochondrial peptides from contiguous regular and swinger RNA.

    Science.gov (United States)

    Seligmann, Hervé

    2016-01-01

    Previous mass spectrometry analyses described human mitochondrial peptides entirely translated from swinger RNAs, RNAs where polymerization systematically exchanged nucleotides. Exchanges follow one among 23 bijective transformation rules, nine symmetric exchanges (X ↔ Y, e.g. A ↔ C) and fourteen asymmetric exchanges (X → Y → Z → X, e.g. A → C → G → A), multiplying by 24 DNA's protein coding potential. Abrupt switches from regular to swinger polymerization produce chimeric RNAs. Here, human mitochondrial proteomic analyses assuming abrupt switches between regular and swinger transcriptions, detect chimeric peptides, encoded by part regular, part swinger RNA. Contiguous regular- and swinger-encoded residues within single peptides are stronger evidence for translation of swinger RNA than previously detected, entirely swinger-encoded peptides: regular parts are positive controls matched with contiguous swinger parts, increasing confidence in results. Chimeric peptides are 200 × rarer than swinger peptides (3/100,000 versus 6/1000). Among 186 peptides with > 8 residues for each regular and swinger parts, regular parts of eleven chimeric peptides correspond to six among the thirteen recognized, mitochondrial protein-coding genes. Chimeric peptides matching partly regular proteins are rarer and less expressed than chimeric peptides matching non-coding sequences, suggesting targeted degradation of misfolded proteins. Present results strengthen hypotheses that the short mitogenome encodes far more proteins than hitherto assumed. Entirely swinger-encoded proteins could exist.

  6. Transfer of C-terminal residues of human apolipoprotein A-I to insect apolipophorin III creates a two-domain chimeric protein with enhanced lipid binding activity.

    Science.gov (United States)

    Horn, James V C; Ellena, Rachel A; Tran, Jesse J; Beck, Wendy H J; Narayanaswami, Vasanthy; Weers, Paul M M

    2017-08-01

    Apolipophorin III (apoLp-III) is an insect apolipoprotein (18kDa) that comprises a single five-helix bundle domain. In contrast, human apolipoprotein A-I (apoA-I) is a 28kDa two-domain protein: an α-helical N-terminal domain (residues 1-189) and a less structured C-terminal domain (residues 190-243). To better understand the apolipoprotein domain organization, a novel chimeric protein was engineered by attaching residues 179 to 243 of apoA-I to the C-terminal end of apoLp-III. The apoLp-III/apoA-I chimera was successfully expressed and purified in E. coli. Western blot analysis and mass spectrometry confirmed the presence of the C-terminal domain of apoA-I within the chimera. While parent apoLp-III did not self-associate, the chimera formed oligomers similar to apoA-I. The chimera displayed a lower α-helical content, but the stability remained similar compared to apoLp-III, consistent with the addition of a less structured domain. The chimera was able to solubilize phospholipid vesicles at a significantly higher rate compared to apoLp-III, approaching that of apoA-I. The chimera was more effective in protecting phospholipase C-treated low density lipoprotein from aggregation compared to apoLp-III. In addition, binding interaction of the chimera with phosphatidylglycerol vesicles and lipopolysaccharides was considerably improved compared to apoLp-III. Thus, addition of the C-terminal domain of apoA-I to apoLp-III created a two-domain protein, with self-association, lipid and lipopolysaccharide binding properties similar to apoA-I. The apoA-I like behavior of the chimera indicate that these properties are independent from residues residing in the N-terminal domain of apoA-I, and that they can be transferred from apoA-I to apoLp-III. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. 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

  8. Bone marrow chimerism as a strategy to produce tolerance in solid organ allotransplantation.

    Science.gov (United States)

    Hu, Min; Alexander, Stephen I; Yi, Shounan

    2016-12-01

    Clinical transplant tolerance has been most successfully achieved combining hematopoietic chimerism with kidney transplantation. This review outlines this strategy in animal models and human transplantation, and possible clinical challenges. Kidney transplant tolerance has been achieved through chimerism in several centers beginning with Massachusetts General Hospital's success with mixed chimerism in human leukocyte antigen (HLA)-mismatched patients and the Stanford group with HLA-matched patients, and the more recent success of the Northwestern protocol achieving full chimerism. This has challenged the original view that stable mixed chimerism is necessary for organ graft tolerance. However, among the HLA-mismatched kidney transplant-tolerant patients, loss of mixed chimerism does not lead to renal-graft rejection, and the development of host Foxp3+ regulatory T cells has been observed. Recent animal models suggest that graft tolerance through bone marrow chimerism occurs through both clonal deletion and regulatory immune cells. Further, Tregs have been shown to improve chimerism in animal models. Animal studies continue to suggest ways to improve our current clinical strategies. Advances in chimerism protocols suggest that tolerance may be clinically achievable with relative safety for HLA-mismatched kidney transplants.

  9. Use of a novel chimeric mouse model with a functionally active human immune system to study human immunodeficiency virus type 1 infection

    NARCIS (Netherlands)

    An, Dong Sung; Poon, Betty; Tsong Fang, Raphael Ho; Weijer, Kees; Blom, Bianca; Spits, Hergen; Chen, Irvin S. Y.; Uittenbogaart, Christel H.

    2007-01-01

    The goal of this study was to develop a small-animal model to study human immunodeficiency virus type 1 (HIV-1) pathogenesis in blood and primary and secondary lymphoid organs. Rag2(-/-)gamma(c)(-/-) mice that are neonatally injected with human CD34(+) cells develop a functional human immune system

  10. Chimeras taking shape: Potential functions of proteins encoded by chimeric RNA transcripts

    Science.gov (United States)

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

    2012-01-01

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

  11. Synthesis and characterization of human recombinant thyrotropin (rec-hTSH) with a chimeric {beta}-subunit (rec-hTSH{beta}-CTPE hCG{beta}); Sintese e caracterizacao do hormonio tireotrofico humano recombinante (rec-hTSH) contendo uma subunidade {beta} quimerica (rec-hTSH{beta}-CTPE hCG{beta})

    Energy Technology Data Exchange (ETDEWEB)

    Murata, Yoko

    1995-12-31

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

  12. Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

    Science.gov (United States)

    Kaneko, Mika K; Abe, Shinji; Ogasawara, Satoshi; Fujii, Yuki; Yamada, Shinji; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Nishioka, Yasuhiko; Kato, Yukinari

    2017-02-01

    Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG 2a , lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG 1 ) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG 1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.

  13. Chimeric anti-tenascin antibody 81C6: Increased tumor localization compared with its murine parent

    International Nuclear Information System (INIS)

    Zalutsky, Michael R.; Archer, Gary E.; Garg, Pradeep K.; Batra, Surinder K.; Bigner, Darell D.

    1996-01-01

    When labeled using the Iodogen method, a chimeric antibody composed of the human IgG 2 constant region and the variable regions of murine anti-tenascin 81C6 exhibited superior uptake in human glioma xenografts compared with its murine parent. In the current study, three paired-label experiments were performed in athymic mice with subcutaneous D-54 MG human glioma xenografts to evaluate further the properties of radioiodinated chimeric 81C6. These studies demonstrated that (a) the enhanced tumor uptake of chimeric 81C6 is specific; (b) when labeling was performed using N-succinimidyl 3-iodobenzoate, chimeric 81C6 again showed preferential accumulation in tumor compared with murine 81C6; and (c) the tumor uptake advantage observed previously with murine 81C6 for N-succinimidyl 3-iodobenzoate compared with Iodogen labeling did not occur with chimeric 81C6

  14. THE TREATMENT EFFECT OF OXYTETRACYCLINE AND VITAMIN C IN AN EPISODE OF PARAINFLUENZA SHEEP IN TIMIS COUNTY

    Directory of Open Access Journals (Sweden)

    Stancu, A

    2017-06-01

    Full Text Available Sheep parainfluenza It is a disease with high diffusibility, sometimes with fatal serious, especially youth. It is caused by parainfluenza 3 virus (PI-3, identical to the bovine parainfluenza virus isolate, in combination with certain bacteria. PI-3 virus was firstly isolated from Hore et al. (1966 in the lungs and nasal mucus of sheep with pneumopathies and Gilmour et al (1968 successfully experimenting with an inactivated vaccine for the prophylaxis of diseases. In our country, parainfluenza sheep was diagnosed in 1977 by pathological examinations. Also by pathological examination was differentiated by Maedi-visna disease and pulmonary adenomatosis.

  15. Porcine induced pluripotent stem cells produce chimeric offspring.

    Science.gov (United States)

    West, Franklin D; Terlouw, Steve L; Kwon, Dae Jin; Mumaw, Jennifer L; Dhara, Sujoy K; Hasneen, Kowser; Dobrinsky, John R; Stice, Steven L

    2010-08-01

    Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology.

  16. Vaccination with Recombinant Parainfluenza Virus 5 Expressing Neuraminidase Protects against Homologous and Heterologous Influenza Virus Challenge.

    Science.gov (United States)

    Mooney, Alaina J; Gabbard, Jon D; Li, Zhuo; Dlugolenski, Daniel A; Johnson, Scott K; Tripp, Ralph A; He, Biao; Tompkins, S Mark

    2017-12-01

    Seasonal human influenza virus continues to cause morbidity and mortality annually, and highly pathogenic avian influenza (HPAI) viruses along with other emerging influenza viruses continue to pose pandemic threats. Vaccination is considered the most effective measure for controlling influenza; however, current strategies rely on a precise vaccine match with currently circulating virus strains for efficacy, requiring constant surveillance and regular development of matched vaccines. Current vaccines focus on eliciting specific antibody responses against the hemagglutinin (HA) surface glycoprotein; however, the diversity of HAs across species and antigenic drift of circulating strains enable the evasion of virus-inhibiting antibody responses, resulting in vaccine failure. The neuraminidase (NA) surface glycoprotein, while diverse, has a conserved enzymatic site and presents an appealing target for priming broadly effective antibody responses. Here we show that vaccination with parainfluenza virus 5 (PIV5), a promising live viral vector expressing NA from avian (H5N1) or pandemic (H1N1) influenza virus, elicited NA-specific antibody and T cell responses, which conferred protection against homologous and heterologous influenza virus challenges. Vaccination with PIV5-N1 NA provided cross-protection against challenge with a heterosubtypic (H3N2) virus. Experiments using antibody transfer indicate that antibodies to NA have an important role in protection. These findings indicate that PIV5 expressing NA may be effective as a broadly protective vaccine against seasonal influenza and emerging pandemic threats. IMPORTANCE Seasonal influenza viruses cause considerable morbidity and mortality annually, while emerging viruses pose potential pandemic threats. Currently licensed influenza virus vaccines rely on the antigenic match of hemagglutinin (HA) for vaccine strain selection, and most vaccines rely on HA inhibition titers to determine efficacy, despite the growing

  17. Chimeric ZHHH neuroglobin acts as a cell membrane-penetrating inducer of neurite outgrowth.

    Science.gov (United States)

    Takahashi, Nozomu; Onozuka, Wataru; Watanabe, Seiji; Wakasugi, Keisuke

    2017-09-01

    Neuroglobin (Ngb) is a heme protein expressed in the vertebrate brain. We previously engineered a chimeric Ngb protein, in which module M1 of human Ngb is replaced by that of zebrafish Ngb, and showed that the chimeric ZHHH Ngb has a cell membrane-penetrating activity similar to that of zebrafish Ngb and also rescues cells from death caused by hypoxia/reoxygenation as does human Ngb. Recently, it was reported that overexpression of mammalian Ngb in neuronal cells induces neurite outgrowth. In this study, we performed neurite outgrowth assays of chimeric Ngb using rat pheochromocytoma PC12 cells. Addition of chimeric Ngb, but not human or zebrafish Ngb, exogenously to the cell medium induces neurite outgrowth. On the other hand, the K7A/K9Q chimeric Ngb double mutant, which cannot translocate into cells, did not induce neurite outgrowth, suggesting that the cell membrane-penetrating activity of the chimeric Ngb is crucial for its neurite outgrowth-promoting activity. We also prepared several site-directed chimeric Ngb mutants and demonstrated that residues crucial for neurite outgrowth-inducing activity of the chimeric Ngb are not exactly the same as those for its neuroprotective activity.

  18. Molecular epidemiology and environmental contamination during an outbreak of parainfluenza virus 3 in a haematology ward.

    Science.gov (United States)

    Kim, T; Jin, C E; Sung, H; Koo, B; Park, J; Kim, S-M; Kim, J Y; Chong, Y P; Lee, S-O; Choi, S-H; Kim, Y S; Woo, J H; Lee, J-H; Lee, J-H; Lee, K-H; Shin, Y; Kim, S-H

    2017-12-01

    Although fomites or contaminated surfaces have been considered as transmission routes, the role of environmental contamination by human parainfluenza virus type 3 (hPIV-3) in healthcare settings is not established. To describe an hPIV-3 nosocomial outbreak and the results of environmental sampling to elucidate the source of nosocomial transmission and the role of environmental contamination. During an hPIV-3 outbreak between May and June 2016, environmental surfaces in contact with clustered patients were swabbed and respiratory specimens used from infected patients and epidemiologically unlinked controls. The epidemiologic relatedness of hPIV-3 strains was investigated by sequencing of the haemagglutinin-neuraminidase and fusion protein genes. Of 19 hPIV-3-infected patients, eight were haematopoietic stem cell recipients and one was a healthcare worker. In addition, four had upper and 12 had lower respiratory tract infections. Of the 19 patients, six (32%) were community-onset infections (symptom onset within environmental swabs up to 12 days after negative respiratory polymerase chain reaction conversion. At least one-third of a peak season nosocomial hPIV-3 outbreak originated from nosocomial transmission, with multiple importations of hPIV-3 from the community, providing experimental evidence for extensive environmental hPIV-3 contamination. Direct contact with the contaminated surfaces and fomites or indirect transmission from infected healthcare workers could be responsible for nosocomial transmission. Copyright © 2017 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

  19. Parainfluenza Virus Type 1 Induces Epithelial IL-8 Production via p38-MAPK Signalling

    Directory of Open Access Journals (Sweden)

    Miguel Ángel Galván Morales

    2014-01-01

    Full Text Available Human parainfluenza virus type 1 (HPIV-1 is the most common cause of croup in infants. The aim of this study was to describe molecular mechanisms associated with IL-8 production during HPIV-1 infection and the role of viral replication in MAPK synthesis and activation. An in vitro model of HPIV-1 infection in the HEp-2 and A549 cell lines was used; a kinetic-based ELISA for IL-8 detection was also used, phosphorylation of the mitogen-activated protein kinases (MAPKs was identified by Western blot analysis, and specific inhibitors for each kinase were used to identify which MAPK was involved. Inactivated viruses were used to assess whether viral replication is required for IL-8 production. Results revealed a gradual increase in IL-8 production at different selected times, when phosphorylation of MAPK was detected. The secretion of IL-8 in the two cell lines infected with the HPIV-1 is related to the phosphorylation of the MAPK as well as viral replication. Inhibition of p38 suppressed the secretion of IL-8 in the HEp-2 cells. No kinase activation was observed when viruses were inactivated.

  20. 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.

  1. Liver transplantation : chimerism, complications and matrix metalloproteinases

    NARCIS (Netherlands)

    Hove, Willem Rogier ten

    2011-01-01

    Chimerism after orthotopic liver transplantation (OLT) is the main focus of the studies described in this thesis. The first study showed that chimerism of different cell lineages within the liver graft does occur after OLT. Subsequently, in allogeneic blood stem cell recipients, chimerism was

  2. Haemophilus parainfluenzae Endocarditis Associated With Maxillary Sinusitis and Complicated by Cerebral Emboli in a Young Man

    Directory of Open Access Journals (Sweden)

    Anthony E. Duzenli MD

    2017-04-01

    Full Text Available HACEK endocarditis is often difficult to diagnose given the slow-growing characteristics of the organisms involved. Haemophilus parainfluenzae, one of the HACEK organisms, is an uncommon cause of endocarditis. We describe a case of a previously healthy young man with H parainfluenzae endocarditis that was associated with maxillary sinusitis and severe systemic complications, including septic cerebral emboli and mitral valve perforation. Previously reported cases have also described a predilection for younger people, cardiac valve pathology, and a high prevalence of stroke.

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

    Science.gov (United States)

    2012-01-01

    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. PMID:22925561

  4. 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.

  5. Radiolabeling and animal experimental studies of anti-breast cancer McAb 6C6 and mouse-human chimeric antibody 6C6CHI

    International Nuclear Information System (INIS)

    Yang Zhi; Hu Xiaoqian; Li Erqiu

    2003-01-01

    The aim of this work is to study bio-distribution and tumor absorption of anti breast cancer monoclonal antibody 6C6 and its chimeric antibody 6C6-CHI. The modified Schwartz method was employed to label 6C6 and 6C6CHI with 99 Tc m . The labeled antibody was injected to mouse via tail vein and the blood clearance and whole body clearance were observed. Nude mice bearing breast cancer MCF7 were injected with 99 Tc m -labeled antibody and the bio-distribution was studied and imaged with γ-ray camera. The yield of the two labeled antibodies was more than 90% and their immunoactivities were more than 80%. The whole body eliminated half-time of 99 Tc m -6C6 was 4.1h, and the half-times in blood were T α =0.55h and T β =12.42h respectively. The whole body half-time of 99 Tc m -6C6CHI was 4.28h and the half-times in blood were T α =0.98h and T β =12.42h respectively. The bio-distribution of nude mice bearing breast cancer MCF7 cells was as follows: the ID%/g of tumor and blood was (7.42±0.85) and (5.67±1.44) respectively at 23h after injection of 99 Tc m -6C6. The T/NT (tumor to non tumor) ratios were all more than 1.0 except kidney and the ID%/g of tumor and blood was (4.23±0.64) and (6.97±0.23) respectively at 23 h after injection of 99 Tc m -6C6CHI. The T/NT (tumor to non tumor) ratios were all more than 1.0 except blood and kidney. The γ imaging results showed that the tumor was imaged clearly at 24h after injection of radiolabeled antibodies and the other organs did not concentrate the antibodies except kidney. Anti-breast cancer monoclonal antibody 6C6 can be well located in tumor. Although ID%/g of tumor of the chimeric antibody 6C6CHI was slightly lower than that of 6C6 antibody, and ID%/g of blood was higher than that of 6C6, the tumor imaging of 6C6-CHI was also clear

  6. A novel self-replicating chimeric lentivirus-like particle.

    Science.gov (United States)

    Jurgens, Christy K; Young, Kelly R; Madden, Victoria J; Johnson, Philip R; Johnston, Robert E

    2012-01-01

    Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4⁺ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.

  7. ENDOGENOUS PYROGEN RELEASE FROM RABBIT BLOOD CELLS INCUBATED IN VITRO WITH PARAINFLUENZA VIRUS.

    Science.gov (United States)

    ATKINS, E; CRONIN, M; ISACSON, P

    1964-12-11

    Rabbit blood cells incubated in vitro with purified parainfluenza-5 virus (DA strain) released a rapidly acting pyrogen. Spleen and lymph node cells were inactive. The pyrogen resembled in behavior a pyrogen extracted from granulocytic exudates. Similar cells in the blood are believed to be activated by virus in vivo to produce the circulating endogenous pyrogen that mediates virus-induced fever.

  8. Virucidal activities of medium- and long-chain fatty alcohols and lipids against respiratory syncytial virus and parainfluenza virus type 2: comparison at different pH levels.

    Science.gov (United States)

    Hilmarsson, H; Traustason, B S; Kristmundsdóttir, T; Thormar, H

    2007-01-01

    Recent studies have shown that some lipids and fatty alcohols have microbicidal activities against a broad variety of pathogens. In this study, virucidal activities of fatty acids, monoglycerides and fatty alcohols were tested against respiratory syncytial virus (RSV) and human parainfluenza virus type 2 (HPIV2) at different concentrations, times and pH levels. The most active compounds were mixed with milk products and fruit juices and the mixtures tested for virucidal effects. The aim was to determine which compounds are the most active against these respiratory viruses and could possibly be used in pharmaceutical formulations or as additives to milk products or juice. Several compounds caused a significant inactivation of virus, and there was generally a good agreement between the activities against RSV and parainfluenza virus. By changing the pH from 7 to 4.2, the virucidal activities of some of the compounds were greatly increased, i.e., they inactivated virus in a shorter time and at lower concentrations. The most active compound tested was 1-monoglyceride of capric acid, monocaprin, which also showed activity against influenza A virus and significant virucidal activities after addition to milk products and fruit juices, even at a concentration as low as 0.06-0.12%. The significant virucidal activities of fatty alcohols and lipids on RSV and parainfluenza virus demonstrated in this in vitro study raise the question of the feasibility of using such compounds as ingredients in pharmaceutical dosage forms against respiratory infections caused by these viruses, and possibly other paramyxo- and myxoviruses.

  9. Epidemiology of parainfluenza infection in England and Wales, 1998-2013: any evidence of change?

    Science.gov (United States)

    Zhao, H; Harris, R J; Ellis, J; Donati, M; Pebody, R G

    2017-04-01

    Human parainfluenza virus (HPIV) infections are one of the commonest causes of upper and lower respiratory tract infections. In order to determine if there have been any recent changes in HPIV epidemiology in England and Wales, laboratory surveillance data between 1998 and 2013 were analysed. The UK national laboratory surveillance database, LabBase, and the newly established laboratory-based virological surveillance system, the Respiratory DataMart System (RDMS), were used. Descriptive analysis was performed to examine the distribution of cases by year, age, sex and serotype, and to examine the overall temporal trend using the χ 2 test. A random-effects model was also employed to model the number of cases. Sixty-eight per cent of all HPIV detections were due to HPIV type 3 (HPIV-3). HPIV-3 infections were detected all year round but peaked annually between March and June. HPIV-1 and HPIV-2 circulated at lower levels accounting for 20% and 8%, respectively, peaking during the last quarter of the year with a biennial cycle. HPIV-4 was detected in smaller numbers, accounting for only 4% and also mainly observed in the last quarter of the year. However, in recent years, HPIV-4 detection has been reported much more commonly with an increase from 0% in 1998 to 3·7% in 2013. Although an overall higher proportion of HPIV infection was reported in infants (43·0%), a long-term decreasing trend in proportion in infants was observed. An increase was also observed in older age groups. Continuous surveillance will be important in tracking any future changes.

  10. Structure of the parainfluenza virus 5 (PIV5 hemagglutinin-neuraminidase (HN ectodomain.

    Directory of Open Access Journals (Sweden)

    Brett D Welch

    Full Text Available Paramyxoviruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G and the fusion protein (F. HN binds sialic acid on host cells (hemagglutinin activity and hydrolyzes these receptors during viral egress (neuraminidase activity, NA. Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain. Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease virus (NDV HN ectodomain revealed the heads (NA domains in a "4-heads-down" conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides. Here we report the x-ray crystal structure of parainfluenza virus 5 (PIV5 HN ectodomain in a "2-heads-up/2-heads-down" conformation where two heads (covalent dimers are in the "down position," forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an "up position." The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F.

  11. Inhibition of interleukin-6 expression by the V protein of parainfluenza virus 5

    International Nuclear Information System (INIS)

    Lin Yuan; Sun Minghao; Fuentes, Sandra M.; Keim, Celia D.; Rothermel, Terri; He Biao

    2007-01-01

    The V protein of parainfluenza virus 5 (PIV5) plays an important role in the evasion of host immune responses. The V protein blocks interferon (IFN) signaling in human cells by causing degradation of the STAT1 protein, a key component of IFN signaling, and blocks IFN-β production by preventing nuclear translocation of IRF3, a key transcription factor for activating IFN-β promoter. Interleukin-6 (IL-6), along with tumor necrosis factor (TNF)-α and IL-1β, is a major proinflammatory cytokine that plays important roles in clearing virus infection through inflammatory responses. Many viruses have developed strategies to block IL-6 expression. Wild-type PIV5 infection induces little, if any, expression of cytokines such as IL-6 or TNF-α, whereas infection by a mutant PIV5 lacking the conserved C-terminal cysteine rich domain (rPIV5VΔC) induced high levels of IL-6 expression. Examination of mRNA levels of IL-6 indicated that the transcription activation of IL-6 played an important role in the increased IL-6 expression. Co-infection with wild-type PIV5 prevented the activation of IL-6 transcription by rPIV5VΔC, and a plasmid encoding the full-length PIV5 V protein prevented the activation of IL-6 promoter-driven reporter gene expression by rPIV5VΔC, indicating that the V protein played a role in inhibiting IL-6 transcription. The activation of IL-6 was independent of IFN-β even though rPIV5VΔC-infected cells produced IFN-β. Using reporter gene assays and chromatin immunoprecipitation (ChIP), it was found that NF-κB played an important role in activating expression of IL-6. We have proposed a model of activating and inhibiting IL-6 transcription by PIV5

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

    International Nuclear Information System (INIS)

    Xiong Jie; Zhou Yunfeng; Sun Wenjie; Wang Weifeng; Liao Zhengkai; Zhou Fuxiang; Xie Conghua

    2010-01-01

    Objective: To combine the radio-inducible CArG element with cancer-specific human telomerase reverse transcriptase (hTERT) gene promoter, and to construct the novel chimeric promoters. Methods: The synthetic hTERT promoters containing different number of radio-inducible CArG elements were constructed, and the activities of the promoters in the cancer cells (HeLa, A549, and MHCC97 cells) and nomal cells (hEL cells) were detected by using luciferase-reporter assays 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)

  13. Perforator chimerism for the reconstruction of complex defects: A new chimeric free flap classification system.

    Science.gov (United States)

    Kim, Jeong Tae; Kim, Youn Hwan; Ghanem, Ali M

    2015-11-01

    Complex defects present structural and functional challenges to reconstructive surgeons. When compared to multiple free flaps or staged reconstruction, the use of chimeric flaps to reconstruct such defects have many advantages such as reduced number of operative procedures and donor site morbidity as well as preservation of recipient vessels. With increased popularity of perforator flaps, chimeric flaps' harvest and design has benefited from 'perforator concept' towards more versatile and better reconstruction solutions. This article discusses perforator based chimeric flaps and presents a practice based classification system that incorporates the perforator flap concept into "Perforator Chimerism". The authors analyzed a variety of chimeric patterns used in 31 consecutive cases to present illustrative case series and their new classification system. Accordingly, chimeric flaps are classified into four types. Type I: Classical Chimerism, Type II: Anastomotic Chimerism, Type III: Perforator Chimerism and Type IV Mixed Chimerism. Types I on specific source vessel anatomy whilst Type II requires microvascular anastomosis to create the chimeric reconstructive solution. Type III chimeric flaps utilizes the perforator concept to raise two components of tissues without microvascular anastomosis between them. Type IV chimeric flaps are mixed type flaps comprising any combination of Types I to III. Incorporation of the perforator concept in planning and designing chimeric flaps has allowed safe, effective and aesthetically superior reconstruction of complex defects. The new classification system aids reconstructive surgeons and trainees to understand chimeric flaps design, facilitating effective incorporation of this important reconstructive technique into the armamentarium of the reconstruction toolbox. Copyright © 2015 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

  14. [A case of pulmonary abscess in which Haemophilus parainfluenzae and Streptococcus intermedius were isolated by percutaneous needle aspiration].

    Science.gov (United States)

    Miyamoto, Atsushi; Tsuboi, Eiyasu; Takaya, Hisashi; Sugino, Keishi; Sakamoto, Susumu; Kawabata, Masateru; Kishi, Kazuma; Narui, Koji; Homma, Sakae; Nakatani, Tatsuo; Nakata, Koichiro; Yoshimura, Kunihiko

    2006-08-01

    Some microbes, including the Bacteroides species, Staphylococcus aureus and Streptococcus milleri groups, can cause pulmonary abscess. Haemophilus parainfluenzae is usually categorized as one of the normal flora which colonizes in the ears and the nasopharynx, and it has been long considered that H. parainfluenzae has little pathogenicity in the lower respiratory tract and lung parenchymal. In this report, we present a case of pulmonary abscess caused by both H. parainfluenzae and Streptococcus intermedius. The patient was a 75-year-old man who had had total esophageo-gastrectomy because of esophageal cancer. He presented with purulent sputum, and chest X-ray film showed a dense consolidation in the right upper lung field. CT-guided transcutaneous fine needle aspiration was performed as a diagnostic procedure. Since both H. parainfluenzae and S. intermedius had been isolated from the lesion, pulmonary abscess caused by these two pathogens was diagnosed. The patient was treated with panipenem/betamipron, and his symptoms and pulmonary infiltrates on the chest X-ray film improved thereafter. So far, very few cases have been reported in which H. parainfluenzae caused lower respiratory tract infection. Although S. intermedius is known as one of the pathogens of pulmonary abscess, it is possible that H. parainfluenzae could also be pathogenic in infectious diseases of the lung.

  15. Estimates of Parainfluenza Virus-Associated Hospitalizations and Cost Among Children Aged Less Than 5 Years in the United States, 1998–2010

    Science.gov (United States)

    Abedi, Glen R.; Prill, Mila M.; Langley, Gayle E.; Wikswo, Mary E.; Weinberg, Geoffrey A.; Curns, Aaron T.; Schneider, Eileen

    2018-01-01

    Background Parainfluenza virus (PIV) is the second leading cause of hospitalization for respiratory illness in young children in the United States. Infection can result in a full range of respiratory illness, including bronchiolitis, croup, and pneumonia. The recognized human subtypes of PIV are numbered 1–4. This study calculates estimates of PIV-associated hospitalizations among US children younger than 5 years using the latest available data. Methods Data from the National Respiratory and Enteric Virus Surveillance System were used to characterize seasonal PIV trends from July 2004 through June 2010. To estimate the number of PIV-associated hospitalizations that occurred annually among US children aged PIV among young children enrolled in the New Vaccine Surveillance Network. Estimates of hospitalization charges attributable to PIV infection were also calculated. Results Parainfluenza virus seasonality follows type-specific seasonal patterns, with PIV-1 circulating in odd-numbered years and PIV-2 and -3 circulating annually. The average annual estimates of PIV-associated bronchiolitis, croup, and pneumonia hospitalizations among children aged PIV-associated bronchiolitis, croup, and pneumonia hospitalizations were approximately $43 million, $58 million, and $158 million, respectively. Conclusions The majority of PIV-associated hospitalizations in young children occur among those aged 0 to 2 years. When vaccines for PIV become available, immunization would be most effective if realized within the first year of life. PMID:26908486

  16. Tolerance and chimerism.

    Science.gov (United States)

    Kolb, Hans-Jochem; Guenther, Wolfgang; Gyurkocza, Boglarka; Hoetzl, Florian; Simoes, Belinda; Falk, Christine; Schleuning, Michael; Ledderose, Georg

    2003-05-15

    Stem-cell transplantation from human leukocyte antigen (HLA)-haploidentical family members carries a high risk of rejection and graft-versus-host disease (GVHD) if donor and recipient differ by more than one HLA antigen. The authors have developed treatment protocols from studies in dog leukocyte antigen-haploidentical dogs that prevent rejection and modify GVHD to the extent that patients with aggressive hematologic neoplasia can be treated with success. Principal improvements have been achieved in the use of cyclophosphamide and total-body irradiation for conditioning and T-cell depletion for prevention of GVHD. More recently, the combination of marrow and CD6-depleted mobilized donor blood cells (MDBC) has been introduced for HLA-haploidentical transplantation on the basis that CD6-depleted MDBC contain immunoregulatory cells besides stem cells and natural killer cells. Clinical results are reported on 36 patients with high-risk hematologic neoplasia. The results encourage the use of HLA-haploidentical stem-cell transplantation at an earlier stage of the disease. This method could also be of use for tolerance induction in organ transplantation.

  17. Replacement of the V3 domain in the surface subunit of the feline immunodeficiency virus envelope glycoprotein with the equivalent region of a T cell-tropic human immunodeficiency virus type 1 results in a chimeric surface protein that efficiently binds to CXCR4.

    Science.gov (United States)

    González, Silvia A; Falcón, Juan I; Affranchino, José L

    2014-03-01

    Feline immunodeficiency virus (FIV) and the T cell-tropic strains of human immunodeficiency virus type 1 (HIV-1) share the use of the chemokine receptor CXCR4 for cell entry. To study this process further we developed a cell surface binding assay based on the expression of a soluble version of the FIV SU C-terminally tagged with the influenza virus hemagglutinin epitope (HA). The specificity of the assay was demonstrated by the following evidence: (1) the SU-HA protein bound to HeLa cells that express CXCR4 but not to MDCK cells that lack this chemokine receptor; and (2) binding of the SU-HA to HeLa cells was blocked by incubation with the CXCR4 antagonist AMD3100 as well as with the anti-CXCR4 monoclonal antibody (MAb) 12G5. Deletion of the V3 region from the FIV SU glycoprotein abolished its ability to bind CXCR4-expressing cells. Remarkably, substitution of the V3 domain of the FIV SU by the equivalent region of the HIV-1 NL4-3 isolate resulted in efficient cell surface binding of the chimeric SU protein to CXCR4. Moreover, transfection of MDCK cells with a plasmid encoding human CXCR4 allowed the association of the chimeric SU-HA glycoprotein to the transfected cells. Interestingly, while cell binding of the chimeric FIV-HIV SU was inhibited by an anti-HIV-1 V3 MAb, its association with CXCR4 was found to be resistant to AMD3100. Of note, the chimeric FIV-HIV Env glycoprotein was capable of promoting CXCR4-dependent cell-to-cell fusion.

  18. Crazy-paving sign in high-resolution computed tomography in parainfluenza virus pneumonia

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    Matsuno, Osamu [Department of Respiratory Disease, NHO National Osaka Minami Medical Center, Kido higashi machi 2-1, Kawachinagano City, Osaka 586-8521 (Japan)], E-mail: matsuno@ommc-hp.jp; Hayama, Yoshitomo; Honda, Hidehiro; Yamane, Hiroyuki; Yamamoto, Suguru; Ueno, Kiyonobu [Department of Respiratory Disease, NHO National Osaka Minami Medical Center, Kido higashi machi 2-1, Kawachinagano City, Osaka 586-8521 (Japan); Saeki, Yukihiko [Department of Clinical Research, NHO National Osaka Minami Medical Center, Kido higashi machi 2-1, Kawachinagano city, Osaka 586-8521 (Japan)

    2010-05-15

    The crazy-paving sign is the appearance of a smooth linear pattern superimposed on an area of ground-glass opacity on thin-section computed tomography (CT). A 69-year-old woman was admitted to our hospital for treatment of pneumonia. Thoracic CT showed a crazy-paving sign in the right lung field on admission. She received ceftriaxone and clarithromycin, and the symptoms and infiltration shadow promptly disappeared. Serologic testing revealed a greater than 4-fold increase in the IgG titer for parainfluenza virus I. To our knowledge, there is no previous report of the crazy-paving sign in associated with viral pneumonia in a non-immunocompromised host or with parainfluenza pneumonia.

  19. Haemophilus parainfluenzae Mural Endocarditis: Case Report and Review of the Literature

    Directory of Open Access Journals (Sweden)

    Luca T. Giurgea

    2016-01-01

    Full Text Available Haemophilus parainfluenzae, which uncommonly causes endocarditis, has never been documented to cause mural involvement. A 62-year-old immunocompetent female without predisposing risk factors for endocarditis except for poor dentition presented with fever, emesis, and dysmetria. Echocardiography found a mass attached to the left ventricular wall with finger-like projections. Computed tomography showed evidence of embolic phenomena to the brain, kidneys, spleen, and colon. Cardiac MRI revealed involvement of the chordae tendineae of the anterior papillary muscles. Blood cultures grew Haemophilus parainfluenzae. The patient was treated successfully with ceftriaxone with resolution of symptoms, including neurologic deficits. After eleven days of antibiotics a worsening holosystolic murmur was discovered. Worsening mitral regurgitation on echocardiography was only found three weeks later. Nine weeks after presentation, intraoperative evaluation revealed chord rupture but no residual vegetation and mitral repair was performed. Four weeks after surgery, the patient was back to her baseline. This case illustrates the ability of Haemophilus parainfluenzae to form large mural vegetations with high propensity of embolization in otherwise normal cardiac tissue among patients with dental risk factors. It also underscores the importance of physical examination in establishing a diagnosis of endocarditis and monitoring for progression of disease.

  20. 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.

  1. Chimeric recombinant antibody fragments in cardiac troponin I immunoassay.

    Science.gov (United States)

    Hyytiä, Heidi; Heikkilä, Taina; Brockmann, Eeva-Christine; Kekki, Henna; Hedberg, Pirjo; Puolakanaho, Tarja; Lövgren, Timo; Pettersson, Kim

    2015-03-01

    To introduce a novel nanoparticle-based immunoassay for cardiac troponin I (cTnI) utilizing chimeric antibody fragments and to demonstrate that removal of antibody Fc-part and antibody chimerization decrease matrix related interferences. A sandwich-type immunoassay for cTnI based on recombinant chimeric (mouse variable/human constant) antigen binding (cFab) antibodies and intrinsically fluorescent nanoparticles was developed. To test whether using chimeric antibody fragments helps to avoid matrix related interferences, samples (n=39) with known amounts of triglycerides, bilirubin, rheumatoid factor (RF) or human anti-mouse antibodies (HAMAs) were measured with the novel assay, along with a previously published nanoparticle-based research assay with the same antibody epitopes. The limit of detection (LoD) was 3.30ng/L. Within-laboratory precision for 29ng/L and 2819ng/L cTnI were 13.7% and 15.9%, respectively. Regression analysis with Siemens ADVIA Centaur® yielded a slope (95% confidence intervals) of 0.18 (0.17-1.19) and a y-intercept of 1.94 (-1.28-3.91) ng/L. When compared to a previously published nanoparticle-based assay, the novel assay showed substantially reduced interference in the tested interference prone samples, 15.4 vs. 51.3%. A rheumatoid factor containing sample was decreased from 241ng/L to

  2. Novel Atlantic bottlenose dolphin parainfluenza virus TtPIV-1 clusters with bovine PIV-3 genotype B strains.

    Science.gov (United States)

    Eberle, Kirsten C; Neill, John D; Venn-Watson, Stephanie K; McGill, Jodi L; Sacco, Randy E

    2015-10-01

    Parainfluenza virus 3 (PIV-3) is a common viral infection not only in humans, but also in many other species. Serological evidence suggests that nearly 100 % of children in the United States have been infected with PIV-3 by 5 years of age. Similarly, in cattle, PIV-3 is commonly associated with bovine respiratory disease complex. A novel dolphin PIV-3 (TtPIV-1) was described by Nollens et al. in 2008 from a dolphin that was diagnosed with an unknown respiratory illness. At that time, TtPIV-1 was found to be most similar to, but distinct from, bovine PIV-3 (BPIV-3). In the present study, similar viral growth kinetics and pro-inflammatory cytokine (IL-1β, IL-6, and CXCL8) production were seen between BPIV-3 and TtPIV-1 in BEAS-2B, MDBK, and Vero cell lines. Initial nomenclature of TtPIV-1 was based on partial sequence of the fusion and RNA polymerase genes. Based on the similarities we saw with the in vitro work, it was important to examine the TtPIV-1 genome in more detail. Full genome sequencing and subsequent phylogenetic analysis revealed that all six viral genes of TtPIV-1 clustered within the recently described BPIV-3 genotype B strains, and it is proposed that TtPIV-1 be re-classified with BPIV-3 genotype B strains.

  3. Human placenta: relative content of antibodies of different classes and subclasses (IgG1-IgG4) containing lambda- and kappa-light chains and chimeric lambda-kappa-immunoglobulins.

    Science.gov (United States)

    Lekchnov, Evgenii A; Sedykh, Sergey E; Dmitrenok, Pavel S; Buneva, Valentina N; Nevinsky, Georgy A

    2015-06-01

    The specific organ placenta is much more than a filter: it is an organ that protects, feeds and regulates the growth of the embryo. Affinity chromatography, ELISA, SDS-PAGE and matrix-assisted laser desorption ionization mass spectrometry were used. Using 10 intact human placentas deprived of blood, a quantitative analysis of average relative content [% of total immunoglobulins (Igs)] was carried out for the first time: (92.7), IgA (2.4), IgM (2.5), kappa-antibodies (51.4), lambda-antibodies (48.6), IgG1 (47.0), IgG2 (39.5), IgG3 (8.8) and IgG4 (4.3). It was shown for the first time that placenta contains sIgA (2.5%). In the classic paradigm, Igs represent products of clonal B-cell populations, each producing antibodies recognizing a single antigen. There is a common belief that IgGs in mammalian biological fluids are monovalent molecules having stable structures and two identical antigen-binding sites. However, similarly to human milk Igs, placenta antibodies undergo extensive half-molecule exchange and the IgG pool consists of 43.5 ± 15.0% kappa-kappa-IgGs and 41.6 ± 17.0% lambda-lambda-IgGs, while 15.0 ± 4.0% of the IgGs contained both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained, respectively (%): IgG1 (47.7 and 34.4), IgG2 (36.3 and 44.5), IgG3 (7.4 and 11.8) and IgG4 (7.5 and 9.1), while chimeric kappa-lambda-IgGs consisted of (%): 43.5 IgG1, 41.0 IgG2, 5.6 IgG3 and 7.9 IgG4. Our data are indicative of the possibility of half-molecule exchange between placenta IgGs of various subclasses, raised against different antigens, which explains a very well-known polyspecificity and cross-reactivity of different human IgGs. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Parainfluenza virus type 5 (PIV-5) morphology revealed by cryo-electron microscopy.

    Science.gov (United States)

    Terrier, Olivier; Rolland, Jean-Paul; Rosa-Calatrava, Manuel; Lina, Bruno; Thomas, Daniel; Moules, Vincent

    2009-06-01

    The knowledge of parainfluenza type 5 (PIV-5) virion morphology is essentially based on the observation of negatively stained preparations in conventional transmission electron microscopy (CTEM). In this study, the ultrastructure of frozen-hydrated intact PIV-5 was examined by cryo-electron microscopy (cryo-EM). Cryo-EM revealed a majority of spherical virions (70%), with a lower pleiomorphy than originally observed in CTEM. Phospholipid bilayer thickness, spike length and glycoprotein spikes density were measured. About 2000 glycoprotein spikes were present in an average-sized spherical virion. Altogether, these data depict a more precise view of PIV-5 morphology.

  5. 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.

  6. Posttransplant chimeric antigen receptor therapy.

    Science.gov (United States)

    Smith, Melody; Zakrzewski, Johannes; James, Scott; Sadelain, Michel

    2018-03-08

    Therapeutic T-cell engineering is emerging as a powerful approach to treat refractory hematological malignancies. Its most successful embodiment to date is based on the use of second-generation chimeric antigen receptors (CARs) targeting CD19, a cell surface molecule found in most B-cell leukemias and lymphomas. Remarkable complete remissions have been obtained with autologous T cells expressing CD19 CARs in patients with relapsed, chemo-refractory B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. Allogeneic CAR T cells may also be harnessed to treat relapse after allogeneic hematopoietic stem cell transplantation. However, the use of donor T cells poses unique challenges owing to potential alloreactivity. We review different approaches to mitigate the risk of causing or aggravating graft-versus-host disease (GVHD), including CAR therapies based on donor leukocyte infusion, virus-specific T cells, T-cell receptor-deficient T cells, lymphoid progenitor cells, and regulatory T cells. Advances in CAR design, T-cell selection and gene editing are poised to enable the safe use of allogeneic CAR T cells without incurring GVHD. © 2018 by The American Society of Hematology.

  7. DAS181 Treatment of Severe Parainfluenza Virus 3 Pneumonia in Allogeneic Hematopoietic Stem Cell Transplant Recipients Requiring Mechanical Ventilation

    Directory of Open Access Journals (Sweden)

    B. Dhakal

    2016-01-01

    Full Text Available Parainfluenza virus (PIV may cause life-threatening pneumonia in allogeneic hematopoietic stem cell transplant (HSCT recipients. Currently, there are no proven effective therapies. We report the use of inhaled DAS181, a novel sialidase fusion protein, for treatment of PIV type 3 pneumonia in two allogeneic hematopoietic SCT recipients with respiratory failure.

  8. 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. Copyright 2001 Wiley-Liss. Inc.

  9. Chimeric polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    Science.gov (United States)

    Wogulis, Mark; Sweeney, Matthew; Heu, Tia

    2017-06-14

    The present invention relates to chimeric GH61 polypeptides having cellulolytic enhancing activity. The present invention also relates to polynucleotides encoding the chimeric GH61 polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the chimeric GH61 polypeptides.

  10. CCR 20th anniversary commentary: a chimeric antibody, C225, inhibits EGFR activation and tumor growth.

    Science.gov (United States)

    Mendelsohn, John; Prewett, Marie; Rockwell, Patricia; Goldstein, Neil I

    2015-01-15

    Murine mAb 225 was effective against the EGFR tyrosine kinase and inhibited tumor growth in preclinical studies. A phase I trial showed safety, tumor localization, and satisfactory pharmacokinetics. Human:murine chimeric C225 retained biologic activity, which was essential for the conduct of subsequent combination therapy trials and eventual regulatory approval. ©2015 American Association for Cancer Research.

  11. 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.

  12. Adenovirus 2, Bordetella bronchiseptica, and Parainfluenza Molecular Diagnostic Assay Results in Puppies After vaccination with Modified Live Vaccines.

    Science.gov (United States)

    Ruch-Gallie, R; Moroff, S; Lappin, M R

    2016-01-01

    Canine adenovirus 2, parainfluenza, and Bordetella bronchiseptica cause respiratory disease in dogs, and each has a modified live intranasal vaccine available. Molecular diagnostic assays to amplify specific nucleic acids are available for each of these agents. If positive molecular diagnostic assay results are common after vaccination, the positive predictive value of the diagnostic assays for disease would be decreased. To determine the impact of administration of commercially available modified live topical adenovirus 2, B. bronchiseptica, and parainfluenza vaccine has on the results of a commercially available PCR panel. Eight puppies from a research breeding facility negative for these pathogens. Blinded prospective pilot study. Puppies were vaccinated with a single dose of modified live topical adenovirus 2, B. bronchiseptica, and parainfluenza and parenteral dose of adenovirus 2, canine distemper virus, and parvovirus. Nasal and pharyngeal swabs were collected on multiple days and submitted for PCR assay. Nucleic acids of all 3 organisms contained in the topical vaccine were detected from both samples multiple times through 28 days after vaccination with higher numbers of positive samples detected between days 3 and 10 after vaccination. Vaccine status should be considered when interpreting respiratory agent PCR results if modified live vaccines have been used. Development of quantitative PCR and wild-type sequencing are necessary to improve positive predictive value of these assays by distinguishing vaccinate from natural infection. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

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

    International Nuclear Information System (INIS)

    Kimikawa, Masaaki; Kawai, Tatsuo; Ota, Kazuo

    1996-01-01

    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)

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

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    Jiping Cai

    2008-01-01

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

  15. Hematopoietic chimerism and transplantation tolerance: a role for regulatory T cells

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    Lise ePasquet

    2011-12-01

    Full Text Available The major obstacle in transplantation medicine is rejection of donor tissues by the host’s immune system. Immunosuppressive drugs can delay but not prevent loss of transplants, and their efficiency is strongly impacted by inter-individual pharmacokinetic differences. Moreover, due to the global immunosuppression induced and to the broad distribution of their targets amongst human tissues, these drugs have severe side effects. Induction of donor-specific non-responsiveness (i.e. immunological tolerance to transplants would solve these problems and would substantially ameliorate patients’ quality of life. It is widely believed that bone marrow or hematopoietic stem cell transplantation, and resulting (mixed hematopoietic chimerism, invariably leads to immunological tolerance to organs of the same donor. A careful analysis of the literature, reviewed here, indeed shows that chimerism consistently prolongs allograft survival. However, in absence of additional conditioning leading to the development of active regulatory mechanisms, it does not prevent chronic rejection. A central role for active tolerance in transplantation-tolerance is also supported by recent data showing that genuine immunological tolerance to organ allografts can be achieved by combining induction of hematopoietic chimerism with infusion of regulatory T lymphocytes. Therefore, conditioning regimens that lead to the establishment of hematopoietic chimerism plus active regulatory mechanisms appear required for induction of genuine tolerance to allogeneic grafts.

  16. 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)

  17. 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...... vaccinated pigs were protected. This new chimeric pestivirus represents a C-strain based DIVA vaccine candidate that can be differentiated based on CSFV E2 specific antibodies....

  18. Chimeric opioid peptides: tools for identifying opioid receptor types.

    OpenAIRE

    Xie, G X; Miyajima, A; Yokota, T; Arai, K; Goldstein, A

    1990-01-01

    We synthesized several chimeric peptides in which the N-terminal nine residues of dynorphin-32, a peptide selective for the kappa opioid receptor, were replaced by opioid peptides selective for other opioid receptor types. Each chimeric peptide retained the high affinity and type selectivity characteristic of its N-terminal sequence. The common C-terminal two-thirds of the chimeric peptides served as an epitope recognized by the same monoclonal antibody. When bound to receptors on a cell surf...

  19. Long-Term Shedding of Influenza Virus, Parainfluenza Virus, Respiratory Syncytial Virus and Nosocomial Epidemiology in Patients with Hematological Disorders.

    Directory of Open Access Journals (Sweden)

    Nicola Lehners

    Full Text Available Respiratory viruses are a cause of upper respiratory tract infections (URTI, but can be associated with severe lower respiratory tract infections (LRTI in immunocompromised patients. The objective of this study was to investigate the genetic variability of influenza virus, parainfluenza virus and respiratory syncytial virus (RSV and the duration of viral shedding in hematological patients. Nasopharyngeal swabs from hematological patients were screened for influenza, parainfluenza and RSV on admission as well as on development of respiratory symptoms. Consecutive swabs were collected until viral clearance. Out of 672 tested patients, a total of 111 patients (17% were infected with one of the investigated viral agents: 40 with influenza, 13 with parainfluenza and 64 with RSV; six patients had influenza/RSV or parainfluenza/RSV co-infections. The majority of infected patients (n = 75/111 underwent stem cell transplantation (42 autologous, 48 allogeneic, 15 autologous and allogeneic. LRTI was observed in 48 patients, of whom 15 patients developed severe LRTI, and 13 patients with respiratory tract infection died. Phylogenetic analysis revealed a variety of influenza A(H1N1pdm09, A(H3N2, influenza B, parainfluenza 3 and RSV A, B viruses. RSV A was detected in 54 patients, RSV B in ten patients. The newly emerging RSV A genotype ON1 predominated in the study cohort and was found in 48 (75% of 64 RSV-infected patients. Furthermore, two distinct clusters were detected for RSV A genotype ON1, identical RSV G gene sequences in these patients are consistent with nosocomial transmission. Long-term viral shedding for more than 30 days was significantly associated with prior allogeneic transplantation (p = 0.01 and was most pronounced in patients with RSV infection (n = 16 with a median duration of viral shedding for 80 days (range 35-334 days. Long-term shedding of respiratory viruses might be a catalyzer of nosocomial transmission and must be considered for

  20. 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...

  1. Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein.

    Science.gov (United States)

    Welch, Brett D; Liu, Yuanyuan; Kors, Christopher A; Leser, George P; Jardetzky, Theodore S; Lamb, Robert A

    2012-10-09

    The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.

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

    International Nuclear Information System (INIS)

    Guo Lizheng; Lu Xiaoyan; Kang, S.-M.; Chen Changyi; Compans, Richard W.; Yao Qizhi

    2003-01-01

    To enhance mucosal immune responses using simian/human immunodeficiency virus-like particles (SHIV VLPs), we have produced novel phenotypically mixed chimeric influenza HA/SHIV VLPs and used them to immunize C57BL/6J mice intranasally. Antibody and cytotoxic T-cell (CTL) responses as well as cytokine production in both systemic and mucosal sites were compared after immunization with SHIV VLPs or chimeric HA/SHIV VLPs. By using enzyme-linked immunosorbent assay (ELISA), the levels of serum IgG and mucosal IgA to the HIV envelope protein (Env) were found to be highest in the group immunized with chimeric HA/SHIV VLPs. Furthermore, the highest titer of serum neutralizing antibody against HIV Env was found with the group immunized with chimeric HA/SHIV VLPs. Analysis of the IgG1/IgG2a ratio indicated that a T H 1-oriented immune response resulted from these VLP immunizations. HA/SHIV VLP-immunized mice also showed significantly higher CTL responses than those observed in SHIV VLP-immunized mice. Moreover, a MHC class I restricted T-cell activation ELISPOT assay showed a mixed type of T H 1/T H 2 cytokines in the HA/SHIV VLP-immunized mice, indicating that the chimeric VLPs can enhance both humoral and cellular immune responses to the HIV Env protein at multiple mucosal and systemic sites. The results indicate that incorporation of influenza HA into heterotypic VLPs may be highly effective for targeting vaccines to mucosal surfaces

  3. Prevalence of chimerism after non-myeloablative hematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Azulamara da Silva Ruiz

    Full Text Available CONTEXT AND OBJECTIVE: Non-myeloablative hematopoietic stem cell transplantation (NMA-HSCT is performed in onco-hematological patients who cannot tolerate ablative conditioning because of older age or comorbidities. This approach does not completely eliminate host cells and initially results in mixed chimerism. Long-term persistence of mixed chimerism results in graft rejection and relapse. Involvement of graft-versus-host disease is concomitant with complete chimerism and graft-versus-tumor effect. The aim of this study was to evaluate the prevalence of chimerism in onco-hematological patients who underwent NMA-HSCT. DESIGN AND SETTING: Observational clinical study on chimerism status after human leukocyte antigen-identical NMA-HSCT at the Discipline of Hematology and Hemotherapy of Universidade Federal de São Paulo. METHODS: We sequentially analyzed the amplification of APO-B, D1S80, DxS52, FVW, 33.6, YNZ-2 and H-ras primers using variable number of tandem repeats (VNTR on 17 pairs and fluorescent in situ hybridization (FISH with the XY probe and SRY primer on 13 sex-unmatched pairs. RESULTS: The informativeness of the primers using VNTR was 60% for APO-B, 75% D1S80, 36% DxS52, 14% FVW, 40% YNZ-22 and 16% H-ras. The SRY primer was informative in female receptors with male donors. The XY-FISH method was informative in 100% of the sex-unmatched pairs. CONCLUSION: These methods were sensitive and informative. In VNTR, the association of APO-B with D1S80 showed 88% informativeness. The quantitative FISH method was more sensitive, but had the disadvantage of only being used for sex-unmatched pairs.

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

    Science.gov (United States)

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

    2015-04-01

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

  5. The L polymerase protein of parainfluenza virus 3 forms an oligomer and can interact with the heterologous Sendai virus L, P and C proteins

    International Nuclear Information System (INIS)

    Smallwood, Sherin; Moyer, Sue A.

    2004-01-01

    We recently showed that the L protein of Sendai virus is present as an oligomer in the active P-L polymerase complex [Smallwood et al., Virology 304 (2002) 235]. We now demonstrate using two different epitope tags that the L protein of a second respirovirus, human parainfluenza type 3 virus (PIV3), also forms an L-L complex. L oligomerization requires the coexpression of the differentially epitope tagged L proteins. By exploiting a series of C-terminal truncations the L-L binding site maps to the N-terminal half of L. There is some complex formation between the heterologous PIV3 and Sendai L and P proteins; however, the heterologous L protein does not function in transcription of either the PIV3 or Sendai template. The PIV3 C protein binds PIV3 L and inhibits RNA synthesis in vitro and in vivo. Significant homology exists between the C proteins of PIV3 and Sendai and complex formation occurs between the PIV3 and Sendai heterologous C and L proteins. In addition, the heterologous C proteins can inhibit transcription at ∼50% of the level of the homologous protein. These data suggest that while the C proteins may be functionally somewhat interchangeable, the L and P proteins are specific for each virus

  6. Genetic analysis of human parainfluenza virus type 3 obtained in Croatia, 2011-2015.

    Science.gov (United States)

    Košutić-Gulija, Tanja; Slovic, Anamarija; Ljubin-Sternak, Sunčanica; Mlinarić-Galinović, Gordana; Forčić, Dubravko

    2017-04-01

    This study investigated the HPIV3 circulating strains in Croatia and whether the other parts of HPIV3 genome (F gene and HN 582 nucleotides fragment) could be equally suitable for genetic and phylogenetic analysis. Clinical materials were collected in period 2011-2015 from children suffering from respiratory illnesses. In positive HPIV3 samples viral genome was partially amplified and sequenced for HN and F genes. Obtained sequences were analysed by phylogenetic analysis and genetic characterization was performed. All samples from this study belonged to subcluster C and over a short period of time, genetic lineage C3a gained prevalence over the other C genetic lineages, from 39 % in 2011 to more than 90 % in 2013 and 2014. Phylogenetic classifications of HPIV3 based on the entire HN gene, HN 582 nt fragment and entire fusion (F) gene showed identical classification results for Croatian strains and the reference strains. Molecular analysis of the F and HN glycoproteins, showed their similar nucleotide diversity (Fcds P=0.0244 and HNcds P=0.0231) and similar Ka/Ks ratios (F Ka/Ks=0.0553 and HN Ka/Ks=0.0428). Potential N-glycosylation sites, cysteine residues and antigenic sites are generally strongly conserved in HPIV3 glycoproteins from both our and the reference samples. The HPIV3 subclaster C3 (genetic lineage C3a) became the most detected circulating HPIV3 strain in Croatia. The results indicated that the HN 582 nt and the entire F gene sequences were as good for phylogenetic analysis as the entire HN gene sequence.

  7. Clinical and Molecular Epidemiology of Human Parainfluenza Viruses 1-4 in Children from Viet Nam.

    Science.gov (United States)

    Linster, Martin; Do, Lien Anh Ha; Minh, Ngo Ngoc Quang; Chen, Yihui; Zhe, Zhu; Tuan, Tran Anh; Tuan, Ha Manh; Su, Yvonne C F; van Doorn, H Rogier; Moorthy, Mahesh; Smith, Gavin J D

    2018-05-01

    HPIVs are serologically and genetically grouped into four species that account for up to 10% of all hospitalizations due to acute respiratory infection in children under the age of five. Genetic and epidemiological data for the four HPIVs derived from two pediatric cohorts in Viet Nam are presented. Respiratory samples were screened for HPIV1-4 by real-time PCR. Demographic and clinical data of patients infected with different HPIV were compared. We used a hemi-nested PCR approach to generate viral genome sequences from HPIV-positive samples and conducted a comprehensive phylogenetic analysis. In total, 170 samples tested positive for HPIV. HPIV3 was most commonly detected in our cohort and 80 co-detections of HPIV with other respiratory viruses were found. Phylogenetic analyses suggest local endemic circulation as well as punctuated introductions of new HPIV lineages. Viral gene flow analysis revealed that Viet Nam is a net importer of viral genetic diversity. Epidemiological analyses imply similar disease severity for all HPIV species. HPIV sequences from Viet Nam formed local clusters and were interspersed with sequences from diverse geographic regions. Combined, this new knowledge will help to investigate global HPIV circulation patterns in more detail and ultimately define more suitable vaccine strains.

  8. Application of 99mTc-labeled chimeric Fab fragments of monoclonal antibody A7 for radioimmunoscintigraphy of pancreatic cancer

    International Nuclear Information System (INIS)

    Matsumura, Hiroomi

    1999-01-01

    Pancreatic cancer is one of the most lethal diseases and its prognosis is still poor. To improve the survival rate, it is essential to develop new technologies for early and definitive diagnosis. In this study, chimeric Fab fragments of monoclonal antibody A7 were successfully radio-labeled with 99m Tc, preventing depression of the antigen-binding activity. 99m Tc-labeled monoclonal antibody A7, 99m Tc-labeled chimeric Fab fragments of monoclonal antibody A7, 99m Tc-labeled normal mouse IgG and 99m Tc-labeled Fab fragments of normal mouse IgG were injected intravenously into nude mice bearing human pancreatic cancer xenografts and the radioactivity was subsequently measured. The tumor accumulation was significantly higher with labeled monoclonal antibody A7 than with normal mouse IgG, and higher with chimeric Fab fragments of monoclonal antibody A7 than with Fab fragments of normal mouse IgG. The tumor/blood ratio of radioactivity increased rapidly over time with chimeric Fab fragments of monoclonal antibody A7. These results suggest that chimeric Fab fragments of monoclonal antibody A7 may be useful for diagnosing pancreatic cancer by means of radioimmunoscintigraphy. (author)

  9. Homologous recombination in hybridoma cells: heavy chain chimeric antibody produced by gene targeting.

    OpenAIRE

    Fell, H P; Yarnold, S; Hellström, I; Hellström, K E; Folger, K R

    1989-01-01

    We demonstrate that murine myeloma cells can efficiently mediate homologous recombination. The murine myeloma cell line J558L was shown to appropriately recombine two transfected DNA molecules in approximately 30% of cells that received and integrated intact copies of both molecules. This activity was then exploited to direct major reconstructions of an endogenous locus within a hybridoma cell line. Production of antigen-specific chimeric heavy chain was achieved by targeting the human IgG1 h...

  10. Chimeric Lyssavirus Glycoproteins with Increased Immunological Potential

    Science.gov (United States)

    Jallet, Corinne; Jacob, Yves; Bahloul, Chokri; Drings, Astrid; Desmezieres, Emmanuel; Tordo, Noël; Perrin, Pierre

    1999-01-01

    The rabies virus glycoprotein molecule (G) can be divided into two parts separated by a flexible hinge: the NH2 half (site II part) containing antigenic site II up to the linear region (amino acids [aa] 253 to 275 encompassing epitope VI [aa 264]) and the COOH half (site III part) containing antigenic site III and the transmembrane and cytoplasmic domains. The structural and immunological roles of each part were investigated by cell transfection and mouse DNA-based immunization with homogeneous and chimeric G genes formed by fusion of the site II part of one genotype (GT) with the site III part of the same or another GT. Various site II-site III combinations between G genes of PV (Pasteur virus strain) rabies (GT1), Mokola (GT3), and EBL1 (European bat lyssavirus 1 [GT5]) viruses were tested. Plasmids pGPV-PV, pGMok-Mok, pGMok-PV, and pGEBL1-PV induced transient expression of correctly transported and folded antigens in neuroblastoma cells and virus-neutralizing antibodies against parental viruses in mice, whereas, pG-PVIII (site III part only) and pGPV-Mok did not. The site III part of PV (GT1) was a strong inducer of T helper cells and was very effective at presenting the site II part of various GTs. Both parts are required for correct folding and transport of chimeric G proteins which have a strong potential value for immunological studies and development of multivalent vaccines. Chimeric plasmid pGEBL1-PV broadens the spectrum of protection against European lyssavirus genotypes (GT1, GT5, and GT6). PMID:9847325

  11. Dissociation between peripheral blood chimerism and tolerance to hindlimb composite tissue transplants: preferential localization of chimerism in donor bone.

    Science.gov (United States)

    Rahhal, Dina N; Xu, Hong; Huang, Wei-Chao; Wu, Shengli; Wen, Yujie; Huang, Yiming; Ildstad, Suzanne T

    2009-09-27

    Mixed chimerism induces donor-specific tolerance to composite tissue allotransplants (CTAs). In the present studies, we used a nonmyeloablative conditioning approach to establish chimerism and promote CTA acceptance. Wistar Furth (RT1A(u)) rats were conditioned with 600 to 300 cGy total body irradiation (TBI, day-1), and 100 x 10(6) T-cell-depleted ACI (RT1A(abl)) bone marrow cells were transplanted on day 0, followed by a 11-day course of tacrolimus and one dose of antilymphocyte serum (day 10). Heterotopic osteomyocutaneous flap transplantation was performed 4 to 6 weeks after bone marrow transplantation. Mixed chimerism was initially achieved in almost all recipients, but long-term acceptance of CTA was only achieved in rats treated with 600 cGy TBI. When anti-alphabeta-T-cell receptor (TCR) monoclonal antibody (mAb) (day-3) was added into the regimens, donor chimerism was similar to recipients preconditioned without anti-alphabeta-TCR mAb. However, the long-term CTA survival was significantly improved in chimeras receiving more than or equal to 300 cGy TBI plus anti-alphabeta-TCR mAb. Higher levels of donor chimerism were associated with CTA acceptance. The majority of flap acceptors lost peripheral blood chimerism within 6 months. However, donor chimerism persisted in the transplanted bone at significantly higher levels compared with other hematopoietic compartments. The compartment donor chimerism may be responsible for the maintenance of tolerance to CTA. Long-term acceptors were tolerant to a donor skin graft challenge even in the absence of peripheral blood chimerism. Mixed chimerism established by nonmyeloablative conditioning induces long-term acceptance of CTA, which is associated with persistent chimerism preferentially in the transplanted donor bone.

  12. Chimeric Amino Acid Rearrangements as Immune Targets in Prostate Cancer

    Science.gov (United States)

    2016-05-01

    COVERED 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Chimeric Amino Acid Rearrangements as Immune Targets in Prostate Cancer 5b. GRANT NUMBER W81XWH...that result from gene rearrangements given their high frequency relative to somatic point mutations. Gene rearrangements can yield novel chimeric

  13. Chimeric opioid peptides: Tools for identifying opioid receptor types

    International Nuclear Information System (INIS)

    Xie, G.; Miyajima, A.; Yokota, T.; Arai, K.; Goldstein, A.

    1990-01-01

    The authors synthesized several chimeric [125J-labelled] peptides in which the N-terminal nine residues of dynorphin-32, a peptide selective for the κ opioid receptor, were replaced by opioid peptides selective for other opioid receptor types. Each chimeric peptide retained the high affinity and type selectivity characteristic of its N-terminal sequence. The common C-terminal two-thirds of the chimeric peptides served as an epitope recognized by the same monoclonal antibody. When bound to receptors on a cell surface or membrane preparation, these peptides could still bind specifically to the monoclonal antibody. These chimeric peptides should be useful for isolating μ, δ, and κ opioid receptors and for identifying opioid receptors on transfected cells in expression cloning procedures. The general approach using chimeric peptides should be applicable to other peptide receptors

  14. Chimeric vaccine composed of viral peptide and mammalian heat-shock protein 60 peptide protects against West Nile virus challenge.

    Science.gov (United States)

    Gershoni-Yahalom, Orly; Landes, Shimon; Kleiman-Shoval, Smadar; Ben-Nathan, David; Kam, Michal; Lachmi, Bat-El; Khinich, Yevgeny; Simanov, Michael; Samina, Itzhak; Eitan, Anat; Cohen, Irun R; Rager-Zisman, Bracha; Porgador, Angel

    2010-08-01

    The protective efficacy and immunogenicity of a chimeric peptide against West Nile virus (WNV) was evaluated. This virus is the aetiological agent of West Nile fever, which has recently emerged in the western hemisphere. The rapid spread of WNV throughout North America, as well as the constantly changing epidemiology and transmission of the virus by blood transfusion and transplantation, have raised major public-health concerns. Currently, there are no effective treatments for WNV or vaccine for human use. We previously identified a novel, continuous B-cell epitope from domain III of the WNV envelope protein, termed Ep15. To test whether this epitope can protect against WNV infection, we synthesized a linear chimeric peptide composed of Ep15 and the heat-shock protein 60 peptide, p458. The p458 peptide is an effective carrier peptide for subunit vaccines against other infectious agents. We now report that mice immunized with the chimeric peptide, p458-Ep15, were resistant to lethal challenges with three different WNV strains. Moreover, their brains were free of viral genome and infectious virus. Mice immunized with Ep15 alone or with p431-Ep15, a control conjugate, were not protected. The chimeric p458-Ep15 peptide induced WNV-specific immunoglobulin G antibodies that neutralized the virus and induced the secretion of interferon-gammain vitro. Challenge of chimeric peptide-immunized mice considerably enhanced WNV-specific neutralizing antibodies. We conclude that this chimeric peptide can be used for formulation of a human vaccine against WNV.

  15. Preparation and characterization of chimeric CD19 monoclonal antibody

    International Nuclear Information System (INIS)

    Zola, H.; Macardle, P.J.; Bradford, T.; Weedon, H.; Yasui, H.; Kurosawa, Y.

    1991-01-01

    CD19 antibodies have been suggested as candidates for immunological attack on leukemic and lymphoma cells of the B lineage because the antigen is restricted to the B lineage. With the potential use of FMC63 in immunotherapy in mind a mouse-human chimera was produced in which the genes coding for the VDJ region of the heavy chain and the VJ region of the light chain derive from the FMC63 mouse hybridoma, while the C region genes code for human IgG1. The genes have been transfected back into a mouse myeloma line, which secretes low levels of immunoglobulin. (Ig). This Ig was purified and biotinylated in order to determine the specificity of the antibody. The chimeric antibody has a reaction profile concordant with the original FMC63 antibody, but has the properties of a human IgG1, including the ability to fix human complement. However, the antibody is not cytotoxic in vitro in the presence of complement or cells capable of mediating antibody-dependent cellular cytotoxicity. Possible reasons for this and ways of using the antibody are discussed. 47 refs., 7 figs

  16. Role of Bibersteinia trehalosi, respiratory syncytial virus, and parainfluenza-3 virus in bighorn sheep pneumonia.

    Science.gov (United States)

    Dassanayake, Rohana P; Shanthalingam, Sudarvili; Subramaniam, Renuka; Herndon, Caroline N; Bavananthasivam, Jegarubee; Haldorson, Gary J; Foreyt, William J; Evermann, James F; Herrmann-Hoesing, Lynn M; Knowles, Donald P; Srikumaran, Subramaniam

    2013-02-22

    Pneumonic bighorn sheep (BHS) have been found to be culture- and/or sero-positive for Bibersteinia trehalosi, respiratory syncytial virus (RSV), and parainfluenza-3 virus (PI-3). The objective of this study was to determine whether these pathogens can cause fatal pneumonia in BHS. In the first study, two groups of four BHS each were intra-tracheally administered with leukotoxin-positive (Group I) or leukotoxin-negative (Group II) B. trehalosi. All four animals in Group I developed severe pneumonia, and two of them died within 3 days. The other two animals showed severe pneumonic lesions on euthanasia and necropsy. Animals in Group II neither died nor showed gross pneumonic lesions on necropsy, suggesting that leukotoxin-positive, but not leukotoxin-negative, B. trehalosi can cause fatal pneumonia in BHS. In the second study, two other groups of four BHS (Groups III and IV) were intra-nasally administered with a mixture of RSV and PI-3. Four days later, RSV/PI-3-inoculated Group IV and another group of four BHS (Group V, positive control) were intra-nasally administered with Mannheimia haemolytica, the pathogen that consistently causes fatal pneumonia in BHS. All four animals in group III developed pneumonia, but did not die during the study period. However all four animals in Group IV, and three animals in Group V developed severe pneumonia and died within two days of M. haemolytica inoculation. The fourth animal in Group V showed severe pneumonic lesions on euthanasia and necropsy. These findings suggest that RSV/PI-3 can cause non-fatal pneumonia, but are not necessary predisposing agents for M. haemolytica-caused pneumonia of BHS. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Production and characterisation of a neutralising chimeric antibody against botulinum neurotoxin A.

    Directory of Open Access Journals (Sweden)

    Julie Prigent

    Full Text Available Botulinum neurotoxins, produced by Clostridium botulinum bacteria, are the causative agent of botulism. This disease only affects a few hundred people each year, thus ranking it among the orphan diseases. However, botulinum toxin type A (BoNT/A is the most potent toxin known to man. Due to their potency and ease of production, these toxins were classified by the Centers for Disease Control and Prevention (CDC as Category A biothreat agents. For several biothreat agents, like BoNT/A, passive immunotherapy remains the only possible effective treatment allowing in vivo neutralization, despite possible major side effects. Recently, several mouse monoclonal antibodies directed against a recombinant fragment of BoNT/A were produced in our laboratory and most efficiently neutralised the neurotoxin. In the present work, the most powerful one, TA12, was selected for chimerisation. The variable regions of this antibody were thus cloned and fused with the constant counterparts of human IgG1 (kappa light and gamma 1 heavy chains. Chimeric antibody production was evaluated in mammalian myeloma cells (SP2/0-Ag14 and insect cells (Sf9. After purifying the recombinant antibody by affinity chromatography, the biochemical properties of chimeric and mouse antibody were compared. Both have the same very low affinity constant (close to 10 pM and the chimeric antibody exhibited a similar capacity to its parent counterpart in neutralising the toxin in vivo. Its strong affinity and high neutralising potency make this chimeric antibody interesting for immunotherapy treatment in humans in cases of poisoning, particularly as there is a probable limitation of the immunological side effects observed with classical polyclonal antisera from heterologous species.

  18. Vectors expressing chimeric Japanese encephalitis dengue 2 viruses.

    Science.gov (United States)

    Wei, Y; Wang, S; Wang, X

    2014-01-01

    Vectors based on self-replicating RNAs (replicons) of flaviviruses are becoming powerful tool for expression of heterologous genes in mammalian cells and development of novel antiviral and anticancer vaccines. We constructed two vectors expressing chimeric viruses consisting of attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) in which the PrM/M-E genes were replaced fully or partially with those of dengue 2 virus (DENV-2). These vectors, named pJED2 and pJED2-1770 were transfected to BHK-21 cells and produced chimeric viruses JED2V and JED2-1770V, respectively. The chimeric viruses could be passaged in C6/36 but not BHK-21 cells. The chimeric viruses produced in C6/36 cells CPE 4-5 days after infection and RT-PCR, sequencing, immunofluorescence assay (IFA) and Western blot analysis confirmed the chimeric nature of produced viruses. The immunogenicity of chimeric viruses in mice was proved by detecting DENV-2 E protein-specific serum IgG antibodies with neutralization titer of 10. Successful preparation of infectious clones of chimeric JEV-DENV-2 viruses showed that JEV-based expression vectors are fully functional.

  19. Recombinant Parainfluenza Virus 5 Expressing Hemagglutinin of Influenza A Virus H5N1 Protected Mice against Lethal Highly Pathogenic Avian Influenza Virus H5N1 Challenge

    Science.gov (United States)

    Li, Zhuo; Mooney, Alaina J.; Gabbard, Jon D.; Gao, Xiudan; Xu, Pei; Place, Ryan J.; Hogan, Robert J.; Tompkins, S. Mark

    2013-01-01

    A safe and effective vaccine is the best way to prevent large-scale highly pathogenic avian influenza virus (HPAI) H5N1 outbreaks in the human population. The current FDA-approved H5N1 vaccine has serious limitations. A more efficacious H5N1 vaccine is urgently needed. Parainfluenza virus 5 (PIV5), a paramyxovirus, is not known to cause any illness in humans. PIV5 is an attractive vaccine vector. In our studies, a single dose of a live recombinant PIV5 expressing a hemagglutinin (HA) gene of H5N1 (rPIV5-H5) from the H5N1 subtype provided sterilizing immunity against lethal doses of HPAI H5N1 infection in mice. Furthermore, we have examined the effect of insertion of H5N1 HA at different locations within the PIV5 genome on the efficacy of a PIV5-based vaccine. Interestingly, insertion of H5N1 HA between the leader sequence, the de facto promoter of PIV5, and the first viral gene, nucleoprotein (NP), did not lead to a viable virus. Insertion of H5N1 HA between NP and the next gene, V/phosphorprotein (V/P), led to a virus that was defective in growth. We have found that insertion of H5N1 HA at the junction between the small hydrophobic (SH) gene and the hemagglutinin-neuraminidase (HN) gene gave the best immunity against HPAI H5N1 challenge: a dose as low as 1,000 PFU was sufficient to protect against lethal HPAI H5N1 challenge in mice. The work suggests that recombinant PIV5 expressing H5N1 HA has great potential as an HPAI H5N1 vaccine. PMID:23077314

  20. Chimeric Plastics : a new class of thermoplastic

    Science.gov (United States)

    Sonnenschein, Mark

    A new class of thermoplastics (dubbed ``Chimerics'') is described that exhibits a high temperature glass transition followed by high performance elastomer properties, prior to melting. These transparent materials are comprised of co-continuous phase-separated block copolymers. One block is an amorphous glass with a high glass transition temperature, and the second is a higher temperature phase transition block creating virtual thermoreversible crosslinks. The material properties are highly influenced by phase separation on the order of 10-30 nanometers. At lower temperatures the polymer reflects the sum of the block copolymer properties. As the amorphous phase glass transition is exceeded, the virtual crosslinks of the higher temperature second phase dominate the plastic properties, resulting in rubber-like elasticity.

  1. Transmission pattern of parainfluenza 3 virus in guinea pig breeding herds.

    Science.gov (United States)

    Blomqvist, Gunilla A M; Martin, Krister; Morein, Bror

    2002-07-01

    In searching for the cause of experimental variations in respiratory research data, serology revealed the prevalence of antibodies against parainfluenza virus type 3 (PIV 3) in guinea pigs. The aim of the present study was to explore the transmission rate, course, and kinetics of enzootic PIV 3 infection in guinea pig breeding units. In the first part of the study, blood samples to be analyzed for PIV 3 antibodies were collected from guinea pigs of a PIV 3-positive breeding colony at different times after birth. In the same breeding unit, 6 of 12 2-week-old guinea pigs were relocated and separately housed. The PIV 3 serum antibody titers of the two groups were compared at various times from birth to 13 weeks after birth. In the second part of the study, the spread of infectious virus and virus persistence were explored by housing seronegative sentinel animals together with 2- to 3-week-old guinea pigs from three different PIV 3-positive breeding units. The guinea pigs remaining in the breeding colony as well as those removed and housed separately showed declining serum antibody titers for about 1 month after birth, thereafter the titers were stable until about 8 weeks after birth. Five weeks later, the mean antibody titer of the guinea pigs remaining in the breeding colony had increased to a markedly higher level than that of the relocated, separately housed guinea pigs. Seroconversion was demonstrated in 7 of the 14 sentinels housed with the 2- to 3-week-old guinea pigs from PIV 3-positive breeding units. Sentinels housed together with PIV 3-positive guinea pigs 24 weeks after the start of the experiment did not seroconvert. We conclude that young guinea pigs born to PIV 3-positive mothers were protected by maternal immunity against infection with PIV 3 during their first 14 days of life. The guinea pig offspring became infected during the period from about 2 weeks until 8 weeks after birth, as demonstrated by seroconversion of sentinel animals and an increasing

  2. Infection of Parainfluenza type 3 (PI-3 as one of the causative agent of pneumonia in sheep and goats

    Directory of Open Access Journals (Sweden)

    Indrawati Sendow

    2002-03-01

    Full Text Available Serological survey was conducted to obtain the prevalence of Parainfluenza type 3 (PI-3 reactor as one of the causative agent of pneumonia in sheep and goats in abatoir at Jakarta and some small holder farms in Indonesia. Serological test using serum neutralization from 724 goat sera and 109 sheep sera indicated that only 1% of goats were serologically reactors and none of sheep sera had antibodies against PI-3 virus. Isolation of the virus from 56 bronchus and trachea swab and 345 lungs indicated that only one sampel from lung showed cythopathic effect (CPE in Madin Darby Bovine Kidney (MDBK cell lines identification of the virus using serum neutralization test indicated that the virus neutralized reference PI-3 antisera. The isolate came from one lung (7% of 24 that showed histopathologically pneumonia intertitialis that usually caused by viral infection.

  3. chimeraviz: a tool for visualizing chimeric RNA.

    Science.gov (United States)

    Lågstad, Stian; Zhao, Sen; Hoff, Andreas M; Johannessen, Bjarne; Lingjærde, Ole Christian; Skotheim, Rolf I

    2017-09-15

    Advances in high-throughput RNA sequencing have enabled more efficient detection of fusion transcripts, but the technology and associated software used for fusion detection from sequencing data often yield a high false discovery rate. Good prioritization of the results is important, and this can be helped by a visualization framework that automatically integrates RNA data with known genomic features. Here we present chimeraviz , a Bioconductor package that automates the creation of chimeric RNA visualizations. The package supports input from nine different fusion-finder tools: deFuse, EricScript, InFusion, JAFFA, FusionCatcher, FusionMap, PRADA, SOAPfuse and STAR-FUSION. chimeraviz is an R package available via Bioconductor ( https://bioconductor.org/packages/release/bioc/html/chimeraviz.html ) under Artistic-2.0. Source code and support is available at GitHub ( https://github.com/stianlagstad/chimeraviz ). rolf.i.skotheim@rr-research.no. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

  4. Chimeric HIV-1 Envelope Glycoproteins with Potent Intrinsic Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Activity*

    Science.gov (United States)

    Boot, Maikel; Cobos Jiménez, Viviana; Kootstra, Neeltje A.; Sanders, Rogier W.

    2013-01-01

    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 EnvGM-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 EnvGM-CSF proteins were able to differentiate human monocytes into cells with a macrophage-like phenotype. Chimeric EnvGM-CSF should be useful for improving humoral immunity against HIV-1 and these studies should inform the design of other chimeric proteins. PMID:23565193

  5. 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.

  6. Chimerism and tolerance without GVHD or engraftment syndrome in HLA-mismatched combined kidney and hematopoietic stem cell transplantation.

    Science.gov (United States)

    Leventhal, Joseph; Abecassis, Michael; Miller, Joshua; Gallon, Lorenzo; Ravindra, Kadiyala; Tollerud, David J; King, Bradley; Elliott, Mary Jane; Herzig, Geoffrey; Herzig, Roger; Ildstad, Suzanne T

    2012-03-07

    The toxicity of chronic immunosuppressive agents required for organ transplant maintenance has prompted investigators to pursue approaches to induce immune tolerance. We developed an approach using a bioengineered mobilized cellular product enriched for hematopoietic stem cells (HSCs) and tolerogenic graft facilitating cells (FCs) combined with nonmyeloablative conditioning; this approach resulted in engraftment, durable chimerism, and tolerance induction in recipients with highly mismatched related and unrelated donors. Eight recipients of human leukocyte antigen (HLA)-mismatched kidney and FC/HSC transplants underwent conditioning with fludarabine, 200-centigray total body irradiation, and cyclophosphamide followed by posttransplant immunosuppression with tacrolimus and mycophenolate mofetil. Subjects ranged in age from 29 to 56 years. HLA match ranged from five of six loci with related donors to one of six loci with unrelated donors. The absolute neutrophil counts reached a nadir about 1 week after transplant, with recovery by 2 weeks. Multilineage chimerism at 1 month ranged from 6 to 100%. The conditioning was well tolerated, with outpatient management after postoperative day 2. Two subjects exhibited transient chimerism and were maintained on low-dose tacrolimus monotherapy. One subject developed viral sepsis 2 months after transplant and experienced renal artery thrombosis. Five subjects experienced durable chimerism, demonstrated immunocompetence and donor-specific tolerance by in vitro proliferative assays, and were successfully weaned off all immunosuppression 1 year after transplant. None of the recipients produced anti-donor antibody or exhibited engraftment syndrome or graft-versus-host disease. These results suggest that manipulation of a mobilized stem cell graft and nonmyeloablative conditioning represents a safe, practical, and reproducible means of inducing durable chimerism and donor-specific tolerance in solid organ transplant recipients.

  7. Chimeric autologous/allogeneic constructs for skin regeneration.

    Science.gov (United States)

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

    2014-08-01

    The ideal treatment for severe cutaneous injuries would eliminate the need for autografts and promote fully functional, aesthetically pleasing autologous skin regeneration. NIKS progenitor cell-based skin tissues have been developed to promote healing by providing barrier function and delivering wound healing factors. Independently, a device has recently been created to "copy" skin by harvesting full-thickness microscopic tissue columns (MTCs) in lieu of autografts traditionally harvested as sheets. We evaluated the feasibility of combining these two technologies by embedding MTCs in NIKS-based skin tissues to generate chimeric autologous/allogeneic constructs. Chimeric constructs have the potential to provide immediate wound coverage, eliminate painful donor site wounds, and promote restoration of a pigmented skin tissue possessing hair follicles, sweat glands, and sebaceous glands. After MTC insertion, chimeric constructs and controls were reintroduced into air-interface culture and maintained in vitro for several weeks. Tissue viability, proliferative capacity, and morphology were evaluated after long-term culture. Our results confirmed successful MTC insertion and integration, and demonstrated the feasibility of generating chimeric autologous/allogeneic constructs that preserved the viability, proliferative capacity, and structure of autologous pigmented skin. These feasibility studies established the proof-of-principle necessary to further develop chimeric autologous/allogeneic constructs for the treatment of complex skin defects. Reprint & Copyright © 2014 Association of Military Surgeons of the U.S.

  8. Construction and expression of a functional monoclonal antibody SZ-51 specific for GMP-140 chimeric fab fragment in Escherichia coli

    International Nuclear Information System (INIS)

    Gu Jianming; Zhang Xiaomin; Xia Lijun; Wan Haiying; Liu Yue; Li Peixia; Ruan Changgeng

    1996-04-01

    The variable region cDNAs of a monoclonal antibody SZ-51 specific for α-granule membrane protein (GMP-140) on the surface of activated human platelets were spliced with the constant region cDNA of the heavy chain CH1 and light chain k of human Ig G by means of the gene recombination techniques. The above recombinant gene was amplified by the polymerase chain reaction (PCR). The expression vector of phage plasmid pHEN1 SZ-51 Fab/Hu was constructed. The pHEN1-51 Fab/Hu was introduced into non-suppressor E. coli HB2151. The amount of expression of SZ-51 chimeric Fab/Hu measured by quantitative ELISA was about 500 μg/L. Western blot demonstrated that the SZ-51 chimeric Fab fragment could specifically bind to GMP-140. (2 figs.)

  9. An infectious bat-derived chimeric influenza virus harbouring the entry machinery of an influenza A virus.

    Science.gov (United States)

    Juozapaitis, Mindaugas; Aguiar Moreira, Étori; Mena, Ignacio; Giese, Sebastian; Riegger, David; Pohlmann, Anne; Höper, Dirk; Zimmer, Gert; Beer, Martin; García-Sastre, Adolfo; Schwemmle, Martin

    2014-07-23

    In 2012, the complete genomic sequence of a new and potentially harmful influenza A-like virus from bats (H17N10) was identified. However, infectious influenza virus was neither isolated from infected bats nor reconstituted, impeding further characterization of this virus. Here we show the generation of an infectious chimeric virus containing six out of the eight bat virus genes, with the remaining two genes encoding the haemagglutinin and neuraminidase proteins of a prototypic influenza A virus. This engineered virus replicates well in a broad range of mammalian cell cultures, human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Importantly, the bat chimeric virus is unable to reassort with other influenza A viruses. Although our data do not exclude the possibility of zoonotic transmission of bat influenza viruses into the human population, they indicate that multiple barriers exist that makes this an unlikely event.

  10. Enhancement of antibody-dependent cellular cytotoxicity of cetuximab by a chimeric protein encompassing interleukin-15.

    Science.gov (United States)

    Ochoa, Maria Carmen; Minute, Luna; López, Ascensión; Pérez-Ruiz, Elisabeth; Gomar, Celia; Vasquez, Marcos; Inoges, Susana; Etxeberria, Iñaki; Rodriguez, Inmaculada; Garasa, Saray; Mayer, Jan-Peter Andreas; Wirtz, Peter; Melero, Ignacio; Berraondo, Pedro

    2018-01-01

    Enhancement of antibody-dependent cellular cytotoxicity (ADCC) may potentiate the antitumor efficacy of tumor-targeted monoclonal antibodies. Increasing the numbers and antitumor activity of NK cells is a promising strategy to maximize the ADCC of standard-of-care tumor-targeted antibodies. For this purpose, we have preclinically tested a recombinant chimeric protein encompassing the sushi domain of the IL15Rα, IL-15, and apolipoprotein A-I (Sushi-IL15-Apo) as produced in CHO cells. The size-exclusion purified monomeric fraction of this chimeric protein was stable and retained the IL-15 and the sushi domain bioactivity as measured by CTLL-2 and Mo-7e cell proliferation and STAT5 phosphorylation in freshly isolated human NK and CD8 + T cells. On cell cultures, Sushi-IL15-Apo increases NK cell proliferation and survival as well as spontaneous and antibody-mediated cytotoxicity. Scavenger receptor class B type I (SR-B1) is the receptor for ApoA-I and is expressed on the surface of tumor cells. SR-B1 can adsorb the chimeric protein on tumor cells and can transpresent IL-15 to NK and CD8 + T cells. A transient NK-humanized murine model was developed to test the increase of ADCC attained by the chimeric protein in vivo . The EGFR + human colon cancer cell line HT-29 was intraperitoneally inoculated in immune-deficient Rag2 -/- γc -/- mice that were reconstituted with freshly isolated PBMCs and treated with the anti-EGFR mAb cetuximab. The combination of the Sushi-IL15-Apo protein and cetuximab reduced the number of remaining tumor cells in the peritoneal cavity and delayed tumor engraftment in the peritoneum. Furthermore, Sushi-IL15-Apo increased the anti-tumor effect of a murine anti-EGFR mAb in Rag1 -/- mice bearing subcutaneous MC38 colon cancer transfected to express EGFR. Thus, Sushi-IL15-Apo is a potent tool to increase the number and the activation of NK cells to promote the ADCC activity of antibodies targeting tumor antigens.

  11. Development and characterization of novel chimeric monoclonal antibodies for broad spectrum neutralization of rabies virus.

    Directory of Open Access Journals (Sweden)

    Pan Kyeom Kim

    Full Text Available Current post-exposure prophylaxis for rabies virus infection has several limitations in terms of supply, cost, safety, and efficacy. Attempts to replace human or equine rabies immune globulins (HRIG or ERIG have been made by several companies and institutes. We developed potent monoclonal antibodies to neutralize a broad spectrum of rabies viruses by screening hybridomas received from the U.S. Centers for Disease Control and Prevention (CDC. Two kinds of chimeric human antibodies (chimeric #7 and #17 were constructed by cloning the variable regions from selected hybridomas and the constant region of a human antibody. Two antibodies were bound to antigenic site III and I/IV, respectively, and were able to neutralize 51 field isolates of rabies virus that were isolated at different times and places such as Asia, Africa, North America, South America, and Australia. These two antibodies neutralize rabies viruses with high efficacy in an in vivo test using Syrian hamster and mouse models and show low risk for adverse immunogenicity.

  12. Development and characterization of novel chimeric monoclonal antibodies for broad spectrum neutralization of rabies virus.

    Science.gov (United States)

    Kim, Pan Kyeom; Keum, Sun Ju; Osinubi, Modupe O V; Franka, Richard; Shin, Ji Young; Park, Sang Tae; Kim, Man Su; Park, Mi Jung; Lee, Soo Young; Carson, William; Greenberg, Lauren; Yu, Pengcheng; Tao, Xiaoyan; Lihua, Wang; Tang, Qing; Liang, Guodong; Shampur, Madhusdana; Rupprecht, Charles E; Chang, Shin Jae

    2017-01-01

    Current post-exposure prophylaxis for rabies virus infection has several limitations in terms of supply, cost, safety, and efficacy. Attempts to replace human or equine rabies immune globulins (HRIG or ERIG) have been made by several companies and institutes. We developed potent monoclonal antibodies to neutralize a broad spectrum of rabies viruses by screening hybridomas received from the U.S. Centers for Disease Control and Prevention (CDC). Two kinds of chimeric human antibodies (chimeric #7 and #17) were constructed by cloning the variable regions from selected hybridomas and the constant region of a human antibody. Two antibodies were bound to antigenic site III and I/IV, respectively, and were able to neutralize 51 field isolates of rabies virus that were isolated at different times and places such as Asia, Africa, North America, South America, and Australia. These two antibodies neutralize rabies viruses with high efficacy in an in vivo test using Syrian hamster and mouse models and show low risk for adverse immunogenicity.

  13. 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....... In comparison, when two blastomeres were aggregated, the developmental competence of the chimeric embryos decreased if the blastomeres were either from PA or from HMC embryos, but not if they were from different sources, i.e. one PA and one HMC blastomere. To evaluate the cell contribution in embryo formation...

  14. 77 FR 3482 - Prospective Grant of Exclusive License: Development of T Cell Receptors and Chimeric Antigen...

    Science.gov (United States)

    2012-01-24

    ... Exclusive License: Development of T Cell Receptors and Chimeric Antigen Receptors Into Therapeutics for.... 61/473,409 entitled ``Anti-epidermal growth factor receptor variant III chimeric antigen receptors... EGFRvIII chimeric antigen (CARs) and methods of using these engineered T cells to treat and/or prevent...

  15. 78 FR 16505 - Prospective Grant of Exclusive License: Chimeric West Nile/Dengue Viruses

    Science.gov (United States)

    2013-03-15

    ... Grant of Exclusive License: Chimeric West Nile/Dengue Viruses AGENCY: Centers for Disease Control and.... Provisional Application 61/049,342, filed 4/30/2008, entitled ``Engineered, Chimeric West Nile/Dengue Viruses;'' PCT Application PCT/US2009/041824, filed 4/27/2009, entitled ``Engineered, Chimeric WN/Flavivirus as...

  16. 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.

  17. Novel fusion genes and chimeric transcripts in ependymal tumors

    DEFF Research Database (Denmark)

    Olsen, Thale Kristin; Panagopoulos, Ioannis; Gorunova, Ludmila

    2016-01-01

    with subsequent Sanger sequencing was used to validate the potential fusions. Fluorescent in situ hybridization (FISH) using locus-specific probes was also performed. A total of 841 candidate chimeric transcripts were identified in the 12 tumors, with an average of 49 unique candidate fusions per tumor. After...... infratentorial anaplastic ependymoma. Our previously reported ALK rearrangements and the RELA and YAP1 fusions found in supratentorial ependymomas were until now the only known fusion genes present in ependymal tumors. The chimeric transcripts presented here are the first to be reported in infratentorial...

  18. Efficacy of a parainfluenza virus 5 (PIV5-based H7N9 vaccine in mice and guinea pigs: antibody titer towards HA was not a good indicator for protection.

    Directory of Open Access Journals (Sweden)

    Zhuo Li

    Full Text Available H7N9 has caused fatal infections in humans. A safe and effective vaccine is the best way to prevent large-scale outbreaks in the human population. Parainfluenza virus 5 (PIV5, an avirulent paramyxovirus, is a promising vaccine vector. In this work, we generated a recombinant PIV5 expressing the HA gene of H7N9 (PIV5-H7 and tested its efficacy against infection with influenza virus A/Anhui/1/2013 (H7N9 in mice and guinea pigs. PIV5-H7 protected the mice against lethal H7N9 challenge. Interestingly, the protection did not require antibody since PIV5-H7 protected JhD mice that do not produce antibody against lethal H7N9 challenge. Furthermore, transfer of anti-H7 serum did not protect mice against H7N9 challenge. PIV5-H7 generated high HAI titers in guinea pigs, however it did not protect against H7N9 infection or transmission. Intriguingly, immunization of guinea pigs with PIV5-H7 and PIV5 expressing NP of influenza A virus H5N1 (PIV5-NP conferred protection against H7N9 infection and transmission. Thus, we have obtained a H7N9 vaccine that protected both mice and guinea pigs against lethal H7N9 challenge and infection respectively.

  19. Preoperative clinical radioimmunodetection of pancreatic cancer by 111In-labeled chimeric monoclonal antibody Nd2

    International Nuclear Information System (INIS)

    Sawada, Tetsuji; Nishihara, Tamahiro; Yamamoto, Atsushi

    1999-01-01

    The present study was carried out with the purpose of evaluating the clinical usefulness of radioimmunodetection (RAID) with 111 In-labeled murine/human chimeric monoclonal antibody, Nd2 (c-Nd2) in patients with pancreatic cancer. Nineteen patients suspected to have pancreatic cancer were administered intravenously 74 MBq/2 mg 111 In-labeled c-Nd2 in 100 ml of saline containing 2% albumin over 30 min. A scintigram was obtained on the 3rd day after infusion by using single photon emission computed tomography (SPECT) imaging. Of the 14 patients finally diagnosed as having pancreatic cancer on the basis of surgical specimens or progress of disease, specific focal uptake at the site of the tumor was detected in 12 (true positive cases), representing a sensitivity of 85.7% (12/14), and liver metastasis was found in one case with metastasis. Of the 5 patients diagnosed with tumor-forming pancreatitis (TFP), 4 patients demonstrated true negative imaging, but one patient whose tumor demonstrated interesting findings in histology and immunostaining, showed false positive imaging. Of patients investigated for human anti-chimeric antibody (HACA) response, none showed HACA response, and no allergic reaction was seen in any of the patients administered c-Nd2. These results suggest that RAID with 111 In-labeled c-Nd2 is useful for differential preoperative diagnosis between invasive pancreatic cancer and TFP. (author)

  20. Mathematical modeling of erythrocyte chimerism informs genetic intervention strategies for sickle cell disease.

    Science.gov (United States)

    Altrock, Philipp M; Brendel, Christian; Renella, Raffaele; Orkin, Stuart H; Williams, David A; Michor, Franziska

    2016-09-01

    Recent advances in gene therapy and genome-engineering technologies offer the opportunity to correct sickle cell disease (SCD), a heritable disorder caused by a point mutation in the β-globin gene. The developmental switch from fetal γ-globin to adult β-globin is governed in part by the transcription factor (TF) BCL11A. This TF has been proposed as a therapeutic target for reactivation of γ-globin and concomitant reduction of β-sickle globin. In this and other approaches, genetic alteration of a portion of the hematopoietic stem cell (HSC) compartment leads to a mixture of sickling and corrected red blood cells (RBCs) in periphery. To reverse the sickling phenotype, a certain proportion of corrected RBCs is necessary; the degree of HSC alteration required to achieve a desired fraction of corrected RBCs remains unknown. To address this issue, we developed a mathematical model describing aging and survival of sickle-susceptible and normal RBCs; the former can have a selective survival advantage leading to their overrepresentation. We identified the level of bone marrow chimerism required for successful stem cell-based gene therapies in SCD. Our findings were further informed using an experimental mouse model, where we transplanted mixtures of Berkeley SCD and normal murine bone marrow cells to establish chimeric grafts in murine hosts. Our integrative theoretical and experimental approach identifies the target frequency of HSC alterations required for effective treatment of sickling syndromes in humans. Our work replaces episodic observations of such target frequencies with a mathematical modeling framework that covers a large and continuous spectrum of chimerism conditions. Am. J. Hematol. 91:931-937, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. Structure of the Paramyxovirus Parainfluenza Virus 5 Nucleoprotein in Complex with an Amino-Terminal Peptide of the Phosphoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Aggarwal, Megha; Leser, George P.; Kors, Christopher A.; Lamb, Robert A.; Sundquist, Wesley I.

    2017-12-13

    Parainfluenza virus 5 (PIV5) belongs to the familyParamyxoviridae, which consists of enveloped viruses with a nonsegmented negative-strand RNA genome encapsidated by the nucleoprotein (N). Paramyxovirus replication is regulated by the phosphoprotein (P) through protein-protein interactions with N and the RNA polymerase (L). The chaperone activity of P is essential to maintain the unassembled RNA-free form of N in order to prevent nonspecific RNA binding and premature N oligomerization. Here, we determined the crystal structure of unassembled PIV5 N in complex with a P peptide (N0P) derived from the N terminus of P (P50) at 2.65 Å. The PIV5 N0P consists of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a hinge region. The cleft at the hinge region of RNA-bound PIV5 N was previously shown to be an RNA binding site. The N0P structure shows that the P peptide binds to the CTD of N and extends toward the RNA binding site to inhibit N oligomerization and, hence, RNA binding. Binding of P peptide also keeps the PIV5 N in the open form. A molecular dynamics (MD) analysis of both the open and closed forms of N shows the flexibility of the CTD and the preference of the N protein to be in an open conformation. The gradual opening of the hinge region, to release the RNA, was also observed. Together, these results advance our knowledge of the conformational swapping of N required for the highly regulated paramyxovirus replication.

    IMPORTANCEParamyxovirus replication is regulated by the interaction of P with N and L proteins. Here, we report the crystal structure of unassembled parainfluenza virus 5 (PIV5) N chaperoned with P peptide. Our results provide a detailed understanding of the binding of P to N. The conformational switching of N between closed and open forms during its initial interaction with P, as well as

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

    International Nuclear Information System (INIS)

    Iri-Sofla, Farnoush Jafari; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud; Rasaee, Mohammad J.

    2011-01-01

    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 (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.

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

    International Nuclear Information System (INIS)

    Aigrain, Louise; Pompon, Denis; Truan, Gilles; Moréra, Solange

    2009-01-01

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

  4. 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.

  5. Therapeutic use of chimeric bacteriophage (phage) lysins in staphylococcal endophthalmitis

    Science.gov (United States)

    Purpose: Phage endolysins are peptidoglycan hydrolases that are produced at the end of the phage lytic cycle to digest the host bacterial cell wall, facilitating the release of mature phage progeny. The aim of this study is to determine the antimicrobial activity of chimeric phage lysins against cli...

  6. Direct observation of hematopoietic progenitor chimerism in fetal freemartin cattle

    Directory of Open Access Journals (Sweden)

    Taponen Juhani

    2007-11-01

    Full Text Available Abstract Background Cattle twins are well known as blood chimeras. However, chimerism in the actual hematopoietic progenitor compartment has not been directly investigated. Here, we analyzed fetal liver of chimeric freemartin cattle by combining a new anti-bovine CD34 antibody and Y-chromosome specific in situ hybridization. Results Bull-derived CD34+ cells were detected in the liver of the female sibling (freemartin at 60 days gestation. The level of bull-derived CD34+ cells was lower in the freemartin than in its male siblings. Bull (Y+ and cow hematopoietic cells often occurred in separate clusters. Around clusters of Y+CD34+ cells, Y+CD34- cells were typically observed. The thymi were also strongly chimeric at 60 days of gestation. Conclusion The fetal freemartin liver contains clusters of bull-derived hematopoietic progenitors, suggesting clonal expansion and differentiation. Even the roots of the hematopoietic system in cattle twins are thus strongly chimeric from the early stages of fetal development. However, the hematopoietic seeding of fetal liver apparently started already before the onset of functional vascular anastomosis.

  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

  8. Chimeric RNAs as potential biomarkers for tumor diagnosis

    Directory of Open Access Journals (Sweden)

    Jianhua Zhou

    2012-03-01

    Full Text Available Cancers claim millions of lives each year. Early detection thatcan enable a higher chance of cure is of paramount importanceto cancer patients. However, diagnostic tools for many forms oftumors have been lacking. Over the last few years, studies ofchimeric RNAs as biomarkers have emerged. Numerous reportsusing bioinformatics and screening methodologies havedescribed more than 30,000 expressed sequence tags (EST orcDNA sequences as putative chimeric RNAs. While cancer cellshave been well known to contain fusion genes derived fromchromosomal translocations, rearrangements or deletions, recentstudies suggest that trans-splicing in cells may be another sourceof chimeric RNA production. Unlike cis-splicing, trans-splicingtakes place between two pre-mRNA molecules, which are inmost cases derived from two different genes, generating achimeric non-co-linear RNA. It is possible that trans-splicingoccurs in normal cells at high frequencies but the resultingchimeric RNAs exist only at low levels. However the levels ofcertain RNA chimeras may be elevated in cancers, leading to theformation of fusion genes. In light of the fact that chimeric RNAshave been shown to be overrepresented in various tumors,studies of the mechanisms that produce chimeric RNAs andidentification of signature RNA chimeras as biomarkers presentan opportunity for the development of diagnoses for early tumordetection. (BMB reports 2012; 45(3: 133-140

  9. Study of allosteric communications in chimeric two-domain proteins

    Czech Academy of Sciences Publication Activity Database

    Boušová, Kristýna

    2017-01-01

    Roč. 26, S1 (2017), s. 74 ISSN 0961-8368. [Annual Symposium of the Protein Society /31./. 24.07.2017-27.07.2017, Montreal] Institutional support: RVO:61388963 Keywords : protein domains * chimeric structures Subject RIV: CE - Biochemistry

  10. (dust, PM10 , and BC) using CHIMERE chemistry tra

    Indian Academy of Sciences (India)

    The objective of this study is to evaluate the ability of a European chemistry transport model,. 'CHIMERE' driven by ..... tive days in May 2008 (12–16 May) to simulate the dust storm ...... Regional Office for Europe, Copenhagen. Zender C, Bian ...

  11. Expression of chimeric HCV peptide in transgenic tobacco plants ...

    African Journals Online (AJOL)

    Expression of chimeric HCV peptide in transgenic tobacco plants infected with recombinant alfalfa mosaic virus for development of a plant-derived vaccine against HCV. AK El Attar, AM Shamloul, AA Shalaby, BY Riad, A Saad, HM Mazyad, JM Keith ...

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

    International Nuclear Information System (INIS)

    Devitt, Gerard; Emerson, Vanessa; Holtkotte, Denise; Pfeiffer, Tanya; Pisch, Thorsten; Bosch, Valerie

    2007-01-01

    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-Env767 stop , which mediates strongly increased incorporation of SIV-Env into SIV particles. In a second strategy, we have employed a truncated HIV-Env protein (Env-Tr752 N750K ) 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-Tr752 N750K 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

  13. Fusion peptides from oncogenic chimeric proteins as putative specific biomarkers of cancer.

    Science.gov (United States)

    Conlon, Kevin P; Basrur, Venkatesha; Rolland, Delphine; Wolfe, Thomas; Nesvizhskii, Alexey I; MacCoss, Michael J; Lim, Megan S; Elenitoba-Johnson, Kojo S J

    2013-10-01

    Chromosomal translocations encoding chimeric fusion proteins constitute one of the most common mechanisms underlying oncogenic transformation in human cancer. Fusion peptides resulting from such oncogenic chimeric fusions, though unique to specific cancer subtypes, are unexplored as cancer biomarkers. Here we show, using an approach termed fusion peptide multiple reaction monitoring mass spectrometry, the direct identification of different cancer-specific fusion peptides arising from protein chimeras that are generated from the juxtaposition of heterologous genes fused by recurrent chromosomal translocations. Using fusion peptide multiple reaction monitoring mass spectrometry in a clinically relevant scenario, we demonstrate the specific, sensitive, and unambiguous detection of a specific diagnostic fusion peptide in clinical samples of anaplastic large cell lymphoma, but not in a diverse array of benign lymph nodes or other forms of primary malignant lymphomas and cancer-derived cell lines. Our studies highlight the utility of fusion peptides as cancer biomarkers and carry broad implications for the use of protein biomarkers in cancer detection and monitoring.

  14. Chimeric peptide-mediated siRNA transduction to inhibit HIV-1 infection.

    Science.gov (United States)

    Bivalkar-Mehla, Shalmali; Mehla, Rajeev; Chauhan, Ashok

    2017-04-01

    Persistent human immunodeficiency virus 1 (HIV-1) infection provokes immune activation and depletes CD4 +  lymphocytes, leading to acquired immunodeficiency syndrome. Uninterrupted administration of combination antiretroviral therapy (cART) in HIV-infected patients suppresses viral replication to below the detectable level and partially restores the immune system. However, cART-unresponsive residual HIV-1 infection and elusive transcriptionally silent but reactivatable viral reservoirs maintain a permanent viral DNA blue print. The virus rebounds within a few weeks after interruption of suppressive therapy. Adjunct gene therapy to control viral replication by ribonucleic acid interference (RNAi) is a post-transcriptional gene silencing strategy that could suppress residual HIV-1 burden and overcome viral resistance. Small interfering ribonucleic acids (siRNAs) are efficient transcriptional inhibitors, but need delivery systems to reach inside target cells. We investigated the potential of chimeric peptide (FP-PTD) to deliver specific siRNAs to HIV-1-susceptible and permissive cells. Chimeric FP-PTD peptide was designed with an RNA binding domain (PTD) to bind siRNA and a cell fusion peptide domain (FP) to enter cells. FP-PTD-siRNA complex entered and inhibited HIV-1 replication in susceptible cells, and could be a candidate for in vivo testing.

  15. Chimeric rhinoviruses displaying MPER epitopes elicit anti-HIV neutralizing responses.

    Directory of Open Access Journals (Sweden)

    Guohua Yi

    Full Text Available The development of an effective AIDS vaccine has been a formidable task, but remains a critical necessity. The well conserved membrane-proximal external region (MPER of the HIV-1 gp41 glycoprotein is one of the crucial targets for AIDS vaccine development, as it has the necessary attribute of being able to elicit antibodies capable of neutralizing diverse isolates of HIV.Guided by X-ray crystallography, molecular modeling, combinatorial chemistry, and powerful selection techniques, we designed and produced six combinatorial libraries of chimeric human rhinoviruses (HRV displaying the MPER epitopes corresponding to mAbs 2F5, 4E10, and/or Z13e1, connected to an immunogenic surface loop of HRV via linkers of varying lengths and sequences. Not all libraries led to viable chimeric viruses with the desired sequences, but the combinatorial approach allowed us to examine large numbers of MPER-displaying chimeras. Among the chimeras were five that elicited antibodies capable of significantly neutralizing HIV-1 pseudoviruses from at least three subtypes, in one case leading to neutralization of 10 pseudoviruses from all six subtypes tested.Optimization of these chimeras or closely related chimeras could conceivably lead to useful components of an effective AIDS vaccine. While the MPER of HIV may not be immunodominant in natural infection by HIV-1, its presence in a vaccine cocktail could provide critical breadth of protection.

  16. Chimeric peptides as modulators of CK2-dependent signaling: Mechanism of action and off-target effects.

    Science.gov (United States)

    Zanin, Sofia; Sandre, Michele; Cozza, Giorgio; Ottaviani, Daniele; Marin, Oriano; Pinna, Lorenzo A; Ruzzene, Maria

    2015-10-01

    Protein kinase CK2 is a tetrameric enzyme composed of two catalytic (α/α') and two regulatory (β) subunits. It has a global prosurvival function, especially in cancer, and represents an attractive therapeutic target. Most CK2 inhibitors available so far are ATP-competitive compounds; however, the possibility to block only the phosphorylation of few substrates has been recently explored, and a compound composed of a Tat cell-penetrating peptide and an active cyclic peptide, selected for its ability to bind to the CK2 substrate E7 protein of human papilloma virus, has been developed [Perea et al., Cancer Res. 2004; 64:7127-7129]. By using a similar chimeric peptide (CK2 modulatory chimeric peptide, CK2-MCP), we performed a study to dissect its molecular mechanism of action and the signaling pathways that it affects in cells. We found that it directly interacts with CK2 itself, counteracting the regulatory and stabilizing functions of the β subunit. Cell treatment with CK2-MCP induces a rapid decrease of the amount of CK2 subunits, as well as of other signaling proteins. Concomitant cell death is observed, more pronounced in tumor cells and not accompanied by apoptotic events. CK2 relocalizes to lysosomes, whose proteases are activated, while the proteasome machinery is inhibited. Several sequence variants of the chimeric peptide have been also synthesized, and their effects compared to those of the parental peptide. Intriguingly, the Tat moiety is essential not only for cell penetration but also for the in vitro efficacy of the peptide. We conclude that this class of chimeric peptides, in addition to altering some properties of CK2 holoenzyme, affects several other cellular targets, causing profound perturbations of cell biology. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Engineered chimeric peptides with antimicrobial and titanium-binding functions to inhibit biofilm formation on Ti implants.

    Science.gov (United States)

    Geng, Hongjuan; Yuan, Yang; Adayi, Aidina; Zhang, Xu; Song, Xin; Gong, Lei; Zhang, Xi; Gao, Ping

    2018-01-01

    Titanium (Ti) implants have been commonly used in oral medicine. However, despite their widespread clinical application, these implants are susceptible to failure induced by microbial infection due to bacterial biofilm formation. Immobilization of chimeric peptides with antibacterial properties on the Ti surface may be a promising antimicrobial approach to inhibit biofilm formation. Here, chimeric peptides were designed by connecting three sequences (hBD-3-1/2/3) derived from human β-defensin-3 (hBD-3) with Ti-binding peptide-l (TBP-l: RKLPDAGPMHTW) via a triple glycine (G) linker to modify Ti surfaces. Using X-ray photoelectron spectroscopy (XPS), the properties of individual domains of the chimeric peptides were evaluated for their binding activity toward the Ti surface. The antimicrobial and anti-biofilm efficacy of the peptides against initial settlers, Streptococcus oralis (S. oralis), Streptococcus gordonii (S. gordonii) and Streptococcus sanguinis (S. sanguinis), was evaluated with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Transmission electron microscopy (TEM) and real-time quantitative PCR (qRT-PCR) were used to study cell membrane changes and the underlying antimicrobial mechanism. Compared with the other two peptides, TBP-1-GGG-hBD3-3 presented stronger antibacterial activity and remained stable in saliva and serum. Therefore, it was chosen as the best candidate to modify Ti surfaces in this study. This peptide inhibited the growth of initial streptococci and biofilm formation on Ti surfaces with no cytotoxicity to MC3T3-E1 cells. Disruption of the integrity of bacterial membranes and decreased expression of adhesion protein genes from S. gordonii revealed aspects of the antibacterial mechanism of TBP-1-GGG-hBD3-3. We conclude that engineered chimeric peptides with antimicrobial activity provide a potential solution for inhibiting biofilm formation on Ti surfaces to reduce or prevent the occurrence of peri

  18. Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells

    OpenAIRE

    Stroncek, David F.; Lee, Daniel W.; Ren, Jiaqiang; Sabatino, Marianna; Highfill, Steven; Khuu, Hanh; Shah, Nirali N.; Kaplan, Rosandra N.; Fry, Terry J.; Mackall, Crystal L.

    2017-01-01

    Background Clinical trials of Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of hematologic malignancies have been promising, but CAR T cell yields have been variable. This variability is due in part to the contamination of the PBMC concentrates with monocytes and granulocytes. Methods Counter-flow elutriation allows for the closed system separation of lymphocytes from monocytes and granulocytes. We ...

  19. [Effect of extracted ZG from gardenia on Hep-2 cell membrane post infected with parainfluenza virus type 1 (PIV-1)].

    Science.gov (United States)

    Guo, Shan-Shan; Huang, Yang; Zhao, Ye; Gao, Ying-Jie; Gong, Wen-Feng; Cui, Xiao-Lan

    2007-09-01

    In order to study the anti-viral mechanism of extracted ZG from Gardenia, the effect of extracted ZG on Hep-2 cell membrane potential, Na -K+-ATPase activity and membrane fluidity post infected with parainfluenza virus type 1 (PIV-1) was observed. Acetylcholine which was fluorescent labeled with DiBAC4 (3) was taken as positive control to observe the changes of membrane potential and was measured by flow cytometer. The phosphorus determination method and spectrophotometer were used to measure the Na+-K+-ATPase activity of Hep-2 cell membrane post PIV-1 infection. Hep-2 cell membrane phospholipids was labeled with fluorescent NBD-C6-HPC and membrane fluidity was measured by confocal laser scanning microscope. The results demonstated that after PIV-1 infection the Hep-2 cell membrane potential decreased significantly and the membrane was in the state of hyperpolarization, Na+-K+-ATPase activity increased and membrane fluidity decreased significantly. There was no apparent interferring effect of extracted ZG on the changes of membrane potential and Na+-K+-ATPase activity post PIV-1 infection, while membrane fluidity was improved significantly. Acetylcholine improved the state of hyperpolarization. The changes of membrane potential, Na -K+-ATPase activity and membrane fluidity might be the biomechanism of PIV-1 infectoin. The extracted ZG improved membrane fluidity to prevent from PIV-1 infection by protecting the cell membrane, which was probably the mechanism of anti-PIV-1 activity of the extracted ZG, but ZG probably had nothing to do with membrane potential and Na+-K+-ATPase activity.

  20. 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.

  1. The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.

    Directory of Open Access Journals (Sweden)

    Yi-Chieh Lin

    Full Text Available Membrane-tethered proteins (mammalian surface display are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

  2. PREVALENCE OF BOVINE HERPESVIRUS-1,PARAINFLUENZA-3,BOVINE ROTAVIRUS, BOVINE VIRAL DIARRHEA, BOVINE ADENOVIRUS-7,BOVINE LEUKEMIA VIRUS AND BLUETONGUE VIRUS ANTIBODIES IN CATTLE IN MEXICO

    OpenAIRE

    SUZAN, Victor M.; ONUMA, Misao; AGUILAR, Romero E.; MURAKAMI, Yosuke

    1983-01-01

    Sera were collected from dairy and beef cattle in 19 different states of Mexico. These sera were tested for bovine herpesvirus-1 (BHV-1), parainfluenza-3 virus (PIV-3), bovine rotavirus (BRV), bovine leukemia virus (BLV), bovine adenovirus-7 (BAV-7), bluetongue virus (BTV) and bovine viral diarrhea virus (BVDV). Seropositive rates for each virus for dairy cattle tested were 158/277(57.0%) for BHV-1,217/286(75.0%) for PIV-3,541/1498(36.1%) for BLV, 134/144(93.1%) for BRV, 39/90(43.3%) for BTV,...

  3. Chimeric microbial rhodopsins for optical activation of Gs-proteins

    Science.gov (United States)

    Yoshida, Kazuho; Yamashita, Takahiro; Sasaki, Kengo; Inoue, Keiichi; Shichida, Yoshinori; Kandori, Hideki

    2017-01-01

    We previously showed that the chimeric proteins of microbial rhodopsins, such as light-driven proton pump bacteriorhodopsin (BR) and Gloeobacter rhodopsin (GR) that contain cytoplasmic loops of bovine rhodopsin, are able to activate Gt protein upon light absorption. These facts suggest similar protein structural changes in both the light-driven proton pump and animal rhodopsin. Here we report two trials to engineer chimeric rhodopsins, one for the inserted loop, and another for the microbial rhodopsin template. For the former, we successfully activated Gs protein by light through the incorporation of the cytoplasmic loop of β2-adrenergic receptor (β2AR). For the latter, we did not observe any G-protein activation for the light-driven sodium pump from Indibacter alkaliphilus (IndiR2) or a light-driven chloride pump halorhodopsin from Natronomonas pharaonis (NpHR), whereas the light-driven proton pump GR showed light-dependent G-protein activation. This fact suggests that a helix opening motion is common to G protein coupled receptor (GPCR) and GR, but not to IndiR2 and NpHR. Light-induced difference FTIR spectroscopy revealed similar structural changes between WT and the third loop chimera for each light-driven pump. A helical structural perturbation, which was largest for GR, was further enhanced in the chimera. We conclude that similar structural dynamics that occur on the cytoplasmic side of GPCR are needed to design chimeric microbial rhodopsins. PMID:29362703

  4. Cord Blood Chimerism And Relapse After Haplo-Cord Transplantation

    Science.gov (United States)

    van Besien, Koen; Koshy, Nebu; Gergis, Usama; Mayer, Sebastian; Cushing, Melissa; Rennert, Hannah; Slotky, Ronit; Mark, Tomer; Pearse, Roger; Rossi, Adriana; Phillips, Adrienne; Vasovic, Liljana; Ferrante, Rosanna; Hsu, Michael; Shore, Tsiporah

    2018-01-01

    Haplo-cord stem cell transplantation combines the infusion of CD34 selected hematopoietic progenitors from a haplo-identical donor with an umbilical cord blood graft from an unrelated donor and allows faster count recovery, with low rates of disease recurrence and chronic GVHD. But the contribution of the umbilical cord blood graft to long-term transplant outcome remains unclear. We analyzed 39 recipients of haplo-cord transplants with AML and MDS, engrafted and in remission at 2 months. Median age was 66 (18-72) and all had intermediate, high, or very high risk disease. Less than 20% UCB chimerism in the CD33 lineage was associated with an increased rate of disease recurrence (54% vs 11% Pdisease recurrence (46% vs 12%, P=0.007) Persistent haplo-chimerism in the CD3 lineage was associated with an increased rate of disease recurrence (40% vs 15%, P=0.009) Chimerism did not predict for treatment related mortality. The cumulative incidence of acute GVHD by day 100 was 43%. The cumulative incidence of moderate/severe chronic GVHD was only 5%. Engraftment of the umbilical cord blood grafts provides powerful GVL effects which protect against disease recurrence and is associated with low risk of chronic GVHD. Engraftment of CD34 selected haplo-identical cells can lead to rapid development of circulating T-cells, but when these cells dominate, GVL-effects are limited and rates of disease recurrence are high. PMID:27333804

  5. Chimeric exchange of coronavirus nsp5 proteases (3CLpro) identifies common and divergent regulatory determinants of protease activity.

    Science.gov (United States)

    Stobart, Christopher C; Sexton, Nicole R; Munjal, Havisha; Lu, Xiaotao; Molland, Katrina L; Tomar, Sakshi; Mesecar, Andrew D; Denison, Mark R

    2013-12-01

    Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp's 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp's at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types.

  6. Expression and purification of toxic anti-breast cancer p28-NRC chimeric protein

    OpenAIRE

    Soleimani, Meysam; Mirmohammad-Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Jahanian-Najafabadi, Ali

    2016-01-01

    Background: Chimeric proteins consisting of a targeting moiety and a cytotoxic moiety are now under intense research focus for targeted therapy of cancer. Here, we report cloning, expression, and purification of such a targeted chimeric protein made up of p28 peptide as both targeting and anticancer moiety fused to NRC peptide as a cytotoxic moiety. However, since the antimicrobial activity of the NRC peptide would intervene expression of the chimeric protein in Escherichia coli, we evaluated...

  7. Chimeric OspA genes, proteins and methods of use thereof

    Science.gov (United States)

    Crowe, Brian A.; Livey, Ian; O'Rourke, Maria; Schwendinger, Michael; Dunn, John J.; Luft, Benjamin J.

    2018-02-20

    The invention relates to the development of chimeric OspA molecules for use in a new Lyme vaccine. More specifically, the chimeric OspA molecules comprise the proximal portion from one OspA serotype, together with the distal portion from another OspA serotype, while retaining antigenic properties of both of the parent polypeptides. The chimeric OspA molecules are delivered alone or in combination to provide protection against a variety of Borrelia genospecies. The invention also provides methods for administering the chimeric OspA molecules to a subject in the prevention and treatment of Lyme disease or borreliosis.

  8. Thionin-D4E1 chimeric protein protects plants against bacterial infections

    Science.gov (United States)

    Stover, Eddie W; Gupta, Goutam; Hao, Guixia

    2017-08-08

    The generation of a chimeric protein containing a first domain encoding either a pro-thionon or thionin, a second domain encoding D4E1 or pro-D4E1, and a third domain encoding a peptide linker located between the first domain and second domain is described. Either the first domain or the second domain is located at the amino terminal of the chimeric protein and the other domain (second domain or first domain, respectively) is located at the carboxyl terminal. The chimeric protein has antibacterial activity. Genetically altered plants and their progeny expressing a polynucleotide encoding the chimeric protein resist diseases caused by bacteria.

  9. [Biological characteristics of a chimeric rabies virus expressing canine parvovirus VP2 protein].

    Science.gov (United States)

    Niu, Xue-Feng; Liu, Xiao-Hui; Sun, Zhao-Jin; Shi, He-He; Chen, Jing; Jiang, Bido; Sun, Jing-Chen; Guo, Xiao-Feng

    2009-09-01

    To obtain a bivalence vaccine against canine rabies virus and canine parvovirus, a chimeric rabies virus expressing canine parvovirus VP2 protein was generated by the technique of reverse genetics. It was shown that the chimeric virus designated as HEP-Flury (VP2) grew well on BHK-21 cells and the VP2 gene could still be stably expressed after ten passages on BHK-21 cells. Experiments on the mice immunized with the chimeric virus HEP-Flury (VP2) demonstrated that specific antibodies against rabies virus and canine parvovirus were induced in immunized mice after vaccination with the live chimeric virus.

  10. Stem cell potency and the ability to contribute to chimeric organisms.

    Science.gov (United States)

    Polejaeva, Irina; Mitalipov, Shoukhrat

    2013-03-01

    Mouse embryonic chimeras are a well-established tool for studying cell lineage commitment and pluripotency. Experimental chimeras were successfully produced by combining two or more preimplantation embryos or by introducing into host embryo cultured pluripotent embryonic stem cells (ESCs). Chimera production using genetically modified ESCs became the method of choice for the generation of knockout or knockin mice. Although the derivation of ESCs or ESC-like cells has been reported for other species, only mouse and rat pluripotent stem cells have been shown to contribute to germline-competent chimeras, which is the defining feature of ESCs. Herein, we describe different approaches employed for the generation of embryonic chimeras, define chimera-competent cell types, and describe cases of spontaneous chimerism in humans. We also review the current state of derivation of pluripotent stem cells in several species and discuss outcomes of various chimera studies when such cells are used.

  11. 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

    International Nuclear Information System (INIS)

    Hoshino, Tomoaki; Kawai, Tatsuo; Ota, Kazuo

    1996-01-01

    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)

  12. 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)

  13. A recombinant chimeric La Crosse virus expressing the surface glycoproteins of Jamestown Canyon virus is immunogenic and protective against challenge with either parental virus in mice or monkeys.

    Science.gov (United States)

    Bennett, R S; Gresko, A K; Nelson, J T; Murphy, B R; Whitehead, S S

    2012-01-01

    La Crosse virus (LACV) and Jamestown Canyon virus (JCV), family Bunyaviridae, are mosquito-borne viruses that are endemic in North America and recognized as etiologic agents of encephalitis in humans. Both viruses belong to the California encephalitis virus serogroup, which causes 70 to 100 cases of encephalitis a year. As a first step in creating live attenuated viral vaccine candidates for this serogroup, we have generated a recombinant LACV expressing the attachment/fusion glycoproteins of JCV. The JCV/LACV chimeric virus contains full-length S and L segments derived from LACV. For the M segment, the open reading frame (ORF) of LACV is replaced with that derived from JCV and is flanked by the untranslated regions of LACV. The resulting chimeric virus retained the same robust growth kinetics in tissue culture as observed for either parent virus, and the virus remains highly infectious and immunogenic in mice. Although both LACV and JCV are highly neurovirulent in 21 day-old mice, with 50% lethal dose (LD₅₀) values of 0.1 and 0.5 log₁₀ PFU, respectively, chimeric JCV/LACV is highly attenuated and does not cause disease even after intracerebral inoculation of 10³ PFU. Parenteral vaccination of mice with 10¹ or 10³ PFU of JCV/LACV protected against lethal challenge with LACV, JCV, and Tahyna virus (TAHV). The chimeric virus was infectious and immunogenic in rhesus monkeys and induced neutralizing antibodies to JCV, LACV, and TAHV. When vaccinated monkeys were challenged with JCV, they were protected against the development of viremia. Generation of highly attenuated yet immunogenic chimeric bunyaviruses could be an efficient general method for development of vaccines effective against these pathogenic viruses.

  14. A chimeric prokaryotic-eukaryotic pentameric ligand gated ion channel reveals interactions between the extracellular and transmembrane domains shape neurosteroid modulation.

    Science.gov (United States)

    Ghosh, Borna; Tsao, Tzu-Wei; Czajkowski, Cynthia

    2017-10-01

    Pentameric ligand-gated ion channels (pLGICs) are the targets of several clinical and endogenous allosteric modulators including anesthetics and neurosteroids. Molecular mechanisms underlying allosteric drug modulation are poorly understood. Here, we constructed a chimeric pLGIC by fusing the extracellular domain (ECD) of the proton-activated, cation-selective bacterial channel GLIC to the transmembrane domain (TMD) of the human ρ1 chloride-selective GABA A R, and tested the hypothesis that drug actions are regulated locally in the domain that houses its binding site. The chimeric channels were proton-gated and chloride-selective demonstrating the GLIC ECD was functionally coupled to the GABAρ TMD. Channels were blocked by picrotoxin and inhibited by pentobarbital, etomidate and propofol. The point mutation, ρ TMD W328M, conferred positive modulation and direct gating by pentobarbital. The data suggest that the structural machinery mediating general anesthetic modulation resides in the TMD. Proton-activation and neurosteroid modulation of the GLIC-ρ chimeric channels, however, did not simply mimic their respective actions on GLIC and GABAρ revealing that across domain interactions between the ECD and TMD play important roles in determining their actions. Proton-induced current responses were biphasic suggesting that the chimeric channels contain an additional proton sensor. Neurosteroid modulation of the GLIC-ρ chimeric channels by the stereoisomers, 5α-THDOC and 5β-THDOC, were swapped compared to their actions on GABAρ indicating that positive versus negative neurosteroid modulation is not encoded solely in the TMD nor by neurosteroid isomer structure but is dependent on specific interdomain connections between the ECD and TMD. Our data reveal a new mechanism for shaping neurosteroid modulation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Chimeric brain: theoretical and clinical aspects.

    Science.gov (United States)

    Saveliev, S V; Lebedev, V V; Evgeniev, M B; Korochkin, L I

    1997-12-01

    Using xeno-transplantation, interactions of neural tissues of vertebrates and insects were studied. Ventral neurogenic primordium of Notch Drosophila melanogaster embryos was transplanted into neural tube of amphibian and mammalian embryos with the aid of microhydrofeeding. Embryos of four different amphibian species, random bred mice and rats were used as graft recipients. It was concluded that there is a possibility to incorporate nerve cells of insects into the brain of vertebrates. Morphological and functional contacts can be established between the transplanted cells and host brain tissues. Transplanted Drosophila cells preserve their viability for a long time, so that a prolonged influence of the transplant upon the recipient can be predicted, which may be used in medical practice. A mixture of human fetal brain and Notch Drosophila melanogaster neural embryonic tissues were transplanted into the ventro-lateral nucleus of the thalamus of the patients of Parkinson' disease. As a result, tremor and constrained movements disappeared. Post-operation patients have been observed within 13-38 months. No side effects were noted during this time.

  16. 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...

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

    Directory of Open Access Journals (Sweden)

    E. Terrenoire

    2015-01-01

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

  18. Chimeric antigen receptor T-cell therapy for solid tumors

    Directory of Open Access Journals (Sweden)

    Kheng Newick

    2016-01-01

    Full Text Available Chimeric antigen receptor (CAR T cells are engineered constructs composed of synthetic receptors that direct T cells to surface antigens for subsequent elimination. Many CAR constructs are also manufactured with elements that augment T-cell persistence and activity. To date, CAR T cells have demonstrated tremendous success in eradicating hematological malignancies (e.g., CD19 CARs in leukemias. This success is not yet extrapolated to solid tumors, and the reasons for this are being actively investigated. Here in this mini-review, we discuss some of the key hurdles encountered by CAR T cells in the solid tumor microenvironment.

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

    International Nuclear Information System (INIS)

    Lapidot, T.; Terenzi, A.; Singer, T.S.; Salomon, O.; Reisner, Y.

    1989-01-01

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

  20. 78 FR 13691 - Prospective Grant of Exclusive License: The Development of m971 and m972 Chimeric Antigen...

    Science.gov (United States)

    2013-02-28

    ... Exclusive License: The Development of m971 and m972 Chimeric Antigen Receptors (CARs) for the Treatment of B... ``M971 Chimeric Antigen Receptors'' [HHS Ref. E-291-2012/0-US-01], and (b) U.S. Patent Application 61/042... malignancies that express CD22 on their cell surface using chimeric antigen receptors which contain the m971 or...

  1. 78 FR 70955 - Prospective Grant of Exclusive Patent License: GMCSF-BclxL-Derived Chimeric Therapeutics for Use...

    Science.gov (United States)

    2013-11-27

    ... Exclusive Patent License: GMCSF-BclxL- Derived Chimeric Therapeutics for Use in Treatment of Cancer...-BclxL-derived chimeric therapeutics and immunotherapeutics, alone or in combination, for restoring...: [email protected] . SUPPLEMENTARY INFORMATION: The subject invention is to a chimeric protein...

  2. 77 FR 62520 - Prospective Grant of Exclusive License: The Development of Anti-CD22 Chimeric Antigen Receptors...

    Science.gov (United States)

    2012-10-15

    ... Exclusive License: The Development of Anti- CD22 Chimeric Antigen Receptors (CARs) for the Treatment of B... ``Anti-CD22 Chimeric Antigen Receptors'' [HHS Ref. E-265-2011/0-US-01], and (b) U.S. Patent Application... CD22 on their cell surface using chimeric antigen receptors which contain the HA22 or BL22 antibody...

  3. Imaging of alpha(v)beta(3) expression by a bifunctional chimeric RGD peptide not cross-reacting with alpha(v)beta(5).

    Science.gov (United States)

    Zannetti, Antonella; Del Vecchio, Silvana; Iommelli, Francesca; Del Gatto, Annarita; De Luca, Stefania; Zaccaro, Laura; Papaccioli, Angela; Sommella, Jvana; Panico, Mariarosaria; Speranza, Antonio; Grieco, Paolo; Novellino, Ettore; Saviano, Michele; Pedone, Carlo; Salvatore, Marco

    2009-08-15

    To test whether a novel bifunctional chimeric peptide comprising a cyclic Arg-Gly-Asp pentapeptide covalently bound to an echistatin domain can discriminate alpha(v)beta(3) from alpha(v)beta(5) integrin, thus allowing the in vivo selective visualization of alpha(v)beta(3) expression by single-photon and positron emission tomography (PET) imaging. The chimeric peptide was preliminarily tested for inhibition of alpha(v)beta(3)-dependent cell adhesion and competition of 125I-echistatin binding to membrane of stably transfected K562 cells expressing alpha(v)beta(3) (Kalpha(v)beta(3)) or alpha(v)beta(5) (Kalpha(v)beta(5)) integrin. The chimeric peptide was then conjugated with diethylenetriaminepentaacetic acid and labeled with 111In for single-photon imaging, whereas a one-step procedure was used for labeling the full-length peptide and a truncated derivative, lacking the last five C-terminal amino acids, with 18F for PET imaging. Nude mice bearing tumors from Kalpha(v)beta(3), Kalpha(v)beta(5), U87MG human glioblastoma, and A431 human epidermoid cells were subjected to single-photon and PET imaging. Adhesion and competitive binding assays showed that the novel chimeric peptide selectively binds to alpha(v)beta(3) integrin and does not cross-react with alpha(v)beta(5). In agreement with in vitro findings, single-photon and PET imaging studies showed that the radiolabeled chimeric peptide selectively localizes in tumor xenografts expressing alphavbeta3 and fails to accumulate in those expressing alpha(v)beta(5) integrin. When 18F-labeled truncated derivative was used for PET imaging, alphavbeta3- and alpha(v)beta(5)-expressing tumors were visualized, indicating that the five C-terminal amino acids are required to differentially bind the two integrins. Our findings indicate that the novel chimeric Arg-Gly-Asp peptide, having no cross-reaction with alphavbeta5 integrin, allows highly selective alphavbeta3 expression imaging and monitoring.

  4. Mixed chimerism to induce tolerance for solid organ transplantation

    International Nuclear Information System (INIS)

    Wren, S.M.; Nalesnik, M.; Hronakes, M.L.; Oh, E.; Ildstad, S.T.

    1991-01-01

    Chimerism, or the coexistence of tissue elements from more than one genetically different strain or species in an organism, is the only experimental state that results in the induction of donor-specific transplantation tolerance. Transplantation of a mixture of T-cell-depleted syngeneic (host-type) plus T-cell-depleted allogeneic (donor) bone marrow into a normal adult recipient mouse (A + B----A) results in mixed allogeneic chimerism. Recipient mice exhibit donor-specific transplantation tolerance, yet have full immunocompetence to recognize and respond to third-party transplantation antigens. After complete hematolymphopoietic repopulation at 28 days, animals accept a donor-specific skin graft but reject major histocompatibility complex (MHC) locus-disparate third-party grafts. We now report that permanent graft acceptance can also be achieved when the graft is placed at the time of bone marrow transplantation. Histologically, grafts were viable and had only minimal inflammatory changes. This model may have potential future clinical application for the induction of donor-specific transplantation tolerance

  5. A multiple multicomponent approach to chimeric peptide-peptoid podands.

    Science.gov (United States)

    Rivera, Daniel G; León, Fredy; Concepción, Odette; Morales, Fidel E; Wessjohann, Ludger A

    2013-05-10

    The success of multi-armed, peptide-based receptors in supramolecular chemistry traditionally is not only based on the sequence but equally on an appropriate positioning of various peptidic chains to create a multivalent array of binding elements. As a faster, more versatile and alternative access toward (pseudo)peptidic receptors, a new approach based on multiple Ugi four-component reactions (Ugi-4CR) is proposed as a means of simultaneously incorporating several binding and catalytic elements into organizing scaffolds. By employing α-amino acids either as the amino or acid components of the Ugi-4CRs, this multiple multicomponent process allows for the one-pot assembly of podands bearing chimeric peptide-peptoid chains as appended arms. Tripodal, bowl-shaped, and concave polyfunctional skeletons are employed as topologically varied platforms for positioning the multiple peptidic chains formed by Ugi-4CRs. In a similar approach, steroidal building blocks with several axially-oriented isocyano groups are synthesized and utilized to align the chimeric chains with conformational constrains, thus providing an alternative to the classical peptido-steroidal receptors. The branched and hybrid peptide-peptoid appendages allow new possibilities for both rational design and combinatorial production of synthetic receptors. The concept is also expandable to other multicomponent reactions. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1998-09-01

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

  7. Efficient delivery and stable gene expression in a hematopoietic cell line using a chimeric serotype 35 fiber pseudotyped helper-dependent adenoviral vector

    International Nuclear Information System (INIS)

    Balamotis, Michael Andrew; Huang, Katie; Mitani, Kohnosuke

    2004-01-01

    Certain human cell populations have remained difficult to infect with human adenovirus (Ad) serotype 5 because of their lack of coxsackievirus B-adenovirus receptor (CAR). Native adenovirus fiber compositions, although diverse, cannot infect all tissue types. Recently, a chimeric Ad5/35 fiber was created, which displays an altered tropism from Ad5. We incorporated this chimeric fiber into a helper-dependent (HD) adenovirus vector system and compared HD to E1-deleted (E1Δ) vectors by transgene expression, cell transduction efficiency, and cytotoxicity. K562 cells were infected ∼50 times more efficiently with the chimeric Ad5/35 fiber compared with the Ad5 fiber. Short-term transgene expression was sustained longer from HD Ad5/35 than E1Δ Ad5/35 vector after in vitro infection of actively dividing K562 cells. Rapid loss of transgene expression from E1Δ Ad5/35 infection was not due to the loss of vector genomes, as determined by quantitative real-time PCR (QRT-PCR), or cytotoxicity, but rather through a putative silencing mechanism

  8. Pathogenesis of a genotype C strain of bovine parainfluenza virus type 3 infection in albino guinea pigs.

    Science.gov (United States)

    Shi, Hong-Fei; Zhu, Yuan-Mao; Dong, Xiu-Mei; Cai, Hong; Ma, Lei; Wang, Shu; Yan, Hao; Wang, Xue-Zhi; Xue, Fei

    2014-08-08

    Bovine parainfluenza virus type 3 (BPIV3) is one of the most important of the known viral respiratory tract agents of both young and adult cattle and widespread among cattle around the world. Up to present, three genotypes A, B and C of BPIV3 have been described on the basis of genetic and phylogenetic analysis and only limited studies on the pathogenesis of the genotype A of BPIV3 infection in calves and laboratory animals have been performed. The report about experimental infections of the genotypes B and C of BPIV3 in laboratory animals and calves was scant. Therefore, an experimental infection of guinea pigs with the Chinese BPIV3 strain SD0835 of the genotype C was performed. Sixteen guinea pigs were intranasally inoculated with the suspension of SD0835, while eight control guinea pigs were also intranasally inoculated with the same volume of supernatant from uninfected MDBK cells. The virus-inoculated guinea pigs displayed a few observable clinical signs that were related to the respiratory tract disease and two of the sixteen experimentally infected guinea pigs died at 2 and 3 days post inoculation (PI), respectively, and apparent gross pneumonic lesions were observed at necropsy. The gross pneumonic lesions in guinea pigs inoculated with SD0835 consisted of dark red, slightly depressed, irregular areas of consolidation in the lung lobes from the second to 9th day of infection at necropsy, and almost complete consolidation and atelectasis of the lung lobes were seen at 7 days PI. Histopathological changes including alveoli septa thickening and focal cellulose pneumonia were also observed in the lungs of guinea pigs experimentally infected with SD0835. Viral replication was detectable by virus isolation and titration, real-time RT-PCR and immunohistochemistry (IHC) staining in the respiratory tissues of guinea pigs as early as 24h after intranasal inoculation with SD0835. The results of virus isolation and titration showed that guinea pigs were permissive for

  9. Engineering of a parainfluenza virus type 5 fusion protein (PIV-5 F): development of an autonomous and hyperfusogenic protein by a combinational mutagenesis approach.

    Science.gov (United States)

    Terrier, O; Durupt, F; Cartet, G; Thomas, L; Lina, B; Rosa-Calatrava, M

    2009-12-01

    The entry of enveloped viruses into host cells is accomplished by fusion of the viral envelope with the target cell membrane. For the paramyxovirus parainfluenza virus type 5 (PIV-5), this fusion involves an attachment protein (HN) and a class I viral fusion protein (F). We investigated the effect of 20 different combinations of 12 amino-acid substitutions within functional domains of the PIV-5 F glycoprotein, by performing cell surface expression measurements, quantitative fusion and syncytia assays. We found that combinations of mutations conferring an autonomous phenotype with mutations leading to an increased fusion activity were compatible and generated functional PIV-5 F proteins. The addition of mutations in the heptad-repeat domains led to both autonomous and hyperfusogenic phenotypes, despite the low cell surface expression of the corresponding mutants. Such engineering approach may prove useful not only for deciphering the fundamental mechanism behind viral-mediated membrane fusion but also in the development of potential therapeutic applications.

  10. Inhibitory effect of 1,2,4-triazole-ciprofloxacin hybrids on Haemophilus parainfluenzae and Haemophilus influenzae biofilm formation in vitro under stationary conditions.

    Science.gov (United States)

    Kosikowska, Urszula; Andrzejczuk, Sylwia; Plech, Tomasz; Malm, Anna

    2016-10-01

    Haemophilus parainfluenzae and Haemophilus influenzae, upper respiratory tract microbiota representatives, are able to colonize natural and artificial surfaces as biofilm. The aim of the present study was to assay the effect of ten 1,2,4-triazole-ciprofloxacin hybrids on planktonic or biofilm-forming haemophili cells in vitro under stationary conditions on the basis of MICs (minimal inhibitory concentrations) and MBICs (minimal biofilm inhibitory concentrations). In addition, anti-adhesive properties of these compounds were examined. The reference strains of H. parainfluenzae and H. influenzae were included. The broth microdilution microtiter plate (MTP) method with twofold dilution of the compounds, or ciprofloxacin (reference agent) in 96-well polystyrene microplates, was used. The optical density (OD) reading was made spectrophotometrically at a wavelength of 570 nm (OD570) both to measure bacterial growth and to detect biofilm-forming cells under the same conditions with 0.1% crystal violet. The following values of parameters were estimated for 1,2,4-triazole-ciprofloxacin hybrids - MIC = 0.03-15.63 mg/L, MBIC = 0.03-15.63 mg/L, MBIC/MIC = 0.125-8, depending on the compound, and for ciprofloxacin - MIC = 0.03-0.06 mg/L, MBIC = 0.03-0.12 mg/L, MBIC/MIC = 1-2. The observed strong anti-adhesive properties (95-100% inhibition) of the tested compounds were reversible during long-term incubation at subinhibitory concentrations. Thus, 1,2,4-triazole-ciprofloxacin hybrids may be considered as starting compounds for designing improved agents not only against planktonic but also against biofilm-forming Haemophilus spp. cells. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  11. Characterization of hemopoietic stem cell chimerism in antibody-facilitated bone marrow chimeras

    International Nuclear Information System (INIS)

    Francescutti, L.H.; Gambel, P.; Wegmann, T.G.

    1985-01-01

    The authors have previously described a model for bone marrow transplantation that involves preparation of the host with monoclonal antibody against class I or class II antigens instead of irradiation or cytotoxic drugs. This allows engraftment and subsequent repopulation of the host by donor tissue. They have previously reported on chimerism in the peripheral blood of P1----(P1 X P2)F1 animals. In this report, the authors describe the examination of the bone marrow and spleen stem cell chimerism of these antibody-facilitated (AF) chimeras, by determining, with an isozyme assay, the phenotype of methylcellulose colonies grown from stem cells. They have found a correlation between peripheral blood chimerism and the stem cell constitution of both spleen and bone marrow. The peripheral blood chimerism also correlates with the level of chimerism in macrophages derived from peritoneal exudate cells. These findings indicate that assaying the peripheral blood of such chimeras provides an excellent indication of the degree of chimerism at the stem cell level and stands in sharp contrast to the level of chimerism in certain lymphoid compartments

  12. Functional analysis of aldehyde oxidase using expressed chimeric enzyme between monkey and rat.

    Science.gov (United States)

    Itoh, Kunio; Asakawa, Tasuku; Hoshino, Kouichi; Adachi, Mayuko; Fukiya, Kensuke; Watanabe, Nobuaki; Tanaka, Yorihisa

    2009-01-01

    Aldehyde oxidase (AO) is a homodimer with a subunit molecular mass of approximately 150 kDa. Each subunit consists of about 20 kDa 2Fe-2S cluster domain storing reducing equivalents, about 40 kDa flavine adenine dinucleotide (FAD) domain and about 85 kDa molybdenum cofactor (MoCo) domain containing a substrate binding site. In order to clarify the properties of each domain, especially substrate binding domain, chimeric cDNAs were constructed by mutual exchange of 2Fe-2S/FAD and MoCo domains between monkey and rat. Chimeric monkey/rat AO was referred to one with monkey type 2Fe-2S/FAD domains and a rat type MoCo domain. Rat/monkey AO was vice versa. AO-catalyzed 2-oxidation activities of (S)-RS-8359 were measured using the expressed enzyme in Escherichia coli. Substrate inhibition was seen in rat AO and chimeric monkey/rat AO, but not in monkey AO and chimeric rat/monkey AO, suggesting that the phenomenon might be dependent on the natures of MoCo domain of rat. A biphasic Eadie-Hofstee profile was observed in monkey AO and chimeric rat/monkey AO, but not rat AO and chimeric monkey/rat AO, indicating that the biphasic profile might be related to the properties of MoCo domain of monkey. Two-fold greater V(max) values were observed in monkey AO than in chimeric rat/monkey AO, and in chimeric monkey/rat AO than in rat AO, suggesting that monkey has the more effective electron transfer system than rat. Thus, the use of chimeric enzymes revealed that 2Fe-2S/FAD and MoCo domains affect the velocity and the quantitative profiles of AO-catalyzed (S)-RS-8359 2-oxidation, respectively.

  13. Generating chimeric mice from embryonic stem cells via vial coculturing or hypertonic microinjection.

    Science.gov (United States)

    Lee, Kun-Hsiung

    2014-01-01

    The generation of a fertile embryonic stem cell (ESC)-derived or F0 (100 % coat color chimerism) mice is the final criterion in proving that the ESC is truly pluripotent. Many methods have been developed to produce chimeric mice. To date, the most popular methods for generating chimeric embryos is well sandwich aggregation between zona pellucida (ZP) removed (denuded) 2.5-day post-coitum (dpc) embryos and ESC clumps, or direct microinjection of ESCs into the cavity (blastocoel) of 3.5-dpc blastocysts. However, due to systemic limitations and the disadvantages of conventional microinjection, aggregation, and coculturing, two novel methods (vial coculturing and hypertonic microinjection) were developed in recent years at my laboratory.Coculturing 2.5-dpc denuded embryos with ESCs in 1.7-mL vials for ~3 h generates chimeras that have significantly high levels of chimerism (including 100 % coat color chimerism) and germline transmission. This method has significantly fewer instrumental and technological limitations than existing methods, and is an efficient, simple, inexpensive, and reproducible method for "mass production" of chimeric embryos. For laboratories without a microinjection system, this is the method of choice for generating chimeric embryos. Microinjecting ESCs into a subzonal space of 2.5-dpc embryos can generate germline-transmitted chimeras including 100 % coat color chimerism. However, this method is adopted rarely due to the very small and tight space between ZP and blastomeres. Using a laser pulse or Piezo-driven instrument/device to help introduce ESCs into the subzonal space of 2.5-dpc embryos demonstrates the superior efficiency in generating ESC-derived (F0) chimeras. Unfortunately, due to the need for an expensive instrument/device and extra fine skill, not many studies have used either method. Recently, ESCs injected into the large subzonal space of 2.5-dpc embryos in an injection medium containing 0.2-0.3 M sucrose very efficiently generated

  14. 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...... 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...

  15. Chimerism representing both paternal alleles detected by HLA typing before kidney transplantation

    DEFF Research Database (Denmark)

    Christiansen, Mette; Petersen, Mikkel Steen; Møller, Bjarne Kuno

    2014-01-01

    trisomy 6p or by chimerism. Flow cytometric analysis, employing antibodies specific for the two paternal HLA-A alleles, clearly showed two distinct populations of cells: 83% expressing HLA-A11 and 12% expressing HLA-A2, suggesting a paternal chimerism. We are studying these cell populations to possibly...... identify the mechanism behind this rather unusual paternally derived chimerism. This exceptional case illustrates that careful scrutiny of HLA-typing results may produce atypical conclusions. Clinically, the father is considered the best donor based on immunogenetics....

  16. New Chimeric Antigen Receptor Design for Solid Tumors

    Directory of Open Access Journals (Sweden)

    Yuedi Wang

    2017-12-01

    Full Text Available In recent years, chimeric antigen receptor (CAR T-cell therapy has become popular in immunotherapy, particularly after its tremendous success in the treatment of lineage-restricted hematologic cancers. However, the application of CAR T-cell therapy for solid tumors has not reached its full potential because of the lack of specific tumor antigens and inhibitory factors in suppressive tumor microenvironment (TME (e.g., programmed death ligand-1, myeloid-derived suppressor cells, and transforming growth factor-β. In this review, we include some limitations in CAR design, such as tumor heterogeneity, indefinite spatial distance between CAR T-cell and its target cell, and suppressive TME. We also summarize some new approaches to overcome these hurdles, including targeting neoantigens and/or multiple antigens at once and depleting some inhibitory factors.

  17. 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.

  18. Synergistic gene and drug tumor therapy using a chimeric peptide.

    Science.gov (United States)

    Han, Kai; Chen, Si; Chen, Wei-Hai; Lei, Qi; Liu, Yun; Zhuo, Ren-Xi; Zhang, Xian-Zheng

    2013-06-01

    Co-delivery of gene and drug for synergistic therapy has provided a promising strategy to cure devastating diseases. Here, an amphiphilic chimeric peptide (Fmoc)2KH7-TAT with pH-responsibility for gene and drug delivery was designed and fabricated. As a drug carrier, the micelles self-assembled from the peptide exhibited a much faster doxorubicin (DOX) release rate at pH 5.0 than that at pH 7.4. As a non-viral gene vector, (Fmoc)(2)KH(7)-TAT peptide could satisfactorily mediate transfection of pGL-3 reporter plasmid with or without the existence of serum in both 293T and HeLa cell-lines. Besides, the endosome escape capability of peptide/DNA complexes was investigated by confocal laser scanning microscopy (CLSM). To evaluate the co-delivery efficiency and the synergistic anti-tumor effect of gene and drug, p53 plasmid and DOX were simultaneously loaded in the peptide micelles to form micelleplexes during the self-assembly of the peptide. Cellular uptake and intracellular delivery of gene and drug were studied by CLSM and flow cytometry respectively. And p53 protein expression was determined via Western blot analysis. The in vitro cytotoxicity and in vivo tumor inhibition effect were also studied. Results suggest that the co-delivery of gene and drug from peptide micelles resulted in effective cell growth inhibition in vitro and significant tumor growth restraining in vivo. The chimeric peptide-based gene and drug co-delivery system will find great potential for tumor therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Preclinical Models in Chimeric Antigen Receptor-Engineered T-Cell Therapy.

    Science.gov (United States)

    Siegler, Elizabeth Louise; Wang, Pin

    2018-05-01

    Cancer immunotherapy has enormous potential in inducing long-term remission in cancer patients, and chimeric antigen receptor (CAR)-engineered T cells have been largely successful in treating hematological malignancies in the clinic. CAR-T therapy has not been as effective in treating solid tumors, in part due to the immunosuppressive tumor microenvironment. Additionally, CAR-T therapy can cause dangerous side effects, including off-tumor toxicity, cytokine release syndrome, and neurotoxicity. Animal models of CAR-T therapy often fail to predict such adverse events and frequently overestimate the efficacy of the treatment. Nearly all preclinical CAR-T studies have been performed in mice, including syngeneic, xenograft, transgenic, and humanized mouse models. Recently, a few studies have used primate models to mimic clinical side effects better. To date, no single model perfectly recapitulates the human immune system and tumor microenvironment, and some models have revealed CAR-T limitations that were contradicted or missed entirely in other models. Careful model selection based on the primary goals of the study is a crucial step in evaluating CAR-T treatment. Advancements are being made in preclinical models, with the ultimate objective of providing safer, more effective CAR-T therapy to patients.

  20. Suppression of Retinal Neovascularization in vivo by Inhibition of Vascular Endothelial Growth Factor (VEGF) Using Soluble VEGF-Receptor Chimeric Proteins

    Science.gov (United States)

    Aiello, Lloyd Paul; Pierce, Eric A.; Foley, Eliot D.; Takagi, Hitoshi; Chen, Helen; Riddle, Lavon; Ferrara, Napoleone; King, George L.; Smith, Lois E. H.

    1995-11-01

    The majority of severe visual loss in the United States results from complications associated with retinal neovascularization in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Intraocular expression of the angiogenic protein vascular endothelial growth factor (VEGF) is closely correlated with neovascularization in these human disorders and with ischemia-induced retinal neovascularization in mice. In this study, we evaluated whether in vivo inhibition of VEGF action could suppress retinal neovascularization in a murine model of ischemic retinopathy. VEGF-neutralizing chimeric proteins were constructed by joining the extracellular domain of either human (Flt) or mouse (Flk) high-affinity VEGF receptors with IgG. Control chimeric proteins that did not bind VEGF were also used. VEGF-receptor chimeric proteins eliminated in vitro retinal endothelial cell growth stimulation by either VEGF (P hypoxic conditioned medium (P < 0.005) without affecting growth under nonstimulated conditions. Control proteins had no effect. To assess in vivo response, animals with bilateral retinal ischemia received intravitreal injections of VEGF antagonist in one eye and control protein in the contralateral eye. Retinal neovascularization was quantitated histologically by a masked protocol. Retinal neovascularization in the eye injected with human Flt or murine Flk chimeric protein was reduced in 100% (25/25; P < 0.0001) and 95% (21/22; P < 0.0001) of animals, respectively, compared to the control treated eye. This response was evident after only a single intravitreal injection and was dose dependent with suppression of neovascularization noted after total delivery of 200 ng of protein (P < 0.002). Reduction of histologically evident neovascular nuclei per 6-um section averaged 47% ± 4% (P < 0.001) and 37% ± 2% (P < 0.001) for Flt and Flk chimeric proteins with maximal inhibitory effects of 77% and 66

  1. 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.

  2. 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)

    Xu, Yu; Liu, Zhengchun; Kong, Haiyan; Sun, Wenjie; Liao, Zhengkai; Zhou, Fuxiang; Xie, Conghua

    2011-01-01

    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.

  3. Upregulation of adhesion molecules on leukemia targets improves the efficacy of cytotoxic T cells transduced with chimeric anti-CD19 receptor.

    Science.gov (United States)

    Laurin, David; Marin, Virna; Biagi, Ettore; Pizzitola, Irene; Agostoni, Valentina; Gallot, Géraldine; Vié, Henri; Jacob, Marie Christine; Chaperot, Laurence; Aspord, Caroline; Plumas, Joël

    2013-04-01

    T lymphocytes engineered to express chimeric antigen receptors (CARs) interact directly with cell surface molecules, bypassing MHC antigen presentation dependence. We generated human anti-CD19ζ CAR cytotoxic T lymphocytes and cytokine-induced killer cells and studied their sensitivity to the expression of adhesion molecules for the killing of primary B-lineage acute lymphoblastic leukemia (B-ALL) targets. Despite a very low basal expression of surface adhesion molecules, B-ALL blasts were lysed by the anti-CD19ζ-CAR transduced effectors as expected. We next investigated the regulatory role of adhesion molecules during CAR-mediated cytolysis. The blocking of these accessory molecules strongly limited the chimeric effector's cytotoxicity. Thereafter, B-ALL cells surface adhesion molecule level expression was induced by IFN-γ or by the combined use of CD40L and IL-4 and the cells were submitted to anti-CD19ζ-CAR transduced effectors lysis. Upregulation of adhesion molecules expression by blasts potentiated their killing. The improved cytotoxicity observed was dependent on target surface expression of adhesion molecules, particularly CD54. Taken together, these results indicate that adhesion molecules, and principally CD54, are involved in the efficiency of recognition by effector chimeric ζ. These observations have potential implications for the design of immunotherapy treatment approaches for hematological malignancies and tumors based on the adoption of CAR effector cells.

  4. Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli.

    Science.gov (United States)

    Wu, Meizhi; Zhao, Lin; Zhu, Lei; Chen, Zhange; Li, Huangjin

    2013-03-01

    Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. M1 chimerism following mutagen treatment of seeds in rice and some other cereals

    International Nuclear Information System (INIS)

    Kawai, T.

    1983-01-01

    Articles reporting on M 1 chimerism following treatment of seed with mutagen in cereals were mostly published in the 1960's. Rice is a good material for making such studies because of its relatively large number of seeds per panicle, rather easily identifiable tillering and panicle branching systems and uniform growth after seedling transplanting. The present article summarizes results of studies on M 1 chimerism in rice and some other cereals which may serve as reference information in discussing M 1 chimerism of those plant species showing different development patterns, as dicotyledonous plants, following treatment of seed with mutagen. Studies on M 1 chimerism provide not only knowledge of the sporophyte development but also basic information for developing methods of harvesting M 2 seed which provide the maximum numbers of mutants of different origins in a limited number of M 2 plants. (author)

  6. Tentative mapping of transcription-induced interchromosomal interaction using chimeric EST and mRNA data.

    Directory of Open Access Journals (Sweden)

    Per Unneberg

    Full Text Available Recent studies on chromosome conformation show that chromosomes colocalize in the nucleus, bringing together active genes in transcription factories. This spatial proximity of actively transcribing genes could provide a means for RNA interaction at the transcript level. We have screened public databases for chimeric EST and mRNA sequences with the intent of mapping transcription-induced interchromosomal interactions. We suggest that chimeric transcripts may be the result of close encounters of active genes, either as functional products or "noise" in the transcription process, and that they could be used as probes for chromosome interactions. We have found a total of 5,614 chimeric ESTs and 587 chimeric mRNAs that meet our selection criteria. Due to their higher quality, the mRNA findings are of particular interest and we hope that they may serve as food for thought for specialists in diverse areas of molecular biology.

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

    International Nuclear Information System (INIS)

    Mourez, Thomas; Mesel-Lemoine, Mariana; Combredet, Chantal; Najburg, Valerie; Cayet, Nadege; Tangy, Frederic

    2011-01-01

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

  8. 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...

  9. Frequency of chimerism in populations of the kelp Lessonia spicata in central Chile.

    Directory of Open Access Journals (Sweden)

    Alejandra V González

    Full Text Available Chimerism occurs when two genetically distinct conspecific individuals fuse together generating a single entity. Coalescence and chimerism in red seaweeds has been positively related to an increase in body size, and the consequent reduction in susceptibility to mortality factors, thus increasing survival, reproductive potential and tolerance to stress in contrast to genetically homogeneous organisms. In addition, they showed that a particular pattern of post-fusion growth maintains higher genetic diversity and chimerism in the holdfast but homogenous axes. In Chilean kelps (brown seaweeds, intraorganismal genetic heterogeneity (IGH and holdfast coalescence has been described in previous research, but the extent of chimerism in wild populations and the patterns of distribution of the genetically heterogeneous thallus zone have scarcely been studied. Since kelps are under continuous harvesting, with enormous social, ecological and economic importance, natural chimerism can be considered a priceless in-situ reservoir of natural genetic resources and variability. In this study, we therefore examined the frequency of IGH and chimerism in three harvested populations of Lessonia spicata. We then evaluated whether chimeric wild-type holdfasts show higher genetic diversity than erect axes (stipe and lamina and explored the impact of this on the traditional estimation of genetic diversity at the population level. We found a high frequency of IGH (60-100% and chimerism (33.3-86.7%, varying according to the studied population. We evidenced that chimerism occurs mostly in holdfasts, exhibiting heterogeneous tissues, whereas stipes and lamina were more homogeneous, generating a vertical gradient of allele and genotype abundance as well as divergence, constituting the first time "within- plant" genetic patterns have been reported in kelps. This is very different from the chimeric patterns described in land plants and animals. Finally, we evidenced that IGH

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

    OpenAIRE

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

    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 ...

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

    Science.gov (United States)

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

    2013-09-24

    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 investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.Molecular Therapy-Nucleic Acids (2013) 2, e124; doi:10.1038/mtna.2013

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

    Directory of Open Access Journals (Sweden)

    HaiFang Yin

    2013-01-01

    Full Text Available 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 investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO and control peptide 3 (B-3-PMO and 3-B-PMO were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO, further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG, indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.

  13. Establishment of donor Chimerism Using Allogeneic Bone Marrow with AMP Cell Co-infusion

    Science.gov (United States)

    2017-09-01

    AWARD NUMBER: W81XWH-15-1-0234 TITLE: Establishment of donor Chimerism Using Allogeneic Bone Marrow with AMP Cell Co-infusion PRINCIPAL...14/2017 4. TITLE AND SUBTITLE Establishment of donor Chimerism Using Allogeneic Bone Marrow with AMP Cell Co-infusion 5a. CONTRACT NUMBER 5b. GRANT...tolerance induction of all types of allografts. In this study, we investigate whether co-infusion of amnion- derived multipotent progenitor (AMP) cells

  14. Achados de tomografia computadorizada de alta resolução em pneumonia pelo vírus parainfluenza pós-transplante de medula óssea: relato de caso

    Directory of Open Access Journals (Sweden)

    Emerson L. Gasparetto

    2004-11-01

    Full Text Available RESUMO: Paciente feminina, de 19 anos, transplantada de medula óssea devido a leucemia mielóide crónica, apresentando tosse seca e coriza no 67.º dia após o procedimento. A radiografia de tórax não evidenciou alterações. A tomografia computadorizada de alta resolução do tórax revelou consolidação subsegmentar na periferia do lobo inferior esquerdo e áreas de redução da atenuação nos terços superior e médio dos pulmões. O lavado broncoalveolar demonstrou pesquisa positiva por imunoflorescência direta para anticorpos anti-vírus parainfluenza. Foi instituído tratamento com ribavirina aerolizada durante 10 dias, havendo melhoria clínico-radiológica do quadro infeccioso.REV PORT PNEUMOL 2004; X (6: 485-489 ABSTRACT: Nineteen year-old female patient, who underwent bone marrow transplantation because of chronic myelogenous leukemia, presented with dry cough and coriza sixty-seven days after the procedure. The chest radiograph was normal. The high resolution computed tomography showed a subsegmental air-space consolidation at the periphery of the left inferior lobe and areas of low attenuation at the superior and middle lung zones. The bronchoalveolar lavage demonstrated positive direct fluorescence antibody testing against parainfluenza virus. Treatment with aerolizated ribavirin was instituted during 10 days and the patient showed clinical-radiological improvement.REV PORT PNEUMOL 2004; X (6: 485-489 Palavras-chave: Vírus parainfluenza, tomografia computadorizada de alta resolução, transplante de medula óssea, Key-words: Parainfluenza virus, high resolution computed tomography, bone marrow transplantation

  15. Reduced immune responses in chimeric mice engrafted with bone marrow cells from mice with airways inflammation.

    Science.gov (United States)

    Scott, Naomi M; Ng, Royce L X; McGonigle, Terence A; Gorman, Shelley; Hart, Prue H

    2015-11-01

    During respiratory inflammation, it is generally assumed that dendritic cells differentiating from the bone marrow are immunogenic rather than immunoregulatory. Using chimeric mice, the outcomes of airways inflammation on bone marrow progenitor cells were studied. Immune responses were analyzed in chimeric mice engrafted for >16 weeks with bone marrow cells from mice with experimental allergic airways disease (EAAD). Responses to sensitization and challenge with the allergen causing inflammation in the bone marrow-donor mice were significantly reduced in the chimeric mice engrafted with bone marrow cells from mice with EAAD (EAAD-chimeric). Responses to intranasal LPS and topical fluorescein isothiocyanate (non-specific challenges) were significantly attenuated. Fewer activated dendritic cells from the airways and skin of the EAAD-chimeric mice could be tracked to the draining lymph nodes, and may contribute to the significantly reduced antigen/chemical-induced hypertrophy in the draining nodes, and the reduced immune responses to sensitizing allergens. Dendritic cells differentiating in vitro from the bone marrow of >16 weeks reconstituted EAAD-chimeric mice retained an ability to poorly prime immune responses when transferred into naïve mice. Dendritic cells developing from bone marrow progenitors during airways inflammation are altered such that daughter cells have reduced antigen priming capabilities.

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

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    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.

  17. Prolonged Survival of Subcutaneous Allogeneic Islet Graft by Donor Chimerism without Immunosuppressive Treatment

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    Brend Ray-Sea Hsu

    2017-01-01

    Full Text Available The aim of this study was to investigate whether tolerance-induced protection of islets in the renal subcapsular space can also prevent subcutaneous allogeneic islets from being rejected. We used bone marrow stem cells from C57BL/6 (H2b mice to construct donor chimerism in conditioned diabetic BALB/c (H2d mice and investigated the effect of donor chimerism on engraftment and survival of subcutaneously transplanted allogeneic islets in streptozotocin-induced diabetic mice. We also studied the anti-inflammatory effect of mesenchymal stem cell on islet engraftment. Full but not low-grade or no donor chimerism was associated with successful engraftment of allogeneic islets and restoration of normoglycemia in the treated diabetic mice. The temporary hyperglycemia was 11 ± 1 versus 19 ± 5 days (p<0.05 for the mice with full donor chimerism with transplanted islets in the renal subcapsular space versus the subcutaneous space, respectively. Cotransplantation of mesenchymal stem cell did not enhance alloislet engraftment. Full multilineage donor chimerism was associated with a higher transient expansion of CD11b+ and Gr-1+ myeloid progenitor cells and effector memory CD4 and CD8 T cells. In conclusion, full donor chimerism protected both renal subcapsular and subcutaneous allogeneic islets in this rodent transplantation model.

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

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    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.

  19. Persistent replication of a hepatitis C virus genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B in a New World monkey.

    Science.gov (United States)

    Suzuki, Saori; Mori, Ken-Ichi; Higashino, Atsunori; Iwasaki, Yuki; Yasutomi, Yasuhiro; Maki, Noboru; Akari, Hirofumi

    2016-01-01

    The development of effective hepatitis C virus (HCV) vaccines is essential for the prevention of further HCV dissemination, especially in developing countries. Therefore the aim of this study is to establish a feasible and immunocompetent surrogate animal model of HCV infection that will help in evaluation of the protective efficacy of newly developing HCV vaccine candidates. To circumvent the narrow host range of HCV, an HCV genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B (GBV-B), which is closely related to HCV, was generated. The chimera between HCV and GBV-B, named HCV/G, replicated more efficiently as compared with the HCV clone in primary marmoset hepatocytes. Furthermore, it was found that the chimera persistently replicated in a tamarin for more than 2 years after intrahepatic inoculation of the chimeric RNA. Although relatively low (chimeric RNA was found in the pellet fraction obtained by ultracentrifugation of the plasma at 73 weeks, indicating production of the chimeric virus. Our results will help establish a novel non-human primate model for HCV infection on the basis of the HCV/G chimera in the major framework of the HCV genome. © 2015 The Societies and John Wiley & Sons Australia, Ltd.

  20. Interleukin 18 secretion and its effect in improving Chimeric Antigen Receptors efficiency

    Science.gov (United States)

    Kim, Jae-Kun

    Clinical trials have shown that chimeric antigen receptor T cells modified to target cancer cells expressing a surface antigen found on immature B-cells. The purpose of this experiment is to take a pro-inflammatory cytokine, and analyze its effect in improving the efficiency of the T cells. IL-18 has been previously shown to recruit T cells to the tumor site and improve their secretion of cytotoxic cytokines. A human model of the proposed armored T cell has been created and has shown success in combating cancer cells in vitro. The next step is to design and produce a murine model to test in vivo in immunocompetent mice. This research project aimed to create two models: one utilizing 2A peptides and another utilizing IRES elements as a multicistronic vector. Both models would require the insertion of the desired genes into SFG backbones. IRES, a DNA element which acts as a binding site for the transcriptional machinery to recognize which part of the DNA to transcribe, commonly found in bicistronic vectors, is large with 500-600 base pairs, and has a lower transgene expression rate. P2A is smaller, only consisting of about 20 amino acids, and typically has a higher transgene expression rate, which may or may not result in higher effectiveness of the model. I would like to thank Dr. Renier Brentjens for being a mentor who cared about giving his interns as much educational value as possible.

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

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    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.

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

    International Nuclear Information System (INIS)

    Sbrana, F; Vassalli, M; Fotia, C; Baldini, N; Ciapetti, G; Bracalello, A; Bochicchio, B; Marletta, G

    2012-01-01

    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)

  3. Chimeric Antigen Receptor- and TCR-Modified T Cells Enter Main Street and Wall Street.

    Science.gov (United States)

    Barrett, David M; Grupp, Stephan A; June, Carl H

    2015-08-01

    The field of adoptive cell transfer (ACT) is currently comprised of chimeric Ag receptor (CAR)- and TCR-engineered T cells and has emerged from principles of basic immunology to paradigm-shifting clinical immunotherapy. ACT of T cells engineered to express artificial receptors that target cells of choice is an exciting new approach for cancer, and it holds equal promise for chronic infection and autoimmunity. Using principles of synthetic biology, advances in immunology, and genetic engineering have made it possible to generate human T cells that display desired specificities and enhanced functionalities. Clinical trials in patients with advanced B cell leukemias and lymphomas treated with CD19-specific CAR T cells have induced durable remissions in adults and children. The prospects for the widespread availability of engineered T cells have changed dramatically given the recent entry of the pharmaceutical industry to this arena. In this overview, we discuss some of the challenges and opportunities that face the field of ACT. Copyright © 2015 by The American Association of Immunologists, Inc.

  4. Clinically compliant spatial and temporal imaging of chimeric antigen receptor T-cells.

    Science.gov (United States)

    Emami-Shahri, Nia; Foster, Julie; Kashani, Roxana; Gazinska, Patrycja; Cook, Celia; Sosabowski, Jane; Maher, John; Papa, Sophie

    2018-03-14

    The unprecedented efficacy of chimeric antigen receptor (CAR) T-cell immunotherapy of CD19 + B-cell malignancy has established a new therapeutic pillar of hematology-oncology. Nonetheless, formidable challenges remain for the attainment of comparable success in patients with solid tumors. To accelerate progress and rapidly characterize emerging toxicities, systems that permit the repeated and non-invasive assessment of CAR T-cell bio-distribution would be invaluable. An ideal solution would entail the use of a non-immunogenic reporter that mediates specific uptake of an inexpensive, non-toxic and clinically established imaging tracer by CAR T cells. Here we show the utility of the human sodium iodide symporter (hNIS) for the temporal and spatial monitoring of CAR T-cell behavior in a cancer-bearing host. This system provides a clinically compliant toolkit for high-resolution serial imaging of CAR T cells in vivo, addressing a fundamental unmet need for future clinical development in the field.

  5. Genetic engineering of chimeric antigen receptors using lamprey derived variable lymphocyte receptors

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    Robert Moot

    2016-01-01

    Full Text Available Chimeric antigen receptors (CARs are used to redirect effector cell specificity to selected cell surface antigens. Using CARs, antitumor activity can be initiated in patients with no prior tumor specific immunity. Although CARs have shown promising clinical results, the technology remains limited by the availability of specific cognate cell target antigens. To increase the repertoire of targetable tumor cell antigens we utilized the immune system of the sea lamprey to generate directed variable lymphocyte receptors (VLRs. VLRs serve as membrane bound and soluble immune effectors analogous but not homologous to immunoglobulins. They have a fundamentally different structure than immunoglobulin (Ig-based antibodies while still demonstrating high degrees of specificity and affinity. To test the functionality of VLRs as the antigen recognition domain of CARs, two VLR-CARs were created. One contained a VLR specific for a murine B cell leukemia and the other contained a VLR specific for the human T cell surface antigen, CD5. The CAR design consisted of the VLR sequence, myc-epitope tag, CD28 transmembrane domain, and intracellular CD3ζ signaling domain. We demonstrate proof of concept, including gene transfer, biosynthesis, cell surface localization, and effector cell activation for multiple VLR-CAR designs. Therefore, VLRs provide an alternative means of CAR-based cancer recognition.

  6. Chimeric antigen receptor T cells: a novel therapy for solid tumors

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    Shengnan Yu

    2017-03-01

    Full Text Available Abstract The chimeric antigen receptor T (CAR-T cell therapy is a newly developed adoptive antitumor treatment. Theoretically, CAR-T cells can specifically localize and eliminate tumor cells by interacting with the tumor-associated antigens (TAAs expressing on tumor cell surface. Current studies demonstrated that various TAAs could act as target antigens for CAR-T cells, for instance, the type III variant epidermal growth factor receptor (EGFRvIII was considered as an ideal target for its aberrant expression on the cell surface of several tumor types. CAR-T cell therapy has achieved gratifying breakthrough in hematological malignancies and promising outcome in solid tumor as showed in various clinical trials. The third generation of CAR-T demonstrates increased antitumor cytotoxicity and persistence through modification of CAR structure. In this review, we summarized the preclinical and clinical progress of CAR-T cells targeting EGFR, human epidermal growth factor receptor 2 (HER2, and mesothelin (MSLN, as well as the challenges for CAR-T cell therapy.

  7. Chimeric Antigen Receptor Expressing Natural Killer Cells for the Immunotherapy of Cancer

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    Rohtesh S. Mehta

    2018-02-01

    Full Text Available Adoptive cell therapy has emerged as a powerful treatment for advanced cancers resistant to conventional agents. Most notable are the remarkable responses seen in patients receiving autologous CD19-redirected chimeric antigen receptor (CAR T cells for the treatment of B lymphoid malignancies; however, the generation of autologous products for each patient is logistically cumbersome and has restricted widespread clinical use. A banked allogeneic product has the potential to overcome these limitations, yet allogeneic T-cells (even if human leukocyte antigen-matched carry a major risk of graft-versus-host disease (GVHD. Natural killer (NK cells are bone marrow-derived innate lymphocytes that can eliminate tumors directly, with their activity governed by the integration of signals from activating and inhibitory receptors and from cytokines including IL-15, IL-12, and IL-18. NK cells do not cause GVHD or other alloimmune or autoimmune toxicities and thus, can provide a potential source of allogeneic “off-the-shelf” cellular therapy, mediating major anti-tumor effects without inducing potentially lethal alloreactivity such as GVHD. Given the multiple unique advantages of NK cells, researchers are now exploring the use of CAR-engineered NK cells for the treatment of various hematological and non-hematological malignancies. Herein, we review preclinical data on the development of CAR-NK cells, advantages, disadvantages, and current obstacles to their clinical use.

  8. Therapeutically Targeting the Inflammasome Product in a Chimeric Model of Endometriosis-Related Surgical Adhesions.

    Science.gov (United States)

    Stocks, Meredith M; Crispens, Marta A; Ding, Tianbing; Mokshagundam, Shilpa; Bruner-Tran, Kaylon L; Osteen, Kevin G

    2017-08-01

    Development of adhesions commonly occurs in association with surgery for endometriosis. Even in the absence of surgery, women with endometriosis appear to be at an enhanced risk of developing adhesions. In the current study, we utilized a chimeric mouse model of experimental endometriosis in order to examine the role of inflammasome activation in the development of postsurgical adhesions. Mice were randomized to receive peritoneal injections of human endometrial tissue fragments or endometrial tissue conditioned media (CM) from women with or without endometriosis 16 hours after ovariectomy and placement of an estradiol-releasing silastic capsule. A subset of mice receiving CM was also treated with interleukin (IL) 1 receptor antagonist (IL-1ra). Our studies demonstrate that peritoneal injection of endometrial tissue fragments near the time of surgery resulted in extensive adhesive disease regardless of tissue origin. However, adhesion scores were significantly higher in mice receiving CM from tissues acquired from patients with endometriosis compared to control tissue CM ( P = .0001). Cytokine bead array analysis of endometrial CM revealed enhanced expression of IL-1β from patients with endometriosis compared to controls ( P endometriosis as a potential causal factor in their increased susceptibility of developing postsurgical adhesions. Thus, targeting inflammasome activation may be an effective strategy for the prevention of surgical adhesions in patients with endometriosis.

  9. Characterization of a switchable chimeric antigen receptor platform in a pre-clinical solid tumor model.

    Science.gov (United States)

    Pishali Bejestani, Elham; Cartellieri, Marc; Bergmann, Ralf; Ehninger, Armin; Loff, Simon; Kramer, Michael; Spehr, Johannes; Dietrich, Antje; Feldmann, Anja; Albert, Susann; Wermke, Martin; Baumann, Michael; Krause, Mechthild; Bornhäuser, Martin; Ehninger, Gerhard; Bachmann, Michael; von Bonin, Malte

    2017-01-01

    The universal modular chimeric antigen receptor (UniCAR) platform redirects CAR-T cells using a separated, soluble targeting module with a short half-life. This segregation allows precise controllability and flexibility. Herein we show that the UniCAR platform can be used to efficiently target solid cancers in vitro and in vivo using a pre-clinical prostate cancer model which overexpresses prostate stem cell antigen (PSCA). Short-term administration of the targeting module to tumor bearing immunocompromised mice engrafted with human UniCAR-T cells significantly delayed tumor growth and prolonged survival of recipient mice both in a low and high tumor burden model. In addition, we analyzed phenotypic and functional changes of cancer cells and UniCAR-T cells in association with the administration of the targeting module to reveal potential immunoevasive mechanisms. Most notably, UniCAR-T cell activation induced upregulation of immune-inhibitory molecules such as programmed death ligands. In conclusion, this work illustrates that the UniCAR platform mediates potent anti-tumor activity in a relevant in vitro and in vivo solid tumor model.

  10. ChimerDB 3.0: an enhanced database for fusion genes from cancer transcriptome and literature data mining.

    Science.gov (United States)

    Lee, Myunggyo; Lee, Kyubum; Yu, Namhee; Jang, Insu; Choi, Ikjung; Kim, Pora; Jang, Ye Eun; Kim, Byounggun; Kim, Sunkyu; Lee, Byungwook; Kang, Jaewoo; Lee, Sanghyuk

    2017-01-04

    Fusion gene is an important class of therapeutic targets and prognostic markers in cancer. ChimerDB is a comprehensive database of fusion genes encompassing analysis of deep sequencing data and manual curations. In this update, the database coverage was enhanced considerably by adding two new modules of The Cancer Genome Atlas (TCGA) RNA-Seq analysis and PubMed abstract mining. ChimerDB 3.0 is composed of three modules of ChimerKB, ChimerPub and ChimerSeq. ChimerKB represents a knowledgebase including 1066 fusion genes with manual curation that were compiled from public resources of fusion genes with experimental evidences. ChimerPub includes 2767 fusion genes obtained from text mining of PubMed abstracts. ChimerSeq module is designed to archive the fusion candidates from deep sequencing data. Importantly, we have analyzed RNA-Seq data of the TCGA project covering 4569 patients in 23 cancer types using two reliable programs of FusionScan and TopHat-Fusion. The new user interface supports diverse search options and graphic representation of fusion gene structure. ChimerDB 3.0 is available at http://ercsb.ewha.ac.kr/fusiongene/. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Science.gov (United States)

    Halstead, Scott B; Russell, Philip K

    2016-03-29

    Clinical observations from the third year of the Sanofi Pasteur chimeric yellow fever dengue tetravalent vaccine (CYD) trials document both protection and vaccination-enhanced dengue disease among vaccine recipients. Children who were 5 years-old or younger when vaccinated experienced a DENV disease resulting in hospitalization at 5 times the rate of controls. On closer inspection, hospitalized cases among vaccinated seropositives, those at highest risk to hospitalized disease accompanying a dengue virus (DENV) infection, were greatly reduced by vaccination. But, seronegative individuals of all ages after being vaccinated were only modestly protected from mild to moderate disease throughout the entire observation period despite developing neutralizing antibodies at high rates. Applying a simple epidemiological model to the data, vaccinated seronegative individuals of all ages were at increased risk of developing hospitalized disease during a subsequent wild type DENV infection. The etiology of disease in placebo and vaccinated children resulting in hospitalization during a DENV infection, while clinically similar are of different origin. The implications of the observed mixture of DENV protection and enhanced disease in CYD vaccinees are discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Radial symmetry in a chimeric glutamate receptor pore

    Science.gov (United States)

    Wilding, Timothy J.; Lopez, Melany N.; Huettner, James E.

    2014-02-01

    Ionotropic glutamate receptors comprise two conformationally different A/C and B/D subunit pairs. Closed channels exhibit fourfold radial symmetry in the transmembrane domain (TMD) but transition to twofold dimer-of-dimers symmetry for extracellular ligand binding and N-terminal domains. Here, to evaluate symmetry in open pores we analysed interaction between the Q/R editing site near the pore loop apex and the transmembrane M3 helix of kainate receptor subunit GluK2. Chimeric subunits that combined the GluK2 TMD with extracellular segments from NMDA receptors, which are obligate heteromers, yielded channels made up of A/C and B/D subunit pairs with distinct substitutions along M3 and/or Q/R site editing status, in an otherwise identical homotetrameric TMD. Our results indicate that Q/R site interaction with M3 occurs within individual subunits and is essentially the same for both A/C and B/D subunit conformations, suggesting that fourfold pore symmetry persists in the open state.

  13. Competitive annealing of multiple DNA origami: formation of chimeric origami

    International Nuclear Information System (INIS)

    Majikes, Jacob M; Nash, Jessica A; LaBean, Thomas H

    2016-01-01

    Scaffolded DNA origami are a robust tool for building discrete nanoscale objects at high yield. This strategy ensures, in the design process, that the desired nanostructure is the minimum free energy state for the designed set of DNA sequences. Despite aiming for the minimum free energy structure, the folding process which leads to that conformation is difficult to characterize, although it has been the subject of much research. In order to shed light on the molecular folding pathways, this study intentionally frustrates the folding process of these systems by simultaneously annealing the staple pools for multiple target or parent origami structures, forcing competition. A surprising result of these competitive, simultaneous anneals is the formation of chimeric DNA origami which inherit structural regions from both parent origami. By comparing the regions inherited from the parent origami, relative stability of substructures were compared. This allowed examination of the folding process with typical characterization techniques and materials. Anneal curves were then used as a means to rapidly generate a phase diagram of anticipated behavior as a function of staple excess and parent staple ratio. This initial study shows that competitive anneals provide an exciting way to create diverse new nanostructures and may be used to examine the relative stability of various structural motifs. (paper)

  14. 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.

  15. A compound chimeric antigen receptor strategy for targeting multiple myeloma.

    Science.gov (United States)

    Chen, K H; Wada, M; Pinz, K G; Liu, H; Shuai, X; Chen, X; Yan, L E; Petrov, J C; Salman, H; Senzel, L; Leung, E L H; Jiang, X; Ma, Y

    2018-02-01

    Current clinical outcomes using chimeric-antigen receptors (CARs) against multiple myeloma show promise in the eradication of bulk disease. However, these anti-BCMA (CD269) CARs observe relapse as a common phenomenon after treatment due to the reemergence of either antigen-positive or -negative cells. Hence, the development of improvements in CAR design to target antigen loss and increase effector cell persistency represents a critical need. Here, we report on the anti-tumor activity of a CAR T-cell possessing two complete and independent CAR receptors against the multiple myeloma antigens BCMA and CS1. We determined that the resulting compound CAR (cCAR) T-cell possesses consistent, potent and directed cytotoxicity against each target antigen population. Using multiple mouse models of myeloma and mixed cell populations, we are further able to show superior in vivo survival by directed cytotoxicity against multiple populations compared to a single-expressing CAR T-cell. These findings indicate that compound targeting of BCMA and CS1 on myeloma cells can potentially be an effective strategy for augmenting the response against myeloma bulk disease and for initiation of broader coverage CAR therapy.

  16. ChimericSeq: An open-source, user-friendly interface for analyzing NGS data to identify and characterize viral-host chimeric sequences

    Science.gov (United States)

    Shieh, Fwu-Shan; Jongeneel, Patrick; Steffen, Jamin D.; Lin, Selena; Jain, Surbhi; Song, Wei

    2017-01-01

    Identification of viral integration sites has been important in understanding the pathogenesis and progression of diseases associated with particular viral infections. The advent of next-generation sequencing (NGS) has enabled researchers to understand the impact that viral integration has on the host, such as tumorigenesis. Current computational methods to analyze NGS data of virus-host junction sites have been limited in terms of their accessibility to a broad user base. In this study, we developed a software application (named ChimericSeq), that is the first program of its kind to offer a graphical user interface, compatibility with both Windows and Mac operating systems, and optimized for effectively identifying and annotating virus-host chimeric reads within NGS data. In addition, ChimericSeq’s pipeline implements custom filtering to remove artifacts and detect reads with quantitative analytical reporting to provide functional significance to discovered integration sites. The improved accessibility of ChimericSeq through a GUI interface in both Windows and Mac has potential to expand NGS analytical support to a broader spectrum of the scientific community. PMID:28829778

  17. ChimericSeq: An open-source, user-friendly interface for analyzing NGS data to identify and characterize viral-host chimeric sequences.

    Directory of Open Access Journals (Sweden)

    Fwu-Shan Shieh

    Full Text Available Identification of viral integration sites has been important in understanding the pathogenesis and progression of diseases associated with particular viral infections. The advent of next-generation sequencing (NGS has enabled researchers to understand the impact that viral integration has on the host, such as tumorigenesis. Current computational methods to analyze NGS data of virus-host junction sites have been limited in terms of their accessibility to a broad user base. In this study, we developed a software application (named ChimericSeq, that is the first program of its kind to offer a graphical user interface, compatibility with both Windows and Mac operating systems, and optimized for effectively identifying and annotating virus-host chimeric reads within NGS data. In addition, ChimericSeq's pipeline implements custom filtering to remove artifacts and detect reads with quantitative analytical reporting to provide functional significance to discovered integration sites. The improved accessibility of ChimericSeq through a GUI interface in both Windows and Mac has potential to expand NGS analytical support to a broader spectrum of the scientific community.

  18. A cooperative interaction between nontranslated RNA sequences and NS5A protein promotes in vivo fitness of a chimeric hepatitis C/GB virus B.

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    Lucile Warter

    Full Text Available GB virus B (GBV-B is closely related to hepatitis C virus (HCV, infects small non-human primates, and is thus a valuable surrogate for studying HCV. Despite significant differences, the 5' nontranslated RNAs (NTRs of these viruses fold into four similar structured domains (I-IV, with domains II-III-IV comprising the viral internal ribosomal entry site (IRES. We previously reported the in vivo rescue of a chimeric GBV-B (vGB/III(HC containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/III(HC genome (within the 3'NTR, upstream of the poly(U tract, and NS5A coding sequence are necessary and sufficient for production of this chimeric virus following intrahepatic inoculation of synthetic RNA in tamarins, and thus apparently compensate for the presence of HCV sequence in domain III. To assess the mechanism(s underlying these compensatory mutations, and to determine whether 5'NTR subdomains participating in genome replication do so in a virus-specific fashion, we constructed and evaluated a series of chimeric subgenomic GBV-B replicons in which various 5'NTR subdomains were substituted with their HCV homologs. Domains I and II of the GBV-B 5'NTR could not be replaced with HCV sequence, indicating that they contain essential, virus-specific RNA replication elements. In contrast, domain III could be swapped with minimal loss of genome replication capacity in cell culture. The 3'NTR and NS5A mutations required for rescue of the related chimeric virus in vivo had no effect on replication of the subgenomic GBneoD/III(HC RNA in vitro. The data suggest that in vivo fitness of the domain III chimeric virus is dependent on a cooperative interaction between the 5'NTR, 3'NTR and NS5A at a step in the viral life cycle subsequent to genome replication, most likely during particle assembly. Such a mechanism may be common to all hepaciviruses.

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

    International Nuclear Information System (INIS)

    Ye Ling; Sun Yuliang; Lin Jianguo; Bu Zhigao; Wu Qingyang; Jiang, Shibo; Steinhauer, David A.; Compans, Richard W.; Yang Chinglai

    2006-01-01

    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

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

    International Nuclear Information System (INIS)

    Wagner, Alexander Y; Holle, Eric; Holle, Lori; Yu, Xianzhong; Schwamberger, Günter

    2008-01-01

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

  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. Attenuation of pathogenic Rift Valley fever virus strain through the chimeric S-segment encoding sandfly fever phlebovirus NSs or a dominant-negative PKR.

    Science.gov (United States)

    Nishiyama, Shoko; Slack, Olga A L; Lokugamage, Nandadeva; Hill, Terence E; Juelich, Terry L; Zhang, Lihong; Smith, Jennifer K; Perez, David; Gong, Bin; Freiberg, Alexander N; Ikegami, Tetsuro

    2016-11-16

    Rift Valley fever is a mosquito-borne zoonotic disease affecting ruminants and humans. Rift Valley fever virus (RVFV: family Bunyaviridae, genus Phlebovirus) causes abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or retinitis in humans. The live-attenuated MP-12 vaccine is conditionally licensed for veterinary use in the US. However, this vaccine lacks a marker for the differentiation of vaccinated from infected animals (DIVA). NSs gene is dispensable for RVFV replication, and thus, rMP-12 strains lacking NSs gene is applicable to monitor vaccinated animals. However, the immunogenicity of MP-12 lacking NSs was not as high as parental MP-12. Thus, chimeric MP-12 strains encoding NSs from either Toscana virus (TOSV), sandfly fever Sicilian virus (SFSV) or Punta Toro virus Adames strain (PTA) were characterized previously. Although chimeric MP-12 strains are highly immunogenic, the attenuation through the S-segment remains unknown. Using pathogenic ZH501 strain, we aimed to demonstrate the attenuation of ZH501 strain through chimeric S-segment encoding either the NSs of TOSV, SFSV, PTA, or Punta Toro virus Balliet strain (PTB). In addition, we characterized rZH501 encoding a human dominant-negative PKR (PKRΔE7), which also enhances the immunogenicity of MP-12. Study done on mice revealed that attenuation of rZH501 occurred through the S-segment encoding either PKRΔE7 or SFSV NSs. However, rZH501 encoding either TOSV, PTA, or PTB NSs in the S-segment uniformly caused lethal encephalitis. Our results indicated that the S-segments encoding PKRΔE7 or SFSV NSs are attenuated and thus applicable toward next generation MP-12 vaccine candidates that encode a DIVA marker.

  3. Secretory overexpression and isotopic labeling of the chimeric relaxin family peptide R3/I5 in Pichia pastoris.

    Science.gov (United States)

    Guo, Yu-Qi; Wu, Qing-Ping; Shao, Xiao-Xia; Shen, Ting; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

    2015-06-01

    Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficient approach for preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression in Pichia pastoris and in vitro enzymatic maturation. A designed single-chain R3/I5 precursor containing the B-chain of human relaxin-3 and the A-chain of human INSL5 was overexpressed in PichiaPink strain 1 by high-density fermentation in a two-liter fermenter, and approximately 200 mg of purified precursor was obtained from one liter of the fermentation supernatant. We also developed an economical approach for preparation of the uniformly (15)N-labeled R3/I5 precursor by culturing in shaking flasks, and approximately 15 mg of purified (15)N-labeled precursor was obtained from one liter of the culture supernatant. After purification by cation ion-exchange chromatography and reverse-phase high performance liquid chromatography, the R3/I5 precursor was converted to the mature two-chain form by sequential treatment with endoproteinase Lys-C and carboxypeptidase B. The mature R3/I5 peptide had an α-helix-dominated conformation and retained full receptor-binding and receptor activation activities. Thus, Pichia overexpression was an efficient approach for sample preparation and isotopic labeling of the chimeric R3/I5 peptide. This approach could also be extended to the preparation of other relaxin family peptides in future studies.

  4. Targeting Multiple Tumors Using T-Cells Engineered to Express a Natural Cytotoxicity Receptor 2-Based Chimeric Receptor

    Directory of Open Access Journals (Sweden)

    Vasyl Eisenberg

    2017-09-01

    Full Text Available Recent developments in cancer treatment are demonstrating the increasing and powerful potential of immunotherapeutic strategies. In this regard, the adoptive transfer of tumor-specific T-lymphocytes approaches can lead to tumor regression in cancer patients. More recently, the use of T-cells genetically engineered to express cancer-specific receptors such as the anti-CD19 chimeric antigen receptor (CAR continues to show promise for the treatment of hematological malignancies. Still, there is a crucial need to develop efficient CAR-T cell approaches for the treatment of solid tumors. It has been shown that other lymphocytes such as natural killer (NK cells can demonstrate potent antitumor function—nonetheless, their use in immunotherapy is rather limited due to difficulties in expanding these cells to therapeutically relevant numbers and to suppression by endogenous inhibitory mechanisms. Cancer recognition by NK cells is partly mediated by molecules termed natural cytotoxicity receptors (NCRs. In the present study, we hypothesize that it is possible to endow T-cells with an NK recognition pattern, providing them with a mean to recognize tumor cells, in a non-MHC restricted way. To test this, we genetically modified human T-cells with different chimeric receptors based on the human NCR2 molecule and then assessed their antitumor activity in vitro and in vivo. Our results show that expression in primary lymphocytes of an NCR2-derived CAR, termed s4428z, confers T-cells with the ability to specifically recognize heterogeneous tumors and to mediate tumor cytotoxicity in a mouse model. This study demonstrates the benefit of combining tumor recognition capability of NK cells with T cell effectiveness to improve cancer immunotherapy.

  5. Relative Contribution of Cellular Complement Inhibitors CD59, CD46, and CD55 to Parainfluenza Virus 5 Inhibition of Complement-Mediated Neutralization

    Directory of Open Access Journals (Sweden)

    Yujia Li

    2018-04-01

    Full Text Available The complement system is a part of the innate immune system that viruses need to face during infections. Many viruses incorporate cellular regulators of complement activation (RCA to block complement pathways and our prior work has shown that Parainfluenza virus 5 (PIV5 incorporates CD55 and CD46 to delay complement-mediated neutralization. In this paper, we tested the role of a third individual RCA inhibitor CD59 in PIV5 interactions with complement pathways. Using a cell line engineered to express CD59, we show that small levels of functional CD59 are associated with progeny PIV5, which is capable of blocking assembly of the C5b-C9 membrane attack complex (MAC. PIV5 containing CD59 (PIV5-CD59 showed increased resistance to complement-mediated neutralization in vitro comparing to PIV5 lacking regulators. Infection of A549 cells with PIV5 and RSV upregulated CD59 expression. TGF-beta treatment of PIV5-infected cells also increased cell surface CD59 expression and progeny virions were more resistant to complement-mediated neutralization. A comparison of individual viruses containing only CD55, CD46, or CD59 showed a potency of inhibiting complement-mediated neutralization, which followed a pattern of CD55 > CD46 > CD59.

  6. Production of infectious chimeric hepatitis C virus genotype 2b harboring minimal regions of JFH-1.

    Science.gov (United States)

    Murayama, Asako; Kato, Takanobu; Akazawa, Daisuke; Sugiyama, Nao; Date, Tomoko; Masaki, Takahiro; Nakamoto, Shingo; Tanaka, Yasuhito; Mizokami, Masashi; Yokosuka, Osamu; Nomoto, Akio; Wakita, Takaji

    2012-02-01

    To establish a cell culture system for chimeric hepatitis C virus (HCV) genotype 2b, we prepared a chimeric construct harboring the 5' untranslated region (UTR) to the E2 region of the MA strain (genotype 2b) and the region of p7 to the 3' UTR of the JFH-1 strain (genotype 2a). This chimeric RNA (MA/JFH-1.1) replicated and produced infectious virus in Huh7.5.1 cells. Replacement of the 5' UTR of this chimera with that from JFH-1 (MA/JFH-1.2) enhanced virus production, but infectivity remained low. In a long-term follow-up study, we identified a cell culture-adaptive mutation in the core region (R167G) and found that it enhanced virus assembly. We previously reported that the NS3 helicase (N3H) and the region of NS5B to 3' X (N5BX) of JFH-1 enabled replication of the J6CF strain (genotype 2a), which could not replicate in cells. To reduce JFH-1 content in MA/JFH-1.2, we produced a chimeric viral genome for MA harboring the N3H and N5BX regions of JFH-1, combined with a JFH-1 5' UTR replacement and the R167G mutation (MA/N3H+N5BX-JFH1/R167G). This chimeric RNA replicated efficiently, but virus production was low. After the introduction of four additional cell culture-adaptive mutations, MA/N3H+N5BX-JFH1/5am produced infectious virus efficiently. Using this chimeric virus harboring minimal regions of JFH-1, we analyzed interferon sensitivity and found that this chimeric virus was more sensitive to interferon than JFH-1 and another chimeric virus containing more regions from JFH-1 (MA/JFH-1.2/R167G). In conclusion, we established an HCV genotype 2b cell culture system using a chimeric genome harboring minimal regions of JFH-1. This cell culture system may be useful for characterizing genotype 2b viruses and developing antiviral strategies.

  7. Kidney Versus Islet Allograft Survival After Induction of Mixed Chimerism With Combined Donor Bone Marrow Transplantation.

    Science.gov (United States)

    Oura, Tetsu; Ko, Dicken S C; Boskovic, Svjetlan; O'Neil, John J; Chipashvili, Vaja; Koulmanda, Maria; Hotta, Kiyohiko; Kawai, Kento; Nadazdin, Ognjenka; Smith, R Neal; Cosimi, A B; Kawai, Tatsuo

    2016-01-01

    We have previously reported successful induction of transient mixed chimerism and long-term acceptance of renal allografts in MHC mismatched nonhuman primates. In this study, we attempted to extend this tolerance induction approach to islet allografts. A total of eight recipients underwent MHC mismatched combined islet and bone marrow (BM) transplantation after induction of diabetes by streptozotocin. Three recipients were treated after a nonmyeloablative conditioning regimen that included low-dose total body and thymic irradiation, horse Atgam (ATG), six doses of anti-CD154 monoclonal antibody (mAb), and a 1-month course of cyclosporine (CyA) (Islet A). In Islet B, anti-CD8 mAb was administered in place of CyA. In Islet C, two recipients were treated with Islet B, but without ATG. The results were compared with previously reported results of eight cynomolgus monkeys that received combined kidney and BM transplantation (Kidney A) following the same conditioning regimen used in Islet A. The majority of kidney/BM recipients achieved long-term renal allograft survival after induction of transient chimerism. However, prolonged islet survival was not achieved in similarly conditioned islet/BM recipients (Islet A), despite induction of comparable levels of chimerism. In order to rule out islet allograft loss due to CyA toxicity, three recipients were treated with anti-CD8 mAb in place of CyA. Although these recipients developed significantly superior mixed chimerism and more prolonged islet allograft survival (61, 103, and 113 days), islet function was lost soon after the disappearance of chimerism. In Islet C recipients, neither prolonged chimerism nor islet survival was observed (30 and 40 days). Significant improvement of mixed chimerism induction and islet allograft survival were achieved with a CyA-free regimen that included anti-CD8 mAb. However, unlike the kidney allograft, islet allograft tolerance was not induced with transient chimerism. Induction of more

  8. Theoretical design of a new chimeric protein for the treatment of breast cancer

    Science.gov (United States)

    Soleimani, Meysam; Mahnam, Karim; Mirmohammad-Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Jahanian-Najafabadi, Ali

    2016-01-01

    p28 and NRC peptides are two anticancer peptides with various mechanisms have shown to be effective against breast cancer. Therefore, it seems that construction of a chimeric protein containing the two peptides might cause synergistic cytotoxic effects. However, since the two peptides bear opposite charges, production of a chimeric protein in which the two moieties do not intervene each other is difficult. In this study, our goal was to find a suitable peptide linker for the new chimeric protein in a manner that none of the peptides intervene the other’s function. We selected some linkers with different characteristics and lengths and created a small library of the chimeric proteins harboring these linkers. Homology modeling and molecular dynamic simulation revealed that (PA)5P and (EAAAK)3 linkers can separate the p28 and NRC peptides effectively. Thus, the chimeric protein linked with (PA)5P or (EAAAK)3 linkers might show synergistic and stronger anticancer effects than the separate peptide moieties because they could exert their cytotoxic effects freely which is not influenced by the other part. PMID:27499788

  9. Hemispheric metacontrol and cerebral dominance in healthy individuals investigated by means of chimeric faces.

    Science.gov (United States)

    Urgesi, Cosimo; Bricolo, Emanuela; Aglioti, Salvatore M

    2005-08-01

    Cerebral dominance and hemispheric metacontrol were investigated by testing the ability of healthy participants to match chimeric, entire, or half faces presented tachistoscopically. The two hemi-faces compounding chimeric or entire stimuli were presented simultaneously or asynchronously at different exposure times. Participants did not consciously detect chimeric faces for simultaneous presentations lasting up to 40 ms. Interestingly, a 20 ms separation between each half-chimera was sufficient to induce detection of conflicts at a conscious level. Although the presence of chimeric faces was not consciously perceived, performance on chimeric faces was poorer than on entire- and half-faces stimuli, thus indicating an implicit processing of perceptual conflicts. Moreover, the precedence of hemispheric stimulation over-ruled the right hemisphere dominance for face processing, insofar as the hemisphere stimulated last appeared to influence the response. This dynamic reversal of cerebral dominance, however, was not caused by a shift in hemispheric specialization, since the level of performance always reflected the right hemisphere specialization for face recognition. Thus, the dissociation between hemispheric dominance and specialization found in the present study hints at the existence of hemispheric metacontrol in healthy individuals.

  10. Reversible Heat-Induced Inactivation of Chimeric β-Glucuronidase in Transgenic Plants1

    Science.gov (United States)

    Almoguera, Concepción; Rojas, Anabel; Jordano, Juan

    2002-01-01

    We compared the expression patterns in transgenic tobacco (Nicotiana tabacum) of two chimeric genes: a translational fusion to β-glucuronidase (GUS) and a transcriptional fusion, both with the same promoter and 5′-flanking sequences of Ha hsp17.7 G4, a small heat shock protein (sHSP) gene from sunflower (Helianthus annuus). We found that immediately after heat shock, the induced expression from the two fusions in seedlings was similar, considering chimeric mRNA or GUS protein accumulation. Surprisingly, we discovered that the chimeric GUS protein encoded by the translational fusion was mostly inactive in such conditions. We also found that this inactivation was fully reversible. Thus, after returning to control temperature, the GUS activity was fully recovered without substantial changes in GUS protein accumulation. In contrast, we did not find differences in the in vitro heat inactivation of the respective GUS proteins. Insolubilization of the chimeric GUS protein correlated with its inactivation, as indicated by immunoprecipitation analyses. The inclusion in another chimeric gene of the 21 amino-terminal amino acids from a different sHSP lead to a comparable reversible inactivation. That effect not only illustrates unexpected post-translational problems, but may also point to sequences involved in interactions specific to sHSPs and in vivo heat stress conditions. PMID:12011363

  11. 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.

  12. Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties

    Science.gov (United States)

    The development of a spider silk manufacturing process is of great interest. piggyBac vectors were used to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk prote...

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

    International Nuclear Information System (INIS)

    Skidmore, B.J.; Miller, L.S.

    1978-01-01

    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 10 5 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.)

  14. Inhibition of tumor growth in syngenetic chimeric mice mediated by a depletion of suppressor T cells

    International Nuclear Information System (INIS)

    Rotter, V.; Trainin, N.

    1975-01-01

    Syngeneic chimeric (lethally irradiated and reconstituted with syngeneic bone marrow cells) mice manifested an increased resistance to the development of Lewis lung carcinoma. In addition, these mice had a higher response to polyvinylpyrrolidone and a reduced reactivity to T mitogens. The present findings suggest that syngeneic chimeric mice lack suppressor T cells shown to regulate the development of Lewis lung tumor and the response to polyvinylpyrrolidone. Other components of the T cell population, such as helper cells responding to sheep red blood cells or cells involved in allograft rejection, assayed in these syngeneic chimeras were found unaffected. The fact that chimeric mice are deficient in a certain suppressor T cell population whereas other T activities are normal suggests the existence of different cell lines within the T cell population. (U.S.)

  15. THE CHIMERIC ALT-VASTUS LATERALIS FREE FLAP IN RECONSTRUCTION OF ADVANCED BRONJ OF THE MAXILLA

    Directory of Open Access Journals (Sweden)

    Francesca Toia

    2015-04-01

    Full Text Available ntroduction Bisphosphonate-related osteonecrosis of the jaw (BRONJ is a dangerous complication of bisphosphonates, a class of pharmaceutical agents used in numerous bone disorders. No gold standard therapy exists, but recent literature suggests that, in advanced stages, the best results are achieved with aggressive debridement. In this paper, we report our experience of treatment of stage 3 BRONJ of the maxilla with extensive surgical debridement and reconstruction with a chimeric ALT-Vastus lateralis flap. Methods Five selected patients with stage 3 BRONJ underwent partial maxillectomy with disease-free margins followed by immediate reconstruction with a chimeric ALT-Vastus lateralis free flap. Results Only two patients experienced minor complications. All other patients healed uneventfully within two weeks and donor site morbidity was minimal. Conclusions Our data suggest that aggressive debridement and reconstruction with a chimeric ALT -Vastus lateralis flap is an effective option for the treatment of stage III BRONJ of the maxilla.

  16. 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

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

    KAUST Repository

    Mahfouz, Magdy M.; Li, Lixin; Piatek, Marek J.; Fang, Xiaoyun; Mansour, Hicham; Bangarusamy, Dhinoth K.; Zhu, Jian-Kang

    2011-01-01

    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

  18. Structural basis for drug and substrate specificity exhibited by FIV encoding a chimeric FIV/HIV protease

    International Nuclear Information System (INIS)

    Lin, Ying-Chuan; Perryman, Alexander L.; Olson, Arthur J.; Torbett, Bruce E.; Elder, John H.; Stout, C. David

    2011-01-01

    Crystal structures of the 6s-98S FIV protease chimera with darunavir and lopinavir bound have been determined at 1.7 and 1.8 Å resolution, respectively. A chimeric feline immunodeficiency virus (FIV) protease (PR) has been engineered that supports infectivity but confers sensitivity to the human immunodeficiency virus (HIV) PR inhibitors darunavir (DRV) and lopinavir (LPV). The 6s-98S PR has five replacements mimicking homologous residues in HIV PR and a sixth which mutated from Pro to Ser during selection. Crystal structures of the 6s-98S FIV PR chimera with DRV and LPV bound have been determined at 1.7 and 1.8 Å resolution, respectively. The structures reveal the role of a flexible 90s loop and residue 98 in supporting Gag processing and infectivity and the roles of residue 37 in the active site and residues 55, 57 and 59 in the flap in conferring the ability to specifically recognize HIV PR drugs. Specifically, Ile37Val preserves tertiary structure but prevents steric clashes with DRV and LPV. Asn55Met and Val59Ile induce a distinct kink in the flap and a new hydrogen bond to DRV. Ile98Pro→Ser and Pro100Asn increase 90s loop flexibility, Gln99Val contributes hydrophobic contacts to DRV and LPV, and Pro100Asn forms compensatory hydrogen bonds. The chimeric PR exhibits a comparable number of hydrogen bonds, electrostatic interactions and hydrophobic contacts with DRV and LPV as in the corresponding HIV PR complexes, consistent with IC 50 values in the nanomolar range

  19. Structural basis for drug and substrate specificity exhibited by FIV encoding a chimeric FIV/HIV protease

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Ying-Chuan; Perryman, Alexander L.; Olson, Arthur J.; Torbett, Bruce E.; Elder, John H.; Stout, C. David, E-mail: dave@scripps.edu [The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States)

    2011-06-01

    Crystal structures of the 6s-98S FIV protease chimera with darunavir and lopinavir bound have been determined at 1.7 and 1.8 Å resolution, respectively. A chimeric feline immunodeficiency virus (FIV) protease (PR) has been engineered that supports infectivity but confers sensitivity to the human immunodeficiency virus (HIV) PR inhibitors darunavir (DRV) and lopinavir (LPV). The 6s-98S PR has five replacements mimicking homologous residues in HIV PR and a sixth which mutated from Pro to Ser during selection. Crystal structures of the 6s-98S FIV PR chimera with DRV and LPV bound have been determined at 1.7 and 1.8 Å resolution, respectively. The structures reveal the role of a flexible 90s loop and residue 98 in supporting Gag processing and infectivity and the roles of residue 37 in the active site and residues 55, 57 and 59 in the flap in conferring the ability to specifically recognize HIV PR drugs. Specifically, Ile37Val preserves tertiary structure but prevents steric clashes with DRV and LPV. Asn55Met and Val59Ile induce a distinct kink in the flap and a new hydrogen bond to DRV. Ile98Pro→Ser and Pro100Asn increase 90s loop flexibility, Gln99Val contributes hydrophobic contacts to DRV and LPV, and Pro100Asn forms compensatory hydrogen bonds. The chimeric PR exhibits a comparable number of hydrogen bonds, electrostatic interactions and hydrophobic contacts with DRV and LPV as in the corresponding HIV PR complexes, consistent with IC{sub 50} values in the nanomolar range.

  20. Bone marrow cell migration to the heart in a chimeric mouse model of acute chagasic disease.

    Science.gov (United States)

    Irion, Camila Iansen; Paredes, Bruno Diaz; Brasil, Guilherme Visconde; Cunha, Sandro Torrentes da; Paula, Luis Felipe; Carvalho, Alysson Roncally; Carvalho, Antonio Carlos Campos de; Carvalho, Adriana Bastos; Goldenberg, Regina Coeli Dos Santos

    2017-08-01

    Chagas disease is a public health problem caused by infection with the protozoan Trypanosoma cruzi. There is currently no effective therapy for Chagas disease. Although there is some evidence for the beneficial effect of bone marrow-derived cells in chagasic disease, the mechanisms underlying their effects in the heart are unknown. Reports have suggested that bone marrow cells are recruited to the chagasic heart; however, studies using chimeric mouse models of chagasic cardiomyopathy are rare. The aim of this study was to investigate the migration of bone marrow cells to the heart after T. cruzi infection in a model of chagasic disease in chimeric mice. To obtain chimerical mice, wild-type (WT) C57BL6 mice were exposed to full body irradiation (7 Gy), causing bone marrow ablation. Then, bone marrow cells from green fluorescent protein (GFP)-transgenic mice were infused into the mice. Graft effectiveness was confirmed by flow cytometry. Experimental mice were divided into four groups: (i) infected chimeric (iChim) mice; (ii) infected WT (iWT) mice, both of which received 3 × 104 trypomastigotes of the Brazil strain; (iii) non-infected chimeric (Chim) mice; and (iv) non-infected WT mice. At one-month post-infection, iChim and iWT mice showed first degree atrioventricular block with decreased heart rate and treadmill exercise parameters compared to those in the non-infected groups. iChim mice showed an increase in parasitaemia, myocarditis, and the presence of amastigote nests in the heart tissue compared to iWT mice. Flow cytometry analysis did not detect haematopoietic progenitor cells in the hearts of infected mice. Furthermore, GFP+ cardiomyocytes were not detected in the tissues of chimeric mice.

  1. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.

    Science.gov (United States)

    Grupp, Stephan A; Kalos, Michael; Barrett, David; Aplenc, Richard; Porter, David L; Rheingold, Susan R; Teachey, David T; Chew, Anne; Hauck, Bernd; Wright, J Fraser; Milone, Michael C; Levine, Bruce L; June, Carl H

    2013-04-18

    Chimeric antigen receptor-modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre-B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×10(6) to 1.2×10(7) CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce antileukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL.

  2. Tumor-Triggered Geometrical Shape Switch of Chimeric Peptide for Enhanced in Vivo Tumor Internalization and Photodynamic Therapy.

    Science.gov (United States)

    Han, Kai; Zhang, Jin; Zhang, Weiyun; Wang, Shibo; Xu, Luming; Zhang, Chi; Zhang, Xianzheng; Han, Heyou

    2017-03-28

    Geometrical shape of nanoparticles plays an important role in cellular internalization. However, the applicability in tumor selective therapeutics is still scarcely reported. In this article, we designed a tumor extracellular acidity-responsive chimeric peptide with geometrical shape switch for enhanced tumor internalization and photodynamic therapy. This chimeric peptide could self-assemble into spherical nanoparticles at physiological condition. While at tumor extracellular acidic microenvironment, chimeric peptide underwent detachment of acidity-sensitive 2,3-dimethylmaleic anhydride groups. The subsequent recovery of ionic complementarity between chimeric peptides resulted in formation of rod-like nanoparticles. Both in vitro and in vivo studies demonstrated that this acidity-triggered geometrical shape switch endowed chimeric peptide with accelerated internalization in tumor cells, prolonged accumulation in tumor tissue, enhanced photodynamic therapy, and minimal side effects. Our results suggested that fusing tumor microenvironment with geometrical shape switch should be a promising strategy for targeted drug delivery.

  3. Translation activity of chimeric ribosomes composed of Escherichia coli and Bacillus subtilis or Geobacillus stearothermophilus subunits

    Directory of Open Access Journals (Sweden)

    Sayaka Tsuji

    2017-07-01

    Full Text Available Ribosome composition, consisting of rRNA and ribosomal proteins, is highly conserved among a broad range of organisms. However, biochemical studies focusing on ribosomal subunit exchangeability between organisms remain limited. In this study, we show that chimeric ribosomes, composed of Escherichia coli and Bacillus subtilis or E. coli and Geobacillus stearothermophilus subunits, are active for β-galactosidase translation in a highly purified E. coli translation system. Activities of the chimeric ribosomes showed only a modest decrease when using E. coli 30 S subunits, indicating functional conservation of the 50 S subunit between these bacterial species.

  4. Radiation induced chimeric rearrangement flower structure of Rhododendron simsii Planch. (Azaleaindica L. ) Use of recurrent irradiation

    Energy Technology Data Exchange (ETDEWEB)

    de Loose, R [IWONL (IRSIA) Irradiation Laboratory, Institute of Ornamental Plant Growing, Melle (Belgium)

    1979-02-01

    A radiation-induced chimeric flower colour sport of vegetatively propagated Rhododendron simsii Planch was recurrently irradiated (up to three times in three consecutive years) with soft X-rays (50kV-30mA), as compared to a single treatment. Because of the low true flower colour mutation frequency the efficiency of the different radiation treatments was compared on the basis of the number of chimeric rearrangements in flower structure i.e. the flower colour change from red with broad white edge towards either homogeneous carminered or white. It is quite clear that recurrent irradiation with appropiate doses is most efficient.

  5. Lym-1 Chimeric Antigen Receptor T Cells Exhibit Potent Anti-Tumor Effects against B-Cell Lymphoma

    Directory of Open Access Journals (Sweden)

    Long Zheng

    2017-12-01

    Full Text Available T cells expressing chimeric antigen receptors (CARs recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgammanull (NSG mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluorescent protein (Raji/Luc-GFP in NSG mice with similar or better reactivity than CD19 CAR T cells. Lym-1 CAR transduction of T cells is a promising immunotherapy for patients with Lym-1 epitope positive B-cell malignancies.

  6. Tubular and endothelial chimerism in renal allografts using fluorescence and chromogenic in situ hybridization (FISH, CISH) technology.

    Science.gov (United States)

    Varga, Zsuzsanna; Gaspert, Ariana; Behnke, Silvia; von Teichman, Adriana; Fritzsche, Florian; Fehr, Thomas

    2012-04-01

    The role of endothelial and tubular chimerism in renal allograft adaptation and rejection varies in different studies. We addressed the correlation between different clinico-pathological settings and sex-chromosomal endothelial and/or tubular chimerism in renal allografts. We examined the presence or absence of the X and Y chromosomes by fluorescence and chromogenic in situ hybridization (FISH, CISH) methodology on paraffin embedded kidney biopsies in 16 gender mismatched renal transplants (1 to 12 years post-transplantation). Twelve patients were male, four female. Four groups were selected: (i) Vascular calcineurin inhibitor toxicity without rejection; (ii) T-cell mediated vascular rejection; (iii) antibody mediated rejection; and (iv) C4d-positivity in AB0-incompatible transplants with or without rejection. Twelve non-transplant kidney biopsies (8 female, 4 male) were used as controls. Tubular chimerism was detected more frequently (69%) than endothelial chimerism (12%) in renal transplants. One of 12 control patients had tubular and endothelial chimeric cells (8%). The Y chromosome occurred in 8/12 male recipients (67%) in tubular epithelial cells and in 5/12 male recipients (42%) in endothelial cells. Double X chromosomes were detected in 3/4 female recipients in tubular epithelium. Tubular chimerism occurred more often with endothelial chimerism and capillaritis without correlation with other parameters, such as rejection. Combined Y chromosomal tubular and lymphatic endothelial chimerism correlated with T-cell mediated vascular rejection in two out of three patients (66%). Combined Y chromosomal tubular and peritubular capillary chimerism correlated with antibody mediated C4d+ rejection in one out of two patients (50%). Tubular and/or endothelial chimerism occur frequently in gender mismatched renal allografts and, when combined, this is associated with T-cell mediated rejection. © 2012 The Authors. Pathology International © 2012 Japanese Society of

  7. The Formation of Chimeric Nanomorphologies, as a Reflection of Naturally Occurring Thermodynamic Processes

    Science.gov (United States)

    Naziris, N.; Demetzos, C.

    2017-11-01

    The self-assembly process of different in nature biomaterials leads to the morphogenesis of various nano-structures, where the individual molecule properties (e.g. hydrophilic-to-hydrophobic balance and elasticity), profoundly affect the intermediate surfaces’ interfacial thermodynamics. Herein, the mixing of a phospholipid and an amphiphilic block copolymer, through the thin-film hydration method, gave different morphologies, among which there were vesicles (i.e. liposomes and polymersomes), micelles and worm-like structures. The formation of such variety of structures is attributed to divergent entropic pathways, which are determined by a number of parameters, such as the lipid:polymer molar ratio and the polymer composition. The developed nanosystems are considered as chimeric/mixed, because of the two different in type biomaterials that compose them. The vesicles also exhibited membrane “irregularities”, which are connected with their biophysical behavior. Nature has “chosen” vesicular forms to be the thermodynamically stable “biological apartments”, in which life was enclosed and additionally, vesicles provided compartmentalized systems, where the intracellular environment was built. Phospholipid properties result in membranes/bilayers that harmonically assimilate other molecules, like proteins and retain their integrity and functionality, while gaining additional features. A cause that alters this relationship might induce changes in the membrane composition and morphology, with respect to lipid rafts/domains, what has been linked with the activation and development of certain human disorders/diseases. The self-assembly of two different biomaterials into various structures that present distinct membrane phenomena is believed to simulate these natural processes.

  8. Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia.

    Science.gov (United States)

    Fraietta, Joseph A; Beckwith, Kyle A; Patel, Prachi R; Ruella, Marco; Zheng, Zhaohui; Barrett, David M; Lacey, Simon F; Melenhorst, Jan Joseph; McGettigan, Shannon E; Cook, Danielle R; Zhang, Changfeng; Xu, Jun; Do, Priscilla; Hulitt, Jessica; Kudchodkar, Sagar B; Cogdill, Alexandria P; Gill, Saar; Porter, David L; Woyach, Jennifer A; Long, Meixiao; Johnson, Amy J; Maddocks, Kami; Muthusamy, Natarajan; Levine, Bruce L; June, Carl H; Byrd, John C; Maus, Marcela V

    2016-03-03

    Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749. © 2016 by The American Society of Hematology.

  9. 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.

  10. Regional inverse modeling for high reactive species with PYVAR-CHIMERE

    Science.gov (United States)

    Fortems-Cheiney, A.; Pison, I.; Dufour, G.; Broquet, G.; Costantino, L.

    2017-12-01

    The degradation of air quality is a worldwide environmental problem: according to the World Health Organization WHO, 92% of the world's population breathe polluted air in 2016. A number of air pollutants associated with respiratory disease and shortened life expectancy play a particularly important role in global outdoor air pollution. In addition to threatening both human health and ecosystems, these gaseous air pollutants including nitrogen oxides (NOx=NO+NO2), sulfur dioxide (SO2), ammonia (NH3), and volatile organic compounds (VOCs) could be precursors of ozone (O3) and Particulate Matter (PM). Without a strong scientific back-up to determine their different sources, the necessary regulations to improve air quality will not be efficient. To date, only chemistry-transport models (CTM) are able to describe pollutant concentrations at any location in the world and their evolution in the atmosphere. Consequently, they have become essential tools for studying air quality. However, CTM are hampered by incomplete information on gaseous precursors and one of the large shortcoming for simulating the gaseous pollutants budgets is the lack of high spatio-temporal variability for the emission estimations provided as inputs for chemistry-transport models. For all these reasons, an inverse system called PYVAR-CHIMERE has been developed, operating in synergy between a CTM and atmospheric observations, and being adjust for the highly reactive species of interest here, as NO2. We present here the first results of this Bayesian variational inverse method for the quantification of NO2 emissions both over Europe (in March 2011) and over China (in January 2015), with a spatial resolution of 0.5°x0.5° and at a weekly temporal resolution, constrained by surface measurements and OMI NO2 satellite observations.

  11. 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. ©2015 American Association for Cancer Research.

  12. Homology-Directed Recombination for Enhanced Engineering of Chimeric Antigen Receptor T Cells

    Directory of Open Access Journals (Sweden)

    Malika Hale

    2017-03-01

    Full Text Available Gene editing by homology-directed recombination (HDR can be used to couple delivery of a therapeutic gene cassette with targeted genomic modifications to generate engineered human T cells with clinically useful profiles. Here, we explore the functionality of therapeutic cassettes delivered by these means and test the flexibility of this approach to clinically relevant alleles. Because CCR5-negative T cells are resistant to HIV-1 infection, CCR5-negative anti-CD19 chimeric antigen receptor (CAR T cells could be used to treat patients with HIV-associated B cell malignancies. We show that targeted delivery of an anti-CD19 CAR cassette to the CCR5 locus using a recombinant AAV homology template and an engineered megaTAL nuclease results in T cells that are functionally equivalent, in both in vitro and in vivo tumor models, to CAR T cells generated by random integration using lentiviral delivery. With the goal of developing off-the-shelf CAR T cell therapies, we next targeted CARs to the T cell receptor alpha constant (TRAC locus by HDR, producing TCR-negative anti-CD19 CAR and anti-B cell maturation antigen (BCMA CAR T cells. These novel cell products exhibited in vitro cytolytic activity against both tumor cell lines and primary cell targets. Our combined results indicate that high-efficiency HDR delivery of therapeutic genes may provide a flexible and robust method that can extend the clinical utility of cell therapeutics.

  13. Isolation of Haemophilus influenzae and Haemophilus parainfluenzae in urethral exudates from men with acute urethritis: a descriptive study of 52 cases.

    Science.gov (United States)

    Deza, Gustavo; Martin-Ezquerra, Gemma; Gómez, Julià; Villar-García, Judit; Supervia, August; Pujol, Ramon M

    2016-02-01

    To describe the clinical characteristics and therapeutic outcomes from male patients diagnosed of Haemophilus spp urethritis. A chart review of patients who presented to our hospital from January 2013 to December 2014 with symptoms of acute urethritis in which Haemophilus spp was isolated in their urethral samples was performed. Haemophilus spp was isolated in 52 out of 413 urethral samples (12.6%) received in our laboratory from patients with symptoms of acute urethritis during the study period. Seven cases corresponded to Haemophilus influenzae and 45 cases to Haemophilus parainfluenzae. The most common clinical presentation was mucopurulent urethral discharge (71%). Eight per cent were HIV-infected patients, and 60% were men who have sex with men. Haemophilus spp was isolated as a single pathogen in 6.8% (28 of 413) of cases. Seventeen per cent of Haemophilus spp were β-lactamase producers. All patients reported having practiced unprotected insertive oral sex the month before consultation, and five of them denied having had another sexual contact apart from this exposure. In all cases in which follow-up was available, empirical treatment achieved a complete clinical resolution. Haemophilus spp was considered a pathogen in at least 6.8% of the patients from the evaluated area. It affected men regardless their sexual orientation or HIV status. Unprotected oral sex could play a role in its transmission. The limitations of the study (small sample size and lack of a representative control group) do not allow to prove the true pathogenic role of Haemophilus spp in acute urethritis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  14. Analysis of microRNAs Expression Profiles in Madin-Darby Bovine Kidney Cells Infected With Caprine Parainfluenza Virus Type 3

    Directory of Open Access Journals (Sweden)

    Jizong Li

    2018-03-01

    Full Text Available Caprine parainfluenza virus type 3 (CPIV3 is a newly emerging pathogenic respiratory agent infecting both young and adult goats, and it was identified in eastern China in 2013. Cellular microRNAs (miRNAs have been reported to be important modulators of the intricate virus-host interactions. In order to elucidate the role of miRNAs in madin-darby bovine kidney (MDBK cells during CPIV3 infection. In this study, we performed high-throughput sequencing technology to analyze small RNA libraries in CPIV3-infected and mock-infected MDBK cells. The results showed that a total of 249 known and 152 novel candidate miRNAs were differentially expressed in MDBK cells after CPIV3 infection, and 22,981 and 22,572 target genes were predicted, respectively. In addition, RT-qPCR assay was used to further confirm the expression patterns of 13 of these differentially expressed miRNAs and their mRNA targets. Functional annotation analysis showed these up- and downregulated target genes were mainly involved in MAPK signaling pathway, Jak-STAT signaling pathway, Toll-like receptor signaling pathway, p53 signaling pathway, focal adhesion, NF-kappa B signaling pathway, and apoptosis, et al. To our knowledge, this is the first report of the comparative expression of miRNAs in MDBK cells after CPIV3 infection. Our finding provides information concerning miRNAs expression profile in response to CPIV3 infection, and offers clues for identifying potential candidates for antiviral therapies against CPIV3.

  15. Duration of serological response to canine parvovirus-type 2, canine distemper virus, canine adenovirus type 1 and canine parainfluenza virus in client-owned dogs in Australia.

    Science.gov (United States)

    Mitchell, S A; Zwijnenberg, R J; Huang, J; Hodge, A; Day, M J

    2012-12-01

    To determine whether client-owned dogs in Australia, last vaccinated with Canvac(®) vaccines containing canine parvovirus-type 2 (CPV-2), canine distemper virus (CDV), canine adenovirus type 2 (CAV-2) ± canine parainfluenza virus (CPiV) at least 18 months ago, were seropositive or responded serologically to revaccination. A total of 235 dogs were recruited from 23 veterinary clinics, representing a variety of breeds, ages and time since last vaccination (TSLV: range 1.5-9 years, mean 2.8 years). Dogs had a blood sample taken and were revaccinated on day 0. A second blood sample was taken 7-14 days later. Blood samples were assessed for antibody titres to CPV-2 (by haemagglutination inhibition) and CDV, CAV type 1 (CAV-1) and CPiV (by virus neutralisation). Dogs with a day 0 titre >10 or a four-fold increase in titre following revaccination were considered to be serological responders. The overall percentage of dogs classified as serological responders was 98.7% for CPV-2, 96.6% for CDV, 99.6% for CAV-1 and 90.3% for CPiV. These results suggest that the duration of serological response induced by modified-live vaccines against CPV-2, CDV, CAV-1 and CPiV, including Canvac(®) vaccines, is beyond 18 months and may extend up to 9 years. Accordingly, these vaccines may be considered for use in extended revaccination interval protocols as recommended by current canine vaccine guidelines. © 2012 The Authors. Australian Veterinary Journal © 2012 Australian Veterinary Association.

  16. Replacement of the Ectodomains of the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Recombinant Parainfluenza Virus Type 3 (PIV3) with Their Counterparts from PIV2 Yields Attenuated PIV2 Vaccine Candidates

    OpenAIRE

    Tao, Tao; Skiadopoulos, Mario H.; Davoodi, Fatemeh; Riggs, Jeffrey M.; Collins, Peter L.; Murphy, Brian R.

    2000-01-01

    We sought to develop a live attenuated parainfluenza virus type 2 (PIV2) vaccine strain for use in infants and young children, using reverse genetic techniques that previously were used to rapidly produce a live attenuated PIV1 vaccine candidate. The PIV1 vaccine candidate, designated rPIV3-1cp45, was generated by substituting the full-length HN and F proteins of PIV1 for those of PIV3 in the attenuated cp45 PIV3 vaccine candidate (T. Tao et al., J. Virol. 72:2955–2961, 1998; M. H. Skiadopoul...

  17. 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.

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

    Science.gov (United States)

    Vernot, Jean-Paul; Perdomo-Arciniegas, Ana María; Pérez-Quintero, Luis Alberto; Martínez, Diego Fernando

    2015-01-01

    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.

  19. Anticorpos neutralizantes contra os vírus da cinomose e da parainfluenza em cães de canis dos municípios de Novo Hamburgo e Porto Alegre, RS, Brasil Neutralizing antibodies to distemper and parainfluenza viruses in dogs in shelter kennels in the municipalities of Novo Hamburgo and Porto Alegre, RS, Brazil

    Directory of Open Access Journals (Sweden)

    Tamahine Larronda Schmidt Hartmann

    2007-08-01

    Full Text Available No presente estudo, foi realizada uma pesquisa em busca de anticorpos neutralizantes contra os vírus da cinomose (CDV e da parainfluenza (CPIV caninos em amostras de soro de 173 cães recolhidos a canis municipais em Novo Hamburgo (n=82 e Porto Alegre (n=91, RS. A pesquisa de anticorpos neutralizantes foi realizada pela técnica de soroneutralização frente a duas amostras vacinais de CDV (Rockborn e Snyder Hill e frente a uma amostra de CPIV (V660. Em relação ao CDV, 95,9% das amostras de soros foram negativas para anticorpos neutralizantes contra a amostra Snyder Hill e 90,7% soronegativas para a amostra Rockborn. Entre os soropositivos (n=20; 11,6%, somente três deles apresentaram anticorpos neutralizantes frente às duas amostras de CDV testadas, indicando pouca reatividade cruzada entre as mesmas. Quanto ao CPIV, a prevalência de anticorpos neutralizantes encontrada frente à amostra V660 foi de 51,4%. Esses achados indicam que a maioria dos cães examinados não teve contato prévio com o CDV, seja por infecção natural ou por imunização prévia. O CPIV, porém, parece estar amplamente difundido na população canina examinada, provavelmente por exposição natural ao vírus.In this report a serological survey was carried out in search for antibodies to canine distemper virus (CDV and canine parainfluenza virus (CPIV in 173 sera from dogs withdraw in kennels of the municipalities of Novo Hamburgo (n=82 and Porto Alegre (n=91, RS, Brazil. Neutralizing antibodies were evaluated against two CDV strains used for vaccine production (Rockborn and Snyder Hill as well as one strain of CPIV (V660. Search for anti-CDV neutralizing antibodies revealed that 95.9% of sera were negative for antibodies to CDV Snyder Hill and 90.7% were negative for antibodies to CDV Rockborn. Among the positive sera (n=20; 11.6 % only three of those had neutralizing antibodies to both CDV strains, indicating a low degree of cross reactivity between those. As

  20. Chimeric Ply187 endolysin kills Staphylococcus aureus more effectively than the parental enzyme.

    Science.gov (United States)

    Peptidoglycan hydrolases are an effective new source of antimicrobials. A chimeric fusion protein of the Ply187 endopeptidase domain and LysK SH3b cell wall binding domain is a potent agent against Staphylococcus aureus in three functional assays....

  1. 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...

  2. Inhibition of HIV-1 replication by chimeric phosphorothioate oligodeoxynucleotides applied in free solution

    DEFF Research Database (Denmark)

    Lund, O S; Hansen, J E

    1998-01-01

    Oligodeoxynucleotides (ODNs) containing a variable number of 3' and 5' terminal phosphorothioate linkages were applied in free solution to cells infected by HIV-1. ODNs of 28 nt length were applied at up to 5 microM concentration. The ODNs were found to inhibit HIV-1 infection in a dose dependent...... by these modified chimers....

  3. Intravitreal injection of a chimeric phage endolysin Ply187 protects mice from Staphylococcus aureus endophthalmitis

    Science.gov (United States)

    Objectives: The treatment of endophthalmitis is becoming very challenging due to the emergence of multidrug-resistant bacteria. Hence, the development of novel therapeutic alternatives for ocular use is essential. Here, we evaluated the therapeutic potential of Ply187AN-KSH3b, a chimeric phage endol...

  4. Isolation of chicken embryonic stem cell and preparation of chicken chimeric model.

    Science.gov (United States)

    Zhang, Yani; Yang, Haiyan; Zhang, Zhentao; Shi, Qingqing; Wang, Dan; Zheng, Mengmeng; Li, Bichun; Song, Jiuzhou

    2013-03-01

    Chicken embryonic stem cells (ESCs) were separated from blastoderms at stage-X and cultured in vitro. Alkaline phosphatase activity and stage-specific embryonic antigen-1 staining was conducted to detect ESCs. Then, chicken ESCs were transfected with linearized plasmid pEGFP-N1 in order to produce chimeric chicken. Firstly, the optimal electrotransfection condition was compared; the results showed the highest transfection efficiency was obtained when the field strength and pulse duration was 280 V and 75 μs, respectively. Secondly, the hatchability of shedding methods, drilling a window at the blunt end of egg and drilling a window at the lateral shell of egg was compared, the results showed that the hatchability was the highest for drilling a window at the lateral shell of egg. Thirdly, the hatchability of microinjection (ESCs was microinjected into chick embryo cavity) was compared too, the results showed there were significant difference between the injection group transfected with ESCs and that of other two groups. In addition, five chimeric chickens were obtained in this study and EGFP gene was expressed in some organs, but only two chimeric chicken expressed EGFP gene in the gonad, indicating that the chimeric chicken could be obtained through chick embryo cavity injection by drilling a window at the lateral shell of egg.

  5. 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...

  6. 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...

  7. Engineered Chimeric Peptides as Antimicrobial Surface Coating Agents toward Infection-Free Implants.

    Science.gov (United States)

    Yazici, Hilal; O'Neill, Mary B; Kacar, Turgay; Wilson, Brandon R; Oren, E Emre; Sarikaya, Mehmet; Tamerler, Candan

    2016-03-02

    Prevention of bacterial colonization and consequent biofilm formation remains a major challenge in implantable medical devices. Implant-associated infections are not only a major cause of implant failures but also their conventional treatment with antibiotics brings further complications due to the escalation in multidrug resistance to a variety of bacterial species. Owing to their unique properties, antimicrobial peptides (AMPs) have gained significant attention as effective agents to combat colonization of microorganisms. These peptides have been shown to exhibit a wide spectrum of activities with specificity to a target cell while having a low tendency for developing bacterial resistance. Engineering biomaterial surfaces that feature AMP properties, therefore, offer a promising approach to prevent implant infections. Here, we engineered a chimeric peptide with bifunctionality that both forms a robust solid-surface coating while presenting antimicrobial property. The individual domains of the chimeric peptides were evaluated for their solid-binding kinetics to titanium substrate as well as for their antimicrobial properties in solution. The antimicrobial efficacy of the chimeric peptide on the implant material was evaluated in vitro against infection by a variety of bacteria, including Streptococcus mutans, Staphylococcus. epidermidis, and Escherichia coli, which are commonly found in oral and orthopedic implant related surgeries. Our results demonstrate significant improvement in reducing bacterial colonization onto titanium surfaces below the detectable limit. Engineered chimeric peptides with freely displayed antimicrobial domains could be a potential solution for developing infection-free surfaces by engineering implant interfaces with highly reduced bacterial colonization property.

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

    Directory of Open Access Journals (Sweden)

    J Roman Arguello

    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. Chimerization of lactoferricin and lactoferrampin peptides strongly potentiates the killing activity against Candida albicans

    NARCIS (Netherlands)

    Bolscher, J.; Nazmi, K.; van Marle, J.; van 't Hof, W.; Veerman, E.

    2012-01-01

    Bovine lactoferrin harbors 2 antimicrobial sequences (LFcin and LFampin), situated in close proximity in the N1-domain. To mimic their semi parallel configuration we have synthesized a chimeric peptide (LFchimera) in which these sequences are linked in a head-to-head fashion to the α- and ε-amino

  10. Chimeric peptide beacons: a direct polypeptide analog of DNA molecular beacons†

    OpenAIRE

    Oh, Kenneth J.; Cash, Kevin J.; Lubin, Arica A.; Plaxco, Kevin W.

    2007-01-01

    We have developed a new biosensor architecture, which is comprised of a polypeptide–peptide nucleic acid tri-block copolymer and which we have termed chimeric peptide beacons (CPB), that generates an optical output via a mechanism analogous to that employed in DNA-based molecular beacons.

  11. Chimeric immune receptors (CIRs) specific to JC virus for immunotherapy in progressive multifocal leukoencephalopathy (PML)

    NARCIS (Netherlands)

    W. Yang; E.L. Beaudoin; L. Lu; R.A. Du Pasquier (Renaud); M.J. Kuroda; R.A. Willemsen (Ralph); I.J. Koralnik; R.P. Junghans

    2007-01-01

    textabstractProgressive multifocal leukoencephalopathy (PML) is a deadly brain disease caused by the polyomavirus JC (JCV). The aim of this study is to develop 'designer T cells' armed with anti-JCV TCR-based chimeric immune receptors (CIRs) by gene modification for PML immunotherapy. Two T cell

  12. 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.

  13. [Immunoreactivity of chimeric proteins carrying poliovirus epitopes on the VP6 of rotavirus as a vector].

    Science.gov (United States)

    Pan, X-X; Zhao, B-X; Teng, Y-M; Xia, W-Y; Wang, J; Li, X-F; Liao, G-Y; Yang, С; Chen, Y-D

    2016-01-01

    Rotavirus and poliovirus continue to present significant risks and burden of disease to children in developing countries. Developing a combined vaccine may effectively prevent both illnesses and may be advantageous in terms of maximizing compliance and vaccine coverage at the same visit. Recently, we sought to generate a vaccine vector by incorporating multiple epitopes into the rotavirus group antigenic protein, VP6. In the present study, a foreign epitope presenting a system using VP6 as a vector was created with six sites on the outer surface of the vector that could be used for insertion of foreign epitopes, and three VP6-based PV1 epitope chimeric proteins were constructed. The chimeric proteins were confirmed by immunoblot, immunofluorescence assay, and injected into guinea pigs to analyze the epitope-specific humoral response. Results showed that these chimeric proteins reacted with anti-VP6F and -PV1 antibodies, and elicited antibodies against both proteins in guinea pigs. Antibodies against the chimeric proteins carrying PV1 epitopes neutralized rotavirus Wa and PV1 infection in vitro. Our study contributes to a better understanding of the use of VP6-based vectors as multiple-epitope delivery vehicles and the epitopes displayed in this form could be considered for development of epitope-based vaccines against rotavirus and poliovirus.

  14. Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule

    KAUST Repository

    Butt, Haroon; Eid, Ayman; Ali, Zahir; Atia, Mohamed A. M.; Mokhtar, Morad M.; Hassan, Norhan; Lee, Ciaran M.; Bao, Gang; Mahfouz, Magdy M.

    2017-01-01

    used CRISPR/Cas9 to generate targeted double-strand breaks and to deliver an RNA repair template for HDR in rice (Oryza sativa). We used chimeric single-guide RNA (cgRNA) molecules carrying both sequences for target site specificity (to generate

  15. 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.

  16. 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.

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

    Science.gov (United States)

    Mishina, Yukiko; Mutoh, Hiroki; Song, Chenchen; Knöpfel, Thomas

    2014-01-01

    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 behaviors. 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 fluorescence resonance energy transfer 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 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.

  18. 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.

  19. Lenalidomide enhances antitumor functions of chimeric antigen receptor modified T cells

    Czech Academy of Sciences Publication Activity Database

    Otáhal, Pavel; Průková, D.; Král, Vlastimil; Fábry, Milan; Vockova, P.; Lateckova, L.; Trněný, M.; Klener, P.

    2016-01-01

    Roč. 5, č. 4 (2016), č. článku e1115940. ISSN 2162-402X R&D Projects: GA MZd(CZ) NT13201 Institutional support: RVO:68378050 Keywords : Chimeric antigenic receptor * lenalidomide * lymphoma * tumor immunotherapy * T cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.719, year: 2016

  20. Extensive chimerism in liver transplants: vascular endothelium, bile duct epithelium, and hepatocytes.

    NARCIS (Netherlands)

    Hove, W.R.; Hoek, B. van; Bajema, I.M.; Ringers, J.; Krieken, J.H.J.M. van; Lagaaij, E.L.

    2003-01-01

    The transplanted liver has been shown to be particularly capable of inducing tolerance. An explanation may be the presence of chimerism. Cells of donor origin have been found in recipient tissues after transplantation of any solid organ. Evidence for the presence of cells of recipient origin within

  1. 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...... that ligand-mediated regulation of receptor-PTPases may have mechanistic similarities with receptor tyrosine kinases....

  2. 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. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  3. Ingraft chimerism in lung transplantation - a study in a porcine model of obliterative bronchiolitis

    Directory of Open Access Journals (Sweden)

    Rubes Jiri

    2011-04-01

    Full Text Available Abstract Background Bronchial epithelium is a target of the alloimmune response in lung transplantation, and intact epithelium may protect allografts from rejection and obliterative bronchiolitis (OB. Herein we study the influence of chimerism on bronchial epithelium and OB development in pigs. Methods A total of 54 immunosuppressed and unimmunosuppressed bronchial allografts were serially obtained 2-90 days after transplantation. Histology (H&E was assessed and the fluorescence in situ hybridization (FISH method for Y chromosomes using pig-specific DNA-label was used to detect recipient derived cells in graft epithelium and bronchial wall, and donor cell migration to recipient organs. Ingraft chimerism was studied by using male recipients with female donors, whereas donor cell migration to recipient organs was studied using female recipients with male donors. Results Early appearance of recipient-derived cells in the airway epithelium appeared predictive of epithelial destruction (R = 0.610 - 0.671 and p R = 0.698 and p p p Conclusions In this study we demonstrate that early appearance of Y chromosomes in the airway epithelium predicts features characteristic of OB. Chimerism occurred in all allografts, including those without features of OB. Therefore we suggest that ingraft chimerism may be a mechanism involved in the repair of alloimmune-mediated tissue injury after transplantation.

  4. 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)

    Kawano, Masaaki; Morikawa, Katsuma; Suda, Tatsuya; Ohno, Naohito; Matsushita, Sho; Akatsuka, Toshitaka; Handa, Hiroshi; Matsui, Masanori

    2014-01-01

    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

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

    Directory of Open Access Journals (Sweden)

    Yu Xianzhong

    2008-12-01

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

  6. Chimeric analysis of EGFP and DsRed2 transgenic mice demonstrates polyclonal maintenance of pancreatic acini.

    Science.gov (United States)

    Ryu, Je-Young; Siswanto, Antoni; Harimoto, Kenichi; Tagawa, Yoh-ichi

    2013-06-01

    The pancreatic islet is an assembly of specific endocrine cells. There are many conflicting reports regarding whether the acinus develops from single or multiple progenitor cells. This study investigated the development and maintenance clonality of the pancreatic acinus and duct using a chimeric analysis with EGFP and DsRed2 transgenic mice. Chimeric mice (G-R mice) were obtained by the aggregation method, using 8-cell stage embryos from EGFP and DsRed2 transgenic mice. The islets from the G-R mice were chimeric and mosaic, consisting of either EGFP- or DsRed2-positive populations, as in previous reports. On the other hand, most acini developed from either EGFP or DsRed2 origin, but some were chimeric. Interestingly, these chimeric acini were clearly separated into two-color regions and were not mosaic. Some large intralobular pancreatic ducts consisting of more than 10 cells were found to be chimeric, but no small ducts made up of less than 9 cells were chimeric. Our histological observations suggest that the pancreatic acinus polyclonally and directionally is maintained by multiple progenitor cells. Pancreatic large ducts also seem to develop polyclonally and might result from the assembly of small ducts that develop from a single origin. These findings provide useful information for further understanding pancreatic maintenance.

  7. Novel deletion alleles carrying CYP21A1P/A2 chimeric genes in Brazilian patients with 21-hydroxylase deficiency

    Directory of Open Access Journals (Sweden)

    Guerra-Júnior Gil

    2010-06-01

    Full Text Available Abstract Background Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is caused by deletions, large gene conversions or mutations in CYP21A2 gene. The human gene is located at 6p21.3 within a locus containing the genes for putative serine/threonine Kinase RP, complement C4, steroid 21-hydroxylase CYP21 tenascin TNX, normally, in a duplicated cluster known as RCCX module. The CYP21 extra copy is a pseudogene (CYP21A1P. In Brazil, 30-kb deletion forming monomodular alleles that carry chimeric CYP21A1P/A2 genes corresponds to ~9% of disease-causing alleles. Such alleles are considered to result from unequal crossovers within the bimodular C4/CYP21 locus. Depending on the localization of recombination breakpoint, different alleles can be generated conferring the locus high degree of allelic variability. The purpose of the study was to investigate the variability of deleted alleles in patients with 21-hydroxylase deficiency. Methods We used different techniques to investigate the variability of 30-kb deletion alleles in patients with 21-hydroxylase deficiency. Alleles were first selected after Southern blotting. The composition of CYP21A1P/A2 chimeric genes was investigated by ASO-PCR and MLPA analyses followed by sequencing to refine the location of recombination breakpoints. Twenty patients carrying at least one allele with C4/CYP21 30-kb deletion were included in the study. Results An allele carrying a CYP21A1P/A2 chimeric gene was found unusually associated to a C4B/C4A Taq I 6.4-kb fragment, generally associated to C4B and CYP21A1P deletions. A novel haplotype bearing both p.P34L and p.H62L, novel and rare mutations, respectively, was identified in exon 1, however p.P30L, the most frequent pseudogene-derived mutation in this exon, was absent. Four unrelated patients showed this haplotype. Absence of p.P34L in CYP21A1P of normal controls indicated that it is not derived from pseudogene. In addition, the combination of different

  8. A Schistosoma japonicum chimeric protein with a novel adjuvant induced a polarized Th1 immune response and protection against liver egg burdens

    Directory of Open Access Journals (Sweden)

    Xue Xiangyang

    2009-05-01

    Full Text Available Abstract Background Schitosomiasis japonica is still a significant public health problem in China. A protective vaccine for human or animal use represents an important strategy for long-term control of this disease. Due to the complex life cycle of schistosomes, different vaccine design approaches may be necessary, including polyvalent subunit vaccines. In this study, we constructed four chimeric proteins (designated SjGP-1~4 via fusion of Sj26GST and four individual paramyosin fragments. We tested these four proteins as vaccine candidates, and investigated the effect of deviating immune response on protection roles in mice. Methods The immunogencity and protection efficacy of chimeric proteins were evaluated in mice. Next, the chimeric protein SjGP-3 was selected and formulated in various adjuvants, including CFA, ISA 206, IMS 1312 and ISA 70M. The titers of antigen-specific IgG, IgE and IgG subclass were measured. The effect of adjuvant on cytokine production and percentages of CD3+CD8-IFN-γ+ cells and CD3+CD8-IL-4+ cells were analyzed at different time points. Worm burdens and liver egg counts in different adjuvant groups were counted to evaluate the protection efficacy against cercarial challenge. Results Immunization of mice with chimeric proteins provided various levels of protection. Among the four proteins, SjGP-3 induced the highest level of protection, and showed enhanced protective efficacy compared with its individual component Sj26GST. Because of this, SjGP-3 was further formulated in various adjuvants to investigate the effect of adjuvant on immune deviation. The results revealed that SjGP-3 formulated in veterinary adjuvant ISA 70M induced a lasting polarized Th1 immune response, whereas the other adjuvants, including CFA, ISA 206 and IMS 1312, generated a moderate mixed Th1/Th2 response after immunization but all except for IMS 1312 shifted to Th2 response after onset of eggs. More importantly, the SjGP-3/70M formulation induced

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

    International Nuclear Information System (INIS)

    Zhang Fanglin; Wu Xingan; Luo Wen; Bai Wentao; Liu Yong; Yan Yan; Wang Haitao; Xu Zhikai

    2007-01-01

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

  10. Development of a mouse-feline chimeric antibody against feline tumor necrosis factor-alpha

    Science.gov (United States)

    DOKI, Tomoyoshi; TAKANO, Tomomi; HOHDATSU, Tsutomu

    2016-01-01

    Feline infectious peritonitis (FIP) is a fatal inflammatory disease caused by FIP virus infection. Feline tumor necrosis factor (fTNF)-alpha is closely involved in the aggravation of FIP pathology. We previously described the preparation of neutralizing mouse anti-fTNF-alpha monoclonal antibody (mAb 2–4) and clarified its role in the clinical condition of cats with FIP using in vitro systems. However, administration of mouse mAb 2–4 to cat may lead to a production of feline anti-mouse antibodies. In the present study, we prepared a mouse-feline chimeric mAb (chimeric mAb 2–4) by fusing the variable region of mouse mAb 2–4 to the constant region of feline antibody. The chimeric mAb 2–4 was confirmed to have fTNF-alpha neutralization activity. Purified mouse mAb 2–4 and chimeric mAb 2–4 were repeatedly administered to cats, and the changes in the ability to induce feline anti-mouse antibody response were investigated. In the serum of cats treated with mouse mAb 2–4, feline anti-mouse antibody production was induced, and the fTNF-alpha neutralization effect of mouse mAb 2–4 was reduced. In contrast, in cats treated with chimeric mAb 2–4, the feline anti-mouse antibody response was decreased compared to that of mouse mAb 2–4-treated cats. PMID:27264736

  11. A Novel Chimeric Endolysin with Antibacterial Activity against Methicillin-Resistant Staphylococcus aureus.

    Science.gov (United States)

    Haddad Kashani, Hamed; Fahimi, Hossein; Dasteh Goli, Yasaman; Moniri, Rezvan

    2017-01-01

    Cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) and amidase are known as catalytic domains of the bacteriophage-derived endolysin LysK and were previously reported to show lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). In the current study, the in silico design and analysis of chimeric CHAP-amidase model was applied to enhance the stability and solubility of protein, which was achieved through improving the properties of primary, secondary and tertiary structures. The coding gene sequence of the chimeric CHAP-amidase was synthesized and subcloned into the pET-22(+) expression vector, and the recombinant protein was expressed in E. coli BL21 (DE3) strain. Subsequent affinity-based purification yielded ~12 mg soluble protein per liter of E. coli culture. Statistical analysis indicated that concentrations of ≥1 μg/mL of the purified protein have significant antibacterial activity against S. aureus MRSA 252 cells. The engineered chimeric CHAP-amidase exhibited 3.2 log reduction of MRSA 252 cell counts at the concentration of 10 μg/mL. A synergistic interaction between CHAP-amidase and vancomycin was detected by using checkerboard assay and calculating the fractional inhibitory concentration (FIC) index. This synergistic effect was shown by 8-fold reduction in the minimum inhibitory concentration of vancomycin. The chimeric CHAP-amidase displayed strong antibacterial activity against S. aureus, S. epidermidis , and enterococcus . However, it did not indicate any significant antibacterial activity against E. coli and Lactococcus lactis . Taken together, these findings suggest that our chimeric CHAP-amidase might represent potential to be used for the development of efficient antibacterial therapies targeting MRSA and certain Gram-positive bacteria.

  12. 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

    . 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...... 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...

  13. 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.

  14. How to train your T cell: genetically engineered chimeric antigen receptor T cells versus bispecific T-cell engagers to target CD19 in B acute lymphoblastic leukemia.

    Science.gov (United States)

    Ruella, Marco; Gill, Saar

    2015-06-01

    Antigen-specific T cell-based immunotherapy is getting its day in the sun. The contemporaneous development of two potent CD19-specific immunotherapeutic modalities for the treatment of B-cell malignancies provides exciting opportunities for patients, physicians and scientists alike. Patients with relapsed, refractory or poor-risk B-cell acute lymphoblastic leukemia (ALL) previously had few therapeutic options and now have two potential new lifelines. Physicians will have the choice between two powerful modalities and indeed could potentially enroll some patients on trials exploring both modalities if needed. For scientists interested in tumor immunology, the advent of chimeric antigen receptor T-cell therapy and of bispecific T-cell engagers (BiTEs) provides unprecedented opportunities to explore the promise and limitations of antigen-specific T-cell therapy in the context of human leukemia. In this article, we compare chimeric antigen receptor T cells and BiTEs targeting CD19 in B-cell ALL in the setting of the available clinical literature.

  15. Exploiting the yeast L-A viral capsid for the in vivo assembly of chimeric VLPs as platform in vaccine development and foreign protein expression.

    Directory of Open Access Journals (Sweden)

    Frank Powilleit

    Full Text Available A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8(+ memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i a heterologous model protein (GFP, (ii a per se toxic protein (K28 alpha-subunit, and (iii a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A. Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.

  16. Long-term persistence and function of hematopoietic stem cell-derived chimeric antigen receptor T cells in a nonhuman primate model of HIV/AIDS.

    Directory of Open Access Journals (Sweden)

    Anjie Zhen

    2017-12-01

    Full Text Available Chimeric Antigen Receptor (CAR T-cells have emerged as a powerful immunotherapy for various forms of cancer and show promise in treating HIV-1 infection. However, significant limitations are persistence and whether peripheral T cell-based products can respond to malignant or infected cells that may reappear months or years after treatment remains unclear. Hematopoietic Stem/Progenitor Cells (HSPCs are capable of long-term engraftment and have the potential to overcome these limitations. Here, we report the use of a protective CD4 chimeric antigen receptor (C46CD4CAR to redirect HSPC-derived T-cells against simian/human immunodeficiency virus (SHIV infection in pigtail macaques. CAR-containing cells persisted for more than 2 years without any measurable toxicity and were capable of multilineage engraftment. Combination antiretroviral therapy (cART treatment followed by cART withdrawal resulted in lower viral rebound in CAR animals relative to controls, and demonstrated an immune memory-like response. We found CAR-expressing cells in multiple lymphoid tissues, decreased tissue-associated SHIV RNA levels, and substantially higher CD4/CD8 ratios in the gut as compared to controls. These results show that HSPC-derived CAR T-cells are capable of long-term engraftment and immune surveillance. This study demonstrates for the first time the safety and feasibility of HSPC-based CAR therapy in a large animal preclinical model.

  17. 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.

  18. Development and Evaluation of a Novel ELISA for Detection of Antibodies against HTLV-I Using Chimeric Peptides.

    Science.gov (United States)

    Mosadeghi, Parvin; Heydari-Zarnagh, Hafez

    2018-04-01

    We aimed to develope a peptide-based indirect ELISA to detect antibodies against Human T-lymphotropic virus type I (HTLV-I). Two chimeric peptides (CP-1 and CP-2) were designed using linear immunodominant epitopes of gp-46-I, and gp21-I proteins, according to the sequence from Uniprot database. These peptides were studied initially in the ELISA using infected sera. The most promising peptideCP-1, was used to develop a peptide ELISA for detection of HTLV-I infected sera. The optimal conditions for CP-1ELISA were: the optimum coating buffer was 100mM NaHCO3, pH 9.6; coating peptide concentration was 10 µg/mL; the optimal blocking buffer was5% fetal bovine serum (FBS); the secondary antibody concentration was 1:2000; and serum dilution was 1:20. 20serum samples from HTLV-I infected patients were evaluated by ELISA developed. CP-1 showed high antigenicity while lacking any cross-reactivity with normal human sera. The results of evaluations indicated that in comparison with commercial ELISA, CP-1 ELISA showed good sensitivity and specificity. With further validation, CP-1as described in the present study could be introduced as novel reliable and cost-effective candidates for the high-specific screening of HTLV-I/-II infections in endemic regions.

  19. Characterization and biodistribution of recombinant and recombinant/chimeric constructs of monoclonal antibody B72.3

    International Nuclear Information System (INIS)

    Colcher, D.; Milenic, D.; Roselli, M.

    1989-01-01

    Radiolabeled B72.3 has been administered both i.v. and i.p. in patients with colorectal and ovarian cancer as well as other carcinomas and has been shown to selectively bind to approximately 70-80% of metastatic lesions. Greater than 50% of the patients that have been treated with B72.3 have developed an immunological response to murine IgG after a single injection. In an attempt to minimize the immune response of these patients to the administered murine monoclonal antibody, we developed a recombinant form of the murine B72.3 as well as a recombinant/chimeric antibody, using the variable regions of the murine B72.3 and human heavy chain (gamma 4) and light chain (kappa) constant regions. We report here that both the recombinant B72.3 [rB72.3] and the recombinant/chimeric B72.3 [cB72.3(gamma 4)] IgGs maintain the tissue binding and idiotypic specificity of the native murine IgG. The native B72.3, rB72.3, and cB72.3(gamma 4) IgGs were radiolabeled and the biodistribution of these IgGs was studied in athymic mice bearing human colon carcinoma xenografts (LS-174T). Differences were observed between the cB72.3(gamma 4) and the native B72.3 in the percentage of injected dose/g that localized in the tumor. The somewhat lower absolute amounts of the cB72.3(gamma 4) in the tumor are mostly likely due to the observed more rapid clearance from the blood and body of the mouse as compared to the native B72.3 and rB72.3. All three forms [native B72.3, rB72.3, and cB72.3(gamma 4)] of the IgG, however, were able to localize the colon tumor with similar radiolocalization indices [percentage of injected dose/g in tumor divided by the percentage of injected dose/g in normal tissue

  20. Variation in N-linked carbohydrate chains in different batches of two chimeric monoclonal IgG1 antibodies produced by different murine SP2/0 transfectoma cell subclones.

    Science.gov (United States)

    Bergwerff, A A; Stroop, C J; Murray, B; Holtorf, A P; Pluschke, G; Van Oostrum, J; Kamerling, J P; Vliegenthart, J F

    1995-06-01

    Two chimeric human/murine monoclonal antibodies were constructed by substitution of the murine constant regions with human gamma 1 and kappa constant regions for heavy and light chains, respectively. The chimeric human/murine molecules are anti-idiotypic antibodies, meaning that they were directed against the antigen binding site in the variable region of another antibody. Antibody batches were produced under identical production conditions, using two selected SP2/0 myeloma cell subclones, which produce chimeric antibodies with different variable regions, but identical constant regions. Several samples were collected during the production of the antibodies in hollow-fibre reactors. The heavy chain, but not the light chain, of the two different chimeric IgG1 antibodies is glycosylated. Structural analysis of the enzymically released N-linked carbohydrate chains by 1H-NMR spectroscopy, as well as by chromatographic profiling, demonstrated that the collection of N-glycans comprises a small amount of monoantennary, and for the greater part diantennary structures. The N-glycans are completely (alpha 1-->6)-fucosylated at the innermost GlcNAc residue. The antennae of the neutral diantennary N-glycans are built up from GlcNAc beta 1-->2, Gal beta 1-->4GlcNAc beta 1-->2 or Gal alpha 1-->3G alpha 1 beta 1-->4GlcNAc beta 1-->2 elements, whereas the antennae of the neutral monoantennary carbohydrate chains have only (beta 1-->2)-linked GlcNAc residues. Galactosylation of the GlcNAc beta 1-->2Man alpha 1-->6 branch occurs four times more frequently than that of the GlcNAc beta 1-->2Man alpha 1-->3 branch, independently of the production batch. A small amount of the diantennary N-glycans are mono- or disialylated, carrying N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), exclusively (alpha 2-->6)-linked to beta Gal. Analysis of the different production batches demonstrates that the structures of the N-linked carbohydrate chains are identical in the two

  1. Validation of chimerism in pediatric recipients of allogeneic hematopoietic stem cell transplantation (HSCT) a comparison between two methods: real-time PCR (qPCR) vs. variable number tandem repeats PCR (VNTR PCR).

    Science.gov (United States)

    Kletzel, Morris; Huang, Wei; Olszewski, Marie; Khan, Sana

    2013-01-01

    Post-hematopoietic stem cell transplantation (HSCT) chimerism monitoring is important to assess relapse and therapeutic intervention. The purpose of our study is to compare two methods variable number tandem repeats (VNTR) vs. quantitative real- time polymerase chain reaction (qPCR) in terms of determining chimerism. 127 (peripheral blood n=112, bone marrow n=15) samples were simultaneously tested by VNTR using APO-B, D1S80, D1S111, D17S30, gene loci SRY and ZP3 and qPCR using 34 assays (CA001-CA034) that are designed to a bi-allelic insertion/deletion (indel) polymorphism in the human genome. Samples were separated in three subsets: total WBC, T-cell and Myeloid cells. Extraction of DNA was performed then quantified. We analyzed column statistics, paired t-test and regression analysis for both methods. There was complete correlation between the two methods. The simplicity and rapidity of the test results from the qPCR method is more efficient and accurate to assess chimerism.

  2. Production and immunogenicity of chimeric virus-like particles containing the spike glycoprotein of infectious bronchitis virus.

    Science.gov (United States)

    Lv, Lishan; Li, Xiaoming; Liu, Genmei; Li, Ran; Liu, Qiliang; Shen, Huifang; Wang, Wei; Xue, Chunyi; Cao, Yongchang

    2014-01-01

    Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.

  3. The use of chimeric vimentin citrullinated peptides for the diagnosis of rheumatoid arthritis.

    Science.gov (United States)

    Malakoutikhah, Morteza; Gómara, María J; Gómez-Puerta, José A; Sanmartí, Raimon; Haro, Isabel

    2011-11-10

    Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes inflammation and, in many cases, destruction of the joints. To prevent progressive and irreversible structural damage, early diagnosis of RA is of paramount importance. The present study addresses the search of new RA citrullinated antigens that could supplement or complement diagnostic/prognostic existing tests. With this aim, the epitope anticitrullinated vimentin antibody response was mapped using synthetic peptides. To improve the sensitivity/specificity balance, a vimentin peptide that was selected, and its cyclic analogue, were combined with fibrin- and filaggrin-related peptides to render chimeric peptides. Our findings highlight the putative application of these chimeric peptides for the design of RA diagnosis systems and imply that more than one serological test is required to classify RA patients based on the presence or absence of ACPAs. Each of the target molecules reported here (fibrin, vimentin, filaggrin) has a specific utility in the identification of a particular subset of RA patients.

  4. Functional evolution and structural conservation in chimeric cytochromes p450: calibrating a structure-guided approach.

    Science.gov (United States)

    Otey, Christopher R; Silberg, Jonathan J; Voigt, Christopher A; Endelman, Jeffrey B; Bandara, Geethani; Arnold, Frances H

    2004-03-01

    Recombination generates chimeric proteins whose ability to fold depends on minimizing structural perturbations that result when portions of the sequence are inherited from different parents. These chimeric sequences can display functional properties characteristic of the parents or acquire entirely new functions. Seventeen chimeras were generated from two CYP102 members of the functionally diverse cytochrome p450 family. Chimeras predicted to have limited structural disruption, as defined by the SCHEMA algorithm, displayed CO binding spectra characteristic of folded p450s. Even this small population exhibited significant functional diversity: chimeras displayed altered substrate specificities, a wide range in thermostabilities, up to a 40-fold increase in peroxidase activity, and ability to hydroxylate a substrate toward which neither parent heme domain shows detectable activity. These results suggest that SCHEMA-guided recombination can be used to generate diverse p450s for exploring function evolution within the p450 structural framework.

  5. 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.

  6. Protein-like Nanoparticles Based on Orthogonal Self-Assembly of Chimeric Peptides.

    Science.gov (United States)

    Jiang, Linhai; Xu, Dawei; Namitz, Kevin E; Cosgrove, Michael S; Lund, Reidar; Dong, He

    2016-10-01

    A novel two-component self-assembling chimeric peptide is designed where two orthogonal protein folding motifs are linked side by side with precisely defined position relative to one another. The self-assembly is driven by a combination of symmetry controlled molecular packing, intermolecular interactions, and geometric constraint to limit the assembly into compact dodecameric protein nanoparticles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    International Nuclear Information System (INIS)

    Hermelin, T.; Brunner, H.; Daskalov, S.; Nakai, H.

    1983-01-01

    One important task of our group at IAEA is to develop procedures aiming to improve sampling of M 2 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 M 1 plants were progeny tested in M 2 . The position of the M 2 seeds on the M 1 plants has been recorded

  8. Bellerophon: a program to detect chimeric sequences in multiple sequence alignments.

    Science.gov (United States)

    Huber, Thomas; Faulkner, Geoffrey; Hugenholtz, Philip

    2004-09-22

    Bellerophon is a program for detecting chimeric sequences in multiple sequence datasets by an adaption of partial treeing analysis. Bellerophon was specifically developed to detect 16S rRNA gene chimeras in PCR-clone libraries of environmental samples but can be applied to other nucleotide sequence alignments. Bellerophon is available as an interactive web server at http://foo.maths.uq.edu.au/~huber/bellerophon.pl

  9. CCR 20th Anniversary Commentary: Chimeric Antigen Receptors-From Model T to the Tesla.

    Science.gov (United States)

    Hwu, Patrick

    2015-07-15

    The research article by Kershaw and colleagues, published in the October 15, 2006, issue of Clinical Cancer Research, presents one of the first clinical trials to utilize chimeric antigen receptors. Subsequent studies have shown promise for the treatment of patients with lymphoid malignancies, but further progress will require optimization, including the identification of more specific antigens for solid tumors. ©2015 American Association for Cancer Research.

  10. Live Zika virus chimeric vaccine candidate based on a yellow fever 17-D attenuated backbone

    OpenAIRE

    Nougairede, Antoine; Klitting, Raphaelle; Aubry, Fabien; Gilles, Magali; Touret, Franck; De Lamballerie, Xavier

    2018-01-01

    Zika virus (ZIKV) recently dispersed throughout the tropics and sub-tropics causing epidemics associated with congenital disease and neurological complications. There is currently no commercial vaccine for ZIKV. Here we describe the initial development of a chimeric virus containing the prM/E proteins of a ZIKV epidemic strain incorporated into a yellow fever 17-D attenuated backbone. Using the versatile and rapid ISA (Infectious Subgenomic Amplicons) reverse genetics method, we compared diff...

  11. EVIR: chimeric receptors that enhance dendritic cell cross-dressing with tumor antigens.

    Science.gov (United States)

    Squadrito, Mario Leonardo; Cianciaruso, Chiara; Hansen, Sarah K; De Palma, Michele

    2018-03-01

    We describe a lentivirus-encoded chimeric receptor, termed extracellular vesicle (EV)-internalizing receptor (EVIR), which enables the selective uptake of cancer-cell-derived EVs by dendritic cells (DCs). The EVIR enhances DC presentation of EV-associated tumor antigens to CD8 + T cells primarily through MHCI recycling and cross-dressing. EVIRs should facilitate exploring the mechanisms and implications of horizontal transfer of tumor antigens to antigen-presenting cells.

  12. Novel chimeric peptide with enhanced cell specificity and anti-inflammatory activity.

    Science.gov (United States)

    Kim, Young-Min; Kim, Nam-Hong; Lee, Jong-Wan; Jang, Jin-Sun; Park, Yung-Hoon; Park, Seong-Cheol; Jang, Mi-Kyeong

    2015-07-31

    An antimicrobial peptide (AMP), Hn-Mc, was designed by combining the N-terminus of HPA3NT3 and the C-terminus of melittin. This chimeric AMP exhibited potent antibacterial activity with low minimal inhibitory concentrations (MICs), ranging from 1 to 2 μM against four drug-susceptible bacteria and ten drug-resistant bacteria. Moreover, the hemolysis and cytotoxicity was reduced significantly compared to those of the parent peptides, highlighting its high cell selectivity. The morphological changes in the giant unilamellar vesicles and bacterial cell surfaces caused by the Hn-Mc peptide suggested that it killed the microbial cells by damaging the membrane envelope. An in vivo study also demonstrated the antibacterial activity of the Hn-Mc peptide in a mouse model infected with drug-resistant bacteria. In addition, the chimeric peptide inhibited the expression of lipopolysaccharide (LPS)-induced cytokines in RAW 264.7 cells by preventing the interaction between LPS and Toll-like receptors. These results suggest that this chimeric peptide is an antimicrobial and anti-inflammatory candidate as a pharmaceutic agent. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Functional rescue of dystrophin-deficient mdx mice by a chimeric peptide-PMO.

    Science.gov (United States)

    Yin, Haifang; Moulton, Hong M; Betts, Corinne; Merritt, Thomas; Seow, Yiqi; Ashraf, Shirin; Wang, Qingsong; Boutilier, Jordan; Wood, Matthew Ja

    2010-10-01

    Splice modulation using antisense oligonucleotides (AOs) has been shown to yield targeted exon exclusion to restore the open reading frame and generate truncated but partially functional dystrophin protein. This has been successfully demonstrated in dystrophin-deficient mdx mice and in Duchenne muscular dystrophy (DMD) patients. However, DMD is a systemic disease; successful therapeutic exploitation of this approach will therefore depend on effective systemic delivery of AOs to all affected tissues. We have previously shown the potential of a muscle-specific/arginine-rich chimeric peptide-phosphorodiamidate morpholino (PMO) conjugate, but its long-term activity, optimized dosing regimen, capacity for functional correction and safety profile remain to be established. Here, we report the results of this chimeric peptide-PMO conjugate in the mdx mouse using low doses (3 and 6 mg/kg) administered via a 6 biweekly systemic intravenous injection protocol. We show 100% dystrophin-positive fibers and near complete correction of the dystrophin transcript defect in all peripheral muscle groups, with restoration of 50% dystrophin protein over 12 weeks, leading to correction of the DMD pathological phenotype and restoration of muscle function in the absence of detectable toxicity or immune response. Chimeric muscle-specific/cell-penetrating peptides therefore represent highly promising agents for systemic delivery of splice-correcting PMO oligomers for DMD therapy.

  14. 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.

  15. Connections between Transcription Downstream of Genes and cis-SAGe Chimeric RNA.

    Science.gov (United States)

    Chwalenia, Katarzyna; Qin, Fujun; Singh, Sandeep; Tangtrongstittikul, Panjapon; Li, Hui

    2017-11-22

    cis-Splicing between adjacent genes (cis-SAGe) is being recognized as one way to produce chimeric fusion RNAs. However, its detail mechanism is not clear. Recent study revealed induction of transcriptions downstream of genes (DoGs) under osmotic stress. Here, we investigated the influence of osmotic stress on cis-SAGe chimeric RNAs and their connection to DoGs. We found,the absence of induction of at least some cis-SAGe fusions and/or their corresponding DoGs at early time point(s). In fact, these DoGs and their cis-SAGe fusions are inversely correlated. This negative correlation was changed to positive at a later time point. These results suggest a direct competition between the two categories of transcripts when total pool of readthrough transcripts is limited at an early time point. At a later time point, DoGs and corresponding cis-SAGe fusions are both induced, indicating that total readthrough transcripts become more abundant. Finally, we observed overall enhancement of cis-SAGe chimeric RNAs in KCl-treated samples by RNA-Seq analysis.

  16. Generation of Chimeric RNAs by cis-splicing of adjacent genes (cis-SAGe) in mammals.

    Science.gov (United States)

    Zhuo, Jian-Shu; Jing, Xiao-Yan; Du, Xin; Yang, Xiu-Qin

    2018-02-20

    Chimeric RNA molecules, possessing exons from two or more independent genes, are traditionally believed to be produced by chromosome rearrangement. However, recent studies revealed that cis-splicing of adjacent genes (cis- SAGe) is one of the major mechanisms underlying the formation of chimeric RNAs. cis-SAGe refers to intergenic splicing of directly adjacent genes with the same transcriptional orientation, resulting in read-through transcripts, termed chimeric RNAs, which contain sequences from two or more parental genes. cis-SAGe was first identified in tumor cells, since then its potential in carcinogenesis has attracted extensive attention. More and more scientists are focusing on it. With the development of research, cis-SAGe was found to be ubiquitous in various normal tissues, and might make a crucial contribution to the formation of novel genes in the evolution of genomes. In this review, we summarize the splicing pattern, expression characteristics, possible mechanisms, and significance of cis-SAGe in mammals. This review will be helpful for general understanding of the current status and development tendency of cis-SAGe.

  17. Design of chimeric expression elements that confer high-level gene activity in chromoplasts.

    Science.gov (United States)

    Caroca, Rodrigo; Howell, Katharine A; Hasse, Claudia; Ruf, Stephanie; Bock, Ralph

    2013-02-01

    Non-green plastids, such as chromoplasts, generally have much lower activity of gene expression than chloroplasts in photosynthetically active tissues. Suppression of plastid genes in non-green tissues occurs through a complex interplay of transcriptional and translational control, with the contribution of regulation of transcript abundance versus translational activity being highly variable between genes. Here, we have investigated whether the low expression of the plastid genome in chromoplasts results from inherent limitations in gene expression capacity, or can be overcome by designing appropriate combinations of promoters and translation initiation signals in the 5' untranslated region (5'-UTR). We constructed chimeric expression elements that combine promoters and 5'-UTRs from plastid genes, which are suppressed during chloroplast-to-chromoplast conversion in Solanum lycopersicum (tomato) fruit ripening, either just at the translational level or just at the level of mRNA accumulation. These chimeric expression elements were introduced into the tomato plastid genome by stable chloroplast transformation. We report the identification of promoter-UTR combinations that confer high-level gene expression in chromoplasts of ripe tomato fruits, resulting in the accumulation of reporter protein GFP to up to 1% of total cellular protein. Our work demonstrates that non-green plastids are capable of expressing genes to high levels. Moreover, the chimeric cis-elements for chromoplasts developed here are widely applicable in basic and applied research using transplastomic methods. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

  18. 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.

  19. Mast Cell Targeted Chimeric Toxin Can Be Developed as an Adjunctive Therapy in Colon Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Shan Wang

    2016-03-01

    Full Text Available The association of colitis with colorectal cancer has become increasingly clear with mast cells being identified as important inflammatory cells in the process. In view of the relationship between mast cells and cancer, we studied the effect and mechanisms of mast cells in the development of colon cancer. Functional and mechanistic insights were gained from ex vivo and in vivo studies of cell interactions between mast cells and CT26 cells. Further evidence was reversely obtained in studies of mast cell targeted Fcε-PE40 chimeric toxin. Experiments revealed mast cells could induce colon tumor cell proliferation and invasion. Cancer progression was found to be related to the density of mast cells in colonic submucosa. The activation of MAPK, Rho-GTPase, and STAT pathways in colon cancer cells was triggered by mast cells during cell-to-cell interaction. Lastly, using an Fcε-PE40 chimeric toxin we constructed, we confirmed the promoting effect of mast cells in development of colon cancer. Mast cells are a promoting factor of colon cancer and thus also a potential therapeutic target. The Fcε-PE40 chimeric toxin targeting mast cells could effectively prevent colon cancer in vitro and in vivo. Consequently, these data may demonstrate a novel immunotherapeutic approach for the treatment of tumors.

  20. A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells.

    Science.gov (United States)

    Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Hamana, Hiroshi; Nakagawa, Hidetoshi; Jin, Aishun; Lin, Zhezhu; Muraguchi, Atsushi

    2014-10-31

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

    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, 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

  2. Reprogramming of rabbit induced pluripotent stem cells toward epiblast and chimeric competency using Krüppel-like factors

    Directory of Open Access Journals (Sweden)

    Yann Tapponnier

    2017-10-01

    Full Text Available Rabbit induced pluripotent stem cells (rbiPSCs possess the characteristic features of primed pluripotency as defined in rodents and primates. In the present study, we reprogrammed rbiPSCs using human Krüppel-like factors (KLFs 2 and 4 and cultured them in a medium supplemented with fetal calf serum and leukemia inhibitory factor. These cells (designated rbEKA were propagated by enzymatic dissociation for at least 30 passages, during which they maintained a normal karyotype. This new culturing protocol resulted in transcriptional and epigenetic reconfiguration, as substantiated by the expression of transcription factors and the presence of histone modifications associated with naïve pluripotency. Furthermore, microarray analysis of rbiPSCs, rbEKA cells, rabbit ICM cells, and rabbit epiblast showed that the global gene expression profile of the reprogrammed rbiPSCs was more similar to that of rabbit ICM and epiblast cells. Injection of rbEKA cells into 8-cell stage rabbit embryos resulted in extensive colonization of ICM in 9% early-blastocysts (E3.5, epiblast in 10% mid-blastocysts (E4.5, and embryonic disk in 1.4% pre-gastrulae (E6. Thus, these results indicate that KLF2 and KLF4 triggered the conversion of rbiPSCs into epiblast-like, embryo colonization-competent PSCs. Our results highlight some of the requirements to achieve bona fide chimeric competency.

  3. Early CD3+/CD15+ peripheral blood leukocyte chimerism patterns correlate with long-term engraftment in non-malignant hematopoietic SCT.

    Science.gov (United States)

    Ketterl, T G; Flesher, M; Shanley, R; Miller, W

    2014-04-01

    Following hematopoietic SCT (HSCT) for non-malignant disorders (NMDs) variable donor chimerism among lympho-hematopoietic lines may be observed. We retrospectively evaluated early post-HSCT, lineage-sorted (CD3+ and CD15+) peripheral blood leukocyte chimerism data to characterize patterns and assess for association with long-term CD15+ engraftment. 'Early' was defined as the first value obtained between days +14 and +42, 'late' as the last recorded value after day +90. 'High' donor chimerism was defined as 80% on either fraction at all time-points. Patients were classified into four subgroups with respect to early CD3+/CD15+ chimerism patterns (high/low) then analyzed for long-term CD15+ chimerism status. A total of 135 transplants were evaluable, with all three time-points available in 97. Underlying disease, graft source, patient age and conditioning intensity varied. 'Split' early chimerism (discordant high/low CD3+/CD15+ status) was common. Multivariable analysis revealed strong association between conditioning regimen and primary disease on early CD3+/CD15+ chimerism patterns and a dominant predictive effect of early CD15+ chimerism on long-term CD15+ donor engraftment (observed at median day +365). These data may guide real-time clinician decisions (restraint vs intervention, when available) when faced with unfavorable or unusual early lympho-hematopoietic chimerism patterns following HSCT for NMD.

  4. Immunogenicity of a modified-live virus vaccine against bovine viral diarrhea virus types 1 and 2, infectious bovine rhinotracheitis virus, bovine parainfluenza-3 virus, and bovine respiratory syncytial virus when administered intranasally in young calves.

    Science.gov (United States)

    Xue, Wenzhi; Ellis, John; Mattick, Debra; Smith, Linda; Brady, Ryan; Trigo, Emilio

    2010-05-14

    The immunogenicity of an intranasally-administered modified-live virus (MLV) vaccine in 3-8 day old calves was evaluated against bovine viral diarrhea virus (BVDV) types 1 and 2, infectious bovine rhinotracheitis (IBR) virus, parainfluenza-3 (PI-3) virus and bovine respiratory syncytial virus (BRSV). Calves were intranasally vaccinated with a single dose of a multivalent MLV vaccine and were challenged with one of the respective viruses three to four weeks post-vaccination in five separate studies. There was significant sparing of diseases in calves intranasally vaccinated with the MLV vaccine, as indicated by significantly fewer clinical signs, lower rectal temperatures, reduced viral shedding, greater white blood cell and platelet counts, and less severe pulmonary lesions than control animals. This was the first MLV combination vaccine to demonstrate efficacy against BVDV types 1 and 2, IBR, PI-3 and BRSV in calves 3-8 days of age. Copyright 2010 Elsevier Ltd. All rights reserved.

  5. Differential impact of respiratory syncytial virus and parainfluenza virus on the frequency of acute otitis media is explained by lower adaptive and innate immune responses in otitis-prone children.

    Science.gov (United States)

    Verhoeven, David; Xu, Qingfu; Pichichero, Michael E

    2014-08-01

    Acute otitis media (AOM) is a leading cause of bacterial pediatric infections associated with viral upper respiratory infections (URIs). We examined the differential impact of respiratory syncytial virus (RSV) and parainfluenza virus URIs on the frequency of AOM caused by Streptococcus pneumoniae (Spn) and nontypeable Haemophilus influenzae (NTHi) in stringently defined otitis-prone (sOP) and non-otitis-prone (NOP) children as a potential mechanism to explain increased susceptibility to AOM. Peripheral blood and nasal washes were obtained from sOP and NOP children (n = 309). Colonization events and antiviral responses consisting of total specific immunoglobulin G (IgG) responses, neutralizing antibody responses, and T-cell responses were determined. Isolated neutrophils were infected with varying multiplicities of infection of both viruses, and opsonophagocytosis potential was measured. A significant increase was found in frequency of AOM events caused by Spn and NTHi, with a concurrent RSV infection in sOP children. These results correlated with diminished total RSV-specific IgG, higher viral nasal burdens, and lower IgG neutralizing capacity. The sOP children had diminished T-cell responses to RSV that correlated with lower Toll-like receptor 3/7 transcript and decreased expression of HLA-DR on antigen-presenting cells. RSV interfered with the Spn phagocytic capacity of neutrophils in a dose-dependent manner. Parainfluenza virus infections did not differentially affect AOM events in sOP and NOP children. Lower innate and adaptive immune responses to RSV in sOP children may slow the kinetics of viral clearance from the nasopharynx and allow for viral interference with antibacterial immune responses, thus contributing to increased frequency of AOMs. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity

    International Nuclear Information System (INIS)

    Qian Weizhu; Wang Ling; Li Bohua; Wang Hao; Hou Sheng; Hong Xueyu; Zhang Dapeng; Guo Yajun

    2008-01-01

    Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application

  7. 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.

  8. Myco-fluidics: The fluid dynamics of fungal chimerism

    Science.gov (United States)

    Roper, Marcus; Hickey, Patrick; Dressaire, Emilie; Roch, Sebastien

    2012-11-01

    Chimeras-fantastical creatures formed as amalgams of many animals-have captured the human imagination since Ancient times. But they are also surprisingly common in Nature. The syncytial cells of filamentous fungi harbor large numbers of nuclei bathed in a single cytoplasm. As a fungus grows these nuclei become genetically diverse, either from mutation or from exchange of nuclei between different fungal individuals, a process that is known to increase the virulence of the fungus and its adaptability. By directly measuring nuclear movement in the model ascomycete fungus Neurospora crassa, we show that the fungus' tolerance for internal genetic diversity is enabled by hydrodynamic mixing of nuclei acting at all length scales within the fungal mycelium. Mathematical modeling and experiments in a mutant with altered mycelial morphology reveal some of the exquisite hydraulic engineering necessary to create these mixing flows from spatially coarse pressure gradients.

  9. An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

    Directory of Open Access Journals (Sweden)

    Marcela V Maus

    2016-01-01

    Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

  10. In-silico design, expression, and purification of novel chimeric Escherichia coli O157:H7 OmpA fused to LTB protein in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Aytak Novinrooz

    Full Text Available E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC, and fatal hemolytic uremic syndrome (HUS and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E. coli O157:H7 containing loop 2-4 of E. coli O157:H7, outer membrane protein A (OmpA, and B subunit of E. coli heat labile enterotoxin (LTB which are connected by a flexible peptide linker. Several online databases and bioinformatics software were utilized to choose the peptide linker among 537 analyzed linkers, design the chimeric protein, and optimize the codon of the relative gene encoding this protein. Subsequently, the recombinant gene encoding OmpA-LTB was synthesized and cloned into pET-24a (+ expression vector and transferred to E. coli BL21(DE3 cells. The expression of OmpA-LTB chimeric protein was then carried out by induction of cultured E. coli Bl21 (DE3 cells with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG. The purification of OmpA-LTB was then performed by nickel affinity chromatography. Expression and purification were analyzed by sodium dodecyl sulphate poly acrylamide gel electrophoresis. Moreover, the identity of the expressed protein was analyzed by western blotting. SDS-PAGE and western immunoblotting confirmed the successful expression of a 27 KDa recombinant protein after 24 hours at 37°C post-IPTG induction. OmpA-LTB was then successfully purified, using nickel affinity chromatography under denaturing conditions. The yield of purification was 12 mg per liter of culture media. Ultimately, we constructed the successful design and efficient

  11. Crystal Structures of Yeast-Produced Enterovirus 71 and Enterovirus 71/Coxsackievirus A16 Chimeric Virus-Like Particles Provide the Structural Basis for Novel Vaccine Design against Hand-Foot-and-Mouth Disease.

    Science.gov (United States)

    Lyu, Ke; He, Ya-Ling; Li, Hao-Yang; Chen, Rong

    2015-06-01

    Human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two major causative agents for hand-foot-and-mouth disease (HFMD). Previously, we demonstrated that a virus-like particle (VLP) for EV71 produced from Saccharomyces cerevisiae is a potential vaccine candidate against EV71 infection, and an EV71/CVA16 chimeric VLP can elicit protective immune responses against both virus infections. Here, we presented the crystal structures of both VLPs, showing that both the linear and conformational neutralization epitopes identified in EV71 are mostly preserved on both VLPs. The replacement of only 4 residues in the VP1 GH loop converted strongly negatively charged surface patches formed by portions of the SP70 epitope in EV71 VLP into a relatively neutral surface in the chimeric VLP, which likely accounted for the additional neutralization capability of the chimeric VLP against CVA16 infection. Such local variations in the amino acid sequences and the surface charge potential are also present in different types of polioviruses. In comparison to EV71 VLP, the chimeric VLP exhibits structural changes at the local site of amino acid replacement and the surface loops of all capsid proteins. This is consistent with the observation that the VP1 GH loop located near the pseudo-3-fold junction is involved in extensive interactions with other capsid regions. Furthermore, portions of VP0 and VP1 in EV71 VLP are at least transiently exposed, revealing the structural flexibility of the VLP. Together, our structural analysis provided insights into the structural basis of enterovirus neutralization and novel vaccine design against HFMD and other enterovirus-associated diseases. Our previous studies demonstrated that the enterovirus 71 (EV71) virus-like particle (VLP) produced from yeast is a vaccine candidate against EV71 infection and that a chimeric EV71/coxsackievirus A16 (CVA16) VLP with the replacement of 4 amino acids in the VP1 GH loop can confer protection against both

  12. Shape-specific nanostructured protein mimics from de novo designed chimeric peptides.

    Science.gov (United States)

    Jiang, Linhai; Yang, Su; Lund, Reidar; Dong, He

    2018-01-30

    Natural proteins self-assemble into highly-ordered nanoscaled architectures to perform specific functions. The intricate functions of proteins have provided great impetus for researchers to develop strategies for designing and engineering synthetic nanostructures as protein mimics. Compared to the success in engineering fibrous protein mimetics, the design of discrete globular protein-like nanostructures has been challenging mainly due to the lack of precise control over geometric packing and intermolecular interactions among synthetic building blocks. In this contribution, we report an effective strategy to construct shape-specific nanostructures based on the self-assembly of chimeric peptides consisting of a coiled coil dimer and a collagen triple helix folding motif. Under salt-free conditions, we showed spontaneous self-assembly of the chimeric peptides into monodisperse, trigonal bipyramidal-like nanoparticles with precise control over the stoichiometry of two folding motifs and the geometrical arrangements relative to one another. Three coiled coil dimers are interdigitated on the equatorial plane while the two collagen triple helices are located in the axial position, perpendicular to the coiled coil plane. A detailed molecular model was proposed and further validated by small angle X-ray scattering experiments and molecular dynamics (MD) simulation. The results from this study indicated that the molecular folding of each motif within the chimeric peptides and their geometric packing played important roles in the formation of discrete protein-like nanoparticles. The peptide design and self-assembly mechanism may open up new routes for the construction of highly organized, discrete self-assembling protein-like nanostructures with greater levels of control over assembly accuracy.

  13. Chimeric immunoglobulin E reactive with tumor-associated antigen activates human Fc epsilon RI bearing cells

    NARCIS (Netherlands)

    Luiten, R. M.; Warnaar, S. O.; Schuurman, J.; Pasmans, S. G.; Latour, S.; Daëron, M.; Fleuren, G. J.; Litvinov, S. V.

    1997-01-01

    Crosslinking of immunoglobulin E molecules that are bound to the Fc epsilon receptors expressed on mast cells or basophils triggers activation of these cells, resulting in the development of a type I hypersensitivity. Targeting this potent immune reaction towards tumors by using IgE that reacts with

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

    DEFF Research Database (Denmark)

    Ekelund, Charlotte Kvist; Skibsted, L.; Søgaard, Kirsten

    2008-01-01

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  16. Predominant or complete recipient T-cell chimerism following alemtuzumab-based allogeneic transplantation is reversed by donor lymphocytes and not associated with graft failure.

    Science.gov (United States)

    Mohamedbhai, Sajir G; Edwards, Noha; Morris, Emma C; Mackinnon, Stephen; Thomson, Kirsty J; Peggs, Karl S

    2012-02-01

    The clinical significance of mixed chimerism following allogeneic haematopoietic stem cell transplantation (HSCT) remains controversial. Its relevance and incidence are probably influenced by the conditioning regimen and incorporation of T-cell depletion. The presence of recipient chimerism levels >40-50% following T-cell replete reduced intensity transplantation correlates with a high risk of graft rejection, regardless of donor-lymphocyte infusions, but it is unclear whether this finding translates to T-cell depleted transplants. We conducted a retrospective single-institution analysis of patients receiving alemtuzumab-based HSCT. 27/152 (18%) evaluable cases had predominantly recipient T-cell chimerism at 3 months or beyond. By contrast, coincident chimerism in the granulocyte lineage was predominantly of donor origin (median 100%) in all but one patient. Donor lymphocyte infusion effectively converted predominantly recipient T-cell chimerism to ful donor chimerism in all evaluable cases including three cases with no detectable donor T cells. The only graft failure occurred in the patient with predominantly recipient myeloid chimerism in whom rejection occurred rapidly before donor lymphocytes could be administered. We conclude that predominant or complete recipient T-cell chimerism following alemtuzumab-based regimens does not have the same clinical implications as that following T-cell replete transplants and can be effectively converted with donor lymphocytes without the need for lympho-depleting agents or re-conditioning. © 2011 Blackwell Publishing Ltd.

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

    DEFF Research Database (Denmark)

    Drent, Esther; Groen, Richard W. J.; Noort, Willy A. Noort

    2016-01-01

    Adoptive transfer of chimeric antigen receptor-transduced T cells is a promising strategy for cancer immunotherapy. The CD38 molecule, with its high expression on multiple myeloma cells, appears a suitable target for antibody therapy. Prompted by this, we used three different CD38 antibody...... sequences to generate second-generation retroviral CD38- chimeric antigen receptor constructs with which we transduced T cells from healthy donors and multiple myeloma patients. We then evaluated the preclinical efficacy and safety of the transduced T cells. Irrespective of the donor and antibody sequence......, CD38-chimeric antigen receptor-transduced T cells proliferated, produced inflammatory cytokines and effectively lysed malignant cell lines and primary malignant cells from patients with acute myeloid leukemia and multi-drug resistant multiple myeloma in a cell-dose, and CD38-dependent manner, despite...

  18. Neurophysiological evidence (ERPs) for hemispheric processing of facial expressions of emotions: Evidence from whole face and chimeric face stimuli.

    Science.gov (United States)

    Damaskinou, Nikoleta; Watling, Dawn

    2018-05-01

    This study was designed to investigate the patterns of electrophysiological responses of early emotional processing at frontocentral sites in adults and to explore whether adults' activation patterns show hemispheric lateralization for facial emotion processing. Thirty-five adults viewed full face and chimeric face stimuli. After viewing two faces, sequentially, participants were asked to decide which of the two faces was more emotive. The findings from the standard faces and the chimeric faces suggest that emotion processing is present during the early phases of face processing in the frontocentral sites. In particular, sad emotional faces are processed differently than neutral and happy (including happy chimeras) faces in these early phases of processing. Further, there were differences in the electrode amplitudes over the left and right hemisphere, particularly in the early temporal window. This research provides supporting evidence that the chimeric face test is a test of emotion processing that elicits right hemispheric processing.

  19. A multiscale model to evaluate the efficacy of anticancer therapies based on chimeric polypeptide nanoparticles

    Science.gov (United States)

    Paiva, L. R.; Martins, M. L.

    2011-01-01

    A multiscale model for tumor growth and its chemotherapy using conjugate nanoparticles is presented, and the corresponding therapeutic outcomes are evaluated. It is found that doxorubicin assembled into chimeric polypeptide nanoparticles cannot eradicate either vascularized primary tumors or avascular micrometastasis even administrated at loads close to their maximum tolerated doses. Furthermore, an effective and safety treatment demands for conjugate nanoparticles targeted to the malignant cells with much higher specificity and affinity than those currently observed in order to leave most of the normal tissues unaffected and to ensure a fast intracellular drug accumulation.

  20. A chimeric measles virus with canine distemper envelope protects ferrets from lethal distemper challenge.

    Science.gov (United States)

    Rouxel, Ronan Nicolas; Svitek, Nicholas; von Messling, Veronika

    2009-08-06

    CDV infects a broad range of carnivores, and over the past decades it has caused outbreaks in a variety of wild carnivore populations. Since the currently available live-attenuated vaccine is not sufficiently safe in these highly susceptible species, we produced a chimeric virus combining the replication complex of the measles Moraten vaccine strain with the envelope of a recent CDV wild type isolate. The resulting virus did not cause disease or immunosuppression in ferrets and conferred protection from challenge with a lethal wild type strain, demonstrating its potential value for wildlife conservation efforts.

  1. High-level expression and characterization of a chimeric lipase from Rhizopus oryzae for biodiesel production.

    Science.gov (United States)

    Yu, Xiao-Wei; Sha, Chong; Guo, Yong-Liang; Xiao, Rong; Xu, Yan

    2013-02-21

    Production of biodiesel from non-edible oils is receiving increasing attention. Tung oil, called "China wood oil" is one kind of promising non-edible biodiesel oil in China. To our knowledge, tung oil has not been used to produce biodiesel by enzymatic method. The enzymatic production of biodiesel has been investigated extensively by using Rhizopus oryzae lipase as catalyst. However, the high cost of R. oryzae lipase remains a barrier for its industrial applications. Through different heterologous expression strategies and fermentation techniques, the highest expression level of the lipase from R. oryzae reached 1334 U/mL in Pichia pastoris, which is still not optimistic for industry applications. The prosequence of lipases from Rhizopus sp. is very important for the folding and secretion of an active lipase. A chimeric lipase from R. oryzae was constructed by replacing the prosequence with that from the R. chinensis lipase and expressed in P. pastoris. The maximum activity of the chimera reached 4050 U/mL, which was 11 fold higher than that of the parent. The properties of the chimera were studied. The immobilized chimera was used successfully for biodiesel production from tung oil, which achieved higher FAME yield compared with the free chimeric lipase, non-chimeric lipase and mature lipase. By response surface methodology, three variables, water content, methanol to tung oil molar ratio and enzyme dosage were proved to be crucial parameters for biosynthesis of FAME and the FAME yield reached 91.9±2.5% at the optimized conditions by adding 5.66 wt.% of the initial water based on oil weight, 3.88 of methanol to tung oil molar ratio and 13.24 wt.% of enzyme concentration based on oil weight at 40°C. This is the first report on improving the expression level of the lipase from R. oryzae by replacing prosequences. The immobilized chimera was used successfully for biodiesel production from tung oil. Using tung oil as non-edible raw material and a chimeric lipase

  2. Transplantation Tolerance through Hematopoietic Chimerism: Progress and Challenges for Clinical Translation

    Directory of Open Access Journals (Sweden)

    Benedikt Mahr

    2017-12-01

    Full Text Available The perception that transplantation of hematopoietic stem cells can confer tolerance to any tissue or organ from the same donor is widely accepted but it has not yet become a treatment option in clinical routine. The reasons for this are multifaceted but can generally be classified into safety and efficacy concerns that also became evident from the results of the first clinical pilot trials. In comparison to standard immunosuppressive therapies, the infection risk associated with the cytotoxic pre-conditioning necessary to allow allogeneic bone marrow engraftment and the risk of developing graft-vs.-host disease (GVHD constitute the most prohibitive hurdles. However, several approaches have recently been developed at the experimental level to reduce or even overcome the necessity for cytoreductive conditioning, such as costimulation blockade, pro-apoptotic drugs, or Treg therapy. But even in the absence of any hazardous pretreatment, the recipients are exposed to the risk of developing GVHD as long as non-tolerant donor T cells are present. Total lymphoid irradiation and enriching the stem cell graft with facilitating cells emerged as potential strategies to reduce this peril. On the other hand, the long-lasting survival of kidney allografts, seen with transient chimerism in some clinical series, questions the need for durable chimerism for robust tolerance. From a safety point of view, loss of chimerism would indeed be favorable as it eliminates the risk of GVHD, but also complicates the assessment of tolerance. Therefore, other biomarkers are warranted to monitor tolerance and to identify those patients who can safely be weaned off immunosuppression. In addition to these safety concerns, the limited efficacy of the current pilot trials with approximately 40–60% patients becoming tolerant remains an important issue that needs to be resolved. Overall, the road ahead to clinical routine may still be rocky but the first successful long

  3. Chimeric antigen receptor T cell therapy in pancreatic cancer: from research to practice.

    Science.gov (United States)

    Jindal, Vishal; Arora, Ena; Masab, Muhammad; Gupta, Sorab

    2018-05-04

    Chimeric antigen receptor (CAR) T cell therapy is genetically engineered tumor antigen-specific anticancer immunotherapy, which after showing great success in hematological malignancies is currently being tried in advanced solid tumors like pancreatic cancer. Immunosuppressive tumor microenvironment and dense fibrous stroma are some of the limitation in the success of this novel therapy. However, genetic modifications and combination therapy is the topic of the research to improve its efficacy. In this article, we summarize the current state of knowledge, limitations, and future prospects for CAR T cell therapy in pancreatic cancer.

  4. Expression of kenaf mitochondrial chimeric genes HM184 causes male sterility in transgenic tobacco plants.

    Science.gov (United States)

    Zhao, Yanhong; Liao, Xiaofang; Huang, Zhipeng; Chen, Peng; Zhou, Bujin; Liu, Dongmei; Kong, Xiangjun; Zhou, Ruiyang

    2015-08-01

    Chimeric genes resulting from the rearrangement of a mitochondrial genome were generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). In the study, earlier we reported that identifying a 47 bp deletion at 3'- flanking of atp9 that was linked to male sterile cytoplasm in kenaf. The truncated fragment was fused with atp9, a mitochondrial transit signal (MTS) and/or GFP, comprised two chimeric genes MTS-HM184-GFP and MTS-HM184. The plant expression vector pBI121 containing chimeric genes were then introduced to tobacco plants by Agrobacterium-mediated T-DNA transformation. The result showed that certain transgenic plants were male sterility or semi-sterility, while some were not. The expression analysis further demonstrated that higher level of expression were showed in the sterility plants, while no expression or less expression in fertility plants, the levels of expression of semi-sterility were in between. And the sterile plant (containing MTS-HM184-GFP) had abnormal anther produced malformed/shriveled pollen grains stained negative that failed to germinate (0%), the corresponding fruits was shrunken, the semi-sterile plants having normal anther shape produced about 10-50% normal pollen grains, the corresponding fruits were not full, and the germination rate was 58%. Meanwhile these transgenic plants which altered on fertility were further analyzed in phenotype. As a result, the metamorphosis leaves were observed in the seedling stage, the plant height of transgenic plants was shorter than wild type. The growth duration of transgenic tobacco was delayed 30-45 days compared to the wild type. The copy numbers of target genes of transgenic tobacco were analyzed using the real-time quantitative method. The results showed that these transgenic plants targeting-expression in mitochondrial containing MTS-HM184-GFP had 1 copy and 2 copies, the other two plants containing MTS-HM184 both had 3 copies, but 0 copy in wild type. In

  5. Quantification of mixed chimerism by real time PCR on whole blood-impregnated FTA cards.

    Science.gov (United States)

    Pezzoli, N; Silvy, M; Woronko, A; Le Treut, T; Lévy-Mozziconacci, A; Reviron, D; Gabert, J; Picard, C

    2007-09-01

    This study has investigated quantification of chimerism in sex-mismatched transplantations by quantitative real time PCR (RQ-PCR) using FTA paper for blood sampling. First, we demonstrate that the quantification of DNA from EDTA-blood which has been deposit on FTA card is accurate and reproducible. Secondly, we show that fraction of recipient cells detected by RQ-PCR was concordant between the FTA and salting-out method, reference DNA extraction method. Furthermore, the sensitivity of detection of recipient cells is relatively similar with the two methods. Our results show that this innovative method can be used for MC assessment by RQ-PCR.

  6. Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity.

    Science.gov (United States)

    Joseph, Christine G; Wilczynski, Andrzej; Holder, Jerry R; Xiang, Zhimin; Bauzo, Rayna M; Scott, Joseph W; Haskell-Luevano, Carrie

    2003-12-01

    Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111-113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7-9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle4-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.

  7. 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

    2009-11-01

    Full Text Available 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.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.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.

  8. Cell surface expression system for the display of heterologous gene products using chimeric flagellin fusions of bacillus halodurans isolate

    CSIR Research Space (South Africa)

    Du Plessis, A

    2006-10-01

    Full Text Available system for the display of heterologous gene products using chimeric flagellin fusions of a Bacillus halodurans isolate Slide 2 © CSIR 2006 www.csir.co.za Bacillus halodurans Alk 36 xrhombus Ability to over-produce cell... for functionality of the His-tag for metal binding. Slide 13 © CSIR 2006 www.csir.co.za PAGE gel showing over-production of chimeric poly-His flagellin proteins 66.2 kDa 45.0 kDa 31.0 kDa 1. LMW ladder 2. NC3 3. NHisC3 4. NC6 5...

  9. Chimeric FimH adhesin of type 1 fimbriae: a bacterial surface display system for heterologous sequences

    DEFF Research Database (Denmark)

    Pallesen, L; Poulsen, LK; Christiansen, Gunna

    1995-01-01

    of heterologous DNA segments encoding two reporter sequences. In the selected positions such insertions did not significantly alter the function of the FimH protein with regard to surface location and adhesive ability. The system seemed to be quite flexible, since chimeric versions of the FimH adhesin containing...... as many as 56 foreign amino acids were transported to the bacterial surface as components of the fimbrial organelles. Furthermore, the foreign protein segments were recognized by insert-specific antibodies when expressed within chimeric proteins on the surface of the bacteria. The results from...

  10. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  11. 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.

  12. High-Level Systemic Expression of Conserved Influenza Epitope in Plants on the Surface of Rod-Shaped Chimeric Particles

    Directory of Open Access Journals (Sweden)

    Natalia V. Petukhova

    2014-04-01

    Full Text Available Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMV‑based viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rod‑shaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.

  13. Pharmacokinetics of chimeric L6 conjugated to indium-111- and yttrium-90-DOTA-peptide in tumor-bearing mice

    International Nuclear Information System (INIS)

    DeNardo, S.J.; Zhong, G.R.; Salako, Q.

    1995-01-01

    A bifunctional chelating agent, DOTA-Gly 3 -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 111 In and 90 Y 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 111 In- and 90 Y-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 125 I-ChL6 obtained in the same mouse model. The whole-body clearance of 125 I-ChL6, 90 Y-and 111 In-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 90 Y-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

  14. 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. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy.

    Science.gov (United States)

    Heczey, Andras; Liu, Daofeng; Tian, Gengwen; Courtney, Amy N; Wei, Jie; Marinova, Ekaterina; Gao, Xiuhua; Guo, Linjie; Yvon, Eric; Hicks, John; Liu, Hao; Dotti, Gianpietro; Metelitsa, Leonid S

    2014-10-30

    Advances in the design of chimeric antigen receptors (CARs) have improved the antitumor efficacy of redirected T cells. However, functional heterogeneity of CAR T cells limits their therapeutic potential and is associated with toxicity. We proposed that CAR expression in Vα24-invariant natural killer T (NKT) cells can build on the natural antitumor properties of these cells while their restriction by monomorphic CD1d limits toxicity. Primary human NKT cells were engineered to express a CAR against the GD2 ganglioside (CAR.GD2), which is highly expressed by neuroblastoma (NB). We compared CAR.GD2 constructs that encoded the CD3ζ chain alone, with CD28, 4-1BB, or CD28 and 4-1BB costimulatory endodomains. CAR.GD2 expression rendered NKT cells highly cytotoxic against NB cells without affecting their CD1d-dependent reactivity. We observed a striking T helper 1-like polarization of NKT cells by 4-1BB-containing CARs. Importantly, expression of both CD28 and 4-1BB endodomains in the CAR.GD2 enhanced in vivo persistence of NKT cells. These CAR.GD2 NKT cells effectively localized to the tumor site had potent antitumor activity, and repeat injections significantly improved the long-term survival of mice with metastatic NB. Unlike T cells, CAR.GD2 NKT cells did not induce graft-versus-host disease. These results establish the potential of NKT cells to serve as a safe and effective platform for CAR-directed cancer immunotherapy. © 2014 by The American Society of Hematology.

  16. Chimeric antigen receptor (CAR)-modified natural killer cell-based immunotherapy and immunological synapse formation in cancer and HIV.

    Science.gov (United States)

    Liu, Dongfang; Tian, Shuo; Zhang, Kai; Xiong, Wei; Lubaki, Ndongala Michel; Chen, Zhiying; Han, Weidong

    2017-12-01

    Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells contribute to the body's immune defenses. Current chimeric antigen receptor (CAR)-modified T cell immunotherapy shows strong promise for treating various cancers and infectious diseases. Although CAR-modified NK cell immunotherapy is rapidly gaining attention, its clinical applications are mainly focused on preclinical investigations using the NK92 cell line. Despite recent advances in CAR-modified T cell immunotherapy, cost and severe toxicity have hindered its widespread use. To alleviate these disadvantages of CAR-modified T cell immunotherapy, additional cytotoxic cell-mediated immunotherapies are urgently needed. The unique biology of NK cells allows them to serve as a safe, effective, alternative immunotherapeutic strategy to CAR-modified T cells in the clinic. While the fundamental mechanisms underlying the cytotoxicity and side effects of CAR-modified T and NK cell immunotherapies remain poorly understood, the formation of the immunological synapse (IS) between CAR-modified T or NK cells and their susceptible target cells is known to be essential. The role of the IS in CAR T and NK cell immunotherapies will allow scientists to harness the power of CAR-modified T and NK cells to treat cancer and infectious diseases. In this review, we highlight the potential applications of CAR-modified NK cells to treat cancer and human immunodeficiency virus (HIV), and discuss the challenges and possible future directions of CAR-modified NK cell immunotherapy, as well as the importance of understanding the molecular mechanisms of CAR-modified T cell- or NK cell-mediated cytotoxicity and side effects, with a focus on the CAR-modified NK cell IS.

  17. Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma.

    Science.gov (United States)

    Li, Nan; Fu, Haiying; Hewitt, Stephen M; Dimitrov, Dimiter S; Ho, Mitchell

    2017-08-08

    Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma.

  18. Anticorpos fixadores de complemento para o vírus respiratório sincicial e adenovírus e inibidores da hemaglutinação para os vírus parainfluenza 1, 2 e 3 numa população infantil brasileira

    Directory of Open Access Journals (Sweden)

    José Alberto Neves Candeias

    1968-06-01

    Full Text Available Apresentaram-se os resultados obtidos na pesquisa de anticorpos fixadores de complemento para o vírus respiratório sincicial e adenovírus, assim como de anticorpos inibidores da hemaglutinação para os vírus parainfluenza dos tipos 1, 2 e 3, num grupo de 972 crianças de idade compreendida entre 3 meses e 14 anos. A técnica de colheita de sangue foi a de embebição em papel de filtro. Do total de crianças examinadas, considerando o conjunto de todas as idades, 34,6% apresentavam anticorpos para o vírus respiratório sincicial; as porcentagens com anticorpos para adenovírus, parainfluenza 1, parainfluenza 2 e parainfluenza 3, foram respectivamente 47,7%, 46,8%, 54,1% e 66,6%. Foram estudadas as distribuições dos anticorpos em função da idade, do sexo e da localização do domicílio. Em relação aos dois últimos atributos obtiveram-se os seguintes resultados: dos indivíduos do sexo masculino, 32,3% apresentavam anticorpos contra o vírus respiratório sincicial, 49,2% contra adenovírus, 60,1%, 65,1% e 78,3%, respectivamente, contra os vírus parainfluenza 1, 2 e 3; nas crianças do sexo feminino as porcentagens de positividade encontradas foram, respectivamente, 37,4%, 45,9%, 31,1%, 41,2% e 52,9%; em relação à localização do domicílio, 44,8% do total de crianças da zona rural mostraram possuir anticorpos contra o vírus respiratório sincicial, 70,1% contra adenovírus, 43,8% contra vírus parainfluenza 1 e 46,8% e 65,4% contra os vírus parainfluenza dos tipos 2 e 3; as porcentagens de positividade na zona urbana foram, respectivamente, 30,5%, 38,7%, 47,9%, 57,1% e 67,1%.The author presents the results of a survey for respiratory syncytial virus and adenovirus complement fixing antibodies and parainfluenza viruses 1, 2, 3 haemagglutination inhibition antibodies in a group of 972 chidren between 3 months and 14 years of age. The filter paper method of collecting whole blood was used. Altogether, the percentage of children

  19. A tale of two sequences: microRNA-target chimeric reads.

    Science.gov (United States)

    Broughton, James P; Pasquinelli, Amy E

    2016-04-04

    In animals, a functional interaction between a microRNA (miRNA) and its target RNA requires only partial base pairing. The limited number of base pair interactions required for miRNA targeting provides miRNAs with broad regulatory potential and also makes target prediction challenging. Computational approaches to target prediction have focused on identifying miRNA target sites based on known sequence features that are important for canonical targeting and may miss non-canonical targets. Current state-of-the-art experimental approaches, such as CLIP-seq (cross-linking immunoprecipitation with sequencing), PAR-CLIP (photoactivatable-ribonucleoside-enhanced CLIP), and iCLIP (individual-nucleotide resolution CLIP), require inference of which miRNA is bound at each site. Recently, the development of methods to ligate miRNAs to their target RNAs during the preparation of sequencing libraries has provided a new tool for the identification of miRNA target sites. The chimeric, or hybrid, miRNA-target reads that are produced by these methods unambiguously identify the miRNA bound at a specific target site. The information provided by these chimeric reads has revealed extensive non-canonical interactions between miRNAs and their target mRNAs, and identified many novel interactions between miRNAs and noncoding RNAs.

  20. NPYFa, A Chimeric Peptide of Met-Enkephalin, and NPFF Induces Tolerance-Free Analgesia.

    Science.gov (United States)

    Mudgal, Annu; Kumar, Krishan; Mollereau, Catherine; Pasha, Santosh

    2016-06-01

    Methionine-enkephalin-Arg-Phe is an endogenous amphiactive analgesic peptide. Neuropeptide FF, on the other hand, is reported for its role in opioid modulation and tolerance development. Based on these reports, in the present study we designed a chimeric peptide NPYFa (YGGFMKKKPQRFamide), having the Met-enkephalin (opioid) and PQRFamide sequence of neuropeptide FF, which can then target both the opioid and neuropeptide FF receptors. We hypothesized that the chimeric peptide so designed would have both analgesic properties and further aid in understanding of the role of neuropeptide FF in the development of opiate tolerance. Our studies