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Sample records for antibody chimeric br96

  1. Radiolabeling and biotinylation of internalizing monoclonal antibody chimeric BR96: Potential use of extracorporeal immunoadsorption with enhanced tumor radioactivity retention of Iodine, Indium and Rhenium

    Energy Technology Data Exchange (ETDEWEB)

    Chen, JianQing

    1997-01-01

    In this thesis, methodology of radiolabeling and simultaneous biotinylation for internalizing monoclonal antibody chimeric BR96 have been investigated by using three element groups of potential therapeutic radionuclides iodine, indium and rhenium, and their different labeling methods. The biodistribution and kinetics of biotinylated and radiolabeled chiBR96 have been studied in colon carcinoma isografted rats. The potential use of ECIA, based on the biotin-avidin concept, has been evaluated and compared with the approach of avidin `chase` in the same animal tumor model with respect to an enhancement of tumor-to-normal tissue (T/N) activity ratio. 131 refs.

  2. Sequential radioimmunotherapy with 177Lu- and 211At-labeled monoclonal antibody BR96 in a syngeneic rat colon carcinoma model

    DEFF Research Database (Denmark)

    Eriksson, Sophie E; Elgström, Erika; Bäck, Tom

    2014-01-01

    for small, established tumors. A combination of such radionuclides may be successful in regimens of radioimmunotherapy. In this study, rats were treated by sequential administration of first a 177Lu-labeled antibody, followed by a 211At-labeled antibody 25 days later. METHODS: Rats bearing solid colon...... carcinoma tumors were treated with 400 MBq/kg body weight 177Lu-BR96. After 25 days, three groups of animals were given either 5 or 10 MBq/kg body weight of 211At-BR96 simultaneously with or without a blocking agent reducing halogen uptake in normal tissues. Control animals were not given any 211At-BR96....... Myelotoxicity, body weight, tumor size, and development of metastases were monitored for 120 days. RESULTS: Tumors were undetectable in 90% of the animals on day 25, independent of treatment. Additional treatment with 211At-labeled antibodies did not reduce the proportion of animals developing metastases...

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

    Institute of Scientific and Technical Information of China (English)

    白银; 王琰; 周丽君; 俞莉章

    2001-01-01

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

  4. [Neutralizing Monoclonal and Chimeric Antibodies to Human IFN-γ].

    Science.gov (United States)

    Larina, M V; Aliev, T K; Solopova, O N; Pozdnyakova, L P; Korobova, S V; Yakimov, S A; Sveshnikov, P G; Dolgikh, D A; Kirpichnikov, M P

    2015-01-01

    Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

  5. Mouse x pig chimeric antibodies expressed in Baculovirus retain the same properties of their parent antibodies.

    Science.gov (United States)

    Jar, Ana M; Osorio, Fernando A; López, Osvaldo J

    2009-01-01

    The development of hybridoma and recombinant DNA technologies has made it possible to use antibodies against cancer, autoimmune disorders, and infectious diseases in humans. These advances in therapy, as well as immunoprophylaxis, could also make it possible to use these technologies in agricultural species of economic importance such as pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus causing very important economic losses to the industry. Passive transfer of antibodies obtained by biotechnology could be used in the future to complement or replace vaccination against this and other pig pathogens. To this end, we constructed and studied the properties of chimeric mouse x pig anti-PRRSV antibodies. We cloned the constant regions of gamma-1 and gamma-2 heavy chains and the lambda light chain of pig antibodies in frame with the variable regions of heavy and light chains of mouse monoclonal antibody ISU25C1, which has neutralizing activity against PRRSV. The coding regions for chimeric IgG1 and IgG2 were expressed in a baculovirus expression system. Both chimeric antibodies recognized PRRSV in ELISA as well as in a Western-blot format and, more importantly, were able to neutralize PRRSV in the same fashion as the parent mouse monoclonal antibody ISU25C1. In addition, we show that both pig IgG1 and IgG2 antibodies could bind complement component C1q, with IgG2 being more efficient than IgG1 in binding C1q. Expressing chimeric pig antibodies with protective capabilities offers a new alternative strategy for infectious disease control in domestic pigs.

  6. 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

  7. A phase II trial of chimeric monoclonal antibody G250 for advanced renal cell carcinoma patients.

    NARCIS (Netherlands)

    Bleumer, I.; Knuth, A.; Oosterwijk, E.; Hofmann, R.; Varga, Z.; Lamers, C.B.H.W.; Kruit, W.; Melchior, S.; Mala, C.; Ullrich, S.; Mulder, P.; Mulders, P.F.A.; Beck, J.L.M.

    2004-01-01

    Chimeric monoclonal antibody G250 (WX-G250) binds to a cell surface antigen found on >90% of renal cell carcinoma (RCC). A multicentre phase II study was performed to evaluate the safety and efficacy of WX-G250 in metastatic RCC (mRCC) patients. In all, 36 patients with mRCC were included. WX-G250 w

  8. 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.

  9. Tetravalent neutralizing antibody response against four dengue serotypes by a single chimeric dengue envelope antigen.

    Science.gov (United States)

    Apt, Doris; Raviprakash, Kanakatte; Brinkman, Alice; Semyonov, Andrey; Yang, Shumin; Skinner, Craig; Diehl, Lori; Lyons, Richard; Porter, Kevin; Punnonen, Juha

    2006-01-16

    We employed DNA shuffling and screening technologies to develop a single recombinant dengue envelope (E) antigen capable of inducing neutralizing antibodies against all four antigenically distinct dengue serotypes. By DNA shuffling of codon-optimized dengue 1-4 E genes, we created a panel of novel chimeric clones expressing C-terminal truncated E antigens that combined epitopes from all four dengue serotypes. DNA vaccines encoding these novel chimeras induced multivalent T cell and neutralizing antibody responses against all four dengue serotypes in mice. By contrast, a mixture of four unshuffled, parental DNA vaccines failed to produce tetravalent neutralizing antibodies in mice. The neutralizing antibody titers for some of these antigens could be further improved by extending the sequences to express full-length pre-membrane and envelope proteins. The chimeric antigens also protected mice against a lethal dengue-2 virus challenge. These data demonstrate that DNA shuffling and associated screening can lead to the selection of multi-epitope antigens against closely related dengue virus serotypes and suggest a broad utility for these technologies in optimizing vaccine antigens.

  10. 嵌合抗体研究进展%Research advances in chimeric antibodies

    Institute of Scientific and Technical Information of China (English)

    张雪

    2012-01-01

    单克隆抗体在疾病的诊断、治疗和预防中发挥着重要作用,但是在临床治疗中人抗鼠抗体反应的出现使鼠源性单克隆抗体的应用受到了很大限制.随着分子生物学、分子免疫学技术的飞速发展,抗体技术已发展到第三代抗体——基因工程抗体阶段,可利用基因工程技术对鼠源性抗体进行改造,保留或增强天然抗体的特异性和主要生物学活性,同时减少鼠源成分,以避免鼠源性单克隆抗体在临床应用方面的缺陷.此文就基因工程抗体中的重要组成部分嵌合抗体的研究进展做一综述.%Monoclonal antibodies play an important role in diagnosis,treatment and prevention of diseases,but the clinical utility of murine monoclonal antibodies has been greatly limited by human anti-mouse antibody responses.With the rapid development of molecular biology and molecular immunology,antibody techniques run to the third generation-genetic engineering antibody.Murine antibodies are reconstructed with genetic engineering techniques,which reserve or increase the specificity and biological activity of natural antibodies,decrease murine components,getting rid of defects of murine monoclonal antibody in clinical application.In this review,research advancement in chimeric antibody which is one of the important constituents of genetic engineering antibodies is described.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

  13. Chimeric monoclonal antibody to tumor necrosis factor alpha (infliximab in psoriasis

    Directory of Open Access Journals (Sweden)

    Sridhar J

    2006-01-01

    Full Text Available Background: Insights into the pathogenesis of psoriasis have provided opportunities to target key steps in the disease process. Tumor necrosis factor-alpha (TNF-a being crucial to the pathogenesis of psoriasis, monoclonal antibodies against this cytokine have proved useful in its treatment. Aim: To study the efficacy of chimeric monoclonal antibody to TNF-a (infliximab in Indian patients with recalcitrant psoriasis vulgaris. Materials and Methods: Three patients with recalcitrant psoriasis vulgaris were studied. Baseline haemogram, biochemical parameters, chest radiograph and Mantoux skin test were performed. A loading dose regimen of 5 mg/kg infliximab was administered at weeks 0, 2 and 6. PASI assessment, adverse drug event monitoring and laboratory assessments were carried out at 2-week intervals until week 10. Patients were followed up until week 22 for relapse. Results: Infliximab was well tolerated. The mean PASI was 25.4 at presentation and declined to 5.5 at 10 weeks. PASI 75 was attained at a mean of 9.6 weeks. Relapse occurred at a mean of 18.6 weeks after the first infusion. Conclusions: This study on Indian patients brings out the importance of cytokine-based therapies in psoriasis. Indigenous production could make these therapies a viable therapeutic option for psoriasis patients in the near future.

  14. Preclinical evaluation of MORAb-009, a chimeric antibody targeting tumor-associated mesothelin

    Science.gov (United States)

    Hassan, Raffit; Ebel, Wolfgang; Routhier, Eric L.; Patel, Rina; Kline, J. Bradford; Zhang, Jingli; Chao, Qimin; Jacob, Sara; Turchin, Howard; Gibbs, Lester; Phillips, Martin D.; Mudali, Shiyama; Iacobuzio-Donahue, Christine; Jaffee, Elizabeth M.; Moreno, Maria; Pastan, Ira; Sass, Philip M.; Nicolaides, Nicholas C.

    2007-01-01

    Novel therapeutic agents that are safe and effective are needed for the treatment of pancreatic, ovarian, lung adenocarcinomas and mesotheliomas. Mesothelin is a glycosyl-phosphatidyl inositol (GPI)-linked membrane protein of 40 kDa over-expressed in all pancreatic adenocarcinoma and mesothelioma, in >70% of ovarian adenocarcinoma, and in non-small cell lung and colorectal cancers. The biological functions of mesothelin are not known, although it appears to be involved in cell adhesion via its interaction with MUC16. We have recently developed MORAb-009, a mouse-human chimeric IgG1κ monoclonal antibody with an affinity of 1.5 nM for human mesothelin. Here we provide evidence that MORAb-009 prevents adhesion of mesothelin-bearing tumor cells to MUC16 positive cells and can elicit cell-mediated cytotoxicity on mesothelin-bearing tumor cells. Treatment that included MORAb-009 in combination with chemotherapy led to a marked reduction in tumor growth of mesothelin-expressing tumors in nude mice compared to chemotherapy or MORAb-009 treatment alone. No adverse effects of MORAb-009 were noted during toxicology studies conducted in non-human primates. The preclinical data obtained from our studies warrants pursuing clinical testing of MORAb-009. We have in fact initiated a Phase I clinical study enrolling patients with mesothelin-positive pancreatic, mesothelioma, non-small cell lung and ovarian cancers. PMID:18088084

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    Despite the preclinical success of adoptive therapy with T cells bearing chimeric nanoconstructed antigen receptors (CARs), certain limitations of this therapeutic approach such as the immunogenicity of the antigen binding domain, the emergence of tumor cell escape variants and the blocking...... capacity of soluble antigen still remain. Here, we address these issues using a novel CAR binding moiety based on the oligoclonal camelid single domain antibodies. A unique set of 13 single domain antibodies were selected from an immunized camel phage library based on their target specificity and binding...... to reverse multiple tumor immune evasion mechanisms, avoid CAR immunogenicity, and overcome problems in cancer gene therapy with engineered nanoconstructs....

  16. Endowing self-binding feature restores the activities of a loss-of-function chimerized anti-GM2 antibody.

    Science.gov (United States)

    Zhao, Yunfeng; Russ, Michael; Retter, Marc; Fanger, Gary; Morgan, Charles; Kohler, Heinz; Muller, Sybille

    2007-02-01

    Our previous studies have described a rare type of antibody that spontaneously binds to itself, or homodimerizes. This self-binding, or autophilic antibody provides stronger protection against bacterial infection than a non-self-binding antibody with identical specificity and affinity, due to an increase of polymeric avidity. Furthermore, we have shown that a peptide derived from the self-binding domain of the autophilic T15 antibody can be crosslinked to the Fc carbohydrate of monoclonal antibodies specific for the B-cell receptor of B-cell tumors. These peptide-crosslinked antibodies can exert self-binding properties, leading to an increase in binding efficiency to the target cells as well as an increase in potential to induce apoptosis. Herein, we report a novel finding that crosslinking of the autophilic T15 peptide rescues a loss-of-function chimerized (ch) anti-GM2 antibody. The parental antibody demonstrates in vivo anti-tumor activity against melanoma xenografts. The T15 peptide-conjugated antibody shows the ability to bind to itself, as well as an increased binding to its antigen, ganglioside GM2. Moreover, the peptide-conjugated antibody also demonstrates an increased ability to bind to two GM2-positive tumor cell lines and notably important, restores its ability to induce apoptosis in two types of tumor cells. These results provide strong support for the clinical potential of the autophilic technology.

  17. Application of {sup 99m}Tc-labeled chimeric Fab fragments of monoclonal antibody A7 for radioimmunoscintigraphy of pancreatic cancer

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Hiroomi [Kyoto Prefectural Univ. of Medicine (Japan)

    1999-06-01

    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 {sup 99m}Tc, preventing depression of the antigen-binding activity. {sup 99m}Tc-labeled monoclonal antibody A7, {sup 99m}Tc-labeled chimeric Fab fragments of monoclonal antibody A7, {sup 99m}Tc-labeled normal mouse IgG and {sup 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)

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2014-12-16

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-01-01

    The authors have cloned the genomic DNA fragments encoding the heavy and light chain variable regions of monoclonal antibody 17-1A, and they have inserted them into mammalian expression vectors containing genomic DNA segments encoding human ..gamma..3 and kappa constant regions. The transfer of these expression vectors containing mouse-human chimeric immunoglobulin genes into Sp2/0 mouse myeloma cells resulted in the production of functional IgG that retained the specific binding to the surface antigen 17-1A expressed on colorectal carcinoma cells.

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

    Directory of Open Access Journals (Sweden)

    Marcela Torres

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

  2. {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)

  3. 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.

  4. Effect of tyrosine kinase inhibitor treatment of renal cell carcinoma on the accumulation of carbonic anhydrase IX-specific chimeric monoclonal antibody cG250

    NARCIS (Netherlands)

    Oosterwijk-Wakka, J.C.; Kats-Ugurlu, G.; Leenders, W.P.J.; Kiemeney, L.A.L.M.; Old, L.J.; Mulders, P.F.A.; Oosterwijk, E.

    2011-01-01

    OBJECTIVE: To investigate the effect of three different tyrosine kinase inhibitors (TKIs) on the biodistribution of chimeric monoclonal antibody (mAb) cG250, which identifies carbonic anhydrase IX (CAIX), in nude mice bearing human renal cell carcinoma (RCC) xenografts. TKIs represent the best, but

  5. Construction of chimeric antibodies: cloning of immunoglobulin genes including their promoter regions by PCR.

    Science.gov (United States)

    Mocikat, R; Kütemeier, G; Harloff, C

    1992-03-01

    In the production of recombinant antibodies, it is necessary to have an immunoglobulin gene promoter for driving the expression of the antibody genes. Here we describe a simple PCR method that allows cloning of the immunoglobulin genes together with their own promoters despite the fact that the sequence of the upstream part of the gene is unknown.

  6. The Anti—tumor Effects of an Anti—CD71 Chimeric Antibody in Vitro and Its Distribution in a Tumor Xenograft Model

    Institute of Scientific and Technical Information of China (English)

    YANGDaofeng; WANGShuo; 等

    2002-01-01

    Objective To investigate the anti-tumor effects in vitro and in vivo distribution of the human/murine chimeric antibody (D2C).Methods The CD71 positive target cells(K562,CEM and SMMC7721) and the effector cells ,freshly isolated human PBMC,with the ratio of target cells to effector cells 1:50,were incubated in various dilutions of D2C antibody(Ab).Antibody dependent cytotoxicity(AD-CC) was tested by using an LDH-release assay.Instead of effector cells,complement was added to the target cells (CEM,SMMC-7721) with various dilutions of D2C Ab.A method of counting death cells was used in complement dependent cytotoxicity(CDC) assay.Tumor localization and distribution of the chimeric antibody(D2C) were observed by labeling the chimeric Ab with radioiodine(131I) and injecting in into nude mice(Balb/c nu/nu) transplanted with human hepatocellular carcinoma cells (SMMC-7721).Results A significant ADCC was observed with the increased concentration of the D2C Ab.Cytolysis of CD71-positive target cells by the D2C Ab was found in the presence of fresh rabbit complement.Labeled D2C administered by intraperitoneal as well as tumor regional in-jection,was visualized by SPECT. The distribution of D2C Ab in murine organs and tissues showed that non-specific binding was lower fol-lowing tumor regional administration than when the antibody was administered by an intraperitoneal injection.The human/murine chimeric antibody(D2C) has in vitro anti-tumor effects and can exert its effects in specific tumor localization.Its distribution and local effects in vi-vo can be detected by radioimmunoimaging. Conclusion CD71 human/murine chimeric antibody showed marked killing of tumor cells in vitro,and specific recognition and high affinity binding to tumor tissue in vivo.

  7. Chimeric Antibody-Binding Vitreoscilla Hemoglobin (VHb Mediates Redox-Catalysis Reaction: New Insight into the Functional Role of VHb

    Directory of Open Access Journals (Sweden)

    Yaneenart Suwanwong, Malin Kvist, Chartchalerm Isarankura-Na-Ayudhya, Natta Tansila, Leif Bulow, Virapong Prachayasittikul

    2006-01-01

    Full Text Available Experimentation was initiated to explore insight into the redox-catalysis reaction derived from the heme prosthetic group of chimeric Vitreoscilla hemoglobin (VHb. Two chimeric genes encoding chimeric VHbs harboring one and two consecutive sequences of Fc-binding motif (Z-domain were successfully constructed and expressed in E. coli strain TG1. The chimeric ZVHb and ZZVHb were purified to a high purity of more than 95% using IgG-Sepharose affinity chromatography. From surface plasmon resonance, binding affinity constants of the chimeric ZVHb and ZZVHb to human IgG were 9.7 x 107 and 49.1 x 107 per molar, respectively. More importantly, the chimeric VHbs exhibited a peroxidase-like activity determined by activity staining on native PAGE and dot blotting. Effects of pH, salt, buffer system, level of peroxidase substrate and chromogen substrate were determined in order to maximize the catalytic reaction. From our findings, the chimeric VHbs displayed their maximum peroxidase-like activity at the neutral pH (~7.0 in the presence of high concentration (20-40 mM of hydrogen peroxide. Under such conditions, the detection limit derived from the calibration curve was at 250 ng for the chimeric VHbs, which was approximately 5-fold higher than that of the horseradish peroxidase. These findings reveal the novel functional role of Vitreoscilla hemoglobin indicating a high trend of feasibility for further biotechnological and medical applications.

  8. Crystal Structure of PG16 and Chimeric Dissection with Somatically Related PG9: Structure-Function Analysis of Two Quaternary-Specific Antibodies That Effectively Neutralize HIV-1

    Energy Technology Data Exchange (ETDEWEB)

    Pancera, Marie; McLellan, Jason S.; Wu, Xueling; Zhu, Jiang; Changela, Anita; Schmidt, Stephen D.; Yang, Yongping; Zhou, Tongqing; Phogat, Sanjay; Mascola, John R.; Kwong, Peter D. (NIH); (Aids Vaccine)

    2010-11-03

    HIV-1 resists neutralization by most antibodies. Two somatically related human antibodies, PG9 and PG16, however, each neutralize 70 to 80% of circulating HIV-1 isolates. Here we present the structure of the antigen-binding fragment of PG16 in monoclinic and orthorhombic lattices at 2.4 and 4.0 {angstrom}, respectively, and use a combination of structural analysis, paratope dissection, and neutralization assessment to determine the functional relevance of three unusual PG9/PG16 features: N-linked glycosylation, extensive affinity maturation, and a heavy chain-third complementarity-determining region (CDR H3) that is one of the longest observed in human antibodies. Glycosylation extended off the side of the light chain variable domain and was not required for neutralization. The CDR H3 formed an axe-shaped subdomain, which comprised 42% of the CDR surface, with the axe head looming {approx}20 {angstrom} above the other combining loops. Comprehensive sets of chimeric swaps between PG9 and PG16 of light chain, heavy chain, and CDR H3 were employed to decipher structure-function relationships. Chimeric swaps generally complemented functionally, with differences in PG9/PG16 neutralization related primarily to residue differences in CDR H3. Meanwhile, chimeric reversions to genomic V genes showed isolate-dependent effects, with affinity maturation playing a significant role in augmenting neutralization breadth (P = 0.036) and potency (P < 0.0001). The structural and functional details of extraordinary CDR H3 and extensive affinity maturation provide insights into the neutralization mechanism of and the elicitation pathway for broadly neutralizing antibodies like PG9 and PG16.

  9. 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.

  10. Purification of chimeric heavy chain monoclonal antibody EG2-hFc using hydrophobic interaction membrane chromatography: an alternative to protein-A affinity chromatography.

    Science.gov (United States)

    Sadavarte, Rahul; Spearman, Maureen; Okun, Natalie; Butler, Michael; Ghosh, Raja

    2014-06-01

    Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc.

  11. Production and characterization of chimeric monoclonal antibodies against Burkholderia pseudomallei and B. mallei using the DHFR expression system.

    Directory of Open Access Journals (Sweden)

    Hyung-Yong Kim

    Full Text Available Burkholderia pseudomallei (BP and B. mallei (BM are closely related gram-negative, facultative anaerobic bacteria which cause life-threatening melioidosis in human and glanders in horse, respectively. Our laboratory has previously generated and characterized more than 100 mouse monoclonal antibodies (MAbs against BP and BM, according to in vitro and in vivo assay. In this study, 3 MAbs (BP7 10B11, BP7 2C6, and BP1 7F7 were selected to develop into chimeric mouse-human monoclonal antibodies (cMAbs against BP and/or BM. For the stable production of cMAbs, we constructed 4 major different vector systems with a dihydrofolate reductase (DHFR amplification marker, and optimized transfection/selection conditions in mammalian host cells with the single-gene and/or double-gene expression system. These 3 cMAbs were stably produced by the DHFR double mutant Chinese hamster ovarian (CHO-DG44 cells. By ELISA and Western blot analysis using whole bacterial antigens treated by heat (65°C/90 min, sodium periodate, and proteinase K, the cMAb BP7 10B11 (cMAb CK1 reacted with glycoproteins (34, 38, 48 kDa in BP; 28, 38, 48 kDa in BM. The cMAb BP7 2C6 (cMAb CK2 recognized surface-capsule antigens with molecular sizes of 38 to 52 kDa, and 200 kDa in BM. The cMAb CK2 was weakly reactive to 14∼28, 200 kDa antigens in BP. The cMAb BP1 7F7 (cMAb CK3 reacted with lipopolysaccharides (38∼52 kDa in BP; 38∼60 kDa in B. thailandensis. Western blot results with the outer surface antigens of the 3 Burkholderia species were consistent with results with the whole Burkholderia cell antigens, suggesting that these immunodominant antigens reacting with the 3 cMAbs were primarily present on the outer surface of the Burkholderia species. These 3 cMAbs would be useful for analyzing the role of the major outer surface antigens in Burkholderia infection.

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

    Science.gov (United States)

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

    2013-12-31

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Karen Knipping

    Full Text Available 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 (chuIgE 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.

  16. Evaluation of chimeric DNA vaccines consisting of premembrane and envelope genes of Japanese encephalitis and dengue viruses as a strategy for reducing induction of dengue virus infection-enhancing antibody response.

    Science.gov (United States)

    Sjatha, Fithriyah; Kuwahara, Miwa; Sudiro, T Mirawati; Kameoka, Masanori; Konishi, Eiji

    2014-02-01

    Neutralizing antibodies induced by dengue virus (DENV) infection show viral infection-enhancing activities at sub-neutralizing doses. On the other hand, preimmunity against Japanese encephalitis virus (JEV), a congener of DENV, does not increase the severity of DENV infection. Several studies have demonstrated that neutralizing epitopes in the genus Flavivirus are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM-E) gene-based expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM-E gene backbone showed high neutralizing antibody titers with less DENV infection-enhancing activity. Our results confirm the applicability of this approach as a new dengue vaccine development strategy.

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

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Weibo; Chen, Kai; He, Lina; Cao, Qizhen; Chen, Xiaoyuan [Stanford University School of Medicine, The Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Stanford, CA (United States); Koong, Albert [Stanford University School of Medicine, Department of Radiation Oncology, Stanford, CA (United States)

    2007-06-15

    Cetuximab, a chimeric monoclonal antibody targeting epidermal growth factor receptor (EGFR) on the surface of cancer cells, was approved by the FDA to treat patients with metastatic colorectal cancer. It is currently also in advanced-stage development for the treatment of several other solid tumors. Here we report for the first time the quantitative positron emission tomography (PET) imaging of EGFR expression in xenograft-bearing mice using {sup 64}Cu-labeled cetuximab. We conjugated cetuximab with macrocyclic chelating agent 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA), labeled with {sup 64}Cu, and tested the resulting {sup 64}Cu-DOTA-cetuximab in seven xenograft tumor models. The tracer uptake measured by PET was correlated with the EGFR expression quantified by western blotting. The estimated human dosimetry based on the PET data in Sprague-Dawley rats was also calculated. MicroPET imaging showed that {sup 64}Cu-DOTA-cetuximab had increasing tumor activity accumulation over time in EGFR-positive tumors but relatively low uptake in EGFR-negative tumors at all times examined (<5%ID/g). There was a good correlation (R {sup 2} = 0.80) between the tracer uptake (measured by PET) and the EGFR expression level (measured by western blotting). Human dosimetry estimation indicated that the tracer may be safely administered to human patients for tumor diagnosis, with the dose-limiting organ being the liver. The success of EGFR-positive tumor imaging using {sup 64}Cu-DOTA-cetuximab can be translated into the clinic to characterize the pharmacokinetics, to select the right population of patients for EGFR-targeted therapy, to monitor the therapeutic efficacy of anti-EGFR treatment, and to optimize the dosage of either cetuximab alone or cetuximab in combination with other therapeutic agents. (orig.)

  18. Clinical significance of chimerism.

    Science.gov (United States)

    Abuelo, Dianne

    2009-05-15

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

  19. Effects of chimeric B7 antibodies on mononuclear cells in ankylosing spondlitis%B7嵌合抗体对强直性脊柱炎中单个核细胞的作用

    Institute of Scientific and Technical Information of China (English)

    徐耀瑜; 位冒冒

    2014-01-01

    目的 研究B7嵌合抗体对强直性脊柱炎(ankylosing spondylitis,AS)中单个核细胞的作用.方法 AS单个核细胞培养体系中,分别加入终浓度为5μg/ml的ch4E5和ch1 D1抗体,补体依赖的细胞毒性试验(MLCT)观察单个核细胞死亡得分的组间差异,流式细胞术(FCM)分析其膜型分子表达变化,ELISA检测其分泌细胞因子的情况.结果 MLCT中嵌合抗体组低于AS组得分(P<0.01),FCM检测嵌合抗体组较AS组CD4/CD8比值下降(P<0.05),CD154表达下降(P<0.01),CD4+CD25high调节性T淋巴细胞增多(P<0.01),加入嵌合抗体的单个核细胞培养体系分泌IL-2和TNF-α减少(P<0.05),IL-4和TGF-β升高(P<0.02).结论 B7嵌合抗体通过作用B7/CD28及CD40/CD154信号通路,改变AS中单个核细胞一些表面分子的表达和细胞因子的分泌,最终影响单个核细胞.%Objective To study the effects of chimeric B7 antibodies on mononuclear cells from patients with ankylosing spondylitis (AS).Methods Chimeric antibodies of ch4E5 and chlD1 against B7 with a final concentration of 5 μg/ml were respectively added into culture media of mononuclear cells from patients with AS.Two other groups including huIgG-treated and untreated culture medium of mononuclear cells from patients with AS were set up as the controls.The culture medium of mononuclear cells from healthy subjects were prepared as normal control group.The disparities of mononuclear cell death in culture among each group were analyzed by using micro-lymphocyte cytotoxicity test (MLCT).The expression of membrane molecules were analyzed by FCM.The concentrations of cytokines in culture media were measured by ELISA.Results Both groups treated with the chimeric antibody showed lower scores for cell death than untreated group as indicated by MLCT (P<0.01).FCM analysis demonstrated that both CD4/CD8 ratio (P <0.05) and membrane expression of CD154 (P<0.01) decreased in chimeric antibody treated groups,but the number of CD4

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ali A Maawy

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

  2. 抗人CD25嵌合抗体基因的构建及其瞬时表达研究%Study on construction and transient expression of human-mouse chimeric antibody gene against human CD25

    Institute of Scientific and Technical Information of China (English)

    胡迪超; 张爱华; 潘勇兵; 詹珊珊; 杨晓明

    2011-01-01

    目的:构建抗人CD25嵌合抗体基因并在哺乳动物细胞中进行瞬时表达和初步鉴定.方法:采用RLM-RACE法克隆WuTac抗体可变区和信号肽序列,并利用基因拼接法构建嵌合抗体基因.用脂质体法瞬时转染三种哺乳动物细胞,并使用ELISA、FCM、WB、Dot blot和免疫荧光法进行检测.结果:成功克隆WuTac抗体可变区和信号肽序列,并构建了抗人CD25嵌合抗体表达质粒.瞬时转染结果表明所表达的嵌合抗体保留了亲本抗体WuTac的抗原结合力.结论:成功构建了抗人CD25嵌合抗体基因,为其进一步研究打下基础.%Objective:To construct chimeric antibody gene against human an CD25 angigen,and prelin inarily identify the expressed prod-ucts produced from transiently transfected mammalian cells in order to facilitate the further study of stable expression.Methods:The RLM-RACE was employed to clone variable region genes and leader sequences,and the Overlap PVR method was used to construct the chimeric anti-body gene.After transiently transfected in three mammalian cells with liposome method, the expressed products were determined by ELISA,FCM,W B,Dotblot and immunofluorescence assay.Results:The variable region genes and leader sequences were successfully amplified,and the eukayotic expression plasmids were constructed.The results from transient transfection indicate the expressed products retain the antigen binding capacity of parental antibody WuTac.Conclusion:The successfully constructed chimeric antibody gene against human CD25 lays sound foun-dation for further study.

  3. Use of Monte Carlo simulations with a realistic rat phantom for examining the correlation between hematopoietic system response and red marrow absorbed dose in Brown Norway rats undergoing radionuclide therapy with {sup 177}Lu- and {sup 90}Y-BR96 mAbs

    Energy Technology Data Exchange (ETDEWEB)

    Larsson, Erik; Ljungberg, Michael; Martensson, Linda; Nilsson, Rune; Tennvall, Jan; Strand, Sven-Erik; Joensson, Bo-Anders [Department of Medical Radiation Physics, Clinical Sciences, Lund University, Lund (Sweden); Department of Oncology, Clinical Sciences, Lund University, Lund (Sweden); Department of Medical Radiation Physics, Clinical Sciences, Lund University, Lund (Sweden)

    2012-07-15

    Purpose: Biokinetic and dosimetry studies in laboratory animals often precede clinical radionuclide therapies in humans. A reliable evaluation of therapeutic efficacy is essential and should be based on accurate dosimetry data from a realistic dosimetry model. The aim of this study was to develop an anatomically realistic dosimetry model for Brown Norway rats to calculate S factors for use in evaluating correlations between absorbed dose and biological effects in a preclinical therapy study. Methods: A realistic rat phantom (Roby) was used, which has some flexibility that allows for a redefinition of organ sizes. The phantom was modified to represent the anatomic geometry of a Brown Norway rat, which was used for Monte Carlo calculations of S factors. Kinetic data for radiolabeled BR96 monoclonal antibodies were used to calculate the absorbed dose. Biological data were gathered from an activity escalation study with {sup 90}Y- and {sup 177}Lu-labeled BR96 monoclonal antibodies, in which blood cell counts and bodyweight were examined up to 2 months follow-up after injection. Reductions in white blood cell and platelet counts and declines in bodyweight were quantified by four methods and compared to the calculated absorbed dose to the bone marrow or the total body. Results: A red marrow absorbed dose-dependent effect on hematological parameters was observed, which could be evaluated by a decrease in blood cell counts. The absorbed dose to the bone marrow, corresponding to the maximal tolerable activity that could safely be administered, was determined to 8.3 Gy for {sup 177}Lu and 12.5 Gy for {sup 90}Y. Conclusions: There was a clear correlation between the hematological effects, quantified with some of the studied parameters, and the calculated red marrow absorbed doses. The decline in body weight was stronger correlated to the total body absorbed dose, rather than the red marrow absorbed dose. Finally, when considering a constant activity concentration, the phantom

  4. CD28嵌合抗体及与抗原分子的3D空间结构模建%Construction of 3D model of CD28 chimeric antibody with its antigen docked

    Institute of Scientific and Technical Information of China (English)

    程钢; 秦媛媛; 程迪; 陈曦; 张学光; 邱玉华

    2012-01-01

    AIM: To construct a 3D model of the chimeric antibodies (AntiCD28: ch-2F5) with corresponding antigen molecuie docked to theoretically verify the rationality of the binding of antibody with its antigen and to provide a method of 3D identification between antigen and antibody and spatial structure analysis. METHODS; We analyzed the sequence by submitting it to http://www. ncbi. nlm. nih. gov/ and made a comparison using integratly the 3 databases of Gen-Bank, Protein data bank and GENO-3D. The 3D model was constructed by Swiss-model homology modeling server and molecular docking online was performed by GRAMM-X Protein Docking Web Server. Chimeric heavy chain, light chain, heavy-light chain complex, heavy-light chain and antigen complex were displayed and photographed by the Chimera Software. Meanwhile, the spatial structures of heavy, light chains, variable region, constant region, CDR and frame area were marked by different colours respectively to exhibit the 3D structure on every side. RESULTS: The 3D structure of the heavy-light chain and antigen complex we constructed was consistent well with the theory of antigen binding to antibody molecules. CONCLUSON; The structure of the chimeric antibody we constructed with the bioinformatic method was in accordance with the general structure of antibody, and its antigen binding site was also consistent with the molecular theory. Thus, the model helps to analyze the 3D structure of antibody and antigen-antibody interaction.%目的:对1株嵌合抗体(antiCD28:ch-2F5)进行3D建模,并与其抗原分子进行对接,验证是否与抗原抗体结合理论相符,并提供一种抗原抗体及其识别的空间结构分析方法.方法:在http://www.ncbi.nlm.nih.gov网站提交序列进行分析,综合运用GenBank、Protein data bank、GENO-3D等数据库比对分析,用Swiss-model同源建模服务器模建,运用GRAMM-X Protein Docking Web Server在线进行对接,结果采用Chimera软件对嵌合抗体的重、轻链、

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

    Institute of Scientific and Technical Information of China (English)

    夏立亮; 吴标; 程亚庭; 蔡家麟; 王颖; 赵国屏

    2012-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  7. Half molecular exchange of IgGs in the blood of healthy humans: chimeric lambda-kappa-immunoglobulins containing HL fragments of antibodies of different subclasses (IgG1-IgG4).

    Science.gov (United States)

    Sedykh, Sergey E; Lekchnov, Evgenii A; Prince, Viktor V; Buneva, Valentina N; Nevinsky, Georgy A

    2016-10-20

    In the classic paradigm, immunoglobulins represent products of clonal B cell populations, each producing antibodies recognizing a single antigen (monospecific). There is a common belief that IgGs in mammalian biological fluids are monospecific molecules having stable structures and two identical antigen-binding sites. But the issue concerning the possibility of exchange by HL-fragments between the antibody molecules in human blood is still unexplored. Different physico-chemical and immunological methods for analysis of half-molecule exchange between human blood IgGs were used. Using eighteen blood samples of healthy humans we have shown unexpected results for the first time: blood antibodies undergo extensive post-transcriptional half-molecule exchange and IgG pools on average consist of 62.4 ± 6.5% IgGs containing kappa light chains (kappa-kappa-IgGs), 29.8.6 ± 5.4% lambda light chains (lambda-lambda-IgGs), and 8.8 ± 2.7% (range 2.6-16.8%) IgGs containing both kappa- and lambda-light chains. Kappa-kappa-IgGs and lambda-lambda-IgGs contained on average (%): IgG1 (36.0 and 32.3), IgG2 (50.9 and 51.4), IgG3 (9.7 and 9.9), and IgG4 (6.5 and 5.7), while chimeric kappa-lambda-IgGs consisted of (%): 25.5 ± 4.2 IgG1, 50.8 ± 3.9 IgG2, 9.1 ± 2.1 IgG3, and 14.5 ± 2.2 IgG4. Our unexpected data are indicative of the possibility of half-molecule exchange between blood IgGs of various subclasses, raised against different antigens. The existence of blood chimeric bifunctional IgGs with different binding sites destroys the classic paradigm. Due to the phenomenon of polyspecificity and cross-reactivity of bifunctional IgGs containing HL-fragments of different types to different antigens, such IgGs may be important in human blood for widening their different biological functions.

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

    Science.gov (United States)

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

    2014-10-01

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

  9. Biodistribution of the chimeric monoclonal antibody U36 radioiodinated with a closo-dodecaborate-containing linker. Comparison with other radioiodination methods.

    NARCIS (Netherlands)

    Nestor, M; Persson, M; Cheng, J.; Tolmachev, V; Dongen, van G.A.M.S.; Anniko, M; Kairemo, K

    2003-01-01

    We have evaluated the applicability of the [(4-isothiocyanatobenzylammonio)undecahydro-closo-dodecaborate (1-)] (DABI) linker molecule for antibody radiohalogenation and compared it to radiohalogenation using the linker N-succinimidyl 4-iodobenzoate (PIB) and to direct radiohalogenation using Chlora

  10. 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.

  11. In vitro characterization of {sup 177}Lu-radiolabelled chimeric anti-CD20 monoclonal antibody and a preliminary dosimetry study

    Energy Technology Data Exchange (ETDEWEB)

    Forrer, Flavio; Mueller-Brand, Jan [University Hospital Basel, Institute of Nuclear Medicine, Basel (Switzerland); Chen, Jianhua; Fani, Melpomeni; Powell, Pia; Maecke, Helmut R. [University Hospital Basel, Division of Radiological Chemistry, Basel (Switzerland); Lohri, Andreas [Basel University Medical Clinic, Liestal (Switzerland); Moldenhauer, Gerhard [German Cancer Research Center, Division of Molecular Immunology, Heidelberg (Germany)

    2009-09-15

    {sup 131}I- and {sup 90}Y-labelled anti-CD20 antibodies have been shown to be effective in the treatment of low-grade, B-cell non-Hodgkin's lymphoma (NHL). However, the most appropriate radionuclide in terms of high efficiency and low toxicity has not yet been established. In this study we evaluated an immunoconjugate formed by the anti-CD20 antibody rituximab and the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). DOTA-rituximab was prepared as a kit formulation and can be labelled in a short time (<20 min) with either {sup 177}Lu or {sup 90}Y. Immunoconjugates with different numbers of DOTA molecules per rituximab were prepared using p-SCN-Bz-DOTA. In vitro immunoreactivity and stability were tested and preliminary dosimetric results were acquired in two patients. The immunological binding properties of DOTA-rituximab to the CD20 antigen were found to be retained after conjugation with up to four chelators. The labelled product was stable against a 10{sup 5} times excess of diethylenetriaminepentaacetic acid (DTPA, 37 C, 7 days). Two patients with relapsed NHL were treated with 740 MBq/m{sup 2} body surface {sup 177}Lu-DOTA-rituximab. Scintigraphic images showed specific uptake at tumour sites and acceptable dosimetric results. The mean whole-body dose was found to be 314 mGy. The administration of {sup 177}Lu-DOTA-rituximab was tolerated well. Our results show that DOTA-rituximab (4:1) can be labelled with {sup 177}Lu with sufficient stability while the immunoconjugate retains its immunoreactivity. {sup 177}Lu-DOTA-rituximab is an interesting, well-tolerated radiolabelled antibody with clinical activity in a low dose range, and provides an approach to the efficient treatment with few side effects for patients with relapsed NHL. (orig.)

  12. Transmission-blocking activity of antibodies to Plasmodium falciparum GLURP.10C chimeric protein formulated in different adjuvants

    DEFF Research Database (Denmark)

    Roeffen, Will; Theisen, Michael; van de Vegte-Bolmer, Marga;

    2015-01-01

    BACKGROUND: Plasmodium falciparum is transmitted from person to person by Anopheles mosquitoes after completing its sexual reproductive cycle within the infected mosquito. An efficacious vaccine holds the potential to interrupt development of the parasite in the mosquito leading to control...... and possibly eradication of malaria. A multi-component, R0.10C, was developed comprising P. falciparum glutamate-rich protein (R0) fused in frame to a correctly folded fragment of Pfs48/45 (10C). Here, a series of novel adjuvants were screened for their ability to elicit transmission-blocking (TB) antibodies...

  13. Antibody

    Science.gov (United States)

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

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

    Institute of Scientific and Technical Information of China (English)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-10-01

    VP7 of group A rotavirus (RVA) contains major neutralizing epitopes. Using the antigenic protein VP6 as the vector, chimeric proteins carrying foreign epitopes have been shown to possess good immunoreactivity and immunogenicity. In the present study, using modified VP6 as the vector, three chimeric proteins carrying epitopes derived from VP7 of RVA were constructed. The results showed that the chimeric proteins reacted with anti-VP6 and with SA11 and Wa virus strains. Antibodies from guinea pigs inoculated with the chimeric proteins recognized VP6 and VP7 of RVA and protected mammalian cells from SA11 and Wa infection in vitro. The neutralizing activities of the antibodies against the chimeric proteins were significantly higher than those against the vector protein VP6F. Thus, development of chimeric vaccines carrying VP7 epitopes using VP6 as a vector could be a promising alternative to enhance immunization against RVAs.

  16. 抗p185erbB2人鼠嵌合抗体慢病毒表达载体的构建%Construction of the Lentiviral Expression Vector for Anti-p185erbB2 Mouse/Human Chimeric Antibody

    Institute of Scientific and Technical Information of China (English)

    刘芳; 李力; 张玮; 王琪

    2013-01-01

    本研究构建抗p185erbB2人鼠嵌合抗体慢病毒表达载体,并将其转染293T细胞,明确转染后嵌合抗体基因的表达情况.采用PCR法扩增抗p185erbB2鼠单抗轻、重链可变区基因(vL和vH)和人IgG1的轻、重链恒定区基因(κ和γ1),再利用三引物PCR法将vL和κ,vH和γ1进行拼接,得到嵌合轻链基因(L)和嵌合重链基因(H),分别插入质粒pVAX1/IRES的IRES元件的下、上游.用内切酶将H-IRES-L从pVAX1/H-IRES-L上切下,插入慢病毒载体pWPI中,经相应酶切和测序鉴定,正确构建了慢病毒表达载体pWPI/H-IRES-L.将其转染293T细胞,48 h后通过荧光显微镜检测绿色荧光蛋白(GFP)的表达,RT-PCR和直接ELISA法检测嵌合抗体的表达.结果显示,转染pWPI/H-IRES-L的293T细胞中有嵌合轻链和嵌合重链基因的共同表达,而且表达的嵌合抗体能够与p185erbB2分子特异性结合,为今后抗p185erbB2工程抗体的研究奠定了基础.%This research was to construct the lentiviral expression vector for anti- pl85erbB2 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-pl85erbB2 mAb and the constant regions of human IgG1 (Κ and γ1) 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

  17. 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.

  18. Expression and biological activity analysis of human-mouse chimeric antibody against anthrax protective antigen%抗炭疽保护性抗原人-鼠嵌合抗体在CHO细胞中的表达和活性研究

    Institute of Scientific and Technical Information of China (English)

    李冰; 陈薇; 李建民; 张军; 徐俊杰; 刘树玲; 任军; 张金龙; 付玲; 侯利华

    2009-01-01

    目的 在CHO细胞中表达抗炭疽芽孢杆菌保护性抗原(rPA)人-鼠嵌合抗体,并对其活性进行分析.方法 RT-PCR克隆具有中和活性鼠源单克隆抗体的轻、重链可变区基因,分别与人Kappa型轻链恒定区、IgG1重链恒定区基因融合后构建了轻重链嵌合抗体基因.将轻重链嵌合抗体基因克隆表达载体上,构建了嵌合抗体的双启动子表达载体pSeeTag-5E1.将pSecTag-5E1转染CHO-dhfr(DG44)细胞,撤掉胸腺嘧啶和次黄嘌呤并不断提高氨甲蝶呤(MTX)的浓度,筛选表达量高的克隆,扩大培养,收集上清,并纯化,用纯化的嵌合抗体进行SDS-PAGE、Western blot、抗原结合活性、体外细胞保护试验和动物试验.结果 在MTX的浓度为5×10~(-8)mol/L时,获得稳定表达细胞株,表达量为18 mg/L.结论 成功在CHO-dhfr(DG44)细胞中表达了具有中和活性的抗rPA人-鼠嵌合抗体,为制备抗炭疽的被动免疫制剂奠定了良好的基础.%Objective To express human-mouse chimeric antibody against anthrax protective anti-gen and to analyze its biological activities. Methods A new mammalian bipromoter expression vector was constructed with dihydrofolate reduetase(DHFR) gene as the selection and complication marker. First, the light and heavy chain variable region gene of the monoclonal antibody 5E1 were cloned by RT-PCR, at the same time the human IgG1 heavy chain constant region gene and kappa type constant region gene were cloned. Next, the human-mouse chimeric antibody genes were synthesized by fusion PCR. Then, the hu-man-mouse chimeric antibody gene were inserted into MCS of pSecTag and B1 to construct pSecTag-5E1L and B1-5E1H, respectively. Finally, heavy chain expression cassette excised from the B1-5E1H with Bgl Ⅱ/BamH Ⅰ was further cloned into the Bgl Ⅱ site of the pSecTag-5E1L to construct pSecTag-5E1. Plasmid pSecTag-5E1 was transfected into CHO(dhfr) engineering cells and high production cell clones that were screened by

  19. 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...

  20. 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)

  1. 抗VEGFR-2嵌合Fab抗体对裸鼠肝癌原位移植瘤的治疗作用%Therapeutical effect of chimeric anti-VEGFR-2 Fab antibody on orthotopic xenograft of hepatocellular carcinoma in BALB/c nude mice

    Institute of Scientific and Technical Information of China (English)

    李倩君; 潘峰; 王丙剑; 朱进; 张建民; 吴亚夫; 周传文

    2013-01-01

    目的 检测抗血管内皮生长因子受体2 (VEGFR-2)嵌合Fab抗体对裸鼠肝癌原位移植瘤的治疗作用.方法 40只4周龄雄性BALB/c裸鼠建立肝癌H22细胞移植瘤模型后随机均分为Fab抗体(A)组和生理盐水对照(B)组.比较两组裸鼠生存时间、移植瘤病理变化及微血管密度(MVD).结果 成功建立裸鼠H22肝癌原位移植瘤模型,HE染色显示肝细胞肝癌.A组裸鼠中位数生存时间明显长于B组(20.0 d vs.14.0 d)(P<0.01);A组肝脏实体瘤内MVD较B组显著减少(8.65±1.79 vs.25.64±1.53)(P<0.05).结论 抗VEGFR-2嵌合Fab抗体对裸鼠肝癌原位移植瘤有治疗作用.%Objective To investigate the therapeutical effect of chimeric anti-vascular endothelial growth factor receptor(VEGFR)-2 Fab antibody on orthotopic xenograft in BALB/c nude mice. Methods The orthotopic xenograft model of hepatocellular carcinoma H22 cells was established in 40 nude mice, which were equally randomized into two groups of A (treated with chimeric anti-VEGFR-2 Fab antibody) and B(treated with normal saline). The survival time, pathological changes and microvessel density(MVD) in H22 xenografts were compared between two groups. Results An orthotopic xenograft model of hepatocellular carcinoma was successfully created in nude mice, which was confermed as hepatocellular carcinoma by hematoxylin-eosin staining. The mean survival time was longer in group A than that in group B(20. 0 d vs. 14. 0 d) (P<0. 05). MVD in the xenografts was less in group A than that in group B(8. 65±1. 79 vs. 25. 64±1. 53) (P<0. 05). Conclusion The chimeric anti-VEGFR-2 Fab antibody has some therapeutical effect on an orthotopic xenografts in nude mice.

  2. 含F/2A序列的抗 P185erbB2人鼠嵌合抗体慢病毒表达载体的构建%Construction of the Lentiviral Expression Vector Containing F/2AFragment for Anti-P185erbB2 Mouse/Human Chimeric Antibody

    Institute of Scientific and Technical Information of China (English)

    刘芳; 李力; 张玮; 王琪

    2012-01-01

    目的 构建含 F/2A 序列的抗 P185erbB2 人鼠嵌合抗体慢病毒表达载体,观察其在 293T 细胞中的表达.方法 用具有自我剪切能力的弗林蛋白酶(Furin)/口蹄疫病毒2A多肽(F/2A)连接人鼠嵌合抗体的重链和轻链,形成一个开放阅读框 (ORF),插入慢病毒表达载体pWPI,构建重组抗P185erbB2全长人鼠嵌合抗体表达载体pWPI/H-F2A-L.以已构建的慢病毒表达载体pWPI/H-IRES-L为对照质粒.应用磷酸钙沉淀法将慢病毒载体 3 质粒系统共转染入 293T 细胞进行包装,测定病毒滴度.再感染 293T 细胞,荧光显微镜下观察 GFP 的表达和转染效率,RT-PCR、ELISA 方法分别检测嵌合抗体 mRNA和蛋白的表达.结果 经测序鉴定,pWPI/H-F2A-L与预期设计一致;pWPI/H-F2A-L组的病毒滴度为4.3×105 TU/ml,而pW-PI/H-IRES-L 组的病毒滴度为3.5×105 TU/ml;两组重组慢病毒的转染效率分别为 87.68%和 79.08%:两组重组慢病毒感染 293T 细胞后,都有嵌合重链和嵌合轻链的表达,由F/2A介导的嵌合抗体的表达水平要高于由 IRES 介导的嵌合抗体.结论 成功构建了含F/2A序列的抗P185erbB2人鼠嵌合抗体慢病毒表达载体,为今后抗P185erbB2工程抗体的研究奠定了基础.%Objective To construct the lentiviral expression vector containing F/2A fragment for anti-P185erbB2 mouse/human chimeric antibody , and detect its expression in 293T cells. Methods The heavy and light chains of chimeric antibody were joined by Furin and 2A ( F/ 2A) self-cleavage peptide and cloned into a lentiviral vector of pWPI , to generate the lentiviral ex-pression vector, pWPI/H-F2A-L Another lentiviral expression vector , pWPI/H4RES-L that had been generated already , was used as control plasmid. 293 T cells were co-transfected with the 3 helper plasmid system by using calcium phosphate precipitation , and then the virus titer was exam-ined. The 293 T cells were infected by the obtained lentiviral particles. The expression of

  3. 抗CD71人-鼠嵌合抗体的体外抗肿瘤效应及在肿瘤异种移植模型中的定位研究%The Anti-tumor Effects of an Anti-CD71 Chimeric Antibody in Vitro and Its Distribution in a Tumor Xenograft Model

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective To investigate the anti-tumor effects in vitro and in vivo distribution of the human/murine chimeric antibody (D2C). Methods The CD71 positive target cells (K562, CEM and SMMC7721) and the effector cells, freshly isolated human PBMC, with the ratio of target cells to effector cells 1∶50,were incubated in various dilutions of D2C antibody (Ab). Antibody dependent cytotoxicity (ADCC) was tested by using an LDH-release assay. Instead of effector cells, complement was added to the target cells (CEM, SMMC-7721) with various dilutions of D2C Ab. A method of counting death cells was used in complement dependent cytotoxicity(CDC) assay. Tumor localization and distribution of the chimeric antibody (D2C) were observed by labeling the chimeric Ab with radioiodine(131I) and injecting it into nude mice (Balb/c nu/nu) transplanted with human hepatocellular carcinoma cells (SMMC-7721). Results A significant ADCC was observed with the increased concentration of the D2C Ab. Cytolysis of CD71-positive target cells by the D2C Ab was found in the presence of fresh rabbit complement. Labeled D2C administered by intraperitoneal as well as tumor regional injection, was visualized by SPECT. The distribution of D2C Ab in murine organs and tissues showed that non-specific binding was lower following tumor regional administration than when the antibody was administered by an intraperitoneal injection. The human/murine chimeric antibody (D2C) has in vitro anti-tumor effects and can exert its effects in specific tumor localization. Its distribution and local effects in vivo can be detected by radioimmunoimaging. Conclusion CD71 human/murine chimeric antibody showed marked killing of tumor cells in vitro, and specific recognition and high affinity binding to tumor tissue in vivo%目的 研究人鼠嵌合抗体D2C的体外抗瘤活性和体内分布。方法 以表达CD71分子的人类肿瘤细胞(K562,CEM和SMMC7721) 为靶细胞,以新鲜分离的正常人外周血单

  4. A Chimeric Pneumovirus Fusion Protein Carrying Neutralizing Epitopes of Both MPV and RSV.

    Directory of Open Access Journals (Sweden)

    Xiaolin Wen

    Full Text Available Respiratory syncytial virus (RSV and human metapneumovirus (HMPV are paramyxoviruses that are responsible for substantial human health burden, particularly in children and the elderly. The fusion (F glycoproteins are major targets of the neutralizing antibody response and studies have mapped dominant antigenic sites in F. Here we grafted a major neutralizing site of RSV F, recognized by the prophylactic monoclonal antibody palivizumab, onto HMPV F, generating a chimeric protein displaying epitopes of both viruses. We demonstrate that the resulting chimeric protein (RPM-1 is recognized by both anti-RSV and anti-HMPV F neutralizing antibodies indicating that it can be used to map the epitope specificity of antibodies raised against both viruses. Mice immunized with the RPM-1 chimeric antigen generate robust neutralizing antibody responses to MPV but weak or no cross-reactive recognition of RSV F, suggesting that grafting of the single palivizumab epitope stimulates a comparatively limited antibody response. The RPM-1 protein provides a new tool for characterizing the immune responses resulting from RSV and HMPV infections and provides insights into the requirements for developing a chimeric subunit vaccine that could induce robust and balanced immunity to both virus infections.

  5. A Chimeric Pneumovirus Fusion Protein Carrying Neutralizing Epitopes of Both MPV and RSV

    Science.gov (United States)

    Wen, Xiaolin; Pickens, Jennifer; Mousa, Jarrod J.; Leser, George P.; Lamb, Robert A.; Crowe, James E.; Jardetzky, Theodore S.

    2016-01-01

    Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are paramyxoviruses that are responsible for substantial human health burden, particularly in children and the elderly. The fusion (F) glycoproteins are major targets of the neutralizing antibody response and studies have mapped dominant antigenic sites in F. Here we grafted a major neutralizing site of RSV F, recognized by the prophylactic monoclonal antibody palivizumab, onto HMPV F, generating a chimeric protein displaying epitopes of both viruses. We demonstrate that the resulting chimeric protein (RPM-1) is recognized by both anti-RSV and anti-HMPV F neutralizing antibodies indicating that it can be used to map the epitope specificity of antibodies raised against both viruses. Mice immunized with the RPM-1 chimeric antigen generate robust neutralizing antibody responses to MPV but weak or no cross-reactive recognition of RSV F, suggesting that grafting of the single palivizumab epitope stimulates a comparatively limited antibody response. The RPM-1 protein provides a new tool for characterizing the immune responses resulting from RSV and HMPV infections and provides insights into the requirements for developing a chimeric subunit vaccine that could induce robust and balanced immunity to both virus infections. PMID:27224013

  6. 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 demons

  7. 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.

  8. [New antibodies in cancer treatment].

    Science.gov (United States)

    Pestalozzi, B C; Knuth, A

    2004-09-22

    Since the development of hybridoma technology in 1975 monoclonal antibodies with pre-defined specificity can be produced. Only twenty years later did it become possible to make therapeutic use of monoclonal antibodies in oncology. To this end it was necessary to attach the antigen-binding site of a mouse antibody onto the scaffold of a human antibody molecule. Such chimeric or "humanized" antibodies may be used in passive immunotherapy without eliciting an immune response. Rituximab and trastuzumab are such humanized antibodies. They are used today routinely in the treatment of malignant lymphoma and breast cancer, respectively. These antibodies are usually used in combination with conventional cytostatic anticancer drugs.

  9. Placental chimerism in early human pregnancy

    Directory of Open Access Journals (Sweden)

    Ashutosh Halder

    2005-01-01

    Full Text Available Background0 : Human chimerism is rare and usually uncovered through investigations of ambiguous genitalia or blood grouping or prenatal diagnosis. Most of the publications on placental chimerism are mainly case reports. There is no systematic search with sensitive techniques for placental chimerism in human. Aim0 : This study was aimed to asses placental chimerism through two sensitive molecular techniques i.e., interphase fluorescent in situ hybridization and quantitative fluorescent PCR. Material and methods0 : Placental chimerism was analyzed using X & Y dual color fluorescent in-situ hybridization onto 154 placentae from natural conceptions, obtained at termination of pregnancy between 7 to 16 weeks of gestation. Results0 : Three cases of placental sex chromosome chimerism were observed (1.95%. Exclusion of maternal contamination and diagnosis was confirmed later by quantitative fluorescent PCR. Conclusion0 : This finding indicates that placental chimerism in early human pregnancy is not rare.

  10. Comparison of immunoglobulin E measurements on IMMULITE and ImmunoCAP in samples consisting of allergen-specific mouse-human chimeric monoclonal antibodies towards allergen extracts and four recombinant allergens

    DEFF Research Database (Denmark)

    Szecsi, Pal B; Stender, Steen

    2013-01-01

    Specific immunoglobulin E (IgE) antibody in vitro tests are performed on enzyme immunoassay systems. Poor agreement among systems has been reported and comparisons have been made exclusively with allergen extracts - not with recombinant allergens. Here we compare the ImmunoCAP and the IMMULITE...

  11. Generation of chimeric rhesus monkeys.

    Science.gov (United States)

    Tachibana, Masahito; Sparman, Michelle; Ramsey, Cathy; Ma, Hong; Lee, Hyo-Sang; Penedo, Maria Cecilia T; Mitalipov, Shoukhrat

    2012-01-20

    Totipotent cells in early embryos are progenitors of all stem cells and are capable of developing into a whole organism, including extraembryonic tissues such as placenta. Pluripotent cells in the inner cell mass (ICM) are the descendants of totipotent cells and can differentiate into any cell type of a body except extraembryonic tissues. The ability to contribute to chimeric animals upon reintroduction into host embryos is the key feature of murine totipotent and pluripotent cells. Here, we demonstrate that rhesus monkey embryonic stem cells (ESCs) and isolated ICMs fail to incorporate into host embryos and develop into chimeras. However, chimeric offspring were produced following aggregation of totipotent cells of the four-cell embryos. These results provide insights into the species-specific nature of primate embryos and suggest that a chimera assay using pluripotent cells may not be feasible.

  12. Effects of platelet glycoprotein IIb/IIIa receptor blockade by a chimeric monoclonal antibody (abciximab) on acute and six-month outcomes after percutaneous transluminal coronary angioplasty for acute myocardial infarction. EPIC investigators.

    Science.gov (United States)

    Lefkovits, J; Ivanhoe, R J; Califf, R M; Bergelson, B A; Anderson, K M; Stoner, G L; Weisman, H F; Topol, E J

    1996-05-15

    Percutaneous transluminal coronary angioplasty (PTCA) for acute myocardial infarction is an attractive alternative to thrombolysis, but is still limited by recurrent ischemia and restenosis. We determined whether adjunctive platelet glycoprotein IIb/IIIa receptor blockade improved outcomes in patients undergoing direct and rescue PTCA in the Evaluation of c7E3 for Prevention of Ischemic Complications (EPIC) trial. Of the 2,099 patients undergoing percutaneous intervention who randomly received chimeric 7E3 Fab (c7E3) as a bolus, a bolus and 12-hour infusion, or placebo, 42 underwent direct PTCA for acute myocardial infarction and 22 patients had rescue PTCA after failed thrombolysis. The primary composite end point comprised death, reinfarction, repeat intervention, or bypass surgery. Outcomes were assessed at 30 days and 6 months. Baseline characteristics were similar in direct and rescue PTCA patients. Pooling the 2 groups, c7E3 bolus and infusion reduced the primary composite end point by 83% (26.1% placebo vs 4.5% c7E3 bolus and infusion, p = 0.06). No reinfarctions or repeat urgent interventions occurred in c7E3 bolus and infusion patients at 30 days, although there was a trend toward more deaths in c7E3-treated patients. Major bleeding was increased with c7E3 (24% vs 13%, p = 0.28). At 6 months, ischemic events were reduced from 47.8% with placebo to 4.5% with c7E3 bolus and infusion (p = 0.002), particularly reinfarction (p = 0.05) and repeat revascularization (p = 0.002). We conclude that adjunctive c7E3 therapy during direct and rescue PTCA decreased acute ischemic events and clinical restenosis in the EPIC trial. These data provide initial evidence of benefit for glycoprotein IIb/IIIa receptor blockade during PTCA for acute myocardial infarction.

  13. CD86嵌合抗体对系统性红斑狼疮患者自身反应性B细胞的作用及影响研究%Effects of an anti-CD86 chimeric antibody (ch1D1) on autoreactive B lymphocytes isolated from pa-tients with SLE

    Institute of Scientific and Technical Information of China (English)

    刘玉华; 陈兆军; 韩杰; 潘峰; 鄢巨振; 张腊红; 郑小银; 刘杨

    2016-01-01

    目的:探讨CD86嵌合抗体ch1D1对系统性红斑狼疮( systemic lupus erythematosus, SLE)患者自身反应性B细胞的作用及影响。方法利用流式细胞术分析SLE患者B细胞表面CD86的表达水平及ch1D1对SLE患者CD4+T细胞活化的影响;利用磁珠法分选出健康志愿者和SLE患者外周血单个核细胞( PBMC)中的B细胞、NK细胞以及CD4+T细胞用于后续实验;利用LDH释放法检测ch1D1介导对SLE患者B细胞的抗体依赖的细胞介导的细胞毒作用( antibody dependent cell medi-ated cytotoxicity,ADCC)及补体依赖的细胞介导的细胞毒作用( complement dependent cell mediated cy-totoxicity,CDC),利用ELISA法检测ch1D1对SLE患者B细胞分泌自身抗体的影响,利用3H掺入法分析ch1D1对SLE患者CD4+T细胞增殖的影响。结果 SLE患者B细胞上CD80(68.08±14.28 vs 46.10±12.14,n=24,P<0.0001)和CD86(44.72±14.90 vs 13.99±10.74,n=24,P<0.0001)的表达水平显著高于健康志愿者,提示B细胞异常活化;与对照组相比,ch1D1能更有效介导对SLE患者B细胞的ADCC和CDC作用(P=0.0172,P=0.0388);活化的T细胞显著增强SLE患者B细胞产生自身抗体,ch1D1显著抑制了SLE患者自身抗体的分泌(P=0.0019);SLE患者CD4+T细胞活化和增殖水平显著高于健康志愿者,而ch1D1显著抑制 SLE患者 CD4+T细胞的增殖和活化( P=0.0024,P=0.0495)。结论 CD86嵌合抗体能更有效地介导对SLE患者自身反应性B细胞的ADCC和CDC作用,抑制其自身抗体的分泌,抑制自身反应性CD4+T细胞的增殖和活化,有望成为治疗SLE的新型免疫制剂。%Objective To investigate the effects of ch1D1, an anti-CD86 chimeric antibody, on autoreactive B lymphocytes isolated from patients with systemic lupus erythematosus ( SLE) . Methods Flow cytometry analysis was performed to measure the expression of CD86 on the surface of B cells isolated from patients with SLE and to analyze the effects of ch1D1 on the activation of CD4+T cells

  14. Antithyroglobulin antibody

    Science.gov (United States)

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

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

    Directory of Open Access Journals (Sweden)

    Jie Zhong

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  17. Manufacture of diploid/tetraploid chimeric mice.

    OpenAIRE

    Lu, T Y; Markert, C L

    1980-01-01

    Tetraploid mouse embryos were produced by cytochalasin B treatment. These embryos usually die before completion of embryonic development and are abnormal morphologically and physiologically. The tetraploid embryos can be rescued to develop to maturity by aggregating them with normal diploid embryos to produce diploid/tetraploid chimeric mice. The diploid/tetraploid chimeric embryos are frequently abnormal: the larger the proportion of tetraploid cells, the greater the abnormality. By karyotyp...

  18. 抗肿瘤坏死因子相关凋亡诱导配体受体2嵌合抗体表达载体的构建、表达及其抗肿瘤活性分析%Construction and stable expression of anti-human tumor necrosis factor-related apoptosis-inducing ligand receptor 2 chimeric antibody in CHO cells

    Institute of Scientific and Technical Information of China (English)

    吕付佳; 史娟; 张亚玺; 刘士廉; 刘彦信; 郑德先

    2011-01-01

    目的:构建抗人肿瘤坏死因子相关凋亡诱导配体(TRAIL)受体2(死亡受体5,DR5)的人-鼠嵌合抗体表达载体,获得稳定表达该嵌合抗体的细胞株,并分析嵌合抗体的抗肿瘤活性.方法:采用DNA重组技术,扩增抗人DR5的鼠源单克隆抗体(mAb)AD5-10的重链(HC)、轻链(LC)可变区基因片段,并将其分别插入含有人IgG重链、轻链恒定区基因的真核表达载体RpCI-neo,以重、轻链表达质粒共转染中国仓鼠卵巢细胞(CHO),筛选稳定表达抗人DR5嵌合抗体(hmAD5-10)的重组细胞.采用Western blot和间接ELISA检测嵌合抗体的表达量及其与抗原DR5的结合活性.采用MTS比色法检测嵌合抗体的生物学活性.并对重组细胞株进行无血清培养驯化.结果:获得了2株稳定表达嵌合抗体的重组细胞株CHO-A5和CHO-B11,抗体的表达水平分别为(0.36±0.11)mg/L和(0.16±0.01)mg/L,嵌合抗体与DR5有较好的结合活性,对体外培养的人T淋巴细胞白血病细胞SVT35有显著的杀伤作用.结论:在真核细胞中表达了具有生物学活性的抗DR5的人-鼠嵌合抗体,为其应用于肿瘤治疗研究奠定了基础.%AIM: To establish an human-mouse chimeric antibody against tumor necrosis factor-related apoptosisinducing ligand (TRAIL) receptor 2 (death receptor 5, DR5) in an eukaryotic cell line and analyse ifs tumoricidal activity. METHODS: The cDNAs encoding for the variable regions of heavy chain (VH) and light chain (VL) of AD5-10 were amplified by PCR and inserted into the human lgG heavy and light chain containing expression vector RpClneo, respectively. The recombinant plasmids were co-transfected into HEK293 and/or CHO cells. The production of anti-DR5 human-mouse chimeric antibody (hmAD5-10) and the antibody affinity for DR5 were identified by ELISA and Western blot assay. The tumoricidal activity of hmAD5-10 was demonstrated by MTS assay. The stable expression cells were selected and cultured in serum-free medium

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

    Directory of Open Access Journals (Sweden)

    Cun Li

    2014-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  1. Chimerism and xenotransplantation. New concepts.

    Science.gov (United States)

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

    1999-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Nazanin Pirooznia

    2011-01-01

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

  3. Generation and preclinical evaluation of a DENV-1/2 prM+E chimeric live attenuated vaccine candidate with enhanced prM cleavage.

    Science.gov (United States)

    Keelapang, Poonsook; Nitatpattana, Narong; Suphatrakul, Amporn; Punyahathaikul, Surat; Sriburi, Rungtawan; Pulmanausahakul, Rojjanaporn; Pichyangkul, Sathit; Malasit, Prida; Yoksan, Sutee; Sittisombut, Nopporn

    2013-10-17

    In the absence of a vaccine or sustainable vector control measures, illnesses caused by dengue virus infection remain an important public health problem in many tropical countries. During the export of dengue virus particles, furin-mediated cleavage of the prM envelope protein is usually incomplete, thus generating a mixture of immature, partially mature and mature extracellular particles. Variations in the arrangement and conformation of the envelope proteins among these particles may be associated with their different roles in shaping the antibody response. In an attempt to improve upon live, attenuated dengue vaccine approaches, a mutant chimeric virus, with enhanced prM cleavage, was generated by introducing a cleavage-enhancing substitution into a chimeric DENV-1/2 virus genome, encoding the prM+E sequence of a recent DENV-1 isolate under an attenuated DENV-2 genetic background. A modest increase in virus specific infectivity observed in the mutant chimeric virus affected neither the attenuation phenotype, when assessed in the suckling mouse neurovirulence model, nor multiplication in mosquitoes. The two chimeric viruses induced similar levels of anti-DENV-1 neutralizing antibody response in mice and rhesus macaques, but more efficient control of viremia during viral challenge was observed in macaques immunized with the mutant chimeric virus. These results indicate that the DENV-1/2 chimeric virus, with enhanced prM cleavage, could be useful as an alternative live, attenuated vaccine candidate for further tests in humans.

  4. Chimerism in health, transplantation and autoimmunity

    NARCIS (Netherlands)

    Koopmans, Marije; Kremer Hovinga, Idske Cornelia Lydia

    2009-01-01

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

  5. Intra-serotype SAT2 chimeric foot-and-mouth disease vaccine protects cattle against FMDV challenge.

    Science.gov (United States)

    Maree, Francois F; Nsamba, Peninah; Mutowembwa, Paidamwoyo; Rotherham, Lia S; Esterhuysen, Jan; Scott, Katherine

    2015-06-09

    The genetic diversity of the three Southern African Territories (SAT) types of foot-and-mouth disease virus (FMDV) reflects high antigenic variation, and indications are that vaccines targeting each SAT-specific topotype may be needed. This has serious implications for control of FMD using vaccines as well as the choice of strains to include in regional antigen banks. Here, we investigated an intra-serotype chimeric virus, vSAT2(ZIM14)-SAT2, which was engineered by replacing the surface-exposed capsid-coding region (1B-1D/2A) of a SAT2 genome-length clone, pSAT2, with that of the field isolate, SAT2/ZIM/14/90. The chimeric FMDV produced by this technique was viable, grew to high titres and stably maintained the 1B-1D/2A sequence upon passage. Chemically inactivated, oil adjuvanted vaccines of both the chimeric and parental immunogens were used to vaccinate cattle. The serological response to vaccination showed the production of strong neutralizing antibody titres that correlated with protection against homologous FMDV challenge. We also predicted a good likelihood that cattle vaccinated with an intra-serotype chimeric vaccine would be protected against challenge with viruses that caused recent outbreaks in southern Africa. These results provide support that chimeric vaccines containing the external capsid of field isolates induce protective immune responses in FMD host species similar to the parental vaccine.

  6. 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.

  7. A CssA, CssB and LTB chimeric protein induces protection against Enterotoxigenic Escherichia coli

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    Samane Bagheri

    2014-06-01

    Full Text Available OBJECTIVES: Enterotoxigenic Escherichia coli (ETEC, a major cause of diarrhea in children under 5, is an important agent for traveler's diarrhea. Heat-labile enterotoxin (LT and colonization factors (CFs are two main virulence mechanisms in ETEC. CS6 is one of the most prevalent CFs consisting of two structural subunits viz., CssA, CssB, necessary for attachment to the intestinal cells. METHODS: In the present research, a chimeric trivalent protein composed of CssB, CssA and LTB was constructed. The chimeric gene was synthesized with codon bias of E. coli for enhanced expression of the protein. Recombinant proteins were expressed and purified. Mice were immunized with the recombinant protein. The antibody titer and specificity of the immune sera were analyzed by ELISA and Western blotting. Efficiency of the immune sera against ETEC was evaluated. RESULTS: Antibody induction was followed by immunization of mice with the chimeric protein. Pretreatment of the ETEC cells with immunized animal antisera remarkably decreased their adhesion to Caco-2 cells. DISCUSSION: The results indicate efficacy of the recombinant chimeric protein as an effective immunogen, which induces strong humoral response as well as protection against ETEC adherence and toxicity.

  8. Biodistribution of iodine-131-labled chimeric monoclonal antibody in patients following intratumoral administraion in advanced lung carcinoma%~(131)碘肿瘤细胞核人鼠嵌合单抗肺癌内直接注射后体内的生物学分布

    Institute of Scientific and Technical Information of China (English)

    李蓓蕾; 陈绍亮; 徐兆强; 于力克; 李田; 石洪成

    2009-01-01

    目的 研究肺癌瘤内直接注射~(131)Ⅰ-chTNT后患者体内分布状况.方法 经病理组织学确诊的原发性肺癌患者11例.根据CT定位,单次瘤内直接注射~(131)Ⅰ-chTNT 18.5~37 MBq/cm~3后,多时点测量血、尿放射性.应用HPLC法检测~(131)Ⅰ-chTNT在体内的稳定性和代谢情况.采用连续显像法估算全身、各主要脏器和肿瘤的放射性,并转换为注射剂量百分率(%ID),以观察~(131)Ⅰ-chTNT在体内的分布.结果 所有11例患者HPLC榆测结果显示,注射后24、48、72、96 h内血清中~(131)Ⅰ-chTNT均以原形存在,原形含量达100%.经计算机拟合,血清药-时曲线符合静脉外给药二室模型,T_(t/2kα)(0.89±0.17)h,T~(131)(1/2β)(86.88±25.97)h.游离~(131)Ⅰ是尿内~(131)Ⅰ-chTNT的唯一代谢产物.264 h累计尿排泄量为注射量的(58.37±17.45)%.瘤内给药后30 min,瘤内~(131)Ⅰ-chTNT量为(51.05±8.41)%ID,瘤/肺比值(T/N)高达(63.87±25.71).264 h时瘤内~(131)Ⅰ-chTNT残留(3.47±3.27)%ID,T/N为(9.61±11.00).全身主要器官中,放射性主要集中在肺、肝、心、肾、脾和甲状腺中.结论 ~(131)Ⅰ-chTNT瘤内直接注射后在瘤内停留时间较长,有利于肿瘤治疗.%Objective To investigate the biodistribution of intratumoral administerd~(131)Ⅰ-labeled human-mouse chimeric monoclonal antibody (chTNT) in patients with advanced lung carcinoma. Methods Eleven patients enrolled had cytological and histological confirmed diagnoses of either stage Ⅲ b or stage Ⅳ inoperable lung carcinoma. Intratumoral injection was directed by thoracic CT-guided catheter using a multi-holed needle. The dose for each patient was 18.5 - 37 MBq/cm~3 tumor mass. Blood samples were drawn at different time intervals for up to 13 days, and urine samples were collected for up to 11 days after injection for pharmacokinetic studies. In vivo stability was examined by HPLC by analyzing serum and urine, which were found to contain~(131)Ⅰ-chTNT. Whole body

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

    Science.gov (United States)

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

    2015-12-10

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

  10. Construction and preliminary investigation of a novel dengue serotype 4 chimeric virus using Japanese encephalitis vaccine strain SA14-14-2 as the backbone.

    Science.gov (United States)

    Li, Zhushi; Yang, Huiqiang; Yang, Jian; Lin, Hua; Wang, Wei; Liu, Lina; Zhao, Yu; Liu, Li; Zeng, Xianwu; Yu, Yongxin; Li, Yuhua

    2014-10-13

    For the purpose of developing a novel dengue vaccine candidate, recombinant plasmids were constructed which contained the full length cDNA clone of Japanese encephalitis (JE) vaccine strain SA14-14-2 with its premembrane (PreM) and envelope (E) genes replaced by the counterparts of dengue virus type 4 (DENV4). By transfecting the in vitro transcription products of the recombinant plasmids into BHK-21 cells, a chimeric virus JEV/DENV4 was successfully recovered. The chimeric virus was identified by complete genome sequencing, Western blot and immunofluorescent staining. Growth characteristics revealed it was well adapted to primary hamster kidney (PHK) cells. Its genetic stability was investigated and only one unintentional mutation in 5'-untranslated region (5'-UTR) was found after 20 passages in PHK cells. Neurotropism, neurovirulence and immunogenicity of the chimeric virus were tested in mice. Besides, the influence of JE vaccine pre-immunization on the neutralizing antibody level induced by the chimeric virus was illuminated. To our knowledge, this is the first chimeric virus incorporating the JE vaccine stain SA14-14-2 and DENV4. It is probably a potential candidate to compose a tetravalent dengue chimeric vaccine.

  11. Immunoreactivity evaluation of a new recombinant chimeric protein against Brucella in the murine model

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    Abbas Abdollahi

    2016-10-01

    Full Text Available Background and Objectives: Brucellosis is an important health problem in developing countries and no vaccine is available for the prevention of infection in humans. Because of clinically infectious diseases and their economic consequences in human and animals, designing a proper vaccine against Brucella is desirable. In this study, we evaluated the immune responses induced by a designed recombinant chimera protein in murine model.Materials and Methods: Three immunodominant antigens of Brucella have been characterized as potential immunogenic and protective antigens including: trigger factor (TF, Omp31 and Bp26 were fused together by EAAAK linkers to produce a chimera (structure were designed in silico, which was synthesized, cloned, and expressed in E. coli BL21 (DE3. The purification of recombinant protein was performed using Ni-NTA agarose. SDS-PAGE and anti-His antibody was used for confirmation purified protein (Western blot. BALB/c immunization was performed by purified protein and adjuvant, and sera antibody levels were measured by ELISA. otted.Results: SDS-PAGE and Western blotting results indicated the similarity of in silico designing and in vitro experiments. ELISA result proved that the immunized sera of mice contain high levels of antibodies (IgG against recombinant chimeric protein.Conclusion: The recombinant chimeric protein could be a potential antigen candidate for the development of a subunit vaccine against Brucella. Keywords: Brucella, Vaccine, Immunity, Recombinant

  12. Multiple chimeric antigen receptors successfully target chondroitin sulfate proteoglycan 4 in several different cancer histologies and cancer stem cells

    OpenAIRE

    Beard, Rachel E; Zheng, Zhili; Lagisetty, Kiran H.; Burns, William R.; Tran, Eric; Hewitt, Stephen M.; Abate-Daga, Daniel; Rosati, Shannon F.; Fine, Howard A.; Ferrone, Soldano; Rosenberg, Steven A.; Morgan, Richard A.

    2014-01-01

    Background The development of immunotherapy has led to significant progress in the treatment of metastatic cancer, including the development of genetic engineering technologies that redirect lymphocytes to recognize and target a wide variety of tumor antigens. Chimeric antigen receptors (CARs) are hybrid proteins combining antibody recognition domains linked to T cell signaling elements. Clinical trials of CAR-transduced peripheral blood lymphocytes (PBL) have induced remission of both solid ...

  13. 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.

  14. Regional atmospheric composition modeling with CHIMERE

    Science.gov (United States)

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

    2013-01-01

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

  15. Regional atmospheric composition modeling with CHIMERE

    Directory of Open Access Journals (Sweden)

    L. Menut

    2013-01-01

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

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

    NARCIS (Netherlands)

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

    2008-01-01

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

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

    NARCIS (Netherlands)

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

    2008-01-01

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

  18. Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

    Science.gov (United States)

    Wu, Jun; Platero-Luengo, Aida; Sakurai, Masahiro; Sugawara, Atsushi; Gil, Maria Antonia; Yamauchi, Takayoshi; Suzuki, Keiichiro; Bogliotti, Yanina Soledad; Cuello, Cristina; Morales Valencia, Mariana; Okumura, Daiji; Luo, Jingping; Vilariño, Marcela; Parrilla, Inmaculada; Soto, Delia Alba; Martinez, Cristina A; Hishida, Tomoaki; Sánchez-Bautista, Sonia; Martinez-Martinez, M Llanos; Wang, Huili; Nohalez, Alicia; Aizawa, Emi; Martinez-Redondo, Paloma; Ocampo, Alejandro; Reddy, Pradeep; Roca, Jordi; Maga, Elizabeth A; Esteban, Concepcion Rodriguez; Berggren, W Travis; Nuñez Delicado, Estrella; Lajara, Jeronimo; Guillen, Isabel; Guillen, Pedro; Campistol, Josep M; Martinez, Emilio A; Ross, Pablo Juan; Izpisua Belmonte, Juan Carlos

    2017-01-26

    Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

  19. Antibody Engineering for Pursuing a Healthier Future

    Science.gov (United States)

    Saeed, Abdullah F. U. H.; Wang, Rongzhi; Ling, Sumei; Wang, Shihua

    2017-01-01

    Since the development of antibody-production techniques, a number of immunoglobulins have been developed on a large scale using conventional methods. Hybridoma technology opened a new horizon in the production of antibodies against target antigens of infectious pathogens, malignant diseases including autoimmune disorders, and numerous potent toxins. However, these clinical humanized or chimeric murine antibodies have several limitations and complexities. Therefore, to overcome these difficulties, recent advances in genetic engineering techniques and phage display technique have allowed the production of highly specific recombinant antibodies. These engineered antibodies have been constructed in the hunt for novel therapeutic drugs equipped with enhanced immunoprotective abilities, such as engaging immune effector functions, effective development of fusion proteins, efficient tumor and tissue penetration, and high-affinity antibodies directed against conserved targets. Advanced antibody engineering techniques have extensive applications in the fields of immunology, biotechnology, diagnostics, and therapeutic medicines. However, there is limited knowledge regarding dynamic antibody development approaches. Therefore, this review extends beyond our understanding of conventional polyclonal and monoclonal antibodies. Furthermore, recent advances in antibody engineering techniques together with antibody fragments, display technologies, immunomodulation, and broad applications of antibodies are discussed to enhance innovative antibody production in pursuit of a healthier future for humans.

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

    Directory of Open Access Journals (Sweden)

    Guohua Yi

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPLE FINDINGS: 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. CONCLUSIONS: 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.

  1. Generation and characterization of a recombinant chimeric protein (rCpLi) consisting of B-cell epitopes of a dermonecrotic protein from Loxosceles intermedia spider venom.

    Science.gov (United States)

    Mendes, T M; Oliveira, D; Figueiredo, L F M; Machado-de-Avila, R A; Duarte, C G; Dias-Lopes, C; Guimarães, G; Felicori, L; Minozzo, J C; Chávez-Olortegui, C

    2013-06-01

    A chimeric protein was constructed expressing three epitopes of LiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. This species is responsible for a large number of accidents involving spiders in Brazil. We demonstrated that the chimeric protein (rCpLi) generated is atoxic and that antibodies previously developed in rabbits against synthetic epitopes reactive with rCpLi in ELISA and immunoblot assays. The antibody response in rabbits against the rCpLi was evaluated by ELISA and we have detected an antibody response in all immunized animals. Overlapping peptides covering the amino acid sequence of the rCpLi were synthesized on a cellulose membrane, and their recognition by rabbit anti-rCpLi serum assessed. Three different antigenic regions were identified. The percentage of inhibition of the dermonecrotic, hemorrhagic and edematogenic activities caused by the recombinant protein LiD1r in naïve rabbits was assessed by pre-incubation with anti-rCpLi antibodies. Anti-rCpLi induced good dermonecrotic and hemorrhagic protection. The levels of protection were similar to the antiboides anti-LiD1r. In summary, we have developed a polyepitope recombinant chimeric protein capable of inducing multiple responses of neutralizing antibodies in a rabbit model. This engineered protein may be a promising candidate for therapeutic serum development or vaccination.

  2. Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society December 7-10, 2015, San Diego, CA, USA.

    Science.gov (United States)

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M; Lund-Johansen, Fridtjof; Bradbury, Andrew R M; Carter, Paul J; Melis, Joost P M

    2016-01-01

    The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6-10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on "Antibodies to watch" in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries.

  3. 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.

  4. 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

    The FimH adhesin of type 1 fimbriae has been tested as a display system for heterologous protein segments on the surface of Escherichia coli. This was carried out by introduction of restriction site handles (BglII sites) in two different positions in the fimH gene, followed by in-frame insertion...... of heterologous DNA segments encoding two reporter sequences. In the selected positions such insertions did not significantly alter the function of the FimH protein with regard to surface location and adhesive ability. The system seemed to be quite flexible, since chimeric versions of the FimH adhesin containing...... 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...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1997-12-01

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

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    A chimeric pestivirus of border disease virus Gifhorn and bovine viral diarrhea virus CP7 (Meyers et al., 1996) was constructed. Virulence, immunogenicity and vaccine properties of the chimeric virus were studied in a vaccination–challenge experiment in pigs. The chimeric virus proved...... to be avirulent and neither chimeric virus nor viral RNA was detected in serum after vaccination. The safety of the vaccine was tested by horizontal transmission to sentinel pigs, which remained uninfected. The vaccine efficacy was examined by challenge infection with classical swine fever virus (CSFV) Eystrup....... In ‘challenge controls’, the viral load of CSFV coincided with the development of pronounced clinical symptoms. In contrast, the vaccinated pigs showed transient and weak clinical signs. Analysis of the viral load in these pigs showed 1000-fold lower viral RNA levels compared to ‘challenge controls...

  7. Chimeric mitochondrial peptides from contiguous regular and swinger RNA

    Directory of Open Access Journals (Sweden)

    Hervé Seligmann

    2016-01-01

    Full Text Available 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.

  8. 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.

  9. T- and B-lymphocyte chimerism in the marmoset

    Energy Technology Data Exchange (ETDEWEB)

    Niblack, G.D.; Kateley, J.R.; Gengozian, N.

    1977-01-01

    Marmosets are natural blood chimeras, this condition resulting from the high frequency of fraternal twinning and the consistent development of placental vascular anastomoses between the two embryos. Identification of chimerism by sex-chromosome analysis of cultured blood lymphocytes provided a means of determining the proportion of chimerism among T and B lymphocytes. Peripheral blood lymphocytes were enriched for T or B cells by filtration through a nylon column (yields >95% T-cells) or inactivation of T lymphocytes by treatment with a goat anti-marmoset thymocyte antiserum in the presence of complement (yields >95% B cells). Mitogenic stimulation of these separated, enriched cell populations yielded metaphase plates which could be scored for percentage male and female cells. Tests on five different blood chimeras showed the T- and B-lymphocyte chimerism to be the same. Stimulation of blood lymphocytes with cells from another species of marmoset in a mixed lymphocyte culture test revealed the chimeric T-cell response (i.e., host and co-twin cells) to be similar to that obtained with a mitogenic lectin. The demonstration of equivalent T- and B-cell chimerism in these animals suggests derivation of these cells from a common stem cell pool and the response of both T-cell populations to an antigenic stimulus in proportions similar to their percentage chimerism suggests complete immunologic tolerance exists in this species for co-twin histocompatibility antigens.

  10. Quantification of mixed chimerism allows early therapeutic interventions

    Directory of Open Access Journals (Sweden)

    Jóice Merzoni

    2014-10-01

    Full Text Available Hematopoietic stem cell transplantation is the curative option for patients with myelodysplastic syndrome; however, it requires a long post-transplantation follow-up. A 53-year-old woman with a diagnosis of myelodysplastic syndrome underwent related donor allogeneic hematopoietic stem cell transplantation in July 2006. Three months after transplantation, a comparative short tandem repeat analysis between donor and recipient revealed full chimerism, indicating complete, healthy bone marrow reconstitution. Three years and ten months after hematopoietic stem cell transplantation, the patient developed leukopenia and thrombocytopenia. Another short tandem repeat analysis was carried out which showed mixed chimerism (52.62%, indicating relapsed disease. A donor lymphocyte infusion was administered. The purpose of donor lymphocyte infusion is to induce a graft-versus-leukemia effect; in fact, this donor's lymphocyte infusion induced full chimerism. Successive short tandem repeat analyses were performed as part of post-transplantation follow-up, and in July 2010, one such analysis again showed mixed chimerism (64.25%. Based on this finding, a second donor lymphocyte infusion was administered, but failed to eradicate the disease. In September 2011, the patient presented with relapsed disease, and a second related donor allogeneic hematopoietic stem cell transplantation was performed. Subsequent short tandem repeat analyses revealed full chimerism, indicating complete bone marrow reconstitution. We conclude that quantitative detection of mixed chimerism is an important diagnostic tool that can guide early therapeutic intervention.

  11. Antimitochondrial antibody

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003529.htm Antimitochondrial antibody To use the sharing features on this page, please enable JavaScript. Antimitochondrial antibodies (AMA) are substances ( antibodies ) that form against mitochondria. ...

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

    Directory of Open Access Journals (Sweden)

    Hamid Sedighian Rad

    2013-09-01

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

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

    Science.gov (United States)

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

    2016-07-19

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

  14. Chimeric hepatitis B virus core particles with parts or copies of the hepatitis C virus core protein.

    OpenAIRE

    Yoshikawa, A.; Tanaka, T; Hoshi, Y.; Kato, N; K. Tachibana; Iizuka, H; Machida, A; Okamoto, H; Yamasaki, M.; Miyakawa, Y

    1993-01-01

    Either parts or multiple copies of the core gene of hepatitis C virus (HCV) were fused to the 3' terminus of the hepatitis B virus (HBV) core gene with 34 codons removed. As many as four copies of HCV core protein (720 amino acids) were fused to the carboxy terminus of truncated HBV core protein (149 amino acids) without preventing the assembly of HBV core particles. Chimeric core particles were sandwiched between monoclonal antibody to HBV core and that to HCV core, thereby indicating that a...

  15. Preparation and Biological Evaluation of 188Re Labeled Monoclonal Antibody TGLA

    Institute of Scientific and Technical Information of China (English)

    WEN; Kai; ZHANG; Jun-li; CHEN; Bao-jun; CUI; Hai-ping

    2012-01-01

    <正>Monoclonal antibody TGLA is a specific targeting CD20 chimeric antibody. It can kill tumor cells and inhibit tumor cells’ growth effectively, which has been applied to clinical therapy of lymphoma cell B. 188 Re is easy to get, and emits both β and γ rays. 188Re labeled monoclonal antibody TGLA can be used for the study of lymphoma therapy and imaging. This work got the product 188Re-TGLA by direct labeling

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

    Science.gov (United States)

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

    2016-01-01

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

  17. Prevention of birch pollen-related food allergy by mucosal treatment with multi-allergen-chimers in mice.

    Directory of Open Access Journals (Sweden)

    Elisabeth Hoflehner

    Full Text Available BACKGROUND: Among birch pollen allergic patients up to 70% develop allergic reactions to Bet v 1-homologue food allergens such as Api g 1 (celery or Dau c 1 (carrot, termed as birch pollen-related food allergy. In most cases, specific immunotherapy with birch pollen extracts does not reduce allergic symptoms to the homologue food allergens. We therefore genetically engineered a multi-allergen chimer and tested if mucosal treatment with this construct could represent a novel approach for prevention of birch pollen-related food allergy. METHODOLOGY: BALB/c mice were poly-sensitized with a mixture of Bet v 1, Api g 1 and Dau c 1 followed by a sublingual challenge with carrot, celery and birch pollen extracts. For prevention of allergy sensitization an allergen chimer composed of immunodominant T cell epitopes of Api g 1 and Dau c 1 linked to the whole Bet v 1 allergen, was intranasally applied prior to sensitization. RESULTS: Intranasal pretreatment with the allergen chimer led to significantly decreased antigen-specific IgE-dependent β-hexosaminidase release, but enhanced allergen-specific IgG2a and IgA antibodies. Accordingly, IL-4 levels in spleen cell cultures and IL-5 levels in restimulated spleen and cervical lymph node cell cultures were markedly reduced, while IFN-γ levels were increased. Immunomodulation was associated with increased IL-10, TGF-β and Foxp3 mRNA levels in NALT and Foxp3 in oral mucosal tissues. Treatment with anti-TGF-β, anti-IL10R or anti-CD25 antibodies abrogated the suppression of allergic responses induced by the chimer. CONCLUSION: Our results indicate that mucosal application of the allergen chimer led to decreased Th2 immune responses against Bet v 1 and its homologue food allergens Api g 1 and Dau c 1 by regulatory and Th1-biased immune responses. These data suggest that mucosal treatment with a multi-allergen vaccine could be a promising treatment strategy to prevent birch pollen-related food allergy.

  18. Monoclonal Antibody-Based Therapeutics for Melioidosis and Glanders

    Directory of Open Access Journals (Sweden)

    Hyung-Yong Kim

    2011-01-01

    Full Text Available Problem statement: Burkholderia Pseudomallei (BP and B. Mallei (BM were two closely related pathogenic gram-negative bacteria. They were the causative agents of melioidosis and glanders, respectively and are recognized by CDC as category B select agents. Significant efforts had been devoted to developing the diagnostic and therapeutic measures against these two pathogens. Monoclonal antibody-based therapeutic was a promising targeted therapy to fight against melioidosis and glanders. Valuable findings have been reported by different groups in their attempt to identify vaccine targets against these two pathogens. Approach: Our group has generated neutralizing Monoclonal Antibodies (MAbs against BP and BM and characterized them by both in vitro and in vivo experiments. We present an overview of the MAb-based therapeutic approaches against BP and BM and demonstrate some of our efforts for developing chimeric and fully human MAbs using antibody engineering. Results: Throughout conventional mouse hybridoma technique and antibody engineering (chimerization and in vitro antibody library techniques, we generated 10 chimeric MAbs (3 stable MAbs and 7 transient MAbs and one fully human MAb against BP and BM. In addition, we present the reactive antigen profiles of these MAbs. Our approaches had potentials to accelerate the development of therapeutics for melioidosis and glanders in humans. Conclusion: Our experience and findings presented here will be valuable for choosing the best antigenic targets and ultimately for the production of effective vaccines for these two pathogens.

  19. Capture ELISA for IgM antibodies against Plasmodium falciparum glutamate rich protein

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Borre, M B; Petersen, E;

    1992-01-01

    This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta-galactos......This report describes a novel mu chain capture ELISA for the detection of IgM antibodies against a Plasmodium falciparum antigen. A fragment of the 220 kDa P. falciparum glutamate rich protein containing amino acid residues 489-1271 was expressed in E. coli as a recombinant chimeric beta...

  20. 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...

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

    Science.gov (United States)

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

    2016-02-01

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

  2. Live attenuated chimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vaccine: Phase I clinical trial for safety and immunogenicity: effect of yellow fever pre-immunity in induction of cross neutralizing antibody responses to all 4 dengue serotypes.

    Science.gov (United States)

    Guirakhoo, Farshad; Kitchener, Scott; Morrison, Dennis; Forrat, Remi; McCarthy, Karen; Nichols, Richard; Yoksan, Sutee; Duan, Xiaochu; Ermak, Thomas H; Kanesa-Thasan, Niranjan; Bedford, Philip; Lang, Jean; Quentin-Millet, Marie-Jose; Monath, Thomas P

    2006-01-01

    A randomized double-blind Phase I Trial was conducted to evaluate safety, tolerability, and immunogenicity of a yellow fever (YF)-dengue 2 (DEN2) chimera (ChimeriVax-DEN2) in comparison to that of YF vaccine (YF-VAX). Forty-two healthy YF naïve adults randomly received a single dose of either ChimeriVax-DEN2 (high dose, 5 log plaque forming units [PFU] or low dose, 3 log PFU) or YF-VAX by the subcutaneous route (SC). To determine the effect of YF preimmunity on the ChimeriVax-DEN2 vaccine, 14 subjects previously vaccinated against YF received a high dose of ChimeriVax-DEN2 as an open-label vaccine. Most adverse events were similar to YF-VAX and of mild to moderate intensity, with no serious side-effects. One hundred percent and 92.3% of YF naïve subjects inoculated with 5.0 and 3.0 log10 PFU of ChimeriVax-DEN2, respectively, seroconverted to wt DEN2 (strain 16681); 92% of subjects inoculated with YF-VAX seroconverted to YF 17D virus but none of YF naïve subjects inoculated with ChimeriVax-DEN2 seroconverted to YF 17D virus. Low seroconversion rates to heterologous DEN serotypes 1, 3 and 4 were observed in YF naïve subjects inoculated with either ChimeriVax-DEN2 or YF-VAX. In contrast, 100% of YF immune subjects inoculated with ChimeriVax-DEN2 seroconverted to all 4 DEN serotypes. Surprisingly, levels of neutralizing antibodies to DEN 1, 2 and 3 viruses in YF immune subjects persisted after 1 year. These data demonstrated that (1) the safety and immunogenicity profile of the ChimeriVax-DEN2 vaccine is consistent with that of YF-VAX, and (2) preimmunity to YF virus does not interfere with ChimeriVax-DEN2 immunization, but induces a long lasting and cross neutralizing antibody response to all 4 DEN serotypes. The latter observation can have practical implications toward development of a dengue vaccine.

  3. 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...

  4. Antibody engineering & therapeutics, the annual meeting of the antibody society December 7–10, 2015, San Diego, CA, USA

    Science.gov (United States)

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M.; Lund-Johansen, Fridtjof; Bradbury, Andrew R.M.; Carter, Paul J.; Melis, Joost P.M.

    2016-01-01

    ABSTRACT The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6–10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on “Antibodies to watch” in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries. PMID:26909869

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

    Science.gov (United States)

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

    2016-01-01

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

  6. Immunogenicity and efficacy of chimeric dengue vaccine (DENVax) formulations in interferon-deficient AG129 mice.

    Science.gov (United States)

    Brewoo, Joseph N; Kinney, Richard M; Powell, Tim D; Arguello, John J; Silengo, Shawn J; Partidos, Charalambos D; Huang, Claire Y-H; Stinchcomb, Dan T; Osorio, Jorge E

    2012-02-14

    Formulations of chimeric dengue vaccine (DENVax) viruses containing the pre-membrane (prM) and envelope (E) genes of serotypes 1-4 expressed in the context of the attenuated DENV-2 PDK-53 genome were tested for safety, immunogenicity and efficacy in interferon receptor knock-out mice (AG129). Monovalent formulations were safe and elicited robust neutralizing antibody responses to the homologous virus and only limited cross-reactivity to other serotypes. A single dose of monovalent DENVax-1, -2, or -3 vaccine provided eighty or greater percent protection against both wild-type (wt) DENV-1 (Mochizuki strain) and DENV-2 (New Guinea C strain) challenge viruses. A single dose of monovalent DENVax-4 also provided complete protection against wt DENV-1 challenge and significantly increased the survival times after challenge with wt DENV-2. In studies using tetravalent mixtures, DENVax ratios were identified that: (i) caused limited viremia, (ii) induced serotype-specific neutralizing antibodies to all four DENV serotypes with different hierarchies, and (iii) conferred full protection against clinical signs of disease following challenge with either wt DENV-1 or DENV-2 viruses. Overall, these data highlight the immunogenic profile of DENVax, a novel candidate tetravalent dengue vaccine and the advantage of sharing a common attenuated genomic backbone among the DENVax monovalent vaccines that confer protection against homologous or heterologous virus challenge.

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Mark S Pearson

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

  9. Serodiagnosis of Toxoplasma gondii infection in farm animals (horses, swine, and sheep) by enzyme-linked immunosorbent assay using chimeric antigens.

    Science.gov (United States)

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

    2015-10-01

    Toxoplasma gondii infects all warm-blooded animals including humans, causing serious public health problems and great economic loss in the animal husbandry. Commonly used serological tests for diagnosis of toxoplasmosis involve preparation of whole Toxoplasma lysate antigen (TLA) from tachyzoites. The production of this antigen is associated with high costs and lengthy preparation and the possibility of staff infection. There are also some difficulties in the standardization of such tests. One approach in order to improve the diagnosis of T. gondii infection is to use recombinant chimeric antigens in place of the TLA, which was confirmed by studies in the serodiagnosis of toxoplasmosis in humans. In this paper, we assess, for the first time, the diagnostic utility of five T. gondii recombinant chimeric antigens (MIC1-MAG1-SAG1S, SAG1L-MIC1-MAG1, SAG2-GRA1-ROP1S, SAG2-GRA1-ROP1L, and GRA1-GRA2-GRA6) in immunoglobulin G (IgG) enzyme-linked immunosorbent assays (IgG ELISAs) with sera from three different groups of livestock animals (horses, pigs, and sheep). The reactivity of individual chimeric antigens was analyzed in relation to the results obtained in IgG ELISAs based on a mixture of three antigens (M1: rSAG1+rMIC1+rMAG1, M2: rSAG2+rGRA1+rROP1, and M3: rGRA1+rGRA2+rGRA6) and referenced to TLA. All chimeric antigens were characterized by high specificity (100%), and the sensitivity of the IgG ELISAs based on chimeric antigens was variable (between 28.4% and 100%) and mainly dependent on the animal species. The chimeric antigens were generally more reactive than mixtures of three antigens. The most effective for the diagnosis of toxoplasmosis was SAG2-GRA1-ROP1L, which can detect specific anti-T. gondii antibodies in 100%, 93.8%, and 100% of positive serum samples from horses, pigs, and sheep, respectively. The present study shows that recombinant chimeric antigens can be successfully used to diagnose T. gondii infection in farm animals, and can replace the commonly

  10. Chimeric classical swine fever (CSF)-Japanese encephalitis (JE) viral replicon as a non-transmissible vaccine candidate against CSF and JE infections.

    Science.gov (United States)

    Yang, Zhenhua; Wu, Rui; Li, Robert W; Li, Ling; Xiong, Zhongliang; Zhao, Haizhong; Guo, Deyin; Pan, Zishu

    2012-04-01

    A trans-complemented chimeric CSF-JE virus replicon was constructed using an infectious cDNA clone of the CSF virus (CSFV) Alfort/187 strain. The CSFV E2 gene was deleted, and a fragment containing the region encoding a truncated envelope protein (tE, amino acid 292-402, domain III) of JE virus (JEV) was inserted into the resultant plasmid, pA187delE2, to generate the recombinant cDNA clone pA187delE2/JEV-tE. Porcine kidney 15 (PK15) cells that constitutively express the CSFV E2p7 proteins were then transfected with in vitro-transcribed RNA from pA187delE2/JEV-tE. As a result, the chimeric CSF-JE virus replicon particle (VRP), rv187delE2/JEV-tE, was rescued. In a mouse model, immunization with the chimeric CSF-JE VRP induced strong production of JEV-specific antibody and conferred protection against a lethal JEV challenge. Pigs immunized with CSF-JE VRP displayed strong anti-CSFV and anti-JEV antibody responses and protection against CSFV and JEV challenge infections. Our evidence suggests that E2-complemented CSF-JE VRP not only has potential as a live-attenuated non-transmissible vaccine candidate against CSF and JE but also serves as a potential DIVA (Differentiating Infected from Vaccinated Animals) vaccine for CSF in pigs. Together, our data suggest that the non-transmissible chimeric VRP expressing foreign antigenic proteins may represent a promising strategy for bivalent DIVA vaccine design.

  11. Comparison of different blood sample processing methods for sensitive detection of low level chimerism by RHD real-time PCR assay.

    Science.gov (United States)

    Javadi, Ahmad; Verduin, Esther P; Brand, Anneke; Schonewille, Henk

    2013-01-01

    The rhesus D blood group, which is expressed on the red blood cells (RBC) of 85% of the Caucasian population, is one of the most immunogenic RBC antigens, inducing D antibody formation in up to 20-80% of D-negative transfusion recipients and about 10% of pregnancies at risk. Pregnancy-induced D-antibodies can persist for many years, but the mechanisms underlying this persistence are unclear. The LOTUS study, a long-term follow-up study of mothers from severely affected children with hemolytic disease of the fetus and newborn investigates, among other endpoints, whether persistent feto-maternal chimerism is associated with long-term maternal anti-D persistence. We questioned which blood sample processing method should be used to detect low levels of RHD chimerism with the highest sensitivity and specificity using qPCR. After optimization of primer and probe concentrations for singleplex RHD exon 5 and 7 qPCR, sensitivity, specificity and efficiency of RHD and DYS1 qPCR were investigated in artificial chimeric samples. Sensitivity of DYS1 was one log higher (0.0001%) in enriched mononuclear cell fractions as compared with whole blood. Comparable linear sensitivity (0.007%) and mean efficiency (84-99%) for RHD qPCR were observed in all samples regardless whether whole blood or pre- or post-mixing of cellular fractions had been used. We conclude that RHD chimerism using singleplex exon 5 and 7 qPCR is linearly detectable down to 1.0 GE, without an advantage of fraction enrichment.

  12. Two courses of rituximab (anti-CD20 monoclonal antibody) for recalcitrant pemphigus vulgaris

    DEFF Research Database (Denmark)

    Faurschou, A.; Gniadecki, R.

    2008-01-01

    Background Pemphigus vulgaris (PV) is a severe autoimmune blistering disease involving the skin and mucous membranes. The response to therapy varies greatly amongst patients and treatment may be challenging. Rituximab is a chimeric monoclonal antibody that selectively targets cell surface antigen...

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

    DEFF Research Database (Denmark)

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

    An advantage of the use of chimeric pestiviruses as modified live vaccines against classical swine fever (CSF) resides in their capacity to be manipulated to achieve the characteristics desired for safe and efficacious DIVA vaccines. We have recently generated a new chimeric virus, Riems26_E2gif...... engineered specifically for this purpose. The E2-substituted Riems26_E2gif was derived by homologues recombination of the complete E2 protein encoding genome region from Border disease strain Gifhorn into a bacterial artificial chromosome (BAC) harbouring the genome of the CSFV vaccine strain C......-Riems. The virulence, immunogenicity and vaccine properties of Riems26_E2gif were tested in a vaccine-challenge experiment in pigs. Riems26_E2gif vaccinated pigs could be differentiated from infected pigs using a CSFV-E2 specific ELISA. Following challenge infection with highly virulent CSFV strain Koslov, all...

  14. Mechanisms of Tolerance Induction by Hematopoietic Chimerism: The Immune Perspective.

    Science.gov (United States)

    Yolcu, Esma S; Shirwan, Haval; Askenasy, Nadir

    2017-03-01

    Hematopoietic chimerism is one of the effective approaches to induce tolerance to donor-derived tissue and organ grafts without administration of life-long immunosuppressive therapy. Although experimental efforts to develop such regimens have been ongoing for decades, substantial cumulative toxicity of combined hematopoietic and tissue transplants precludes wide clinical implementation. Tolerance is an active immunological process that includes both peripheral and central mechanisms of mutual education of coresident donor and host immune systems. The major stages include sequential suppression of early alloreactivity, establishment of hematopoietic chimerism and suppressor cells that sustain the state of tolerance, with significant mechanistic and temporal overlap along the tolerization process. Efforts to devise less toxic transplant strategies by reduction of preparatory conditioning focus on modulation rather than deletion of residual host immunity and early reinstitution of regulatory subsets at the central and peripheral levels. Stem Cells Translational Medicine 2017;6:700-712.

  15. Long-term experience of plasmapheresis in antibody-mediated rejection in renal transplantation.

    LENUS (Irish Health Repository)

    Brown, C M

    2009-11-01

    Antibody-mediated rejection (AMR) continues to pose a serious challenge in renal transplantation with potentially devastating consequences. Treatment options for this condition include plasmapheresis, high-dose intravenous immunoglobulin (IVIG), plasmapheresis with low-dose IVIG, and the use of rituximab (anti-CD20 chimeric antibody). We previously reported on the short-term outcome of plasmapheresis as a rescue therapy for AMR in our centre. We now report on the long-term follow up.

  16. Novel nanocomposites from spider silk–silica fusion (chimeric) proteins

    OpenAIRE

    Wong Po Foo, Cheryl; Patwardhan, Siddharth V.; Belton, David J.; Kitchel, Brandon; Anastasiades, Daphne; Huang, Jia; Naik, Rajesh R.; Perry, Carole C.; Kaplan, David L.

    2006-01-01

    Silica skeletal architectures in diatoms are characterized by remarkable morphological and nanostructural details. Silk proteins from spiders and silkworms form strong and intricate self-assembling fibrous biomaterials in nature. We combined the features of silk with biosilica through the design, synthesis, and characterization of a novel family of chimeric proteins for subsequent use in model materials forming reactions. The domains from the major ampullate spidroin 1 (MaSp1) protein of Neph...

  17. 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.

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

    Institute of Scientific and Technical Information of China (English)

    何畏; 梁志清; 史常旭; 李玉清

    2001-01-01

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

  19. Novel in-ovo chimeric recombinant Newcastle disease vaccine protects against both Newcastle disease and infectious bursal disease.

    Science.gov (United States)

    Ge, Jinying; Wang, Xijun; Tian, Meijie; Wen, Zhiyuan; Feng, Qiulin; Qi, Xiaole; Gao, Honglei; Wang, Xiaomei; Bu, Zhigao

    2014-03-14

    Development of a safe and efficient in-ovo vaccine against Newcastle disease (NDV) and very virulent infectious bursal disease virus (vvIBDV) is of great importance. In this study, a chimeric NDV LaSota virus with the L gene of Clone-30 (rLaC30L) was used to generate a recombinant chimeric virus expressing the VP2 protein of vvIBDV (rLaC30L-VP2). The safety and efficacy of rLaC30L-VP2 in-ovo vaccination was then evaluated in 18-day-old special pathogen free (SPF) chicken embryos and commercial broiler embryos for prevention of NDV and vvIBDV. Hatchability and global survival rate of the hatched birds was not affected by in-ovo rLaC30L-VP2 vaccination. However, rLaC30L-VP2 in-ovo vaccination induced significant anti-IBDV and anti-NDV antibodies in SPF birds and commercial broilers, and 100% of vaccinated chickens were protected against a lethal NDV challenge. In-ovo rLaC30L-VP2 vaccination also provided resistance against vvIBDV challenge in a significant amount of animals. These results suggest that rLaC30L-VP2 is a safe and efficient bivalent live in-ovo vaccine against NDV and vvIBDV.

  20. Structure-based approach to rationally design a chimeric protein for an effective vaccine against Group B Streptococcus infections

    Science.gov (United States)

    Nuccitelli, Annalisa; Cozzi, Roberta; Gourlay, Louise J.; Donnarumma, Danilo; Necchi, Francesca; Norais, Nathalie; Telford, John L.; Rappuoli, Rino; Bolognesi, Martino; Maione, Domenico; Grandi, Guido; Rinaudo, C. Daniela

    2011-01-01

    Structural vaccinology is an emerging strategy for the rational design of vaccine candidates. We successfully applied structural vaccinology to design a fully synthetic protein with multivalent protection activity. In Group B Streptococcus, cell-surface pili have aroused great interest because of their direct roles in virulence and importance as protective antigens. The backbone subunit of type 2a pilus (BP-2a) is present in six immunogenically different but structurally similar variants. We determined the 3D structure of one of the variants, and experimentally demonstrated that protective antibodies specifically recognize one of the four domains that comprise the protein. We therefore constructed a synthetic protein constituted by the protective domain of each one of the six variants and showed that the chimeric protein protects mice against the challenge with all of the type 2a pilus-carrying strains. This work demonstrates the power of structural vaccinology and will facilitate the development of an optimized, broadly protective pilus-based vaccine against Group B Streptococcus by combining the uniquely generated chimeric protein with protective pilin subunits from two other previously identified pilus types. In addition, this work describes a template procedure that can be followed to develop vaccines against other bacterial pathogens. PMID:21593422

  1. Chimeric RNA Oligonucleotides with Triazole and Phosphate Linkages: Synthesis and RNA Interference.

    Science.gov (United States)

    Fujino, Tomoko; Kogashi, Kanako; Okada, Koudai; Mattarella, Martin; Suzuki, Takeru; Yasumoto, Kenichi; Sogawa, Kazuhiro; Isobe, Hiroyuki

    2015-12-01

    Chimeric RNA oligonucleotides with an artificial triazole linker were synthesized using solution-phase click chemistry and solid-phase automated synthesis. Scalable synthesis methods for jointing units for the chimeric structure have been developed, and after click-coupling of the jointing units with triazole linkers, a series of chimeric oligonucleotides was prepared by utilizing the well-established phosphoramidite method for the elongation. The series of chimeric 21-mer oligonucleotides that possessed the triazole linker at different strands and positions allowed for a screening study of the RNA interference to clarify the preference of the triazole modifications in small-interfering RNA molecules.

  2. CD19-Targeted chimeric antigen receptor-modified T-cell immunotherapy for B-cell malignancies.

    Science.gov (United States)

    Turtle, C J; Riddell, S R; Maloney, D G

    2016-09-01

    Chimeric antigen receptors (CARs) comprise a tumor-targeting moiety, often in the form of a single chain variable fragment derived from a monoclonal antibody, fused to one or more intracellular T-cell signaling sequences. Lymphodepletion chemotherapy followed by infusion of T cells that are genetically modified to express a CD19-specific CAR is a promising therapy for patients with refractory CD19(+) B-cell malignancies, producing rates of complete remission that are remarkably high in acute lymphoblastic leukemia and encouraging in non-Hodgkin lymphoma and chronic lymphocytic leukemia. Responses are often durable, although additional studies are needed to define the role of CAR-T cell immunotherapy in the context of other treatments. CAR-modified T-cell immunotherapy can be complicated by cytokine release syndrome and neurologic toxicity, which in most cases are manageable and reversible. Here we review recent clinical trial data and discuss issues for the field.

  3. 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

    with a dose regimen of 375 mg/m2 i.v. approximately once weekly for 4 consecutive weeks. Six patients received a fixed dose of 500 mg disregarding their weight supplemented by 100 mg of methylprednisone i.v. or 50-100 mg of Pred given as premedication together with an antihistamine just before infusion...

  4. Thyroid Antibodies

    Science.gov (United States)

    ... e.g., at regular intervals after thyroid cancer treatment) Thyroid stimulating hormone receptor antibody, Thyroid Stimulating Immunoglobulin TRAb, TSHR Ab, TSI Graves disease When a person has symptoms of hyperthyroidism If a pregnant woman has a known autoimmune ...

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

    Science.gov (United States)

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

    2014-05-01

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

  6. Chimeric antigen receptor (CAR)-directed adoptive immunotherapy: a new era in targeted cancer therapy.

    Science.gov (United States)

    Chen, Yamei; Liu, Delong

    2014-01-01

    As a result of the recent advances in molecular immunology, virology, genetics, and cell processing, chimeric antigen receptor (CAR)-directed cancer therapy has finally arrived for clinical application. CAR-directed adoptive immunotherapy represents a novel form of gene therapy, cellular therapy, and immunotherapy, a combination of three in one. Early phase clinical trial was reported in patients with refractory chronic lymphoid leukemia with 17p deletion. Accompanying the cytokine storm and tumor lysis syndrome was the shocking disappearance of the leukemia cells refractory to chemotherapy and monoclonal antibodies. CAR therapy was reproduced in both children and adults with refractory acute lymphoid leukemia. The CAR technology is being explored for solid tumor therapy, such as glioma. Close to 30 clinical trials are underway in the related fields (www.clinicaltrials.gov). Further improvement in gene targeting, cell expansion, delivery constructs (such as using Sleeping Beauty or Piggyback transposons) will undoubtedly enhance clinical utility. It is foreseeable that CAR-engineered T cell therapy will bring targeted cancer therapy into a new era.

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

    Science.gov (United States)

    Alrifai, Doraid; Sarker, Debashis; Maher, John

    2016-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  9. Solution structure of a Plasmodium falciparum AMA-1/MSP 1 chimeric protein vaccine candidate (PfCP-2.9 for malaria

    Directory of Open Access Journals (Sweden)

    Jin Changwen

    2010-03-01

    Full Text Available Abstract Background The Plasmodium falciparum chimeric protein PfCP-2.9 is a promising asexual-stage malaria vaccine evaluated in clinical trials. This chimeric protein consists of two cysteine-rich domains: domain III of the apical membrane antigen 1 (AMA-1 [III] and the C-terminal region of the merozoite surface protein 1 (MSP1-19. It has been reported that the fusion of these two antigens enhanced their immunogenicity and antibody-mediated inhibition of parasite growth in vitro. Methods The 15N-labeled and 13C/15N-labeled PfCP-2.9 was produced in Pichia pastoris for nuclear magnetic resonance (NMR structure analysis. The chemical shift assignments of PfCP-2.9 were compared with those previously reported for the individual domains (i.e., PfAMA-1(III or PfMSP 1-19. The two-dimensional spectra and transverse relaxation rates (R2 of the PfMSP1-19 alone were compared with that of the PfCP-2.9. Results Confident backbone assignments were obtained for 122 out of 241 residues of PfCP-2.9. The assigned residues in PfCP-2.9 were very similar to those previously reported for the individual domains. The conformation of the PfMSP1-19 in different constructs is essentially the same. Comparison of transverse relaxation rates (R2 strongly suggests no weak interaction between the domains. Conclusions These data indicate that the fusion of AMA-1(III and MSP1-19 as chimeric protein did not change their structures, supporting the use of the chimeric protein as a potential malaria vaccine.

  10. 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.

  11. Novel nanocomposites from spider silk–silica fusion (chimeric) proteins

    Science.gov (United States)

    Wong Po Foo, Cheryl; Patwardhan, Siddharth V.; Belton, David J.; Kitchel, Brandon; Anastasiades, Daphne; Huang, Jia; Naik, Rajesh R.; Perry, Carole C.; Kaplan, David L.

    2006-01-01

    Silica skeletal architectures in diatoms are characterized by remarkable morphological and nanostructural details. Silk proteins from spiders and silkworms form strong and intricate self-assembling fibrous biomaterials in nature. We combined the features of silk with biosilica through the design, synthesis, and characterization of a novel family of chimeric proteins for subsequent use in model materials forming reactions. The domains from the major ampullate spidroin 1 (MaSp1) protein of Nephila clavipes spider dragline silk provide control over structural and morphological details because it can be self-assembled through diverse processing methods including film casting and fiber electrospinning. Biosilica nanostructures in diatoms are formed in aqueous ambient conditions at neutral pH and low temperatures. The R5 peptide derived from the silaffin protein of Cylindrotheca fusiformis induces and regulates silica precipitation in the chimeric protein designs under similar ambient conditions. Whereas mineralization reactions performed in the presence of R5 peptide alone form silica particles with a size distribution of 0.5–10 μm in diameter, reactions performed in the presence of the new fusion proteins generate nanocomposite materials containing silica particles with a narrower size distribution of 0.5–2 μm in diameter. Furthermore, we demonstrate that composite morphology and structure could be regulated by controlling processing conditions to produce films and fibers. These results suggest that the chimeric protein provides new options for processing and control over silica particle sizes, important benefits for biomedical and specialty materials, particularly in light of the all aqueous processing and the nanocomposite features of these new materials. PMID:16769898

  12. Characterization of chimeric Bacillus thuringiensis Vip3 toxins.

    Science.gov (United States)

    Fang, Jun; Xu, Xiaoli; Wang, Ping; Zhao, Jian-Zhou; Shelton, Anthony M; Cheng, Jiaan; Feng, Ming-Guang; Shen, Zhicheng

    2007-02-01

    Bacillus thuringiensis vegetative insecticidal proteins (Vip) are potential alternatives for B. thuringiensis endotoxins that are currently utilized in commercial transgenic insect-resistant crops. Screening a large number of B. thuringiensis isolates resulted in the cloning of vip3Ac1. Vip3Ac1 showed high insecticidal activity against the fall armyworm Spodoptera frugiperda and the cotton bollworm Helicoverpa zea but very low activity against the silkworm Bombyx mori. The host specificity of this Vip3 toxin was altered by sequence swapping with a previously identified toxin, Vip3Aa1. While both Vip3Aa1 and Vip3Ac1 showed no detectable toxicity against the European corn borer Ostrinia nubilalis, the chimeric protein Vip3AcAa, consisting of the N-terminal region of Vip3Ac1 and the C-terminal region of Vip3Aa1, became insecticidal to the European corn borer. In addition, the chimeric Vip3AcAa had increased toxicity to the fall armyworm. Furthermore, both Vip3Ac1 and Vip3AcAa are highly insecticidal to a strain of cabbage looper (Trichoplusia ni) that is highly resistant to the B. thuringiensis endotoxin Cry1Ac, thus experimentally showing for the first time the lack of cross-resistance between B. thuringiensis Cry1A proteins and Vip3A toxins. The results in this study demonstrated that vip3Ac1 and its chimeric vip3 genes can be excellent candidates for engineering a new generation of transgenic plants for insect pest control.

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

    Science.gov (United States)

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

    2009-08-13

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

  14. Chimeric mitochondrial peptides from contiguous regular and swinger RNA

    OpenAIRE

    Hervé Seligmann

    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 m...

  15. 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

    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...... antibody, anti-Gal IgE did not induce mediator release, potentially reflecting the delayed type of anaphylaxis. The alpha-1,3-Gal epitope fine structure of both the recombinant IgE and affinity-purified serum were defined by STD NMR revealing similar contributions of carbohydrate residues and participation...

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    L. Menut

    2013-07-01

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

  18. Serotype Chimeric Human Adenoviruses for Cancer GeneTherapy

    Directory of Open Access Journals (Sweden)

    Akseli Hemminki

    2010-09-01

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

  19. Chimeric behavior of excited thioxanthone in protic solvents: II. Theory.

    Science.gov (United States)

    Rai-Constapel, Vidisha; Villnow, Torben; Ryseck, Gerald; Gilch, Peter; Marian, Christel M

    2014-12-18

    The chimeric behavior of thioxanthone in protic solvents has been investigated employing computational chemistry methods. In particular, methanol and 2,2,2-trifluoroethanol have been chosen in this study. The solvent environment has been modeled using microsolvation in combination with a conductor-like screening model. The vertical excitation spectrum within the same solvent is seen to depend on the number of specific bonds formed between the chromophore and the solvent molecules. Two different models have been discussed in this work, namely, one and two H-bond models. In particular, the formation of the second H-bond causes the energy gap between the πHπL* and nOπL* states to increase further. Excited-state absorption spectra for the photophysically relevant electronic states have been theoretically determined for comparison with the time-resolved spectra recorded experimentally [Villnow, T.; Ryseck, G.; Rai-Constapel, V.; Marian, C. M.; Gilch, P. J. Phys. Chem. A 2014]. The equilibration of the 1(πHπL*) and 3(nOπL*) states holds responsible for the chimeric behavior. This equilibrium sets in with a calculated time constant of 23 ps in methanol and 14 ps in TFE (5 and 10 ps in experiment, respectively). The radiative decay from the optically bright 1(πHπL*) state is computed to occur with a time constant of 25 ns in both solvents (14–25 ns in experiment).

  20. Functional Characterization of an scFv-Fc Antibody that Immunotherapeutically Targets the Common Cancer Cell Surface Proteoglycan CSPG4

    OpenAIRE

    Wang, Xinhui; Katayama, Akihiro; Wang, Yangyang; YU Ling; Favoino, Elvira; Sakakura, Koichi; Favole, Alessandra; Tsuchikawa, Takahiro; Silver, Susan; Watkins, Simon C.; Kageshita, Toshiro; Ferrone, Soldano

    2011-01-01

    Cell surface chondroitin sulfate proteoglycan 4 (CSPG4) is an attractive target for antibody-based cancer immunotherapy because of its role in tumor cell biology, its high expression on malignant cells including cancer-initiating cells, and its restricted distribution in normal tissues. The clinical use of CSPG4 has been hampered by the lack of a CSPG4-specific chimeric, humanized, or fully human monoclonal antibody. To overcome this limitation, we generated a CSPG4-specific fully human singl...

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

    Directory of Open Access Journals (Sweden)

    Zheng Zhili

    2012-02-01

    Full Text Available Abstract Background There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymphocytes (PBL with CARs directed against a variety of tumor associated antigens. Despite the improvements in the design of CARs and expansion of the number of target antigens, there is no universal flow cytometric method available to detect the expression of CARs on the surface of transduced lymphocytes. Methods Currently anti-fragment antigen binding (Fab conjugates are most widely used to determine the expression of CARs on gene-modified lymphocytes by flow cytometry. The limitations of these reagents are that many of them are not commercially available, generally they are polyclonal antibodies and often the results are inconsistent. In an effort to develop a simple universal flow cytometric method to detect the expression of CARs, we employed protein L to determine the expression of CARs on transduced lymphocytes. Protein L is an immunoglobulin (Ig-binding protein that binds to the variable light chains (kappa chain of Ig without interfering with antigen binding site. Protein L binds to most classes of Ig and also binds to single-chain antibody fragments (scFv and Fab fragments. Results We used CARs derived from both human and murine antibodies to validate this novel protein L based flow cytometric method and the results correlated well with other established methods. Activated human PBLs were transduced with retroviral vectors expressing two human antibody based CARs (anti-EGFRvIII, and anti-VEGFR2, two murine antibody derived CARs (anti-CSPG4, and anti

  2. The synergistic effect of combined immunization with a DNA vaccine and chimeric yellow fever/dengue virus leads to strong protection against dengue.

    Directory of Open Access Journals (Sweden)

    Adriana S Azevedo

    Full Text Available The dengue envelope glycoprotein (E is the major component of virion surface and its ectodomain is composed of domains I, II and III. This protein is the main target for the development of a dengue vaccine with induction of neutralizing antibodies. In the present work, we tested two different vaccination strategies, with combined immunizations in a prime/booster regimen or simultaneous inoculation with a DNA vaccine (pE1D2 and a chimeric yellow fever/dengue 2 virus (YF17D-D2. The pE1D2 DNA vaccine encodes the ectodomain of the envelope DENV2 protein fused to t-PA signal peptide, while the YF17D-D2 was constructed by replacing the prM and E genes from the 17D yellow fever vaccine virus by those from DENV2. Balb/c mice were inoculated with these two vaccines by different prime/booster or simultaneous immunization protocols and most of them induced a synergistic effect on the elicited immune response, mainly in neutralizing antibody production. Furthermore, combined immunization remarkably increased protection against a lethal dose of DENV2, when compared to each vaccine administered alone. Results also revealed that immunization with the DNA vaccine, regardless of the combination with the chimeric virus, induced a robust cell immune response, with production of IFN-γ by CD8+ T lymphocytes.

  3. The synergistic effect of combined immunization with a DNA vaccine and chimeric yellow fever/dengue virus leads to strong protection against dengue.

    Science.gov (United States)

    Azevedo, Adriana S; Gonçalves, Antônio J S; Archer, Marcia; Freire, Marcos S; Galler, Ricardo; Alves, Ada M B

    2013-01-01

    The dengue envelope glycoprotein (E) is the major component of virion surface and its ectodomain is composed of domains I, II and III. This protein is the main target for the development of a dengue vaccine with induction of neutralizing antibodies. In the present work, we tested two different vaccination strategies, with combined immunizations in a prime/booster regimen or simultaneous inoculation with a DNA vaccine (pE1D2) and a chimeric yellow fever/dengue 2 virus (YF17D-D2). The pE1D2 DNA vaccine encodes the ectodomain of the envelope DENV2 protein fused to t-PA signal peptide, while the YF17D-D2 was constructed by replacing the prM and E genes from the 17D yellow fever vaccine virus by those from DENV2. Balb/c mice were inoculated with these two vaccines by different prime/booster or simultaneous immunization protocols and most of them induced a synergistic effect on the elicited immune response, mainly in neutralizing antibody production. Furthermore, combined immunization remarkably increased protection against a lethal dose of DENV2, when compared to each vaccine administered alone. Results also revealed that immunization with the DNA vaccine, regardless of the combination with the chimeric virus, induced a robust cell immune response, with production of IFN-γ by CD8+ T lymphocytes.

  4. Novel fusion genes and chimeric transcripts in ependymal tumors

    DEFF Research Database (Denmark)

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

    2016-01-01

    We have previously identified two ALK rearrangements in a subset of ependymal tumors using a combination of cytogenetic data and RNA sequencing. The aim of this study was to perform an unbiased search for fusion transcripts in our entire series of ependymal tumors. Fusion analysis was performed...... using the FusionCatcher algorithm on 12 RNA-sequenced ependymal tumors. Candidate transcripts were prioritized based on the software's filtering and manual visualization using the BLAST (Basic Local Alignment Search Tool) and BLAT (BLAST-like alignment tool) tools. Genomic and reverse transcriptase PCR...... 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...

  5. Modeling cognition and disease using human glial chimeric mice

    DEFF Research Database (Denmark)

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

    2015-01-01

    As new methods for producing and isolating human glial progenitor cells (hGPCs) have been developed, the disorders of myelin have become especially compelling targets for cell-based therapy. Yet as animal modeling of glial progenitor cell-based therapies has progressed, it has become clear...... cognition and information processing. In addition, the cellular humanization of these brains permits their use in studying glial infectious and inflammatory disorders unique to humans, and the effects of those disorders on the glial contributions to cognition. Perhaps most intriguingly, by pairing our...... 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...

  6. Chimeric Antigen Receptor T Cell Therapy in Hematology.

    Science.gov (United States)

    Ataca, Pınar; Arslan, Önder

    2015-12-01

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

  7. 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.

  8. Preclinical and Clinical Development of a YFV 17 D-Based Chimeric Vaccine against West Nile Virus

    Directory of Open Access Journals (Sweden)

    Gustavo H. Dayan

    2013-12-01

    Full Text Available Substantial success has been achieved in the development and implementation of West Nile (WN vaccines for horses; however, no human WN vaccines are approved. This review focuses on the construction, pre-clinical and clinical characterization of ChimeriVax-WN02 for humans, a live chimeric vaccine composed of a yellow fever (YF 17D virus in which the prM-E envelope protein genes are replaced with the corresponding genes of the WN NY99 virus. Pre-clinical studies demonstrated that ChimeriVax-WN02 was significantly less neurovirulent than YF 17D in mice and rhesus and cynomolgus monkeys. The vaccine elicited neutralizing antibody titers after inoculation in hamsters and monkeys and protected immunized animals from lethal challenge including intracerebral inoculation of high dose of WN NY99 virus. Safety, viremia and immunogenicity of ChimeriVax-WN02 were assessed in one phase I study and in two phase II clinical trials. No safety signals were detected in the three clinical trials with no remarkable differences in incidence of adverse events (AEs between vaccine and placebo recipients. Viremia was transient and the mean viremia levels were low. The vaccine elicited strong and durable neutralizing antibody and cytotoxic T cell responses. WN epidemiology impedes a classical licensure pathway; therefore, innovative licensure strategies should be explored.

  9. Antiparietal cell antibody test

    Science.gov (United States)

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; ...

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

    Science.gov (United States)

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

    2016-08-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

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

    Science.gov (United States)

    2017-01-01

    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 affected

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

    OpenAIRE

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

    2016-01-01

    Dramatic genome dynamics, such as chromosome instability, contribute to the remarkable genomic heterogeneity among the blastomeres comprising a single embryo during human preimplantation development. This heterogeneity, when compatible with life, manifests as constitutional mosaicism, chimerism, and mixoploidy in live-born individuals. Chimerism and mixoploidy are defined by the presence of cell lineages with different parental genomes or different ploidy states in a single individual, respec...

  14. Optimization of radioimmunotherapy of renal cell carcinoma: labeling of monoclonal antibody cG250 with 131I, 90Y, 177Lu, or 186Re.

    NARCIS (Netherlands)

    Brouwers, A.H.; Eerd-Vismale, J.E.M. van; Frielink, C.; Oosterwijk, E.; Oyen, W.J.G.; Corstens, F.H.M.; Boerman, O.C.

    2004-01-01

    Radioimmunotherapy (RIT) can be performed with various radionuclides. We tested the stability, biodistribution, and therapeutic efficacy of various radioimmunoconjugates ((131)I, (88/90)Y, (177)Lu, and (186)Re) of chimeric antirenal cell cancer monoclonal antibody G250 (mAb cG250) in nude mice with

  15. Pre-existing IgG antibodies cross-reacting with the Fab region of infliximab predict efficacy and safety of infliximab therapy in inflammatory bowel disease

    DEFF Research Database (Denmark)

    Steenholdt, Casper; Palarasah, Yaseelan; Bendtzen, Klaus

    2013-01-01

    BACKGROUND: Infliximab (IFX) is a chimeric murine/human anti-TNF antibody (Ab) used for the treatment of Crohn's disease (CD) and ulcerative colitis (UC). Loss of response is common and associated with development of anti-IFX Abs during ongoing therapy. However, human anti-murine immunoglobulin Abs...

  16. Antistaphylococcal activity of bacteriophage derived chimeric protein P128

    Directory of Open Access Journals (Sweden)

    Vipra Aradhana A

    2012-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Helena Storchova

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

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Seyed Hossein Khaleghinejad

    2016-06-01

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

  20. Dosimetry of chimeric TNT in lung tumor patients

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2016-11-01

    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.

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Science.gov (United States)

    Brudno, Jennifer N; Kochenderfer, James N

    2016-06-30

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

  4. 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.

  5. The impact of MM5 and WRF meteorology over complex terrain on CHIMERE model calculations

    Directory of Open Access Journals (Sweden)

    A. de Meij

    2009-01-01

    Full Text Available The objective of this study is to evaluate the impact of meteorological input data on calculated gas and aerosol concentrations. We use two different meteorological models (MM5 and WRF together with the chemistry transport model CHIMERE. We focus on the Po valley area (Italy for January and June 2005.

    Firstly we evaluate the meteorological parameters with observations. The analysis shows that the performance of both models is similar, however some small differences are still noticeable.

    Secondly, we analyze the impact of using MM5 and WRF on calculated PM10 and O3 concentrations. In general CHIMERE/MM5 and CHIMERE/WRF underestimate the PM10 concentrations for January. The difference in PM10 concentrations for January between CHIMERE/MM5 and CHIMERE/WRF is around a factor 1.6 (PM10 higher for CHIMERE/MM5. This difference and the larger underestimation in PM10 concentrations by CHIMERE/WRF are related to the differences in heat fluxes and the resulting PBL heights calculated by WRF. In general the PBL height by WRF meteorology is a factor 2.8 higher at noon in January than calculated by MM5. This study showed that the difference in microphysics scheme has an impact on the profile of cloud liquid water (CLW calculated by the meteorological driver and therefore on the production of SO4 aerosol.

    A sensitivity analysis shows that changing the Noah Land Surface Model (LSM for the 5-layer soil temperature model, the calculated monthly mean PM10 concentrations increase by 30%, due to the change in the heat fluxes and the resulting PBL heights.

    For June, PM10 calculated concentrations by CHIMERE/MM5 and CHIMERE/WRF are similar and agree with the observations. Calculated O3 values for June are in general overestimated by a factor 1.3 by CHIMERE/MM5 and CHIMRE/WRF. The reason for this is that daytime NO2

  6. IgE recognition of chimeric isoforms of the honeybee (Apis mellifera) venom allergen Api m 10 evaluated by protein array technology.

    Science.gov (United States)

    Van Vaerenbergh, Matthias; De Smet, Lina; Rafei-Shamsabadi, David; Blank, Simon; Spillner, Edzard; Ebo, Didier G; Devreese, Bart; Jakob, Thilo; de Graaf, Dirk C

    2015-02-01

    Api m 10 has recently been established as novel major allergen that is recognized by more than 60% of honeybee venom (HBV) allergic patients. Previous studies suggest Api m 10 protein heterogeneity which may have implications for diagnosis and immunotherapy of HBV allergy. In the present study, RT-PCR revealed the expression of at least nine additional Api m 10 transcript isoforms by the venom glands. Two distinct mechanisms are responsible for the generation of these isoforms: while the previously known variant 2 is produced by an alternative splicing event, novel identified isoforms are intragenic chimeric transcripts. To the best of our knowledge, this is the first report of the identification of chimeric transcripts generated by the honeybee. By a retrospective proteomic analysis we found evidence for the presence of several of these isoforms in the venom proteome. Additionally, we analyzed IgE reactivity to different isoforms by protein array technology using sera from HBV allergic patients, which revealed that IgE recognition of Api m 10 is both isoform- and patient-specific. While it was previously demonstrated that the majority of HBV allergic patients display IgE reactivity to variant 2, our study also shows that some patients lacking IgE antibodies for variant 2 display IgE reactivity to two of the novel identified Api m 10 variants, i.e. variants 3 and 4.

  7. Effects of genetic engineering on the pharmacokinetics of antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Colcher, D.; Goel, A.; Pavlinkova, G.; Beresford, G.; Booth, B.; Batra, S.K. [University of Nebraska Medical Center, Omaha NE (United States). Dept. of Pathology and Microbiology and Molecular Biology

    1999-06-01

    Monoclonal antibodies (MAbs) may be considered 'magic bullets' due to their ability to recognize and eradicate malignant cells. MAbs, however, have practical limitations for their rapid application in the clinics. The structure of the antibody molecules can be engineered to modify functional domains such as antigen-binding sites and/or effectors functions. Advanced in genetic engineering have provided rapid progress the development of new immunoglobulin constructs of MAbs with defined research and therapeutic application. Recombinant antibody constructs are being engineered, such as human mouse chimeric, domain-dispositioned, domain-deleted, humanized and single-chain Fv fragments. Genetically-engineered antibodies differ in size and rate of catabolism. Pharmacokinetics studies show that the intact IgG (150 kD), enzymatically derived fragments Fab' (50 kD) and single chain Fv (28 kD) have different clearance rates. These antibody forms clear 50% from the blood pool in 2.1 days, 30 minutes and 10 minutes, respectively. Genetically-engineered antibodies make a new class of immunotherapeutic tracers for cancer treatment.

  8. Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection.

    Directory of Open Access Journals (Sweden)

    Rami Sommerstein

    2015-11-01

    Full Text Available Arenaviruses such as Lassa virus (LASV can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein's globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy.

  9. Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection.

    Science.gov (United States)

    Sommerstein, Rami; Flatz, Lukas; Remy, Melissa M; Malinge, Pauline; Magistrelli, Giovanni; Fischer, Nicolas; Sahin, Mehmet; Bergthaler, Andreas; Igonet, Sebastien; Ter Meulen, Jan; Rigo, Dorothée; Meda, Paolo; Rabah, Nadia; Coutard, Bruno; Bowden, Thomas A; Lambert, Paul-Henri; Siegrist, Claire-Anne; Pinschewer, Daniel D

    2015-11-01

    Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein's globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy.

  10. T-regulatory cell treatment prevents chronic rejection of heart allografts in a murine mixed chimerism model

    OpenAIRE

    Pilat, Nina; Farkas, Andreas M.; Mahr, Benedikt; Schwarz, Christoph; Unger, Lukas; Hock, Karin; Oberhuber, Rupert; Aumayr, Klaus; Wrba, Fritz; Wekerle, Thomas

    2014-01-01

    Background The mixed chimerism approach induces donor-specific tolerance in both pre-clinical models and clinical pilot trials. However, chronic rejection of heart allografts and acute rejection of skin allografts were observed in some chimeric animals despite persistent hematopoietic chimerism and tolerance toward donor antigens in vitro. We tested whether additional cell therapy with regulatory T cells (Tregs) is able to induce full immunologic tolerance and prevent chronic rejection. Metho...

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

    Science.gov (United States)

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

    2014-02-01

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

  12. A sensitive enzyme immunoassay for amygdalin in food extracts using a recombinant antibody.

    Science.gov (United States)

    Cho, A-Yeon; Shin, Kum-Joo; Chung, Junho; Oh, Sangsuk

    2008-10-01

    Amygdalin (laterile) is a cyanogenic glycoside commonly found in the pits of many fruits and raw nuts. When amygdalin-containing seeds are crushed and moistened, free cyanide is formed. Pits and nuts containing unusually high levels of amygdalin can therefore cause cyanide poisoning, and detection of amygdalin in food extracts can be a life-saving measure. In this study, we generated recombinant antibodies against amygdalin from a phage display of a combinatorial rabbit/human chimeric antibody library and used it in a sensitive competition enzyme immunoassay system to detect amygdalin in extracts of pits and nuts. The detection limit was determined to be 1 x 10(-9) M.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Nayoun Kim

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

  15. 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

    2017-01-01

    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/. PMID:27899563

  16. Human papillomavirus 16 L1-E7 chimeric virus like particles show prophylactic and therapeutic efficacy in murine model of cervical cancer.

    Science.gov (United States)

    Sharma, Chandresh; Dey, Bindu; Wahiduzzaman, Mohammed; Singh, Neeta

    2012-08-03

    Cervical cancer is found to be associated with human papillomavirus (HPV) infection, with HPV16 being the most prevalent. An effective vaccine against HPV can thus, be instrumental in controlling cervical cancer. An ideal HPV vaccine should aim to generate both humoral immune response to prevent new infection as well as cell-mediated immunity to eliminate established infection. In this study, we have generated a potential preventive and therapeutic candidate vaccine against HPV16. We expressed and purified recombinant HPV16 L1(ΔN26)-E7(ΔC38) protein in E. coli which was assembled into chimeric virus like particles (CVLPs) in vitro. These CVLPs were able to induce neutralizing antibodies and trigger cell-mediated immune response, in murine model of cervical cancer, exhibiting antitumor efficacy. Hence, this study has aimed to provide a vaccine candidate possessing both, prophylactic and therapeutic efficacy against HPV16 associated cervical cancer.

  17. Polyclonal immunoglobulins from a chronic hepatitis C virus patient protect human liver-chimeric mice from infection with a homologous hepatitis C virus strain

    DEFF Research Database (Denmark)

    Vanwolleghem, Thomas; Bukh, Jens; Meuleman, Philip

    2008-01-01

    The role of the humoral immune response in the natural course of hepatitis C virus (HCV) infection is widely debated. Most chronically infected patients have immunoglobulin G (IgG) antibodies capable of neutralizing HCV pseudoparticles (HCVpp) in vitro. It is, however, not clear whether these Ig...... were loaded with chronic phase polyclonal IgG and challenged 3 days later with a 100% infectious dose of the acute phase H77C virus, both originating from patient H. Passive immunization induced sterilizing immunity in five of eight challenged animals. In the three nonprotected animals, the HCV...... chimeric mice, the inoculum was pre-incubated in vitro at an IgG concentration normally observed in humans. Conclusion: Polyclonal IgG from a patient with a long-standing HCV infection not only displays neutralizing activity in vitro using the HCVpp system, but also conveys sterilizing immunity toward...

  18. Reduction of porcine circovirus type 2 (PCV2 viremia by a reformulated inactivated chimeric PCV1-2 vaccine-induced humoral and cellular immunity after experimental PCV2 challenge

    Directory of Open Access Journals (Sweden)

    Seo Hwi

    2012-10-01

    Full Text Available Abstract Background The objective of the present study was to elucidate the humoral and cellular immune response mechanisms by which a reformulated inactivated chimeric PCV1-2 vaccine reduces the PCV2 viremia. Forty PCV2 seronegative 3-week-old pigs were randomly divided into the following four groups: vaccinated challenged (T01, vaccinated non-challenged (T02, non-vaccinated challenged (T03, and non-vaccinated non-challenged (T04 animals. The pigs in groups T01 and T02 were immunized with a reformulated inactivated chimeric PCV1-2 vaccine (Fostera™ PCV; Pfizer Animal Health administered as a 2.0 ml dose at 21 days of age. At 35 days of age (0 days post-challenge, the pigs in groups T01 and T03 were inoculated intranasally with 2 ml each of PCV2b. Results A reduction of PCV2 viremia coincided with the appearance of both PCV2-specific neutralizing antibodies (NA and interferon-γ-secreting cells (IFN-γ-SCs in the vaccinated animals. However, the presence of anti-PCV2 IgG antibodies did not correlate with the reduction of PCV2 viremia. Lymphocyte subset analysis indicated that the numbers of CD3+ and CD4+ cells increased in vaccinated animals but the numbers of CD4+ cells decreased transiently in non-vaccinated animals. The observation of a delayed type hypersensitivity response in only the vaccinated animals also supports a CD4+ cell-associated protective cellular immune response induced by the reformulated inactivated chimeric PCV1-2 vaccine. Conclusions The induction of PCV2-specific NA and IFN-γ-SCs, and CD4+ cells by the reformulated inactivated chimeric PCV1-2 vaccine is the important protective immune response leading to reduction of the PCV2 viremia and control of the PCV2 infection. To our knowledge this is the first demonstration of protective humoral and cellular immunity induced by the reformulated inactivated chimeric PCV1-2 vaccine and its effect on reduction of PCV2 viremia by vaccination.

  19. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  1. [Antinuclear antibodies].

    Science.gov (United States)

    Cabiedes, Javier; Núñez-Álvarez, Carlos A

    2010-01-01

    Anti-nuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. Besides the autoimmune ANA there are other ANA that can be detected in circulation, like natural and infectious ANA. Because of its high sensibility, detection of the ANA must be done by indirect immunofluorescence (IIF) as screening test and all of those positive samples are convenient to confirm its specificity by ELISA, western blot or other techniques. Positive ANA detected by IIF must be evaluated taking in to account the pattern and titer. The following recommended step is the specificity characterization (reactivity against extractable nuclear antigens [ENA], dsDNA, etc.) which is useful for the diagnosis and follow up of patients with autoimmune diseases, and by such reasoning, its detection must be performed in an orderly and reasonable way using guides or strategies focused to the good use and interpretation of the autoantibodies. The objective of this review is to present a compilation of the literature and our experience in the detection and study of the ANA.

  2. Catastrophic relapse of Evans syndrome five years after allogeneic BMT notwithstanding full donor chimerism. Terminal hemolytic-uremic syndrome.

    Science.gov (United States)

    Marmont, A M; Gualandi, F; Occhini, D; Morandi, F; Ferretti, E; Pezzolo, A; Strada, P; Ravetti, J L; Pistoia, V; Falanga, A; Bacigalupo, A

    2006-09-01

    A patient with severe Evans syndrome received an allo-BMT from his HLA-identical sister on November, 2000. Full marrow and blood donor chimerism were achieved only after 5 donor lymphocyte infusions (DLI), and coincided with complete clinical remission and disappearence of auto-antibodies. Five years later, hemolytic anemia recurred with rapid increase of serum bilirubin to over 50 mg%: he responded to combined therapy, but died on day +17 from admission of an acute hemolytic uremic syndrome (HUS). All circulating blood cells, including erythrocytes, were 100% donor. Ex vivo cultured and expanded T and B cells from the peripheral blood were also 100% donor. The supernatants from B cell cultures, containing either IgM or IgG, did not react with a panel of erythrocytes. Thus in this typical autoimmune disease with a predominant B cell pathogenesis the donor immune system resulted "innocent of autoimmunity". The persistence of long-lived recipient autoreactive plasma-cell lines in survival niches, still producing autoantibodies, may be hypothesized for this and similar cases. The postulated graft-versus-autoimmunity (GVA) effect was apparently not sufficient to eradicate autoimmunity in this patient.

  3. PRELIMINARY STUDY OF A NOVEL HUMAN PAPILLOMAVIRUS TYPE 16 L1/E6-E7 CHIMERIC RECOMBINANT DNA VACCINE

    Institute of Scientific and Technical Information of China (English)

    郑瑾; 马军; 张福萍; 杨筱凤; 董小平; 司履生; 王一理

    2004-01-01

    Objective Preparations of HPV16 L1/E6 and L1/E7 prophylactic and therapeutic DNA vaccines. Methods The nucleotides within HPV16 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by mage primer site-directed mutagenesis method. The correctly mutated E6 and E7 fragments were separately cloned into an eukaryotic expression vector pVAX1, together with HPV16 L1 gene, generating chimeric recombinants plasmids 1MpVAX1-L1E6, 2MpVAX1-L1E6, 1MpVAX1-L1E7, 2MpVAX1-L1E7 and 3MpVAX1-L1E7. CHO cells were transiently transfected with the individual DNA vaccines by calcium phosphate method. Target protein expressions in the extracts of the transfected cell lines were measured by ELISA and immunohistochemistry, with HPV16 L1 and E6 specific monoclonal antibodies. Results ELISA assays showed the P/N ratios in the cell extracts transfected with L1E6 and L1E7 plasmids were more than 2.1. Immunohistochemistry revealed brownish precipitant signal in cytoplasm and nuclei of the transfected cells. Conclusion Successful constructions of prophylactic and therapeutic DNA vaccine plasmids lay solid foundation for future animal experiment and clinical trial.

  4. Monitoring of chimerism using fluorescence in situ hybridization in a child with severe combined immune deficiency following bone marrow transplant

    Energy Technology Data Exchange (ETDEWEB)

    Wenger, S.L.; Chen, X.O.; Katz, A.J. [Children`s Hospital of Pittsburgh, PA (United States)]|[Univ. of Pittsburgh, PA (United States)

    1994-09-01

    A boy with severe combined immunodeficiency received a bone marrow transplant from his sister when he was approximately 3 years of age. His peripheral blood karyotype at age 3 and 4 years was 46,XX (20 cells analyzed). Because of a decline in antibody production at 19 years of age, the patient`s peripheral blood was analyzed again for suspected chimerism. His karyotype in phytohemagglutinin (PHA)-stimulated culture was 46,XX in 49 cells and 46,XY in one cell. Both metaphase and interphase cells were examined for sex chromosome constitution using X and Y dual-color alpha-satellite probes for fluorescence in situ hybridization (FISH). FISH results for metaphase cells showed 1/50 XY cells, but 38% of interphase cells showed the presence of both X and Y centromere. Pokeweed mitogen (PWM)-stimulated cultures grew poorly and were therefore analyzed using FISH only: 81% of interphase cells were 46,XX. The discrepancy between metaphase and interphase in the PHA-stimulated cultures most likely represents a failure of this boy`s own XY T-cells to be stimulated.

  5. Chimeric virus-like particles containing influenza HA antigen and GPI-CCL28 induce long-lasting mucosal immunity against H3N2 viruses

    Science.gov (United States)

    Mohan, Teena; Berman, Zachary; Luo, Yuan; Wang, Chao; Wang, Shelly; Compans, Richard W.; Wang, Bao-Zhong

    2017-01-01

    Influenza virus is a significant cause of morbidity and mortality, with worldwide seasonal epidemics. The duration and quality of humoral immunity and generation of immunological memory to vaccines is critical for protective immunity. In the current study, we examined the long-lasting protective efficacy of chimeric VLPs (cVLPs) containing influenza HA and GPI-anchored CCL28 as antigen and mucosal adjuvant, respectively, when immunized intranasally in mice. We report that the cVLPs induced significantly higher and sustainable levels of virus-specific antibody responses, especially IgA levels and hemagglutination inhibition (HAI) titers, more than 8-month post-vaccination compared to influenza VLPs without CCL28 or influenza VLPs physically mixed with sCCL28 (soluble) in mice. After challenging the vaccinated animals at month 8 with H3N2 viruses, the cVLP group also demonstrated strong recall responses. On day 4 post-challenge, we measured increased antibody levels, ASCs and HAI titers with reduced viral load and inflammatory responses in the cVLP group. The animals vaccinated with the cVLP showed 20% cross-protection against drifted (Philippines) and 60% protection against homologous (Aichi) H3N2 viruses. Thus, the results suggest that the GPI-anchored CCL28 induces significantly higher mucosal antibody responses, involved in providing long-term cross-protection against H3N2 influenza virus when compared to other vaccination groups. PMID:28067290

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

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

    Directory of Open Access Journals (Sweden)

    George Shilpa E

    2012-06-01

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

  9. Selection of antibodies from synthetic antibody libraries.

    Science.gov (United States)

    Harel Inbar, Noa; Benhar, Itai

    2012-10-15

    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.

  10. 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

  11. Stoichiometry of monoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1

    DEFF Research Database (Denmark)

    Schønning, Kristian; Lund, O; Lund, O S;

    1999-01-01

    In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes...... are coexpressed. By the coexpression of Env glycoproteins that either can or cannot bind a neutralizing MAb in an env transcomplementation assay, virions were generated in which the proportion of MAb binding sites could be regulated. As the proportion of MAb binding sites in Env chimeric virus increased, MAb...... neutralization gradually increased. Virus neutralization by virion aggregation was minimal, as MAb binding to HIV-1 Env did not interfere with an AMLV Env-mediated infection by HIV-1(AMLV/HIV-1) pseudotypes of CD4(-) HEK293 cells. MAb neutralization of chimeric virions could be described as a third...

  12. Generation and developmental characteristics of porcine tetraploid embryos and tetraploid/diploid chimeric embryos.

    Science.gov (United States)

    He, Wenteng; Kong, Qingran; Shi, Yongqian; Xie, Bingteng; Jiao, Mingxia; Huang, Tianqing; Guo, Shimeng; Hu, Kui; Liu, Zhonghua

    2013-10-01

    The aim of this study was to optimize electrofusion conditions for generating porcine tetraploid (4n) embryos and produce tetraploid/diploid (4n/2n) chimeric embryos. Different electric field intensities were tested and 2 direct current (DC) pulses of 0.9 kV/cm for 30 μs was selected as the optimum condition for electrofusion of 2-cell embryos to produce 4n embryos. The fusion rate of 2-cell embryos and the development rate to blastocyst of presumably 4n embryos, reached 85.4% and 28.5%, respectively. 68.18% of the fused embryos were found to be 4n as demonstrated by fluorescent in situ hybridization (FISH). Although the number of blastomeres in 4n blastocysts was significantly lower than in 2n blastocysts (P0.05), suggesting that the blastocyst forming capacity in 4n embryos is similar to those in 2n embryos. Moreover, 4n/2n chimeric embryos were obtained by aggregation of 4n and 2n embryos. We found that the developmental rate and cell number of blastocysts of 4-cell (4n)/4-cell (2n) chimeric embryos were significantly higher than those of 2-cell (4n)/4-cell (2n), 4-cell (4n)/8-cell (2n), 4-cell (4n)/2-cell (2n) chimeric embryos (P<0.05). Consistent with mouse chimeras, the majority of 4n cells contribute to the trophectoderm (TE), while the 2n cells are mainly present in the inner cell mass (ICM) of porcine 4n/2n chimeric embryos. Our study established a feasible and efficient approach to produce porcine 4n embryos and 4n/2n chimeric embryos.

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

    Science.gov (United States)

    Shih, Hung-Jui; Jones, Corbin D.

    2008-01-01

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

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

    NARCIS (Netherlands)

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

    2004-01-01

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

  15. 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...

  16. 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.

  17. The impact of chimerism in DNA-based forensic sex determination analysis.

    Science.gov (United States)

    George, Renjith; Donald, Preethy Mary; Nagraj, Sumanth Kumbargere; Idiculla, Jose Joy; Hj Ismail, Rashid

    2013-01-01

    Sex determination is the most important step in personal identification in forensic investigations. DNA-based sex determination analysis is comparatively more reliable than the other conventional methods of sex determination analysis. Advanced technology like real-time polymerase chain reaction (PCR) offers accurate and reproducible results and is at the level of legal acceptance. But still there are situations like chimerism where an individual possess both male and female specific factors together in their body. Sex determination analysis in such cases can give erroneous results. This paper discusses the phenomenon of chimerism and its impact on sex determination analysis in forensic investigations.

  18. Lymphadenectomy prior to rat hind limb allotransplantation prevents graft-versus-host disease in chimeric hosts

    NARCIS (Netherlands)

    Brouha, PCR; Perez-Abadia, G; Francois, CG; Laurentin-Perez, LA; Gorantla, [No Value; Vossen, M; Tai, C; Pidwell, D; Anderson, GL; Stadelmann, WK; Hewitt, CW; Kon, M; Barker, JH; Maldonado, C

    2004-01-01

    In previous rat studies, the use of mixed allogeneic chimerism (MAC) to induce host tolerance to hind limb allografts has resulted in severe graft-versus-host disease (GVHD). The purpose of this study was to determine if immunocompetent cells in bone marrow (BM) and/or lymph nodes (LNs) of transplan

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

    Directory of Open Access Journals (Sweden)

    Paulina eRuiz

    2015-11-01

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

  20. Multipaddled anterolateral thigh chimeric flap for reconstruction of complex defects in head and neck.

    Directory of Open Access Journals (Sweden)

    Canhua Jiang

    Full Text Available The anterolateral thigh flap has been the workhouse flap for coverage of soft-tissue defects in head and neck for decades. However, the reconstruction of multiple and complex soft-tissue defects in head and neck with multipaddled anterolateral thigh chimeric flaps is still a challenge for reconstructive surgeries. Here, a clinical series of 12 cases is reported in which multipaddled anterolateral thigh chimeric flaps were used for complex soft-tissue defects with several separately anatomic locations in head and neck. Of the 12 cases, 7 patients presented with trismus were diagnosed as advanced buccal cancer with oral submucous fibrosis, 2 tongue cancer cases were found accompanied with multiple oral mucosa lesions or buccal cancer, and 3 were hypopharyngeal cancer with anterior neck skin invaded. All soft-tissue defects were reconstructed by multipaddled anterolateral thigh chimeric flaps, including 9 tripaddled anterolateral thigh flaps and 3 bipaddled flaps. The mean length of skin paddle was 19.2 (range: 14-23 cm and the mean width was 4.9 (range: 2.5-7 cm. All flaps survived and all donor sites were closed primarily. After a mean follow-up time of 9.1 months, there were no problems with the donor or recipient sites. This study supports that the multipaddled anterolateral thigh chimeric flap is a reliable and good alternative for complex and multiple soft-tissue defects of the head and neck.

  1. 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.

  2. 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...

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

    Science.gov (United States)

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

    2016-05-01

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

  5. Versatile bio-ink for covalent immobilization of chimeric avidin on sol-gel substrates.

    Science.gov (United States)

    Heikkinen, Jarkko J; Kivimäki, Liisa; Määttä, Juha A E; Mäkelä, Inka; Hakalahti, Leena; Takkinen, Kristiina; Kulomaa, Markku S; Hytönen, Vesa P; Hormi, Osmo E O

    2011-10-15

    A bio-ink for covalent deposition of thermostable, high affinity biotin-binding chimeric avidin onto sol-gel substrates was developed. The bio-ink was prepared from heterobifunctional crosslinker 6-maleimidohexanoic acid N-hydroxysuccinimide which was first reacted either with 3-aminopropyltriethoxysilane or 3-aminopropyldimethylethoxysilane to form silane linkers 6-maleimide-N-(3-(triethoxysilyl)propyl)hexanamide or -(ethoxydimethylsilyl)propyl)-hexanamide. C-terminal cysteine genetically engineered to chimeric avidin was reacted with the maleimide group of silane linker in methanol/PBS solution to form a suspension, which was printed on sol-gel modified PMMA film. Different concentrations of chimeric avidin and ratios between silane linkers were tested to find the best properties for the bio-ink to enable gravure or inkjet printing. Bio-ink prepared from 3-aminopropyltriethoxysilane was found to provide the highest amount of active immobilized chimeric avidin. The developed bio-ink was shown to be valuable for automated fabrication of avidin-functionalized polymer films.

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

    Science.gov (United States)

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

  7. 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.

  8. 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 o...

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

    Directory of Open Access Journals (Sweden)

    Sarah Djebali

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

  10. 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.

  11. 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)

  12. Low levels of allogeneic but not syngeneic hematopoietic chimerism reverse autoimmune insulitis in prediabetic NOD mice.

    Science.gov (United States)

    Kaminitz, Ayelet; Mizrahi, Keren; Yaniv, Isaac; Farkas, Daniel L; Stein, Jerry; Askenasy, Nadir

    2009-09-01

    The relative efficiencies of allogeneic and syngeneic bone marrow transplantation and the threshold levels of donor chimerism required to control autoimmune insulitis were evaluated in prediabetic NOD mice. Male and female NOD mice were conditioned by radiation and grafted with bone marrow cells from allogeneic and syngeneic sex-mismatched donors. Establishment of full allogeneic chimerism in peripheral blood reversed insulitis and restored glucose tolerance despite persistence of residual host immune cells. By contrast, sublethal total body irradiation (with or without syngeneic transplant) reduced the incidence and delayed the onset of diabetes. The latter pattern was also seen in mice that rejected the bone marrow allografts. Low levels of stable allogeneic hematopoietic chimerism (>1%) were sufficient to prevent the evolution of diabetes following allogeneic transplantation. The data indicate that immunomodulation attained at low levels of allogeneic, but not syngeneic, hematopoietic chimerism is effective in resolution of islet inflammation at even relatively late stages in the evolution of the prediabetic state in a preclinical model. However, our data question the efficacy and rationale behind syngeneic (autologous-like) immuno-hematopoietic reconstitution in type 1 diabetes.

  13. Understanding Zika Virus Stability and Developing a Chimeric Vaccine through Functional Analysis

    Science.gov (United States)

    Yang, Yujiao; Muruato, Antonio E.; Zou, Jing; Shan, Chao; Nunes, Bruno T. D.; Medeiros, Daniele B. A.; Vasconcelos, Pedro F. C.; Weaver, Scott C.; Rossi, Shannan L.

    2017-01-01

    ABSTRACT Compared with other flaviviruses, Zika virus (ZIKV) is uniquely associated with congenital diseases in pregnant women. One recent study reported that (i) ZIKV has higher thermostability than dengue virus (DENV [a flavivirus closely related to ZIKV]), which might contribute to the disease outcome; (ii) the higher thermostability of ZIKV could arise from an extended loop structure in domain III of the viral envelope (E) protein and an extra hydrogen-bond interaction between E molecules (V. A. Kostyuchenko, E. X. Y. Lim, S. Zhang, G. Fibriansah, T.-S. Ng, J. S. G. Ooi, J. Shi, and S.-M. Lok, Nature 533:425–428, 2016, https://doi.org/10.1038/nature17994). Here we report the functional analysis of the structural information in the context of complete ZIKV and DENV-2 virions. Swapping the prM-E genes between ZIKV and DENV-2 switched the thermostability of the chimeric viruses, identifying the prM-E proteins as the major determinants for virion thermostability. Shortening the extended loop of the E protein by 1 amino acid was lethal for ZIKV assembly/release. Mutations (Q350I and T351V) that abolished the extra hydrogen-bond interaction between the E proteins did not reduce ZIKV thermostability, indicating that the extra interaction does not increase the thermostability. Interestingly, mutant T351V was attenuated in A129 mice defective in type I interferon receptors, even though the virus retained the wild-type thermostability. Furthermore, we found that a chimeric ZIKV with the DENV-2 prM-E and a chimeric DENV-2 with the ZIKV prM-E were highly attenuated in A129 mice; these chimeric viruses were highly immunogenic and protective against DENV-2 and ZIKV challenge, respectively. These results indicate the potential of these chimeric viruses for vaccine development. PMID:28174309

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

    Science.gov (United States)

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

    2016-09-01

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

  15. 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

  16. Acetylcholine receptor antibody

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003576.htm Acetylcholine receptor antibody To use the sharing features on this page, please enable JavaScript. Acetylcholine receptor antibody is a protein found in the blood ...

  17. Antinuclear antibody panel

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003535.htm Antinuclear antibody panel To use the sharing features on this page, please enable JavaScript. The antinuclear antibody panel is a blood test that looks at ...

  18. Lyme disease antibody

    Science.gov (United States)

    ... JavaScript. The Lyme disease blood test looks for antibodies in the blood to the bacteria that causes ... needed. A laboratory specialist looks for Lyme disease antibodies in the blood sample using the ELISA test . ...

  19. The antibody mining toolbox

    OpenAIRE

    D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D.; Shen, Xiaohong; Bradbury, Andrew RM; Kiss, Csaba

    2013-01-01

    In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput ...

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

    Science.gov (United States)

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

    2007-12-17

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

  1. Radioimmunotherapy of carcinoma of colon with [131I]-labeled recombinant chimeric monoclonal antibodies to carcinoembryonic antigen

    Institute of Scientific and Technical Information of China (English)

    Qiu-jun LU; Guang-xing BIAN; Yuan-yuan CHEN; Min ZHANG; Shao-ming GUO; Li-qing WEN

    2005-01-01

    Aim: To study the distribution of [131I]-labeled anti-CEA MoAbs and its therapeutic effect on the human colonic cancer model in nude mice. Methods: A nude mice model of human colonic cancer was established. [131I]-labeled anti-CEA MoAbs were injected intravenously into mice. The distribution of the MoAbs was then determined and the effect of RIT on human colonic cancer was observed. Results:The [131I]-labeled anti-CEA MoAbs had a specific distribution after injection.Tumor/non-tumor ratios for [131I]-labeled anti-CEA MoAbs were 10-20 times higher than [131I]-labeled IgG 96 h after injection. Thirty days after injection, significant inhibition of the volume and weight of tumor was observed in the treated mice compared with the control. The tumor growth inhibition rate of 3.1 mCi/kg CEA MoAbs group (LS 180, LS 174T, SW1116) was 47.8%-64.0%. This was 69.6%-78.6%in the 6.25 mCi/kg CEA MoAbs group, and 81.8%-86.2% in the 12.5 mCi/kg [131I]-labeled anti-CEA MoAbs group. The plasma CEA level was also lower in treated mice. Conclusion: The results indicate that [131I]-labeled anti-CEA MoAbs can be effective in RIT on colonic cancers.

  2. The Complexity of a Dengue Vaccine: A Review of the Human Antibody Response.

    Directory of Open Access Journals (Sweden)

    Jacky Flipse

    Full Text Available Dengue is the most prevalent mosquito-borne viral disease worldwide. Yet, there are no vaccines or specific antivirals available to prevent or treat the disease. Several dengue vaccines are currently in clinical or preclinical stages. The most advanced vaccine is the chimeric tetravalent CYD-TDV vaccine of Sanofi Pasteur. This vaccine has recently cleared Phase III, and efficacy results have been published. Excellent tetravalent seroconversion was seen, yet the protective efficacy against infection was surprisingly low. Here, we will describe the complicating factors involved in the generation of a safe and efficacious dengue vaccine. Furthermore, we will discuss the human antibody responses during infection, including the epitopes targeted in humans. Also, we will discuss the current understanding of the assays used to evaluate antibody response. We hope this review will aid future dengue vaccine development as well as fundamental research related to the phenomenon of antibody-dependent enhancement of dengue virus infection.

  3. The Complexity of a Dengue Vaccine: A Review of the Human Antibody Response.

    Science.gov (United States)

    Flipse, Jacky; Smit, Jolanda M

    2015-01-01

    Dengue is the most prevalent mosquito-borne viral disease worldwide. Yet, there are no vaccines or specific antivirals available to prevent or treat the disease. Several dengue vaccines are currently in clinical or preclinical stages. The most advanced vaccine is the chimeric tetravalent CYD-TDV vaccine of Sanofi Pasteur. This vaccine has recently cleared Phase III, and efficacy results have been published. Excellent tetravalent seroconversion was seen, yet the protective efficacy against infection was surprisingly low. Here, we will describe the complicating factors involved in the generation of a safe and efficacious dengue vaccine. Furthermore, we will discuss the human antibody responses during infection, including the epitopes targeted in humans. Also, we will discuss the current understanding of the assays used to evaluate antibody response. We hope this review will aid future dengue vaccine development as well as fundamental research related to the phenomenon of antibody-dependent enhancement of dengue virus infection.

  4. Anti-CD20 monoclonal antibodies as novel treatments for non-Hodgkin's lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    White, C.A.; Larocca, A.; Grillo-Lopez, A.J. [IDEC Pharmaceuticals, 3030 Callan Road, San Diego, CA (United States)

    1999-03-01

    Anti-CD20 monoclonal antibodies (MAbs) offer new options for patients with non-Hodgkin's lymphoma, needed because existing therapies have many limitations. The unconjugated, chimeric anti-CD20 antibody, Rituximab (MabThera, Rituxan), has recently been approved in the USA for patients with relapsed or refractory, low-grade or follicular, B-cell non-Hodgkin's lymphoma, and in Europe for therapy of relapsed stage III/IV follicular lymphoma. In the pivotal study of Rituximab, an overall response rate of 50% was achieved with median time to progressionin responders of 13.2 months. Studies are ongoing with the {sup 90}Y-labelled murine anti-CD20 antibody, IDEC-Y2B8. The response rate in a Phase I/II study in low-grade and intermediate-grade patients was 67%. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  5. Structural basis for EGF receptor inhibition by the therapeutic antibody IMC-11F8.

    Science.gov (United States)

    Li, Shiqing; Kussie, Paul; Ferguson, Kathryn M

    2008-02-01

    Therapeutic anticancer strategies that target and inactivate the epidermal growth factor receptor (EGFR) are under intense study in the clinic. Here we describe the mechanism of EGFR inhibition by an antibody drug IMC-11F8. IMC-11F8 is a fully human antibody that has similar antitumor potency as the chimeric cetuximab/Erbitux and might represent a safer therapeutic alternative. We report the X-ray crystal structure of the Fab fragment of IMC-11F8 (Fab11F8) in complex with the entire extracellular region and with isolated domain III of EGFR. We compare this to our previous study of the cetuximab/EGFR interaction. Fab11F8 interacts with a remarkably similar epitope, but through a completely different set of interactions. Both the similarities and differences in binding of these two antibodies have important implications for the development of inhibitors that could exploit this same mechanism of EGFR inhibition.

  6. BRAF and MEK inhibition variably affect GD2-specific chimeric antigen receptor (CAR) T-cell function in vitro.

    Science.gov (United States)

    Gargett, Tessa; Fraser, Cara K; Dotti, Gianpietro; Yvon, Eric S; Brown, Michael P

    2015-01-01

    Cancer immunotherapy has long been used in the treatment of metastatic melanoma, and an anti-CTLA-4 monoclonal antibody treatment has recently been approved by the US Food and Drug Administration. Targeted therapies such as small molecule kinase inhibitors targeting deregulated mitogen-activated protein kinase (MAPK) signaling have markedly improved melanoma control in up to 50% of metastatic disease patients and have likewise been recently approved. Combination therapies for melanoma have been proposed as a way to exploit the high-level but short-term responses associated with kinase inhibitor therapies and the low-level but longer-term responses associated with immunotherapy. Cancer immunotherapy now includes adoptive transfer of autologous tumor-specific chimeric antigen receptor (CAR) T cells and this mode of therapy is a candidate for combination with small molecule drugs. This paper describes CART cells that target GD2-expressing melanoma cells and investigates the effects of approved MAPK pathway-targeted therapies for melanoma [vemurafenib (Vem), dabrafenib (Dab), and trametinib (Tram)] on the viability, activation, proliferation, and cytotoxic T lymphocyte activity of these CAR T cells, as well as on normal peripheral blood mononuclear cells. We report that, although all these drugs lead to inhibition of stimulated T cells at high concentrations in vitro, only Vem inhibited T cells at concentrations equivalent to reported plasma concentrations in treated patients. Although the combination of Dab and Tram also resulted in inhibition of T-cell effector functions at some therapeutic concentrations, Dab itself had little adverse effect on CAR T-cell function. These findings may have implications for novel therapeutic combinations of adoptive CAR T-cell immunotherapy and MAPK pathway inhibitors.

  7. [VGKC-complex antibodies].

    Science.gov (United States)

    Watanabe, Osamu

    2013-04-01

    Various antibodies are associated with voltage-gated potassium channels (VGKCs). Representative antibodies to VGKCs were first identified by radioimmunoassays using radioisotope-labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were detected only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in patients with Morvan's syndrome and in those with a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins (for example LGI-1 and CASPR-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now commonly known as VGKC-complex antibodies. In general, LGI-1 antibodies are most commonly detected in patients with limbic encephalitis with syndrome of inappropriate secretion of antidiuretic hormone. CASPR-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability. Furthermore, VGKC-complex antibodies are tightly associated with chronic idiopathic pain. Hyperexcitability of nociceptive pathways has also been implicated. These antibodies may be detected in sera of some patients with neurodegenerative diseases (for example, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease).

  8. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    Directory of Open Access Journals (Sweden)

    Tal Noy-Porat

    2016-03-01

    Full Text Available Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1 that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  9. In vivo evaluation of the cross-genotype neutralizing activity of polyclonal antibodies against hepatitis C virus

    DEFF Research Database (Denmark)

    Meuleman, Philip; Bukh, Jens; Verhoye, Lieven

    2011-01-01

    in the kinetics of viral infection. Sequence analysis of the recovered viruses did not suggest antibody-induced viral escape. Conclusion: Polyclonal anti-HCV antibodies isolated from a chronic HCV patient can protect against an in vivo challenge with different HCV genotypes. However, the in vivo protective...... HCV infection still remains elusive. We have previously shown that purified polyclonal antibodies isolated from plasma obtained in 2003 from a chronic HCV patient (Patient H) can protect human liver chimeric mice from a subsequent challenge with the autologous HCV strain isolated from Patient H...... in 1977 (H77). In this study we investigated whether polyclonal antibodies isolated from Patient H in 2006 (H06), which display high cross-genotype neutralizing activity in both the HCV pseudoparticle (HCVpp) and HCV cell culture (HCVcc) systems, were also able to prevent HCV infection of different...

  10. Adjuvants and immunization strategies to induce influenza virus hemagglutinin stalk antibodies.

    Directory of Open Access Journals (Sweden)

    Peter H Goff

    Full Text Available The global population remains vulnerable in the face of the next pandemic influenza virus outbreak, and reformulated vaccinations are administered annually to manage seasonal epidemics. Therefore, development of a new generation of vaccines is needed to generate broad and persistent immunity to influenza viruses. Here, we describe three adjuvants that enhance the induction of stalk-directed antibodies against heterologous and heterosubtypic influenza viruses when administered with chimeric HA proteins. Addavax, an MF59-like nanoemulsion, poly(I:C, and an RNA hairpin derived from Sendai virus (SeV Cantell were efficacious intramuscularly. The SeV RNA and poly(I:C also proved to be effective respiratory mucosal adjuvants. Although the quantity and quality of antibodies induced by the adjuvants varied, immunized mice demonstrated comparable levels of protection against challenge with influenza A viruses on the basis of HA stalk reactivity. Finally, we present that intranasally, but not intramuscularly, administered chimeric HA proteins induce mucosal IgA antibodies directed at the HA stalk.

  11. Functional balance between T cell chimeric receptor density and tumor associated antigen density: CTL mediated cytolysis and lymphokine production.

    Science.gov (United States)

    Weijtens, M E; Hart, E H; Bolhuis, R L

    2000-01-01

    Genetically engineered expression of tumor-specific single chain antibody chimeric receptors (ch-Rec) on human T lymphocytes endow these cells with the parental monoclonal antibody (mAb) dictated tumor specificity and may be useful for clinical immuno-genetherapy. Therefore it was of importance to assess how the densities of tumor-specific receptors and tumor associated antigens (TAA), respectively, affect primary human T lymphocyte functions in relation to target cell susceptibilities to lysis. We therefore studied the functional balance between ch-Rec densities on human T lymphocytes and TAA on tumor cells. The gene construct encoding a ch-Rec derived from (1) a renal carcinoma cell (RCC) specific mouse mAb (G250), and (2) the human signal transducing Fc(epsilon)RI gamma-chain was used. To obtain ch-RecHIGH-POS and ch-RecLOW-POS T lymphocytes, two distinct retroviral vectors were used to introduce the gene constructs into primary human T lymphocytes. Levels of ch-Rec-redirected T lymphocyte mediated tumor cell lysis, as well as lymphokine production were determined using RCC lines as target/stimulator cells, which express either no or increasing densities of the TAA. A functional and dynamic balance between ch-Rec densities on cytotoxic T lymphocytes (CTLs) on the one hand and TAA densities on RCCs on the other, was found. In short, ch-RecHIGH-POS CTLs are triggered by TAAHIGH-POS as well as TAALOW-POS RCCs to lyse tumor cells and produce (IFN-gamma and TNF-alpha) lymphokine. In contrast, ch-RecLOW-POS T lymphocytes are only triggered for cytolysis and lymphokine production by relatively TAAHIGH-POS RCCs. In conclusion, (1) the activation of T lymphocyte responses is co-determined by the expression levels of the ch-Rec on T lymphocytes and the TAA on tumor cells and (2) at relatively high T lymphocyte ch-Rec expression levels the CTLs lyse tumor cells with a wide range of TAA densities. Gene Therapy (2000) 7, 35-42.

  12. Induction of Chimerism Permits Low-Dose Islet Grafts in the Liver or Pancreas to Reverse Refractory Autoimmune Diabetes

    OpenAIRE

    Zhang, Chunyan; Wang, Miao; Racine, Jeremy J.; Liu, Hongjun; Lin, Chia-Lei; Nair, Indu; Lau, Joyce; Cao, Yu-An; Todorov, Ivan; Atkinson, Mark; Zeng, Defu

    2010-01-01

    OBJECTIVE To test whether induction of chimerism lowers the amount of donor islets required for reversal of diabetes and renders the pancreas a suitable site for islet grafts in autoimmune diabetic mice. RESEARCH DESIGN AND METHODS The required donor islet dose for reversal of diabetes in late-stage diabetic NOD mice after transplantation into the liver or pancreas was compared under immunosuppression or after induction of chimerism. Recipient mice were monitored for blood glucose levels and ...

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    Live modified vaccines to protect against classical swine fever virus (CSFV), based on chimeric pestiviruses, have been developed to enable serological Differentiation of Infected from Vaccinated Animals (DIVA). In this context, the chimeric virus CP7_E2gif vaccine candidate is unique as it does...... not include any CSFV components. In the present study, the DIVA vaccine properties of CP7_E2gif were evaluated in comparison to the conventional live attenuated Riemser C-strain vaccine. Sera and tonsil samples obtained from pigs immunised with these two vaccines were analysed. No viral RNA was found in serum...... after vaccination with CP7_E2gif, whereas some serum samples from C-strain vaccinated animals were positive. In both vaccinated groups, individual viral RNA-positive tonsil samples were detected in animals euthanised between 7 and 21 days post vaccination. Furthermore, serum samples from these animals...

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

    Science.gov (United States)

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

    2016-08-01

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

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

    Science.gov (United States)

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

    2013-12-01

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

  17. Spotlight on chimeric antigen receptor engineered T cell research and clinical trials in China.

    Science.gov (United States)

    Luo, Can; Wei, Jianshu; Han, Weidong

    2016-04-01

    T cell mediated adoptive immune response has been characterized as the key to anti-tumor immunity. Scientists around the world including in China, have been trying to harness the power of T cells against tumors for decades. Recently, the biosynthetic chimeric antigen receptor engineered T cell (CAR-T) strategy was developed and exhibited encouraging clinical efficacy, especially in hematological malignancies. Chimeric antigen receptor research reports began in 2009 in China according to our PubMed search results. Clinical trials have been ongoing in China since 2013 according to the trial registrations on clinicaltrials. gov.. After years of assiduous efforts, research and clinical scientists in China have made their own achievements in the CAR-T therapy field. In this review, we aim to highlight CAR-T research and clinical trials in China, to provide an informative reference for colleagues in the field.

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

    Science.gov (United States)

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

    1997-04-01

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

  19. 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.

  20. Conformational influence of the ribose 2'-hydroxyl group: crystal structures of DNA-RNA chimeric duplexes

    Science.gov (United States)

    Egli, M.; Usman, N.; Rich, A.

    1993-01-01

    We have crystallized three double-helical DNA-RNA chimeric duplexes and determined their structures by X-ray crystallography at resolutions between 2 and 2.25 A. The two self-complementary duplexes [r(G)d(CGTATACGC)]2 and [d(GCGT)r(A)d(TACGC)]2, as well as the Okazaki fragment d(GGGTATACGC).r(GCG)d(TATACCC), were found to adopt A-type conformations. The crystal structures are non-isomorphous, and the crystallographic environments for the three chimeras are different. A number of intramolecular interactions of the ribose 2'-hydroxyl groups contribute to the stabilization of the A-conformation. Hydrogen bonds between 2'-hydroxyls and 5'-oxygens or phosphate oxygens, in addition to the previously observed hydrogen bonds to 1'-oxygens of adjacent riboses and deoxyriboses, are observed in the DNA-RNA chimeric duplexes. The crystalline chimeric duplexes do not show a transition between the DNA A- and B-conformations. CD spectra suggest that the Okazaki fragment assumes an A-conformation in solution as well. In this molecule the three RNA residues may therefore lock the complete decamer in the A-conformation. Crystals of an all-DNA strand with the same sequence as the self-complementary chimeras show a morphology which is different from those of the chimera crystals. Moreover, the oligonucleotide does not match any of the sequence characteristics of DNAs usually adopting the A-conformation in the crystalline state (e.g., octamers with short alternating stretches of purines and pyrimidines). In DNA-RNA chimeric duplexes, it is therefore possible that a single RNA residue can drive the conformational equilibrium toward the A-conformation.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2006-01-01

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

  3. 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.

  4. The impact of chimerism in DNA-based forensic sex determination analysis

    OpenAIRE

    George, Renjith; Donald, Preethy Mary; Nagraj, Sumanth Kumbargere; Idiculla, Jose Joy; Hj Ismail, Rashid

    2013-01-01

    Sex determination is the most important step in personal identification in forensic investigations. DNA-based sex determination analysis is comparatively more reliable than the other conventional methods of sex determination analysis. Advanced technology like real-time polymerase chain reaction (PCR) offers accurate and reproducible results and is at the level of legal acceptance. But still there are situations like chimerism where an individual possess both male and female specific factors t...

  5. Chimeric antigen receptor (CAR)-directed adoptive immunotherapy: a new era in targeted cancer therapy

    OpenAIRE

    Chen, Yamei; Liu, Delong

    2014-01-01

    As a result of the recent advances in molecular immunology, virology, genetics, and cell processing, chimeric antigen receptor (CAR)-directed cancer therapy has finally arrived for clinical application. CAR-directed adoptive immunotherapy represents a novel form of gene therapy, cellular therapy, and immunotherapy, a combination of three in one. Early phase clinical trial was reported in patients with refractory chronic lymphoid leukemia with 17p deletion. Accompanying the cyto...

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

    Directory of Open Access Journals (Sweden)

    Febbraio Maria

    2007-03-01

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

  7. 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.

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

    Science.gov (United States)

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

    2016-06-29

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

  9. Construction of a photo-responsive chimeric histidine kinase in Escherichia coli.

    Science.gov (United States)

    Hori, Mayuko; Oka, Shyunsuke; Sugie, Yoshimi; Ohtsuka, Hokuto; Aiba, Hirofumi

    2017-01-31

    Two-component signal transduction systems (TCS), that are also referred to as His to Asp phosphorelay systems, are involved in widespread cellular responses to diverse signals from bacteria to plants. Previously, we succeeded in reconstructing a cyanobacterial photo-perception system in Escherichia coli by employing a CcaS-CcaR two-component system from Nostoc punctiforme. In this study, we have added a photo-responsive ability to ArcB-ArcA (anoxic redox control) TCS of E. coli by fusing a cyanobacterial photoreceptor domain of CcaS with an intracellular histidine kinase (HK) domain of ArcB. For this, we constructed several chimeric HKs between CcaS and ArcB and found that one chimeric HK, named ArcaS9, has a photo-responsive ability. When ArcaS9 was expressed with an ArcA response regulator in E. coli expressing phycocyanobilin (PCB)-producing enzymes, the expression of sdh, a target gene of ArcB-ArcA TCS was regulated in a light-color-dependent manner. Thus we succeeded in endowing E. coli HK with a photo-responsive ability. This provides an insight into how the sensing ability of HK can be manipulated by a chimeric construct.

  10. 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.

  11. Sensitivity of HIV type 1 primary isolates to human anti-CD40 antibody-mediated suppression is related to coreceptor use.

    Science.gov (United States)

    Abayneh, Sisay A; Ellmark, Peter; Karlsson, Ulf; Andersson, Henrik; Borrebaeck, Carl A K; Karlsson, Ingrid; Fenyö, Eva Maria

    2008-03-01

    The effect of CD40 ligation on infection by HIV-1 primary isolates with different R5 phenotypes was evaluated with a novel set of anti-CD40 monoclonal antibodies originating from a human phage display library. Five human monoclonal anti-CD40 antibodies of IgG1 subtype characterized by the ability to activate B cells via CD40 were tested for induction of the CC-chemokines RANTES and MIP-1alpha and inhibition of HIV-1 replication in primary monocyte-derived macrophages (MDM). All activating anti-CD40 antibodies were able to induce CC-chemokines in MDM. We chose the most potent antibody, clone B44, for further experiments. This antibody had a suppressive effect on HIV-1 isolates of the R5 phenotype with limited use of CCR5/CXCR4 chimeric receptors. In comparison, HIV-1 isolates with broader use of CCR5/CXCR4 chimeric receptors or with CXCR4 use were less sensitive to anti-CD40-induced suppression. The results indicate that HIV-1 replication is inhibited by human anti-CD40 monoclonal antibodies through the mechanism of CC-chemokine induction. This effect is thus restricted to HIV-1 isolates sensitive to inhibition by CC-chemokines.

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

    Science.gov (United States)

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

    2013-02-01

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

  13. Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors.

    Science.gov (United States)

    Thokala, Radhika; Olivares, Simon; Mi, Tiejuan; Maiti, Sourindra; Deniger, Drew; Huls, Helen; Torikai, Hiroki; Singh, Harjeet; Champlin, Richard E; Laskowski, Tamara; McNamara, George; Cooper, Laurence J N

    2016-01-01

    Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR's in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies.

  14. Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors

    Science.gov (United States)

    Olivares, Simon; Mi, Tiejuan; Maiti, Sourindra; Deniger, Drew; Huls, Helen; Torikai, Hiroki; Singh, Harjeet; Champlin, Richard E.; Laskowski, Tamara; McNamara, George; Cooper, Laurence J. N.

    2016-01-01

    Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR’s in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies. PMID:27548616

  15. Expression of recombinant antibodies.

    Science.gov (United States)

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  16. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  17. Recombinant renewable polyclonal antibodies.

    Science.gov (United States)

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  18. Recombinant anti-tenascin antibody constructs

    Energy Technology Data Exchange (ETDEWEB)

    ZALUTSKY, MICHAEL R

    2006-08-29

    The general objective of this research is to combine genetically derived molecular constructs reactive with tenascin, with appropriate radionuclides and labeling methods in order to generate more effective diagnostic and therapeutic reagents for oncologic nuclear medicine. Tenascin, a polymorphic extracellular matrix glycoprotein, is of interest because of its high expression on glioma, melanoma, as well as prostate and breast carcinoma. Recently, we have also documented high levels of tenascin in lymphomas, particularly those of higher grade, making the potential clinical impact of tenascin-specific radiodiagnostics and therapeutics even greater. An essential feature of our work plan is the ability to exploit our extensive clinical experience in order to design second-generation constructs with properties which could improve clinical efficacy. To date, we have treated over 150 brain tumor patients with 131I-labeled murine 81C6, an antibody which binds specifically to the alternatively spliced fibronectin type III repeats CD of the tenascin molecule. During the current grant period, we have made several observations which form the basis for our proposed specific aims. First, tissue distribution and catabolism experiments in animal models have demonstrated enhanced stability for a chimeric construct composed of murine variable regions and human IgG2 constant domains. Furthermore, pharmacokinetic studies in patients with 131I-labeled chimeric 81C6 have shown significantly longer retention in glioma tumor resection cavities compared with its murine parent. Second, we have initiated the first clinical trial of an endoradiotherapeutic labeled with the 7.2-hr -particle emitter 211At. Twelve glioma patients have received 211At-labeled chimeric 81C6 directly into their brain tumor resection cavity, and very encouraging results have been obtained. Now that the feasibility of human studies with 211At, has been demonstrated, the development and evaluation of anti

  19. Anti-insulin antibody test

    Science.gov (United States)

    Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... You appear to have an allergic response to insulin Insulin no longer seems to control your diabetes

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

    Science.gov (United States)

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

    2014-12-01

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

  1. Molecular imaging of rheumatoid arthritis by radiolabelled monoclonal antibodies: new imaging strategies to guide molecular therapies

    Energy Technology Data Exchange (ETDEWEB)

    Malviya, G.; Dierckx, R.A. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Conti, F. [Rheumatology Unit, I Faculty of Medicine and Surgery, Sapienza University of Rome (Italy); Chianelli, M. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Unit of Nuclear Medicine, Regina apostolorum Hospital, Albano, Rome (Italy); Scopinaro, F. [Nuclear Medicine Department, Sapienza University of Rome, St. Andrea Hospital, Rome (Italy); Signore, A. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Nuclear Medicine Department, Sapienza University of Rome, St. Andrea Hospital, Rome (Italy)

    2010-02-15

    The closing of the last century opened a wide variety of approaches for inflammation imaging and treatment of patients with rheumatoid arthritis (RA). The introduction of biological therapies for the management of RA started a revolution in the therapeutic armamentarium with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion molecules, activation markers, antigens or receptors, to interfere with specific inflammation pathways at the molecular level, leading to immune-modulation of the underlying pathogenic process. These new generation of mAbs can also be radiolabelled by using direct or indirect method, with a variety of nuclides, depending upon the specific diagnostic application. For studying rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-{alpha}, anti-CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody, have been radiolabelled mainly with {sup 99m}Tc or {sup 111}In. Scintigraphy with these radiolabelled antibodies may offer an exciting possibility for the study of RA patients and holds two types of information: (1) it allows better staging of the disease and diagnosis of the state of activity by early detection of inflamed joints that might be difficult to assess; (2) it might provide a possibility to perform 'evidence-based biological therapy' of arthritis with a view to assessing whether an antibody will localise in an inflamed joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection of patients to be treated since biological therapies can be associated with severe side-effects and are considerably expensive. This article reviews the use of radiolabelled mAbs in the study of RA with particular emphasis on the use of different radiolabelled monoclonal antibodies for

  2. Construction and immunogenic properties of a chimeric protein comprising CfaE, CfaB and LTB against Enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Gheibi Hayat, Seyed-Mohammad; Mousavi Gargari, Seyed-Latif; Nazarian, Shahram

    2016-11-01

    ETEC (Enterotoxigenic Escherichia coli) is a major cause of diarrhea in developing countries and children. ETEC has two virulence factors including colonization factors antigen (CFA) and labile enterotoxins (LTs). CFA/I consists the major pilin subunit CfaB and a minor adhesive subunit, CfaE. In this study a tripartite fusion protein containing CfaB, CfaE and LTB was designed. In silico analysis of the tertiary structure of the chimeric protein showed a protein with three main domains linked together with linkers. Linear and conformational B-cell epitopes were identified. A chimera consisting cfaB, cfaE and ltB(BET)was then synthesized with E. coli codon bias in pUC57 and sub cloned into pET32 vector. Recombinant protein was expressed and purified by affinity chromatography and confirmed by western blotting. Mice were immunized with recombinant protein and the antibody titer and specificity of the sera were analyzed by ELISA. The efficiency of the immune sera against ETEC was evaluated by binding assay and GM1-ELISA. VaxiJen analysis of the protein showed high antigenicity. Post-immune sera contained high titers of anti-BET IgG. Pretreatment of ETEC cells with sera from immunized mice decreased their ability to adhere to cells of the human colon adenocarcinoma cell line HT29.

  3. B-cell epitope of beta toxin of Clostridium perfringens genetically conjugated to a carrier protein: expression, purification and characterization of the chimeric protein.

    Science.gov (United States)

    Bhatia, Bharti; Solanki, Amit Kumar; Kaushik, Himani; Dixit, Aparna; Garg, Lalit C

    2014-10-01

    Beta toxin (btx) is the prime virulence factor for the pathogenesis of Clostridium perfringens type C strain, known to cause necrotic enteritis and enterotoxaemia in mammalian species. The existing vaccines targeting btx are formaldehyde inactivated culture filtrates of Clostridium. These filtrates raise antigenic load in the host leading to nonspecific and poor responses. The present study aimed to overcome these drawbacks and generate a chimeric protein carrying in silico identified B-cell epitope of btx fused with a carrier protein as a vaccine candidate. Using bioinformatic tools, three stretches of amino acids were predicted as putative B-cell epitopes. One of the epitopes spanning 140-156 amino acid residues was genetically conjugated with B-subunit of heat labile enterotoxin (LTB) of Escherichia coli and expressed as a translational fusion in Vibrio cholerae secretory expression system. High level expression of the recombinant fusion protein rLTB-Btx140-156 was obtained and the protein was successfully purified. The recombinant protein retained the native LTB property to pentamerize and bind to GM1 ganglioside receptor of LTB. The antigenicity of both the epitope and the carrier protein was maintained in fusion protein as indicated by immunoblotting against anti-LTB and anti-btx antibody. The rLTB-Btx140-156 fusion protein therefore can be evaluated as a potential vaccine candidate against C. perfringens.

  4. New recombinant chimeric antigens, P35-MAG1, MIC1-ROP1, and MAG1-ROP1, for the serodiagnosis of human toxoplasmosis.

    Science.gov (United States)

    Drapała, Dorota; Holec-Gąsior, Lucyna; Kur, Józef

    2015-05-01

    The aim of the study was to evaluate the usefulness of 3 chimeric Toxoplasma gondii antigens, P35-MAG1, MIC1-ROP1 and MAG1-ROP1, in the serodiagnosis of an acute toxoplasmosis in humans. Proteins were produced as fusion proteins containing His tags ends and then further purified by metal affinity chromatography. Their application for the diagnosis of recently acquired T. gondii infection was tested in IgG and IgM enzyme-linked immunosorbent assays (ELISAs). At 100%, 77.3%, and 86.4%, respectively, the reactivity of the IgG ELISA using P35-MAG1, MIC1-ROP1, and MAG1-ROP1 for sera from patients where acute toxoplasmosis was suspected was significantly higher than for the samples from people with a chronic infection, at 26.2%, 36.1%, and 32.8%, respectively. Moreover, P35-MAG1, MIC1-ROP1, and MAG1-ROP1 detected IgM antibodies with a reactivity at 81.8%, 72.7%, and 59.1%, respectively. The results presented in the article show that, particularly, P35-MAG1 may be useful in the preliminary detection of recent T. gondii infection.

  5. Tumor Antigen Specific Activation of Primary Human T-Cells Expressing a Virally Encoded Chimeric T-Cell Receptor Specific for p185HER2

    Institute of Scientific and Technical Information of China (English)

    杨建民; MichaelSFRIEDMAN; ChristopherMREYNOLDS; MarianneTHUBEN; LeeWILKE; JenniferFULLER; 李桥; ZeligESHHAR; JamesJMULE; KevimTMCDONAGH

    2004-01-01

    We have developed and tested chimeric T-cell receptors (TCR) specific for p185HER2. In these experiments,retroviral vectors expressing the N297 or N29ξ receptors were constructed in pRET6. Amphotropic viral producer cells were established in the GALV-based PG13 packaging cell line. Ficoll purified human peripheral blood lymphocytes (PBL) were vitally transduced using an optimized protocol incorporating activation with immobilized anti-CD3/anti-CD28 monoclonal antibodies, followed by viral infection in the presence of fibronectin fragment CH296. Transduced cells were co-cultured with human tumor cell lines that overexpress (SK-OV-3) or underexpress (MCF7) p185HER2 to assay for antigen specific immune responses. Both CD4+ and CD8+ T-cells transduced with the N297 or N29ξ chTCR demonstrated HER2-specific antigen responses, as determined by release of Th1 like cytokines, and cellular cytotoxicity assays. Our results support the feasibility of adoptive immunothempy with genetically modified T-cells expressing a chTCR specific for p185HER2.

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

    Science.gov (United States)

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

    2015-11-01

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

  7. High-dose radioimmunotherapy in refractory b-celI non-Hodgikin's lymphoma with I-131-labeled chimeric anti CD-20 C2B8 (I-131 rituximab): pilot trial

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sung Eun; Park, Yeon Hee; Cheon, Gi Jeong; Ryoo, Baek Yeol; Lee, Seung Sook; Choi, Chang Woon; Lim, Sang Moo [Korea Institute of Radiological and Medical Science, Seoul (Korea, Republic of)

    2004-07-01

    The native chimeric human-mouse anti CD-20 antibody IDEC-C2B8 (rituximab) is therapeutically applied in relapsed or refractory NHL. This ongoing pilot study was to evaluate whether high-dose radioimmunotherapy (RIT) with I-131 rituximab is therapeutically effective in refractory B-cell NHL. 5 patients (5 male, aged 50.89{+-}16.89) with chemorefractory NHL of B-cell origin (2 diffuse large B cell, 1 burkitt's lymphoma, and 2 mantle cell Iymphoma) oe, with a life expectancy of at least 3 months, and with a Kamofsky performance score of 60 and above were studied. The chimeric IgG1 anti CD 20 monoclonal antibody rituximab (mabthera, Roche) was radiolabelled with iodine-131 (I-131) using a modified chloaramine T method with high radiochemical purity (95%{+-}0.9) and preservation of immunoreactivity. All patients received therapeutic loading doses of unlabelled rituximab (18.5 MBq/kg) immediately prior to administration of therapeutic dose (3.7 GBq-8.5 GBq), and then underwent gamma camera scan and pre-and post-RIT FDG PET (within 7 day and day 30). Blood cell nadirs were reached at 2-3 weeks after therapy infusion, but all patients recovered at 6 weeks after treatment. Non hematologic toxicity was restricted to mild-to moderate nausea, fever, transient bilirubin, or liver enzyme elevation. Two (8.5 GBq) with mantle cell lymphoma and one with burkitt's lymphoma experienced good partial remissions, and one (5.5 GBq, DLBL) with bulky disease had a partial remission, and one patient (3.7 GBq, DLBL) with bulky disease had a mixed response. High-dose RIT with I-131 labelled rituximab seems to be effective and moderate toxicity. Further follow-up to monitor the long-term outcome are indicated.

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

    Directory of Open Access Journals (Sweden)

    Ewa C S Ellis

    Full Text Available OBJECTIVE: 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. DESIGN: 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. RESULTS: 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. CONCLUSION: 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.

  9. Monoclonal antibody "gold rush".

    Science.gov (United States)

    Maggon, Krishan

    2007-01-01

    The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush.

  10. 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.

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

    Science.gov (United States)

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

    2006-09-01

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

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

    Science.gov (United States)

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

    1997-11-14

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

  13. Antiphospholipid antibody syndrome.

    Science.gov (United States)

    Kutteh, William H; Hinote, Candace D

    2014-03-01

    Antiphospholipid antibodies (aPLs) are acquired antibodies directed against negatively charged phospholipids. Obstetric antiphospholipid antibody syndrome (APS) is diagnosed in the presence of certain clinical features in conjunction with positive laboratory findings. Obstetric APS is one of the most commonly identified causes of recurrent pregnancy loss. Thus, obstetric APS is distinguished from APS in other organ systems where the most common manifestation is thrombosis. Several pathophysiologic mechanisms of action of aPLs have been described. This article discusses the diagnostic and obstetric challenges of obstetric APS, proposed pathophysiologic mechanisms of APS during pregnancy, and the management of women during and after pregnancy.

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

    Directory of Open Access Journals (Sweden)

    Tamborrini Marco

    2011-12-01

    Full Text Available Abstract Background In clinical trials, immunopotentiating reconstituted influenza virosomes (IRIVs have shown great potential as a versatile antigen delivery platform for synthetic peptides derived from Plasmodium falciparum antigens. This study describes the immunogenicity of a virosomally-formulated recombinant fusion protein comprising domains of the two malaria vaccine candidate antigens MSP3 and GLURP. Methods The highly purified recombinant protein GMZ2 was coupled to phosphatidylethanolamine and the conjugates incorporated into the membrane of IRIVs. The immunogenicity of this adjuvant-free virosomal formulation was compared to GMZ2 formulated with the adjuvants Montanide ISA 720 and Alum in three mouse strains with different genetic backgrounds. Results Intramuscular injections of all three candidate vaccine formulations induced GMZ2-specific antibody responses in all mice tested. In general, the humoral immune response in outbred NMRI mice was stronger than that in inbred BALB/c and C57BL/6 mice. ELISA with the recombinant antigens demonstrated immunodominance of the GLURP component over the MSP3 component. However, compared to the Al(OH3-adjuvanted formulation the two other formulations elicited in NMRI mice a larger proportion of anti-MSP3 antibodies. Analyses of the induced GMZ2-specific IgG subclass profiles showed for all three formulations a predominance of the IgG1 isotype. Immune sera against all three formulations exhibited cross-reactivity with in vitro cultivated blood-stage parasites. Immunofluorescence and immunoblot competition experiments showed that both components of the hybrid protein induced IgG cross-reactive with the corresponding native proteins. Conclusion A virosomal formulation of the chimeric protein GMZ2 induced P. falciparum blood stage parasite cross-reactive IgG responses specific for both MSP3 and GLURP. GMZ2 thus represents a candidate component suitable for inclusion into a multi-valent virosomal

  15. Interaction of antibodies to synthetic peptides of proNGF with in vitro synthesized NGF precursors.

    Science.gov (United States)

    Dicou, E

    1989-09-25

    Sera raised against three synthetic peptides that reproduce sequences of the pro-nerve growth factor (proNGF) protein were tested in immunoprecipitation experiments using in vitro translation products of SP6-directed NGF mRNA in a rabbit reticulocyte lysate. The interaction of these antibodies with bacterially synthesized chimeric preproNGF was also examined. Digestion of the translation products by the gamma-subunit generated the 22 and 18 kDa intermediates. A predominant 13 kDa intermediate was obtained after digestion of translation products in wheat germ extract. This is shown to be the N-terminal peptide by immunoprecipitation with an anti-peptide serum. These antibodies may be used to detect NGF precursor cleavage products in vivo.

  16. Anti-ALK Antibodies in Patients with ALK-Positive Malignancies Not Expressing NPM-ALK.

    Science.gov (United States)

    Damm-Welk, Christine; Siddiqi, Faraz; Fischer, Matthias; Hero, Barbara; Narayanan, Vignesh; Camidge, David Ross; Harris, Michael; Burke, Amos; Lehrnbecher, Thomas; Pulford, Karen; Oschlies, Ilske; Siebert, Reiner; Turner, Suzanne; Woessmann, Wilhelm

    2016-01-01

    Patients with Nucleophosmin (NPM)- Anaplastic Lymphoma Kinase (ALK) fusion positive Anaplastic Large Cell Lymphoma produce autoantibodies against ALK indicative of an immune response against epitopes of the chimeric fusion protein. We asked whether ALK-expression in other malignancies induces specific antibodies. Antibodies against ALK were detected in sera of one of 50 analysed ALK-expressing neuroblastoma patients, 13 of 21 ALK positive non-small cell lung carcinoma (NSCLC) patients, 13 of 22 ALK translocation-positive, but NPM-ALK-negative lymphoma patients and one of one ALK-positive rhabdomyosarcoma patient, but not in 20 healthy adults. These data suggest that boosting a pre-existent anti-ALK immune response may be more feasible for patients with ALK-positive NSCLC, lymphomas and rhabdomyosarcomas than for tumours expressing wild-type ALK.

  17. Functional capabilities of marmoset T and B lymphocytes in primary in vitro antibody formation

    Energy Technology Data Exchange (ETDEWEB)

    Nickerson, D.A.; Gengozian, N.

    1981-01-15

    In vitro tests of T- and B-lymphocyte function of two marmoset species, Saguinus fuscicollis and Saguinus oedipus, were examined to explore the lower immune response profile previously reported for S. o. oedipus. Experiments with trinitrophenyl-lipopolysaccharide (TNP-LPS) revealed peripheral blood leukocytes (PBL) from both species capable of antibody formation. This response was both T cell and monocyte independent; indeed, removal of T cells led to an enhanced response, indicating a regulatory role for this cell in each species. Studies with the nonmitogenic form of TNP-LPS, trinitrophenyl-base-hydrolyzed-lipopolysaccharide, revealed that plaque-forming cells could be obtained from S. fuscicollis PBL while S. o. oedipus PBL were unresponsive. This report also demonstrates that hemopoietic chimerism, a feature common to all marmosets, has a negative influence on antibody-forming capabilities.

  18. Rapid obtention of stable, bioluminescent tumor cell lines using a tCD2-luciferase chimeric construct

    Directory of Open Access Journals (Sweden)

    Gourzones Claire

    2011-03-01

    Full Text Available Abstract Background Bioluminescent tumor cell lines are experimental tools of major importance for cancer investigation, especially imaging of tumors in xenografted animals. Stable expression of exogenous luciferase in tumor cells combined to systemic injection of luciferin provides an excellent signal/background ratio for external optical imaging. Therefore, there is a need to rationalize and speed up the production of luciferase-positive tumor cell lines representative of multiple tumor phenotypes. For this aim we have designed a fusion gene linking the luciferase 2 protein to the c-terminus of a truncated form of the rat CD2 protein (tCD2-luc2. To allow simultaneous assessment of the wild-type luciferase 2 in a context of tCD2 co-expression, we have made a bicistronic construct for concomitant but separate expression of these two proteins (luc2-IRES-tCD2. Both the mono- and bi-cistronic constructs were transduced in lymphoid and epithelial cells using lentiviral vectors. Results The tCD2-luc2 chimera behaves as a type I membrane protein with surface presentation of CD2 epitopes. One of these epitopes reacts with the OX34, a widely spread, high affinity monoclonal antibody. Stably transfected cells are sorted by flow cytometry on the basis of OX34 staining. In vitro and, moreover, in xenografted tumors, the tCD2-luc2 chimera retains a substantial and stable luciferase activity, although not as high as the wild-type luciferase expressed from the luc2-IRES-tCD2 construct. Expression of the tCD2-luc2 chimera does not harm cell and tumor growth. Conclusion Lentiviral transduction of the chimeric tCD2-luc2 fusion gene allows selection of cell clones with stable luciferase expression in less than seven days without antibiotic selection. We believe that it will be helpful to increase the number of tumor cell lines available for in vivo imaging and assessment of novel therapeutic modalities. On a longer term, the tCD2-luc2 chimera has the potential to

  19. Comparison of the immunoreactivity of rituximab antibody labeled with either I-125 or Re-188 for radioimmunotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Tae Hyun; Chung, Hye Kyung; Lee, Tae Sup; Chung, Wee Sup; Woo, Kwang Sun; Lee, Myung Jin; Kim, So Yeon; Chung, Jae Ho; CHoi, Chang Woon; Lim, Sang Moo [KIRAMS, Seoul (Korea, Republic of); Darwati, Siti [National Nuclear Energy Agency, Tangerang (Indonesia)

    2004-07-01

    Monoclonal antibodies against tumor-associated antigens can be applied as delivery vehicles for radionuclides to treat tumors. The specificity of MAbs for tumor-associated antigens can be exploited to direct radionuclides selectively to tumor cells after systemic administration. In radioimmunotherapy, therapeutic efficacy depends on the choice of the radionuclide. The chemical characteristics of radioiodine and radiometals (Re-188) differ significantly with respect to labeling procedure and consequently the specificity of monoclonal antibody can be affected due to discrepancy of labeling condition. Rituximab is a genetically engineered, chimeric anti-CD20 monoclonal antibody with mouse variable and human constant region. The CD-20 itself plays an important role in human B-cell proliferation and is an effective target for immunotherapy. In the present study, we compared the immunoreactivity of I-125-labeled Rituximab with Re-188-labeled Rituximab according to radionuclide-optimized labeling condition in cell binding assay of Lindmo method.

  20. Construction of a chimeric hepatitis C virus replicon based on a strain isolated from a chronic hepatitis C patient.

    Science.gov (United States)

    Cao, Huang; Zhu, Wandi; Han, Qingxia; Pei, Rongjuan; Chen, Xinwen

    2014-02-01

    Subgenomic replicons of hepatitis C virus (HCV) have been widely used for studying HCV replication. Here, we report a new subgenomic replicon based on a strain isolated from a chronically infected patient. The coding sequence of HCV was recovered from a Chinese chronic hepatitis C patient displaying high serum HCV copy numbers. A consensus sequence designated as CCH strain was constructed based on the sequences of five clones and this was classified by sequence alignment as belonging to genotype 2a. The subgenomic replicon of CCH was replication-deficient in cell culture, due to dysfunctions in NS3 and NS5B. Various JFH1/CCH chimeric replicons were constructed, and specific mutations were introduced. The introduction of mutations could partially restore the replication of chimeric replicons. A replication-competent chimeric construct was finally obtained by the introduction of NS3 from JFH1 into the backbone of the CCH strain.

  1. Anti-cartilage antibody.

    Science.gov (United States)

    Greenbury, C L; Skingle, J

    1979-08-01

    Antibody to cartilage has been demonstrated by indirect immunofluorescence on rat trachea in the serum of about 3% of 1126 patients with rheumatoid arthritis. Titres ranged from 1:20 to 1:640. The antibody was not found in 284 patients with primary or secondary osteoarthritis or in 1825 blood donors, nor, with the exception of two weak reactors, in 1314 paraplegic patients. In most cases the antibody appears to be specific for native type II collagen. Using this as an antigen in a haemagglutination test 94% of anti-cartilage sera were positive, whereas among 100 rheumatoid control sera there were only three weak positives. More than 80% of patients with antibody had some erosion of articular cartilage, but there was no correlation with age, sex, duration of disease, nor any recognisable clinical event or change.

  2. Antithyroid microsomal antibody

    Science.gov (United States)

    ... to confirm the cause of thyroid problems, including Hashimoto thyroiditis . The test is also used to find ... positive test may be due to: Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also ...

  3. Serum herpes simplex antibodies

    Science.gov (United States)

    ... 2. HSV-1 most often causes cold sores (oral herpes). HSV-2 causes genital herpes. How the Test ... whether a person has ever been infected with oral or genital herpes . It looks for antibodies to herpes simplex virus ...

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

    Directory of Open Access Journals (Sweden)

    Eneour Puill-Stephan

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

  5. 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...

  6. Click-chemistry strategy for labeling antibodies with copper-64 via a cross-bridged tetraazamacrocyclic chelator scaffold.

    Science.gov (United States)

    Kumar, Amit; Hao, Guiyang; Liu, Li; Ramezani, Saleh; Hsieh, Jer-Tsong; Öz, Orhan K; Sun, Xiankai

    2015-04-15

    We report a click-chemistry based modular strategy for antibody labeling with (64)Cu (t1/2 = 12.7 h; β(+) 0.656 MeV, 17.4%; β(-) 0.573 MeV, 39%; EC 43%) under ambient condition utilizing a cross-bridged tetraazamacrocyclic (CB-TE2A) analogue, which otherwise requires harsh conditions that make the CB-TE2A analogues under-utilized for protein labeling despite the fact that they form kinetically inert copper complexes with high in vivo stability. Our strategy involves prelabeling a CB-TE2A based scaffold (CB-TE2A-1C) with (64)Cu and its subsequent reaction with an antibody via the tetrazine-norbornene mediated click chemistry. The effectiveness of this strategy was demonstrated by labeling two monoclonal antibodies, an anti-PSMA antibody (YPSMA-1) and a chimeric anti-phosphatidylserine antibody (Bavituximab). The immunoreactivity of the antibodies remained unchanged after the tetrazine modification and click-chemistry (64)Cu labeling. To further demonstrate the practicality of the modular (64)Cu labeling strategy, we tested positron emission tomography (PET) imaging of tumor with the (64)Cu-labeled bavituximab in a mouse xenograft model. The tumor visualization and uptake of the labeled antibody exhibited the versatility of the click-chemistry strategy.

  7. Tumor scintigraphy by the method for subtracting the initial image with technetium-99m labeled antibody

    Energy Technology Data Exchange (ETDEWEB)

    Karube, Yoshiharu; Katsuno, Kentaro; Ito, Sanae; Matsunaga, Kazuhisa; Takata, Jiro [Fukuoka Univ. (Japan). Faculty of Pharmaceutical Sciences; Kuroki, Masahide; Murakami, Masaaki; Matsuoka, Yuji

    1999-12-01

    The method for subtracting the initial image from the localization image was evaluated for radioimmunoscintigraphy of tumors with technetium-99m (Tc-99m) labeled antibodies. Monoclonal antibodies were parental mouse and mouse-human chimeric antibodies to carcinoembryonic antigen (CEA), designated F11-39 and ChF11-39, respectively, both of which have been found to discriminate CEA in tumor tissues from the CEA-related antigens. After reduction of the intrinsic disulfide bonds, these antibodies were labeled with Tc-99m. In vivo studies were performed on athymic nude mice bearing the human CEA-producing gastric carcinoma xenografts. Though biodistribution results showed selective and progressive accumulation of Tc-99m labeled antibodies at the tumor site, high radioactivity in blood was inappropriate for scintigraphic visualization of the tumors within a few hours. We examined the subtraction of the initial Tc-99m image from the Tc-99m localization image after a few hours. Subtracted images of the same count reflected the in vivo behavior of the Tc-99m radioactivity. The subtracted scintigrams revealed excellent tumor images with no significant extrarenal background. Visualization of the tumor site was dependent on antigen-specific binding and nonspecific exudation. These results demonstrate that a method of subtraction of the initial image may serve as a potentially useful diagnostic method for an abnormal site for agents with a low pharmacokinetic value. (author)

  8. Mapping of the C3b-binding site of CR1 and construction of a (CR1)2-F(ab')2 chimeric complement inhibitor.

    Science.gov (United States)

    Kalli, K R; Hsu, P H; Bartow, T J; Ahearn, J M; Matsumoto, A K; Klickstein, L B; Fearon, D T

    1991-12-01

    CR1/CR2 chimeric receptors in which various short consensus repeats (SCRs) of CR1 were attached to CR2 were transiently expressed on COS cells, and assessed for the binding of polymerized C3b (pC3b) and anti-CR2 by immunofluorescence. Of COS cells expressing chimeras containing SCR 1-4, 1-3, 2-4, 1-2, and 2-3 of the long homologous repeats (LHRs) -B or -C, 96%, 66%, 23%, 0%, and 0%, respectively, bound pC3b. K562 cells were stably transfected with wild-type CR1, deletion mutants of CR1, and the CR1/CR2 chimeras, respectively, and assayed for binding of 125I-pC3b. The dissociation constants (Kd) for pC3b of wild-type CR1 and the LHR-BD and -CD constructs were in the range of 1.0-2.7 nM, and of the CR1/CR2 chimeras containing SCRs 1-4, 1-3, and 2-4 of LHR-B or -C were 1.8-2.4, 6-9, and 22-36 nM, respectively. The factor I-cofactor function of the CR1/CR2 chimeras paralleled the C3b-binding function of the constructs. A CR1/immunoglobulin (Ig) chimeric protein was prepared by fusing SCRs 1-4 of LHR-B to the heavy chains of a murine F(ab')2 anti-nitrophenacetyl (NP) monoclonal antibody. The (CR1)2-F(ab')2 chimera, which retained its specificity for NP, was as effective as soluble, full-length CR1 in binding pC3b, serving as a cofactor for factor I-mediated cleavage of C3b, and inhibiting activation of the alternative pathway, indicating that the bivalent expression of these SCRs reconstitutes the alternative pathway inhibitory function of CR1. The feasibility of creating CR1/Ig chimeras makes possible a new strategy of targeting complement inhibition by the use of Ig fusion partners having particular antigenic specificities.

  9. Potato virus X movement in Nicotiana benthamiana: new details revealed by chimeric coat protein variants.

    Science.gov (United States)

    Betti, Camilla; Lico, Chiara; Maffi, Dario; D'Angeli, Simone; Altamura, Maria Maddalena; Benvenuto, Eugenio; Faoro, Franco; Baschieri, Selene

    2012-02-01

    Potato virus X coat protein is necessary for both cell-to-cell and phloem transfer, but it has not been clarified definitively whether it is needed in both movement phases solely as a component of the assembled particles or also of differently structured ribonucleoprotein complexes. To clarify this issue, we studied the infection progression of a mutant carrying an N-terminal deletion of the coat protein, which was used to construct chimeric virus particles displaying peptides selectively affecting phloem transfer or cell-to-cell movement. Nicotiana benthamiana plants inoculated with expression vectors encoding the wild-type, mutant and chimeric viral genomes were examined by microscopy techniques. These experiments showed that coat protein-peptide fusions promoting cell-to-cell transfer only were not competent for virion assembly, whereas long-distance movement was possible only for coat proteins compatible with virus particle formation. Moreover, the ability of the assembled PVX to enter and persist into developing xylem elements was revealed here for the first time.

  10. Structure-Activity Relationship and Signaling of New Chimeric CXCR4 Agonists.

    Science.gov (United States)

    Mona, Christine E; Besserer-Offroy, Élie; Cabana, Jérôme; Lefrançois, Marilou; Boulais, Philip E; Lefebvre, Marie-Reine; Leduc, Richard; Lavigne, Pierre; Heveker, Nikolaus; Marsault, Éric; Escher, Emanuel

    2016-08-25

    The CXCR4 receptor binds with meaningful affinities only CXCL12 and synthetic antagonists/inverse agonists. We recently described high affinity synthetic agonists for this chemokine receptor, obtained by grafting the CXCL12 N-terminus onto the inverse agonist T140. While those chimeric molecules behave as agonists for CXCR4, their binding and activation mode are unknown. The present SAR of those CXCL12-oligopeptide grafts reveals the key determinants involved in CXCR4 activation. Position 3 (Val) controls affinity, whereas position 7 (Tyr) acts as an efficacy switch. Chimeric molecules bearing aromatic residues in position 3 possess high binding affinities for CXCR4 and are Gαi full agonists with robust chemotactic properties. Fine-tuning of electron-poor aromatic rings in position 7 enhances receptor activation. To rationalize these results, a homology model of a receptor-ligand complex was built using the published crystal structures of CXCR4. Molecular dynamics simulations reveal further details accounting for the observed SAR for this series.

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

    Science.gov (United States)

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

    2008-02-01

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

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

    Science.gov (United States)

    Work, Thierry M.; Forsman, Zac H.; Szabo, Zoltan; Lewis, Teresa D.; Aeby, Greta S.; Toonen, Robert J.

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Thierry M Work

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

  14. Going viral: chimeric antigen receptor T-cell therapy for hematological malignancies.

    Science.gov (United States)

    Gill, Saar; June, Carl H

    2015-01-01

    On July 1, 2014, the United States Food and Drug Administration granted 'breakthrough therapy' designation to CTL019, the anti-CD19 chimeric antigen receptor T-cell therapy developed at the University of Pennsylvania. This is the first personalized cellular therapy for cancer to be so designated and occurred 25 years after the first publication describing genetic redirection of T cells to a surface antigen of choice. The peer-reviewed literature currently contains the outcomes of more than 100 patients treated on clinical trials of anti-CD19 redirected T cells, and preliminary results on many more patients have been presented. At last count almost 30 clinical trials targeting CD19 were actively recruiting patients in North America, Europe, and Asia. Patients with high-risk B-cell malignancies therefore represent the first beneficiaries of an exciting and potent new treatment modality that harnesses the power of the immune system as never before. A handful of trials are targeting non-CD19 hematological and solid malignancies and represent the vanguard of enormous preclinical efforts to develop CAR T-cell therapy beyond B-cell malignancies. In this review, we explain the concept of chimeric antigen receptor gene-modified T cells, describe the extant results in hematologic malignancies, and share our outlook on where this modality is likely to head in the near future.

  15. HLA Chimerism in allogenic haplo-identical peripheral blood stem cell transplant

    Directory of Open Access Journals (Sweden)

    Chhaya Sonal

    2004-01-01

    Full Text Available HLA antigens were used as markers to establish the presence of chimerism (i.e. simultaneous presence of two lymphocyte populations from recipient as well as donor in a patient with chronic granulomatous disease treated with one haplotype matched stem cell transplant. Neutrophil engraftment occurred on Day 6 post peripheral blood stem cell transplant (PBSCT. Platelet counts were maintained above 20x10[9]/L. Six months after the allogenic PBSCT, lymphocyte population was chimeric and cells of both donor (father and host HLA type were present. The patient revealed a shift in his HLA antigen profile and there was evidence of donor cell engraftment. The HLA phenotype A26,CwXX,B8,DRB1FNx0103//A32,Cw4,B35,DRB1FNx0116// represented his true phenotype whereas A11,Cw7,B62,DRB1FNx0114 represented donor (father origin.. HLA system as a genetic marker is a useful additional approach to determine engraftment following an allogenic haplo-identical stem cell transplantation.

  16. Formation of chimeric genes by copy-number variation as a mutational mechanism in schizophrenia.

    Science.gov (United States)

    Rippey, Caitlin; Walsh, Tom; Gulsuner, Suleyman; Brodsky, Matt; Nord, Alex S; Gasperini, Molly; Pierce, Sarah; Spurrell, Cailyn; Coe, Bradley P; Krumm, Niklas; Lee, Ming K; Sebat, Jonathan; McClellan, Jon M; King, Mary-Claire

    2013-10-03

    Chimeric genes can be caused by structural genomic rearrangements that fuse together portions of two different genes to create a novel gene. We hypothesize that brain-expressed chimeras may contribute to schizophrenia. Individuals with schizophrenia and control individuals were screened genome wide for copy-number variants (CNVs) that disrupted two genes on the same DNA strand. Candidate events were filtered for predicted brain expression and for frequency genes in localization, regulation, or function. Subcellular localizations of DNAJA2-NETO2 and MAP3K3-DDX42 differed from their parent genes. On the basis of the expression profile of the MATK promoter, MATK-ZFR2 is likely to be far more highly expressed in the brain during development than the ZFR2 parent gene. MATK-ZFR2 includes a ZFR2-derived isoform that we demonstrate localizes preferentially to neuronal dendritic branch sites. These results suggest that the formation of chimeric genes is a mechanism by which CNVs contribute to schizophrenia and that, by interfering with parent gene function, chimeras may disrupt critical brain processes, including neurogenesis, neuronal differentiation, and dendritic arborization.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Huang Qiman; Yang Qunying; Elisabeth Martinez; Guo Sandui

    2000-01-01

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

  19. Heparin-Induced Thrombocytopenia Antibody Test

    Science.gov (United States)

    ... Global Sites Search Help? Heparin-induced Thrombocytopenia PF4 Antibody Share this page: Was this page helpful? Also known as: Heparin-PF4 Antibody; HIT Antibody; HIT PF4 Antibody; Heparin Induced Antibody; ...

  20. The imaging and the fractal metrology of chimeric liposomal Drug Delivery nano Systems: the role of macromolecular architecture of polymeric guest.

    Science.gov (United States)

    Pippa, Natassa; Pispas, Stergios; Demetzos, Costas

    2014-09-01

    The major advance of mixed liposomes (the so-called chimeric systems) is to control the size, structure, and morphology of these nanoassemblies, and therefore, system colloidal properties, with the aid of a large variety of parameters, such as chemical architecture and composition. The goal of this study is to investigate the alterations of the physicochemical and morphological characteristics of chimeric dipalmitoylphosphatidylcholine (DPPC) liposomes, caused by the incorporation of block and gradient copolymers (different macromolecular architecture) with different chemical compositions (different amounts of hydrophobic component). Light scattering techniques were utilized in order to characterize physicochemically and to delineate the fractal morphology of chimeric liposomes. In this study, we also investigated the structural differences between the prepared chimeric liposomes as are visualized by scanning electron microscopy (SEM). It could be concluded that all the chimeric liposomes have regular structure, as SEM images revealed, while their fractal dimensionality was found to be dependent on the macromolecular architecture of the polymeric guest.

  1. Engineering antibodies for cancer therapy.

    Science.gov (United States)

    Boder, Eric T; Jiang, Wei

    2011-01-01

    The advent of modern antibody engineering has led to numerous successes in the application of these proteins for cancer therapy in the 13 years since the first Food and Drug Administration approval, which has stimulated active interest in developing more and better drugs based on these molecules. A wide range of tools for discovering and engineering antibodies has been brought to bear on this challenge in the past two decades. Here, we summarize mechanisms of monoclonal antibody therapeutic activity, challenges to effective antibody-based treatment, existing technologies for antibody engineering, and current concepts for engineering new antibody formats and antibody alternatives as next generation biopharmaceuticals for cancer treatment.

  2. Affinity Maturation of Monoclonal Antibody 1E11 by Targeted Randomization in CDR3 Regions Optimizes Therapeutic Antibody Targeting of HER2-Positive Gastric Cancer.

    Science.gov (United States)

    Ko, Bong-Kook; Choi, Soyoung; Cui, Lei Guang; Lee, Young-Ha; Hwang, In-Sik; Kim, Kyu-Tae; Shim, Hyunbo; Lee, Jong-Seo

    2015-01-01

    Anti-HER2 murine monoclonal antibody 1E11 has strong and synergistic anti-tumor activity in HER2-overexpressing gastric cancer cells when used in combination with trastuzumab. We presently optimized this antibody for human therapeutics. First, the complementarity determining regions (CDRs) of the murine antibody were grafted onto human germline immunoglobulin variable genes. No difference in affinity and biological activity was observed between chimeric 1E11 (ch1E11) and humanized 1E11 (hz1E11). Next, affinity maturation of hz1E11 was performed by the randomization of CDR-L3 and H3 residues followed by stringent biopanning selection. Milder selection pressure favored the selection of more diverse clones, whereas higher selection stringency resulted in the convergence of the panning output to a smaller number of clones with improved affinity. Clone 1A12 had four amino acid substitutions in CDR-L3, and showed a 10-fold increase in affinity compared to the parental clone and increased potency in an in vitro anti-proliferative activity assay with HER2-overepxressing gastric cancer cells. Clone 1A12 inhibited tumor growth of NCI-N87 xenograft model with similar efficacy to trastuzumab alone, and the combination treatment of 1A12 and trastuzumab completely removed the established tumors. These results suggest that humanized and affinity matured monoclonal antibody 1A12 is a highly optimized molecule for future therapeutic development against HER2-positive tumors.

  3. Protective Immunity Induced by a Chimeric Porcine Circovirus in Piglets%嵌合猪圆环病毒对仔猪的免疫保护研究

    Institute of Scientific and Technical Information of China (English)

    邵小雪; 谢俊岭; 高峰; 周双海; 高洁; 王婷; 谭伟

    2012-01-01

    为分析嵌合猪圆环病毒(PCV 1-2)对仔猪的免疫效力,检测了PCV1-2免疫诱导仔猪产生抗猪圆环病毒Ⅱ型(PCV2)感染的保护性免疫.将10头28日龄健康普通仔猪随机平均分成两组,一组肌注免疫106.05 TCID50 PCV 1-2作为免疫组;另一组不予免疫作为对照.到免疫后28天,免疫组仔猪血清中PCV2ORF2蛋白抗体全部转为阳性.在免疫后28天,所有仔猪经口鼻予以106.95 TCID50 PCV2攻击.攻毒后,免疫组仔猪全部保持临床健康,而对照组仔猪全部出现了PCV2疾病的临床症状;免疫组血清和淋巴组织中PCV2 DNA载量均显著(P<0.05)减少;对照组仔猪淋巴组织普遍出现中度到重度的淋巴细胞减少与组织细胞浸润,而免疫组没有.研究数据表明:PCV1-2免疫诱发了仔猪对PCV2感染的保护性免疫,有望成为候选PCV2弱毒疫苗.%To explore the immunity efficacy of a chimeric porcine circovirus (PCV1-2) to piglets, the protective immunity induced by the PCV1-2 against porcine circovirus type 2 (PCV2) infections in piglets were investigated. A total of 10 healthy conventional piglets were randomly and equally assigned to two groups of 5 piglets each. Piglets in group 1 were vaccinated by intramuscular injection with 106.05 50% tissue culture infective doses (TCID50) of the chimeric PCV1-2 live virus. Piglets in group 2 were not vaccinated and served as controls. By 28 days post-vaccination (DPV), all piglets in group 1 had seroconverted to PCV2 ORF2 antibody. At 28 DPV, all piglets were challenged orally and intranasally with 50625 TCID50 wild pathogenic PCV2 viruses. All vaccinated piglets remained clinically healthy after challenge, while unvaccinated animals developed clinical signs relative to PCV2 diseases. The PCV2 DNA loads in sera and lymphoid tissues were significantly (P<0.05) reduced in vaccinated piglets. Moderate to severe lymphoid depletion and histiocytic replacement were detected in most lymphoid tissues of unvaccinated

  4. Natural and Man-made Antibody Repertories for Antibody Discovery

    Directory of Open Access Journals (Sweden)

    Juan C eAlmagro

    2012-11-01

    Full Text Available Antibodies are the fastest-growing segment of the biologics market. The success of antibody-based drugs resides in their exquisite specificity, high potency, stability, solubility, safety and relatively inexpensive manufacturing process in comparison with other biologics. We outline here the structural studies and fundamental principles that define how antibodies interact with diverse targets. We also describe the antibody repertoires and affinity maturation mechanisms of human, mice and chickens, plus the use of novel single-domain antibodies in camelids and sharks. These species all utilize diverse evolutionary solutions to generate specific and high affinity antibodies and illustrate the plasticity of natural antibody repertoires. In addition, we discuss the multiple variations of man-made antibody repertoires designed and validated in the last two decades, which have served as tools to explore how the size, diversity and composition of a repertoire impact the antibody discovery process.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Danielle R. Lucon

    2006-01-01

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

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

    Science.gov (United States)

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

    2015-06-01

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

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

    NARCIS (Netherlands)

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

    2009-01-01

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

  9. Immune response and protective profile elicited by a multi-epitope chimeric protein derived from Leptospira interrogans

    Directory of Open Access Journals (Sweden)

    Luis G.V. Fernandes

    2017-04-01

    Conclusions: Although a complete characterization of the immune response elicited by rChi/adjuvant in hamsters is required, it is believed that the construction of chimeric genes is an important attempt towards the generation of an effective vaccine against leptospirosis.

  10. Chimeric virus-like particles for the delivery of an inserted conserved influenza A-specific CTL epitope.

    Science.gov (United States)

    Cheong, Wan-Shoo; Reiseger, Jessica; Turner, Stephen John; Boyd, Richard; Netter, Hans-Jürgen

    2009-02-01

    The small hepatitis B virus surface antigens (HBsAg-S) have the ability to self-assemble with host-derived lipids into empty non-infectious virus-like particles (VLPs). HBsAg-S VLPs are the sole component of the licensed hepatitis B vaccine, and they are a useful delivery platform for foreign epitopes. To develop VLPs capable of transporting foreign cytotoxic T lymphocyte (CTL) epitopes, HBsAg-S specific CTL epitopes at various sites were substituted with a conserved CTL epitope derived from the influenza matrix protein. Depending on the insertion site, the introduction of the MHC class I A2.1-restricted influenza epitope was compatible with the secretion competence of HBsAg-S indicating that chimeric VLPs were assembled. Immunizations of transgenic HHDII mice with chimeric VLPs induced anti-influenza CTL responses proving that the inserted foreign epitope can be correctly processed and cross-presented. Chimeric VLPs in the absence of adjuvant were able to induce memory T cell responses, which could be recalled by influenza virus infections in the mouse model system. The ability of chimeric HBsAg-S VLPs to induce anti-foreign CTL responses and also with the proven ability to induce humoral immune responses constitute a highly versatile platform for the delivery of selected multiple epitopes to target disease associated infectious agents.

  11. [An analysis of chimeric mice obtained by the injection of the inner cell mass into the blastocyst].

    Science.gov (United States)

    Mitalipov, Sh M; Fedorov, L M; Strel'chenko, N S

    1993-01-01

    Mouse chimeras were produced using injections of ICM cells into blastocysts. Chimerism of resulting animals was determined by their coat color and spectrum of glucosephosphate isomerase isoenzymes. The use of modifications of the injection method for solving different genetic and embryological problems is discussed.

  12. Induction of partial protection against infection with Toxoplasma gondii genotype II by DNA vaccination with recombinant chimeric tachyzoite antigens

    DEFF Research Database (Denmark)

    Rosenberg, Carina Agerbo; De Craeye, S.; Jongert, E.

    2009-01-01

    complications. Although several strategies have been suggested for making a vaccine, none is currently available. Here, we investigate the protection conferred by DNA vaccination with two constructs, pcEC2 (MIC2-MIC3-SAG1) and pcEC3 (GRA3-GRA7-M2AP), encoding chimeric proteins containing multiple antigenic...

  13. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    surface expression of various antibody formats in the generated knockout strain. Functional scFv and scFab fragments were efficiently displayed on yeast whereas impaired chain assembly and heavy chain degradation was observed for display of full-length IgG molecules. To identify the optimal polypeptide...... linker for yeast surface display of scFv and scFab fragments, we compared a series of different Gly-Ser-based linkers in display and antigen binding proficiency. We show that these formats of the model antibody can accommodate linkers of different lengths and that introduction of alanine or glutamate...... fragments by in vivo homologous recombination large combinatorial antibody libraries can easily be generated. We have optimized ordered assembly of three CDR fragments into a gapped vector and observed increased transformation efficiency in a yeast strain carrying a deletion of the SGS1 helicase...

  14. Antibody informatics for drug discovery

    DEFF Research Database (Denmark)

    Shirai, Hiroki; Prades, Catherine; Vita, Randi;

    2014-01-01

    to the antibody science in every project in antibody drug discovery. Recent experimental technologies allow for the rapid generation of large-scale data on antibody sequences, affinity, potency, structures, and biological functions; this should accelerate drug discovery research. Therefore, a robust bioinformatic...... infrastructure for these large data sets has become necessary. In this article, we first identify and discuss the typical obstacles faced during the antibody drug discovery process. We then summarize the current status of three sub-fields of antibody informatics as follows: (i) recent progress in technologies...... for antibody rational design using computational approaches to affinity and stability improvement, as well as ab-initio and homology-based antibody modeling; (ii) resources for antibody sequences, structures, and immune epitopes and open drug discovery resources for development of antibody drugs; and (iii...

  15. Adoptive immunotherapy to increase the level of donor hematopoietic chimerism after nonmyeloablative marrow transplantation for severe canine hereditary hemolytic anemia.

    Science.gov (United States)

    Takatu, Alessandra; Nash, Richard A; Zaucha, Jan M; Little, Marie-Terese; Georges, George E; Sale, George E; Zellmer, Eustacia; Kuhr, Christian S; Lothrop, Clinton D; Storb, Rainer

    2003-11-01

    Severe hemolytic anemia in Basenji dogs secondary to pyruvate kinase deficiency can be corrected by allogeneic hematopoietic cell transplantation (HCT) from littermates with normal hematopoiesis after conventional myeloablative or nonmyeloablative conditioning regimens. If the levels of donor chimerism were low (after nonmyeloablative HCT, there was only partial correction of the hemolytic anemia. We next addressed whether allogeneic cell therapy after nonmyeloablative HCT would convert mixed to full hematopoietic chimerism, achieve sustained remission from hemolysis, and prevent progression of marrow fibrosis and liver cirrhosis. Three pyruvate kinase-deficient dogs were given HCT from their respective dog leukocyte antigen-identical littermates after nonmyeloablative conditioning with 200 cGy of total body irradiation. Postgrafting immunosuppression consisted of mycophenolate mofetil and cyclosporine. All 3 dogs engrafted and had mixed hematopoietic chimerism with donor levels ranging from 12% to 55% in bone marrow. In 2 of the 3 dogs, there were decreases in the levels of donor chimerism so that at 25 weeks after nonmyeloablative HCT, hemolysis recurred that was associated with increased reticulocyte counts. All 3 dogs then had 2 serial infusions of donor lymphocytes (DLI) from their respective donors at least 20 weeks apart to convert from mixed to full donor chimerism. Both dogs with recurrence of hemolytic anemia after nonmyeloablative HCT achieved higher levels of donor chimerism, with donor contributions ranging from 47% to 62% in the bone marrow and 50% to 69% and 16% to 25% in the granulocyte and mononuclear cell fractions of the peripheral blood, respectively, and with remission of the hemolytic anemia. One dog responded after the first DLI, and 5 weeks after the second DLI, the other dog converted to full donor chimerism. At last follow-up, all these dogs showed clinical improvement, as determined by increasing hematocrits and normal reticulocyte counts

  16. Antithyroglobulin Antibodies and Antimicrosomal Antibodies in Various Thyroid Diseases

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Gwon Jun; Hong, Key Sak; Choi, Kang Won; Lee, Kyu; Koh, Chang Soon; Lee, Mun Ho; Park, Sung Hoe; Chi, Je Geun; Lee, Sang Kook [Seoul National University College of Medicine, Seoul (Korea, Republic of)

    1979-03-15

    The authors investigated the incidence of antithyroglobulin antibodies and antibodies and antimicrosomal antibodies measured by tanned red cell hemagglutination method in subjects suffering from various thyroid disorders. 1) In 15 normal patients, neither suffering from any thyroid diseases nor from any other autoimmune disorders, the antithyroglobulin antibodies were all negative, but the antimicrosomal antibody was positive only in one patient (6.7%). 2) The antithyroglobulin antibodies were positive in 31.5% (34 patients) of 108 patients with various thyroid diseases, and the antimicrosomal antibodies were positive in 37.0% (40 patients). 3) of the 25 patients with Graves' diseases, 7 patients (28.0%) showed positive for the antithyroglobulin antibodies, and 9 (36.0%) for the antimicrosomal antibodies. There was no definite differences in clinical and thyroid functions between the groups with positive and negative results. 4) Both antibodies were positive in 16 (88.9%) and 17 (94.4%) patients respectively among 18 patients with Hashimoto's thyroiditis, all of them were diagnosed histologically. 5) Three out of 33 patients with thyroid adenoma showed positive antibodies, and 3 of 16 patients with thyroid carcinoma revealed positive antibodies. 6) TRCH antibodies demonstrated negative results in 2 patients with subacute thyroiditis, but positive in one patient with idiopathic primary myxedema. 7) The number of patients with high titers(>l:802) was 16 for antithyroglobulin antibody, and 62.5% (10 patients) of which was Hashimoto's thyroiditis. Thirteen (65.0) of 20 patients with high titers (>l:802) for antimicrosomal antibody was Hashimoto's thyroiditis. TRCH test is a simple, sensitive method, and has high reliability and reproducibility. The incidences and titers of antithyroglobulin antibody and antimicrosomal antibody are especially high in Hashimoto's thyroiditis.

  17. Prediction of Antibody Epitopes

    DEFF Research Database (Denmark)

    Nielsen, Morten; Marcatili, Paolo

    2015-01-01

    Antibodies recognize their cognate antigens in a precise and effective way. In order to do so, they target regions of the antigenic molecules that have specific features such as large exposed areas, presence of charged or polar atoms, specific secondary structure elements, and lack of similarity...... to self-proteins. Given the sequence or the structure of a protein of interest, several methods exploit such features to predict the residues that are more likely to be recognized by an immunoglobulin.Here, we present two methods (BepiPred and DiscoTope) to predict linear and discontinuous antibody...

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

    Science.gov (United States)

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

    2012-12-01

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

  19. Insight into Substrate Preference of Two Chimeric Esterases by Combining Experiment and Molecular Simulation

    Institute of Scientific and Technical Information of China (English)

    ZHOU Xiao-li; HAN Wei-wei; ZHENG Bai-song; FENG Yan

    2013-01-01

    Better understanding of the relationship between the substrate preference and structural module of esterases is helpful to novel enzyme development.For this purpose,two chimeric esterases AAM7 and PAR,constructed via domain swapping between two ancient thermophilic esterases,were investigated on their molecular simulation(including homology modeling,substrates docking and substrate binding affinity validation) and enzymatic assay(specific activities and activation energies calculating).Our results indicate that the factors contributing to the substrate preference of many enzymes especially the broad-specificity enzymes like esterases are multiple and complicated,the substrate binding domains or binding pockets are important but not the only factor for substrate preference.

  20. Reducing ulcerogenic potential of biphenyl acetic acid: Design and development of chimeric derivatives with amino acids

    Directory of Open Access Journals (Sweden)

    Suneela Dhaneshwar

    2016-09-01

    Full Text Available In an attempt to minimize the ulcerogenic potential and associated gastro-intestinal toxicity of bioprecursor fenbufen and its active metabolite biphenyl acetic acid, carrier-linked chimeric derivatives of the latter were designed and synthesized using amino acids as the promoities. DCC coupling method was used for the synthesis of these amides. The chimeras were characterized by IR and 1H NMR. Pharmacological investigations were carried out in animal models for analgesic, anti-inflammatory, anti-arthritic and ulcerogenic activities. The chimeras exhibited high gastro-sparing effect; quick onset and longer duration of analgesia; enhanced/prolonged anti-inflammatory activity and better anti-arthritic effect than fenbufen or biphenyl acetic acid. These derivatives could be useful as a chronotherapy for rheumatoid arthritis due to their prolonged analgesic and anti-inflammatory effects.

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

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

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

  2. 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

    The potential of the major structural protein of type 1 fimbriae as a display system for heterologous sequences was tested. As a reporter-epitope, a heterologous sequence mimicking a neutralizing epitope of the cholera toxin B chain was inserted, in one or two copies, into four different positions...... in the fimA gene. This was carried out by introduction of new restriction sites by PCR-mediated site-directed mutagenesis of fimA in positions predicted to correspond to optimally surface-located regions of the subunit protein. Subsequently, the synthetic cholera-toxin-encoding DNA segment was inserted...... 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...

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

    Directory of Open Access Journals (Sweden)

    Pinar Ataca

    2015-12-01

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

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

    Science.gov (United States)

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

    2015-10-16

    There is growing interest in using engineered cells as therapeutic agents. For example, synthetic chimeric antigen receptors (CARs) can redirect T cells to recognize and eliminate tumor cells expressing specific antigens. Despite promising clinical results, these engineered T cells can exhibit excessive activity that is difficult to control and can cause severe toxicity. We designed "ON-switch" CARs that enable small-molecule control over T cell therapeutic functions while still retaining antigen specificity. In these split receptors, antigen-binding and intracellular signaling components assemble only in the presence of a heterodimerizing small molecule. This titratable pharmacologic regulation could allow physicians to precisely control the timing, location, and dosage of T cell activity, thereby mitigating toxicity. This work illustrates the potential of combining cellular engineering with orthogonal chemical tools to yield safer therapeutic cells that tightly integrate cell-autonomous recognition and user control.

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

    Science.gov (United States)

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

    2009-12-01

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

  6. 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.

  7. Chikungunya, Influenza, Nipah, and Semliki Forest Chimeric Viruses with Vesicular Stomatitis Virus: Actions in the Brain.

    Science.gov (United States)

    van den Pol, Anthony N; Mao, Guochao; Chattopadhyay, Anasuya; Rose, John K; Davis, John N

    2017-03-15

    Recombinant vesicular stomatitis virus (VSV)-based chimeric viruses that include genes from other viruses show promise as vaccines and oncolytic viruses. However, the critical safety concern is the neurotropic nature conveyed by the VSV glycoprotein. VSVs that include the VSV glycoprotein (G) gene, even in most recombinant attenuated strains, can still show substantial adverse or lethal actions in the brain. Here, we test 4 chimeric viruses in the brain, including those in which glycoprotein genes from Nipah, chikungunya (CHIKV), and influenza H5N1 viruses were substituted for the VSV glycoprotein gene. We also test a virus-like vesicle (VLV) in which the VSV glycoprotein gene is expressed from a replicon encoding the nonstructural proteins of Semliki Forest virus. VSVΔG-CHIKV, VSVΔG-H5N1, and VLV were all safe in the adult mouse brain, as were VSVΔG viruses expressing either the Nipah F or G glycoprotein. In contrast, a complementing pair of VSVΔG viruses expressing Nipah G and F glycoproteins were lethal within the brain within a surprisingly short time frame of 2 days. Intranasal inoculation in postnatal day 14 mice with VSVΔG-CHIKV or VLV evoked no adverse response, whereas VSVΔG-H5N1 by this route was lethal in most mice. A key immune mechanism underlying the safety of VSVΔG-CHIKV, VSVΔG-H5N1, and VLV in the adult brain was the type I interferon response; all three viruses were lethal in the brains of adult mice lacking the interferon receptor, suggesting that the viruses can infect and replicate and spread in brain cells if not blocked by interferon-stimulated genes within the brain.IMPORTANCE Vesicular stomatitis virus (VSV) shows considerable promise both as a vaccine vector and as an oncolytic virus. The greatest limitation of VSV is that it is highly neurotropic and can be lethal within the brain. The neurotropism can be mostly attributed to the VSV G glycoprotein. Here, we test 4 chimeric viruses of VSV with glycoprotein genes from Nipah

  8. Compositions, antibodies, asthma diagnosis methods, and methods for preparing antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hongjun; Zangar, Richard C.

    2017-01-17

    Methods for preparing an antibody are provided with the method including incorporating 3-bromo-4-hydroxy-benzoic acid into a protein to form an antigen, immunizing a mammalian host with the antigen, and recovering an antibody having an affinity for the antigen from the host. Antibodies having a binding affinity for a monohalotyrosine are provided as well as composition comprising an antibody bound with monohalotyrosine. Compositions comprising a protein having a 3-bromo-4-hydroxy-benzoic acid moiety are also provided. Methods for evaluating the severity of asthma are provide with the methods including analyzing sputum of a patient using an antibody having a binding affinity for monohalotyrosine, and measuring the amount of antibody bound to protein. Methods for determining eosinophil activity in bodily fluid are also provided with the methods including exposing bodily fluid to an antibody having a binding affinity for monohalotyrosine, and measuring the amount of bound antibody to determine the eosinophil activity.

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

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

    Science.gov (United States)

    Stromatas, Stavros

    2010-05-01

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

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

    Science.gov (United States)

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

    2016-01-01

    Background: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA (cfDNA) is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control (EDC) is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for monitoring stability of cfDNA concentration in blood samples over time. Methods: A0 DNA fragment of 167 bp chimeric sequence of parvovirus B19 and pBHA designated as EDC fragment was designed. To determine the impact of different factors during DNA extraction processing on quantification of cfDNA, blood samples were collected from normal subjects and divided into aliquots with and without specific treatment. In time intervals, the plasma samples were isolated. The amplicon of 167 bp EDC fragment in final concentration of 1.1 pg/500 μl was added to each plasma sample and total DNA was extracted by an in house method. Relative and absolute quantification real time PCR was performed to quantify both EDC fragment and cfDNA in extracted samples. Results: Comparison of real time PCR threshold cycle (Ct) for cfDNA fragment in tubes with and without specific treatment indicated a decrease in untreated tubes. In contrast, the threshold cycle was constant for EDC fragment in treated and untreated tubes, indicating the difference in Ct values of the cfDNA is because of specific treatments that were made on them. Conclusions: Spiking of DNA fragment size relevant to cfDNA into the plasma sample can be useful to minimize the bias due to sample preparation and extraction processing. Therefore, it is highly recommended that standard external DNA control be employed for the extraction and quantification of cfDNA for accurate data analysis. PMID:27141267

  12. Repeated evolution of chimeric fusion genes in the β-globin gene family of laurasiatherian mammals.

    Science.gov (United States)

    Gaudry, Michael J; Storz, Jay F; Butts, Gary Tyler; Campbell, Kevin L; Hoffmann, Federico G

    2014-05-09

    The evolutionary fate of chimeric fusion genes may be strongly influenced by their recombinational mode of origin and the nature of functional divergence between the parental genes. In the β-globin gene family of placental mammals, the two postnatally expressed δ- and β-globin genes (HBD and HBB, respectively) have a propensity for recombinational exchange via gene conversion and unequal crossing-over. In the latter case, there are good reasons to expect differences in retention rates for the reciprocal HBB/HBD and HBD/HBB fusion genes due to thalassemia pathologies associated with the HBD/HBB "Lepore" deletion mutant in humans. Here, we report a comparative genomic analysis of the mammalian β-globin gene cluster, which revealed that chimeric HBB/HBD fusion genes originated independently in four separate lineages of laurasiatherian mammals: Eulipotyphlans (shrews, moles, and hedgehogs), carnivores, microchiropteran bats, and cetaceans. In cases where an independently derived "anti-Lepore" duplication mutant has become fixed, the parental HBD and/or HBB genes have typically been inactivated or deleted, so that the newly created HBB/HBD fusion gene is primarily responsible for synthesizing the β-type subunits of adult and fetal hemoglobin (Hb). Contrary to conventional wisdom that the HBD gene is a vestigial relict that is typically inactivated or expressed at negligible levels, we show that HBD-like genes often encode a substantial fraction (20-100%) of β-chain Hbs in laurasiatherian taxa. Our results indicate that the ascendancy or resuscitation of genes with HBD-like coding sequence requires the secondary acquisition of HBB-like promoter sequence via unequal crossing-over or interparalog gene conversion.

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

    Directory of Open Access Journals (Sweden)

    Ye Xiaoting

    2012-09-01

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

  14. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    Energy Technology Data Exchange (ETDEWEB)

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Luque, Daniel; Terrón, María C. [Unidad de Microscopía Electrónica y Confocal, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Calder, Lesley J. [National Institute for Medical Research, MRC, Mill Hill, London NW7 1AA (United Kingdom); Melero, José A., E-mail: jmelero@isciii.es [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain)

    2014-07-15

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.

  15. Human germline antibody gene segments encode polyspecific antibodies.

    Science.gov (United States)

    Willis, Jordan R; Briney, Bryan S; DeLuca, Samuel L; Crowe, James E; Meiler, Jens

    2013-04-01

    Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.

  16. Prediction of antibody persistency from antibody titres to natalizumab

    DEFF Research Database (Denmark)

    Jensen, Poul Erik H; Koch-Henriksen, Nils; Sellebjerg, Finn Thorup;

    2012-01-01

    In a subgroup of patients with multiple sclerosis natalizumab therapy causes generation of anti-natalizumab antibodies that may be transient or persistent. It is recommended to discontinue natalizumab therapy in persistently antibody-positive patients.......In a subgroup of patients with multiple sclerosis natalizumab therapy causes generation of anti-natalizumab antibodies that may be transient or persistent. It is recommended to discontinue natalizumab therapy in persistently antibody-positive patients....

  17. Monoclonal antibodies in myeloma

    DEFF Research Database (Denmark)

    Sondergeld, P.; van de Donk, N. W. C. J.; Richardson, P. G.;

    2015-01-01

    The development of monoclonal antibodies (mAbs) for the treatment of disease goes back to the vision of Paul Ehrlich in the late 19th century; however, the first successful treatment with a mAb was not until 1982, in a lymphoma patient. In multiple myeloma, mAbs are a very recent and exciting add...

  18. Antibody Blood Tests

    Science.gov (United States)

    ... What do I do if I have a negative blood test (or panel) but I’m still having symptoms? While it is rare, it is possible for patients to have a negative antibody test results and still have celiac disease. ...

  19. RBC Antibody Screen

    Science.gov (United States)

    ... test also may be used to help diagnose autoimmune-related hemolytic anemia in conjunction with a DAT. This condition may be caused when a person produces antibodies against his or her own RBC antigens. This can happen with some autoimmune disorders , such as lupus , with diseases such as ...

  20. Improved humoral and cellular immune responses against the gp120 V3 loop of HIV-1 following genetic immunization with a chimeric DNA vaccine encoding the V3 inserted into the hepatitis B surface antigen

    DEFF Research Database (Denmark)

    Fomsgaard, A; Nielsen, H V; Bryder, K

    1998-01-01

    with the HIV MN gp160 envelope plasmid induced a slow and low titred anti-MN V3 antibody response at 12 weeks post-inoculation (p.i.) and a late appearing (7 weeks), weak and variable CTL response. In contrast, DNA vaccination with the HBsAg-encoding plasmid induced a rapid and high titred anti-HBsAg antibody...... response and a uniform strong anti-HBs CTL response already 1 week p.i. in all mice. DNA vaccination with the chimeric MN V3/HBsAg plasmid elicited humoral responses against both viruses within 3-6 weeks which peaked at 6-12 weeks and remained stable for at least 25 weeks. In addition, specific CTL......-2d-restricted cytotoxic T lymphocyte (CTL) epitope. In an attempt to improve the immunogenicity of V3 in DNA vaccines, a plasmid expressing MN V3 as a fusion protein with the highly immunogenic middle (pre-S2 + S) surface antigen of hepatitis B virus (HBsAg) was constructed. Epidermal inoculation...

  1. Improved humoral and cellular immune response against the gp120 V3 loop of HIV-1 following genetic immunization with a chimeric DNA vaccine encoding the V3 inserted into the hepatites B surface antigen

    DEFF Research Database (Denmark)

    Fomsgaard, A.; Nielsen, H.V.; Bryder, K.

    1998-01-01

    MN gp160 envelope plasmid induced a slow and low titred anti-MN V3 antibody response at 12 weeks post-inoculation (p.i.) and a late appearing (7 weeks), weak and variable CTL response. In contrast, DNA vaccination with the HBsAg-encoding plasmid induced a rapid and high titred anti-HBsAg antibody...... response and a uniform strong anti-HBs CTL response already 1 week p.i. in all mice. DNA vaccination with the chimeric MN V2/HBsAg plasmid elicited humoral responses against both viruses within 3-6 weeks which peaked at 6-12 weeks and remained stable for at least 25 weeks. In addition, specific CTL......-2d-restricted cytotoxic T lymphocyte (CTL) epitope. In an attempt to improve the immunogenicity of V3 in DNA vaccines, a plasmid expressing MN V3 as a fusion protein with the highly immunogenic middle (pre-S2+S) surface antigen of hepatitis B virus (HBsAg) was constructed. Epidermal inoculation...

  2. What Is Antiphospholipid Antibody Syndrome?

    Science.gov (United States)

    ... page from the NHLBI on Twitter. What Is Antiphospholipid Antibody Syndrome? Antiphospholipid (AN-te-fos-fo-LIP-id) antibody ... weeks or months. This condition is called catastrophic antiphospholipid syndrome (CAPS). People who have APS also are at ...

  3. Red Blood Cell Antibody Identification

    Science.gov (United States)

    ... ID, RBC; RBC Ab ID Formal name: Red Blood Cell Antibody Identification Related tests: Direct Antiglobulin Test ; RBC ... I should know? How is it used? Red blood cell (RBC) antibody identification is used as a follow- ...

  4. Lupus anticoagulants and antiphospholipid antibodies

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/000547.htm Lupus anticoagulants and antiphospholipid antibodies To use the sharing features on this page, please enable JavaScript. Lupus anticoagulants are antibodies against substances in the lining ...

  5. Anti-smooth muscle antibody

    Science.gov (United States)

    ... gov/ency/article/003531.htm Anti-smooth muscle antibody To use the sharing features on this page, please enable JavaScript. Anti-smooth muscle antibody is a blood test that detects the presence ...

  6. Antibody Engineering and Therapeutics Conference

    OpenAIRE

    Larrick, James W; Parren, Paul WHI; Huston, James S; Plückthun, Andreas; Bradbury, Andrew; Tomlinson, Ian M; Chester, Kerry A.; Burton, Dennis R.; Adams, Gregory P.; Weiner, Louis M.; Scott, Jamie K.; Alfenito, Mark R; Veldman, Trudi; Reichert, Janice M.

    2013-01-01

    The Antibody Engineering and Therapeutics conference, which serves as the annual meeting of The Antibody Society, will be held in Huntington Beach, CA from Sunday December 8 through Thursday December 12, 2013. The scientific program will cover the full spectrum of challenges in antibody research and development, and provide updates on recent progress in areas from basic science through approval of antibody therapeutics. Keynote presentations will be given by Leroy Hood (Institute of System Bi...

  7. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo

    2015-01-01

    The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides...... can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery...

  8. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    Zegers, N.D.

    1995-01-01

    Synthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps that lead to the uni

  9. Radioimmunotherapy in refractory b-cell nonhodgkins lymphoma with I-131-labeled chimeric anti cd-20 c2b8 (I-131 rituximab): preliminary result

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Hye Jin; Park, Yeon Hee; Kim, Sung Eun and others [Korea University Medical School, Seoul (Korea, Republic of)

    2005-07-01

    Recently, the native chimeric human-mouse anti CD-20 antibody IDEC-C2B8 (Rituximab) has been widely applied in NHL. This ongoing phase study was to evaluate whether radioimmunotherapy (RIT) with I-131 rituximab is effective in refractory B-cell NHL. Inclusion criteria were as follows: B-cell NHL with relapsed or refractory to primary standard therapy, measurable disease, adequate hematologic, renal, and hepatic function, informed consent. The rituximab (Mabthera, Roach) was radiolabeled with iodine-131(I-131) using a modified chloramine T method with high radiochemical purity (95%) and preservation of immuno-reactivity. All patients received loading doses of unlabeled rituximab (median, 40 mg: range, 20{approx}70 mg) immediately prior to administration of therapeutic dose (51.4{approx}152.2 MBq/kg), and then underwent gamma camera scan. 11 patients were enrolled (4 low-grade B-cell NHL, 7 DLBCL, median age 63 years). Patients had received a median of three prior chemotherapy regimens. The objective response rate was 36.4% (1 CR, 3 PRs). These all responses were observed in low-grade B-cell NHL, except one with DLBCL. Adverse events were primarily hematologic toxicities; the incidence of grade 3/4 neutropenia, thrombocytopenia, and anemia was 27.3%, 45.5%, and 18.2%, respectively. The treatment-related mortality was observed in one patient, who had been previously treated with high-dose chemotherapy plus TBI with autologous stem cell transplantation. RIT with I-131 rituximab seems to be effective tolerable in refractory low-grade B-cell NHL, although modest activity in refractory DLBCL. Further studies to define the efficacy of I-131 rituximab in DLBCL are warranted.

  10. A Chimeric Cetuximab-Functionalized Corona as a Potent Delivery System for Microtubule-Destabilizing Nanocomplexes to Hepatocellular Carcinoma Cells: A Focus on EGFR and Tubulin Intracellular Dynamics.

    Science.gov (United States)

    Poojari, Radhika; Kini, Sudarshan; Srivastava, Rohit; Panda, Dulal

    2015-11-01

    In this study, we have developed microtubule destabilizing agents combretastatin A4 (CA4) or 2-methoxyestradiol (2ME) encapsulated poly(d,l-lactide-co-glycolide)-b-poly(ethylene glycol) (PLGA-b-PEG) nanocomplexes for targeted delivery to human hepatocellular carcinoma (HCC) cells. An epidermal growth factor receptor (EGFR) is known to be overexpressed in HCC cells. Therefore, the targeting moiety cetuximab (Cet), an anti-EGFR chimeric monoclonal antibody, is functionalized on the surface of these diblock copolymeric coronas. Cetuximab is associated with the extracellular domain of the EGFR; therefore, the uptake of the cetuximab conjugated nanocomplexes occurred efficiently in EGFR overexpressing HCC cells indicating potent internalization of the complex. The cetuximab targeted-PLGA-b-PEG nanocomplexes encapsulating CA4 or 2ME strongly inhibited phospho-EGFR expression, depolymerized microtubules, produced spindle abnormalities, stalled mitosis, and induced apoptosis in Huh7 cells compared to the free drugs, CA4 or 2ME. Further, the combinatorial strategy of targeted nanocomplexes, Cet-PLGA-b-PEG-CA4 NP and Cet-PLGA-b-PEG-2ME NP, significantly reduced the migration of Huh7 cells, and markedly enhanced the anticancer effects of the microtubule-targeted drugs in Huh7 cells compared to the free drugs, CA4 or 2ME. The results indicated that EGFR receptor-mediated internalization via cetuximab facilitated enhanced uptake of the nanocomplexes leading to potent anticancer efficacy in Huh7 cells. Cetuximab-functionalized PLGA-b-PEG nanocomplexes possess a strong potential for the targeted delivery of CA4 or 2ME in EGFR overexpressed HCC cells, and the strategy may be useful for selectively targeting microtubules in these cells.

  11. Dendritic cells primed with a chimeric plasmid containing HIV-1-gag associated with lysosomal-associated protein-1 (LAMP/gag) is a potential therapeutic vaccine against HIV.

    Science.gov (United States)

    Lucas, Carolina G D O; Matassoli, Flavio L; Peçanha, Ligia M T; Santillo, Bruna Tereso; Oliveira, Luanda Mara da Silva; Oshiro, Telma Miyuki; Marques, Ernesto T D A; Oxenius, Annette; de Arruda, Luciana B

    2016-08-01

    The decline in number and function of T cells is a hallmark of HIV infection, and preservation or restoration of HIV-specific cellular immune response is a major goal of AIDS treatment. Dendritic cells (DCs) play a key role in the initiation and maintenance of the immune response, and their use as a vaccine vehicle is a promising strategy for enhancing vaccine efficacy. We evaluated the potential of DC-mediated immunization with a DNA vaccine consisting of HIV-1-p55gag (gag, group-specific antigen) associated to lysosomal associated protein (LAMP) sequence (LAMP/gag vaccine). Immunization of mice with mouse DCs transfected with LAMP/gag (Lg-mDCs) stimulated more potent B- and T-cell responses than naked DNA or DCs pulsed with inactivated HIV. Anti-Gag antibody levels were sustained for at least 3 mo after immunization, and recall T-cell responses were also strongly detected at this time point. Human DCs transfected with LAMP/gag (Lg-hDCs) were also activated and able to stimulate greater T-cell response than native gag-transfected DCs. Coculture between Lg-hDCs and T lymphocytes obtained from patients with HIV resulted in upregulation of CD38, CD69, HLA-DR, and granzyme B by CD4(+) and CD8(+) T cells, and increased IFN-γ and TNF-α production. These results indicate that the use of LAMP/gag-DC may be an efficient strategy for enhancing immune function in patients with HIV.-Lucas, C. G. D. O., Matassoli, F. L., Peçanha, L. M. T., Santillo, B. T., Oliveira, L. M. D. S., Oshiro, T. M., Marques, E. T. D. A., Jr., Oxenius, A., de Arruda, L. B. Dendritic cells primed with a chimeric plasmid containing HIV-1-gag associated with lysosomal-associated protein-1 (LAMP/gag) is a potential therapeutic vaccine against HIV.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

    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 5×106 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 humanCC 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

  13. Antiphospholipid Antibody and Antiphospholipid Syndrome

    Institute of Scientific and Technical Information of China (English)

    吴竞生

    2008-01-01

    @@ Antiphospholipid antibodies (APA) APA is a big category for all kinds of negative charge phospholipid or lecithin - a protein complex autoantibodies or the same antibody, through its recognition of antigen (target protein) different, and phospholipids or lecithin - protein complex combination of various rely on the interference Phospholipid clotting and anti-coagulation factor, and promote endothelial cells, platelets, complement activation and play a role. APA including lupus anticoagulant(LA) and anticardiolipin antibody (ACA), In addition, there are anti-β2 glycoprotein-I (β2-GPI) antibody, anti-prothrombin (a- PT) antibody, anti-lysophosphatidic acid antibody and anti-phosphatidylserine antibody, and so on. APA as the main target of phospholipid-binding protein, including β2-GPI, prothrombin, annexin, protein C (PC) and protein S (PS), plasminogen, and so on.

  14. Engineering antibodies by yeast display.

    Science.gov (United States)

    Boder, Eric T; Raeeszadeh-Sarmazdeh, Maryam; Price, J Vincent

    2012-10-15

    Since its first application to antibody engineering 15 years ago, yeast display technology has been developed into a highly potent tool for both affinity maturing lead molecules and isolating novel antibodies and antibody-like species. Robust approaches to the creation of diversity, construction of yeast libraries, and library screening or selection have been elaborated, improving the quality of engineered molecules and certainty of success in an antibody engineering campaign and positioning yeast display as one of the premier antibody engineering technologies currently in use. Here, we summarize the history of antibody engineering by yeast surface display, approaches used in its application, and a number of examples highlighting the utility of this method for antibody engineering.

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

    Science.gov (United States)

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

    2015-09-01

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

  16. Development and Characterization of a Humanized Anti-HER2 Antibody HuA21 with Potent Anti-Tumor Properties in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ruilin Li

    2016-04-01

    Full Text Available Human epidermal growth factor receptor 2 (HER2 is one of the most studied tumor-associated antigens for cancer immunotherapy. An engineered anti-HER-2 chimeric A21 antibody (chA21 is a chimeric antibody targeted to subdomain I of the HER2 extracellular domain. Here, we report the anti-tumor activity of the novel engineered monoclonal antibody humanized chA21 (HuA21 that targets HER2 on the basis of chA21, and we describe the underlying mechanisms. Our results reveal that HuA21 markedly inhibits the proliferation and migration of HER2-overexpressing breast cancer cells and causes enhanced antibody-dependent cell-mediated cytotoxicity potency against HER2-overexpressing tumor cells. In particular, HuA21, but not trastuzumab (Tra, markedly suppresses growth and enhances the internalization of the antibody in Tra-resistant BT-474 breast cancer cells. These characteristics are highly associated with the intrinsic ability of HuA21 to down-regulate HER2 activation and inhibit the extracellular signal-regulated kinase 1/2 (ERK1/2 and protein kinase B (Akt signaling pathways. Furthermore, the combination of HuA21 with Tra synergistically enhances the anti-tumor effects in vitro and in vivo and inhibits HER2 activation and the ERK1/2 and Akt signaling pathways. Altogether, our results suggest that HuA21 may represent a unique anti-HER2 antibody with potential as a therapeutic candidate alone or in combination with other anti-HER2 reagents in cancer therapy.

  17. Development and Characterization of a Humanized Anti-HER2 Antibody HuA21 with Potent Anti-Tumor Properties in Breast Cancer Cells.

    Science.gov (United States)

    Li, Ruilin; Hu, Siyi; Chang, Yan; Zhang, Zhihui; Zha, Zhao; Huang, Hui; Shen, Guodong; Liu, Jing; Song, Lihua; Wei, Wei

    2016-04-15

    Human epidermal growth factor receptor 2 (HER2) is one of the most studied tumor-associated antigens for cancer immunotherapy. An engineered anti-HER-2 chimeric A21 antibody (chA21) is a chimeric antibody targeted to subdomain I of the HER2 extracellular domain. Here, we report the anti-tumor activity of the novel engineered monoclonal antibody humanized chA21 (HuA21) that targets HER2 on the basis of chA21, and we describe the underlying mechanisms. Our results reveal that HuA21 markedly inhibits the proliferation and migration of HER2-overexpressing breast cancer cells and causes enhanced antibody-dependent cell-mediated cytotoxicity potency against HER2-overexpressing tumor cells. In particular, HuA21, but not trastuzumab (Tra), markedly suppresses growth and enhances the internalization of the antibody in Tra-resistant BT-474 breast cancer cells. These characteristics are highly associated with the intrinsic ability of HuA21 to down-regulate HER2 activation and inhibit the extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (Akt) signaling pathways. Furthermore, the combination of HuA21 with Tra synergistically enhances the anti-tumor effects in vitro and in vivo and inhibits HER2 activation and the ERK1/2 and Akt signaling pathways. Altogether, our results suggest that HuA21 may represent a unique anti-HER2 antibody with potential as a therapeutic candidate alone or in combination with other anti-HER2 reagents in cancer therapy.

  18. Differences in Glycoprotein Complex Receptor Binding Site Accessibility Prompt Poor Cross-Reactivity of Neutralizing Antibodies between Closely Related Arenaviruses.

    Science.gov (United States)

    Brouillette, Rachel B; Phillips, Elisabeth K; Ayithan, Natarajan; Maury, Wendy

    2017-04-01

    The glycoprotein complex (GPC) of arenaviruses, composed of stable signal peptide, GP1, and GP2, is the only antigen correlated with antibody-mediated neutralization. However, despite strong cross-reactivity of convalescent antisera between related arenavirus species, weak or no cross-neutralization occurs. Two closely related clade B viruses, Machupo virus (MACV) and Junín virus (JUNV), have nearly identical overall GPC architecture and share a host receptor, transferrin receptor 1 (TfR1). Given structural and functional similarities of the GP1 receptor binding site (RBS) of these viruses and the recent demonstration that the RBS is an important target for neutralizing antibodies, it is not clear how these viruses avoid cross-neutralization. To address this, MACV/JUNV chimeric GPCs were assessed for interaction with a group of α-JUNV GPC monoclonal antibodies (MAbs) and mouse antisera against JUNV or MACV GPC. All six MAbs targeted GP1, with those that neutralized JUNV GPC-pseudovirions competing with each other for RBS binding. However, these MAbs were unable to bind to a chimeric GPC composed of JUNV GP1 containing a small disulfide bonded loop (loop 10) unique to MACV GPC, suggesting that this loop may block MAbs interaction with the GP1 RBS. Consistent with this loop causing interference, mouse anti-JUNV GPC antisera that solely neutralized pseudovirions bearing autologous GP1 provided enhanced neutralization of MACV GPC when this loop was removed. Our studies provide evidence that loop 10, which is unique to MACV GP1, is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of α-JUNV antisera against MACV.IMPORTANCE Multiple New World arenaviruses can cause severe disease in humans, and some geographic overlap exists among these viruses. A vaccine that protects against a broad range of New World arenaviruses is desirable for purposes of simplicity, cost, and broad protection against multiple National

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

    Science.gov (United States)

    Teulé, Florence; Miao, Yun-Gen; Sohn, Bong-Hee; Kim, Young-Soo; Hull, J Joe; Fraser, Malcolm J; Lewis, Randolph V; Jarvis, Donald L

    2012-01-17

    The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors 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 proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental silkworm silk fibers.

  20. Correlative scanning-transmission electron microscopy reveals that a chimeric flavivirus is released as individual particles in secretory vesicles.

    Science.gov (United States)

    Burlaud-Gaillard, Julien; Sellin, Caroline; Georgeault, Sonia; Uzbekov, Rustem; Lebos, Claude; Guillaume, Jean-Marc; Roingeard, Philippe

    2014-01-01

    The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-03-19

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

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

    Science.gov (United States)

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

    2005-01-01

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

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

    Science.gov (United States)

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

    2016-01-26

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

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

    Directory of Open Access Journals (Sweden)

    Mallya Meera

    2008-09-01

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

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

    Science.gov (United States)

    Huber, Bettina; Schellenbacher, Christina; Shafti-Keramat, Saeed; Jindra, Christoph; Christensen, Neil; Kirnbauer, Reinhard

    2017-01-01

    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 cutaneous HPV

  6. ChiTaRS-3.1—the enhanced chimeric transcripts and RNA-seq database matched with protein–protein interactions

    Science.gov (United States)

    Gorohovski, Alessandro; Tagore, Somnath; Palande, Vikrant; Malka, Assaf; Raviv-Shay, Dorith; Frenkel-Morgenstern, Milana

    2017-01-01

    Discovery of chimeric RNAs, which are produced by chromosomal translocations as well as the joining of exons from different genes by trans-splicing, has added a new level of complexity to our study and understanding of the transcriptome. The enhanced ChiTaRS-3.1 database (http://chitars.md.biu.ac.il) is designed to make widely accessible a wealth of mined data on chimeric RNAs, with easy-to-use analytical tools built-in. The database comprises 34 922 chimeric transcripts along with 11 714 cancer breakpoints. In this latest version, we have included multiple cross-references to GeneCards, iHop, PubMed, NCBI, Ensembl, OMIM, RefSeq and the Mitelman collection for every entry in the ‘Full Collection’. In addition, for every chimera, we have added a predicted chimeric protein–protein interaction (ChiPPI) network, which allows for easy visualization of protein partners of both parental and fusion proteins for all human chimeras. The database contains a comprehensive annotation for 34 922 chimeric transcripts from eight organisms, and includes the manual annotation of 200 sense-antiSense (SaS) chimeras. The current improvements in the content and functionality to the ChiTaRS database make it a central resource for the study of chimeric transcripts and fusion proteins. PMID:27899596

  7. Monitoring of hematopoietic chimerism after transplantation for pediatric myelodysplastic syndrome: real-time or conventional short tandem repeat PCR in peripheral blood or bone marrow?

    Science.gov (United States)

    Willasch, Andre M; Kreyenberg, Hermann; Shayegi, Nona; Rettinger, Eva; Meyer, Vida; Zabel, Marion; Lang, Peter; Kremens, Bernhard; Meisel, Roland; Strahm, Brigitte; Rossig, Claudia; Gruhn, Bernd; Klingebiel, Thomas; Niemeyer, Charlotte M; Bader, Peter

    2014-12-01

    Quantitative real-time PCR (qPCR) has been proposed as a highly sensitive method for monitoring hematopoietic chimerism and may serve as a surrogate marker for the detection of minimal residual disease minimal residual disease in myelodysplastic syndrome (MDS), until specific methods of detection become available. Because a systematic comparison of the clinical utility of qPCR with the gold standard short tandem repeat (STR)-PCR has not been reported, we retrospectively measured chimerism by qPCR in 54 children transplanted for MDS in a previous study. Results obtained by STR-PCR in the initial study served as comparison. Because the detection limit of qPCR was sufficiently low to detect an autologous background, we defined the sample as mixed chimera if the proportion of recipient-derived cells exceeded .5%. The true positive rates were 100% versus 80% (qPCR versus STR-PCR, not significant), and mixed chimerism in most cases was detected earlier by qPCR than by STR-PCR (median, 31 days) when chimerism was quantified concurrently in peripheral blood and bone marrow. Both methods revealed a substantial rate of false positives (22.7% versus 13.6%, not significant), indicating the importance of serial testing of chimerism to monitor its progression. Finally, we propose criteria for monitoring chimerism in pediatric MDS with regard to the subtypes, specimens, PCR method, and timing of sampling.

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

    OpenAIRE

    Teulé, Florence; Miao, Yun-Gen; Sohn, Bong-Hee; Kim, Young-Soo; Hull, J. Joe; Fraser, Malcolm J.; Lewis, Randolph V.; Jarvis, Donald L.

    2012-01-01

    The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials t...

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

    DEFF Research Database (Denmark)

    Lorentzen, C L; thor Straten, Per

    2015-01-01

    Adoptive cell therapy (ACT) for cancer represents a promising new treatment modality. ACT based on the administration of cytotoxic T cells genetically engineered to express a chimeric antigen receptor (CAR) recognizing CD19 expressed by B cell malignancies has been shown to induce complete lasting......-associated toxicities, which needs attention. Herein we review current data and discuss key aspects of this powerful approach to treat and potentially cure B cell malignancies....

  10. Origin and ascendancy of a chimeric fusion gene: the beta/delta-globin gene of paenungulate mammals.

    Science.gov (United States)

    Opazo, Juan C; Sloan, Angela M; Campbell, Kevin L; Storz, Jay F

    2009-07-01

    The delta-globin gene (HBD) of eutherian mammals exhibits a propensity for recombinational exchange with the closely linked beta-globin gene (HBB) and has been independently converted by the HBB gene in multiple lineages. Here we report the presence of a chimeric beta/delta fusion gene in the African elephant (Loxodonta africana) that was created by unequal crossing-over between misaligned HBD and HBB paralogs. The recombinant chromosome that harbors the beta/delta fusion gene in elephants is structurally similar to the "anti-Lepore" duplication mutant of humans (the reciprocal exchange product of the hemoglobin Lepore deletion mutant). However, the situation in the African elephant is unique in that the chimeric beta/delta fusion gene supplanted the parental HBB gene and is therefore solely responsible for synthesizing the beta-chain subunits of adult hemoglobin. A phylogenetic survey of beta-like globin genes in afrotherian and xenarthran mammals revealed that the origin of the chimeric beta/delta fusion gene and the concomitant inactivation of the HBB gene predated the radiation of "Paenungulata," a clade of afrotherian mammals that includes three orders: Proboscidea (elephants), Sirenia (dugongs and manatees), and Hyracoidea (hyraxes). The reduced fitness of the human Hb Lepore deletion mutant helps to explain why independently derived beta/delta fusion genes (which occur on an anti-Lepore chromosome) have been fixed in a number of mammalian lineages, whereas the reciprocal delta/beta fusion gene (which occurs on a Lepore chromosome) has yet to be documented in any nonhuman mammal. This illustrates how the evolutionary fates of chimeric fusion genes can be strongly influenced by their recombinational mode of origin.

  11. A tailor-made chimeric thiamine diphosphate dependent enzyme for the direct asymmetric synthesis of (S)-benzoins.

    Science.gov (United States)

    Westphal, Robert; Vogel, Constantin; Schmitz, Carlo; Pleiss, Jürgen; Müller, Michael; Pohl, Martina; Rother, Dörte

    2014-08-25

    Thiamine diphosphate dependent enzymes are well known for catalyzing the asymmetric synthesis of chiral α-hydroxy ketones from simple prochiral substrates. The steric and chemical properties of the enzyme active site define the product spectrum. Enzymes catalyzing the carboligation of aromatic aldehydes to (S)-benzoins have not so far been identified. We were able to close this gap by constructing a chimeric enzyme, which catalyzes the synthesis of various (S)-benzoins with excellent enantiomeric excess (>99%) and very good conversion.

  12. Alemtuzumab levels impact acute GVHD, mixed chimerism, and lymphocyte recovery following alemtuzumab, fludarabine, and melphalan RIC HCT.

    Science.gov (United States)

    Marsh, Rebecca A; Lane, Adam; Mehta, Parinda A; Neumeier, Lisa; Jodele, Sonata; Davies, Stella M; Filipovich, Alexandra H

    2016-01-28

    Reduced intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (HCT) with alemtuzumab, fludarabine, and melphalan is an effective approach for patients with nonmalignant disorders. Mixed chimerism and graft-versus-host-disease (GVHD) remain limitations on success. We hypothesized that higher levels of alemtuzumab at day 0 would result in a low risk of acute GVHD, a higher risk of mixed chimerism, and delayed early lymphocyte recovery and that alemtuzumab level thresholds for increased risks of these outcomes would be definable. We collected data from 105 patients to examine the influence of peritransplant alemtuzumab levels on acute GVHD, mixed chimerism, and lymphocyte recovery. The cumulative incidences of initial grades I-IV, II-IV, and III-IV acute GVHD in patients with alemtuzumab levels ≤0.15 vs ≥0.16 μg/mL were 68% vs 18% (P alemtuzumab level ≤0.15 μg/mL was 21%, vs 42% with levels of 0.16 to 4.35 μg/mL, and 100% with levels >4.35 μg/mL (P = .003). Patients with alemtuzumab levels ≤0.15 or 0.16 to 0.56 μg/mL had higher lymphocyte counts at day +30 and higher T-cell counts at day +100 compared with patients with levels ≥0.57 μg/mL (all P alemtuzumab levels impact acute GVHD, mixed chimerism, and lymphocyte recovery following RIC HCT with alemtuzumab, fludarabine, and melphalan. Precision dosing trials are warranted. We recommend a day 0 therapeutic range of 0.2 to 0.4 μg/mL.

  13. Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

    Directory of Open Access Journals (Sweden)

    Sandra Arenhart

    Full Text Available Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3. The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-06-15

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

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

    Science.gov (United States)

    Pippa, Natassa; Kaditi, Eleni; Pispas, Stergios; Demetzos, Costas

    2013-06-01

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

  17. 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.

  18. Task demands modulate decision and eye movement responses in the chimeric face test: examining the right hemisphere processing account

    Directory of Open Access Journals (Sweden)

    Jason eCoronel

    2014-03-01

    Full Text Available A large and growing body of work, conducted in both brain-intact and brain-damaged populations, has used the free viewing chimeric face test as a measure of hemispheric dominance for the extraction of emotional information from faces. These studies generally show that normal right-handed individuals tend to perceive chimeric faces as more emotional if the emotional expression is presented on the half of the face to the viewer’s left (left hemiface. However, the mechanisms underlying this lateralized bias remain unclear. Here, we examine the extent to which this bias is driven by right hemisphere processing advantages versus default scanning biases in a unique way -- by changing task demands. In particular, we compare the original task with one in which right-hemisphere-biased processing cannot provide a decision advantage. Our behavioral and eye-movement data are inconsistent with the predictions of a default scanning bias account and support the idea that the left hemiface bias found in the chimeric face test is largely due to strategic use of right hemisphere processing mechanisms.

  19. Anti-neutrophil cytoplasmic antibodies stimulate release of neutrophil microparticles.

    LENUS (Irish Health Repository)

    Hong, Ying

    2012-01-01

    The mechanisms by which anti-neutrophil cytoplasmic antibodies (ANCAs) may contribute to the pathogenesis of ANCA-associated vasculitis are not well understood. In this study, both polyclonal ANCAs isolated from patients and chimeric proteinase 3-ANCA induced the release of neutrophil microparticles from primed neutrophils. These microparticles expressed a variety of markers, including the ANCA autoantigens proteinase 3 and myeloperoxidase. They bound endothelial cells via a CD18-mediated mechanism and induced an increase in endothelial intercellular adhesion molecule-1 expression, production of endothelial reactive oxygen species, and release of endothelial IL-6 and IL-8. Removal of the neutrophil microparticles by filtration or inhibition of reactive oxygen species production with antioxidants abolished microparticle-mediated endothelial activation. In addition, these microparticles promoted the generation of thrombin. In vivo, we detected more neutrophil microparticles in the plasma of children with ANCA-associated vasculitis compared with that in healthy controls or those with inactive vasculitis. Taken together, these results support a role for neutrophil microparticles in the pathogenesis of ANCA-associated vasculitis, potentially providing a target for future therapeutics.

  20. Anti-CD137 monoclonal antibodies and adoptive T cell therapy: a perfect marriage?

    Science.gov (United States)

    Weigelin, Bettina; Bolaños, Elixabet; Rodriguez-Ruiz, Maria E; Martinez-Forero, Ivan; Friedl, Peter; Melero, Ignacio

    2016-05-01

    CD137(4-1BB) costimulation and adoptive T cell therapy strongly synergize in terms of achieving maximal efficacy against experimental cancers. These costimulatory biological functions of CD137 have been exploited by means of introducing the CD137 signaling domain in clinically successful chimeric antigen receptors and to more efficiently expand T cells in culture. In addition, immunomagnetic sorting of CD137-positive T cells among tumor-infiltrating lymphocytes selects for the fittest antitumor T lymphocytes for subsequent cultures. In mouse models, co-infusion of both agonist antibodies and T cells attains marked synergistic effects that result from more focused and intense cytolytic activity visualized under in vivo microscopy and from more efficient entrance of T cells into the tumor through the vasculature. These several levels of dynamic interaction between adoptive T cell therapy and CD137 offer much opportunity to raise the efficacy of current cancer immunotherapies.

  1. The initial antibody response to HIV-1: induction of ineffective early B cell responses against GP41 by the transmitted/founder virus

    Energy Technology Data Exchange (ETDEWEB)

    Chavez, Leslie L [Los Alamos National Laboratory; Perelson, Alan [Los Alamos National Laboratory

    2008-01-01

    A window of opportunity for immune responses to extinguish HIV -1 exists from the moment of transmission through establishment of the latent pool of HIV -I-infected cells. A critical time to study the initial immune responses to the transmitted/founder virus is the eclipse phase of HIV-1 infection (time from transmission to the first appearance of plasma virus) but, to date, this period has been logistically difficult to analyze. Studies in non-human primates challenged with chimeric simianhuman immunodeficiency virus have shown that neutralizing antibodies, when present at the time of infection, can prevent virus infection.

  2. How antibodies use complement to regulate antibody responses.

    Science.gov (United States)

    Sörman, Anna; Zhang, Lu; Ding, Zhoujie; Heyman, Birgitta

    2014-10-01

    Antibodies, forming immune complexes with their specific antigen, can cause complete suppression or several 100-fold enhancement of the antibody response. Immune complexes containing IgG and IgM may activate complement and in such situations also complement components will be part of the immune complex. Here, we review experimental data on how antibodies via the complement system upregulate specific antibody responses. Current data suggest that murine IgG1, IgG2a, and IgG2b upregulate antibody responses primarily via Fc-receptors and not via complement. In contrast, IgM and IgG3 act via complement and require the presence of complement receptors 1 and 2 (CR1/2) expressed on both B cells and follicular dendritic cells. Complement plays a crucial role for antibody responses not only to antigen complexed to antibodies, but also to antigen administered alone. Lack of C1q, but not of Factor B or MBL, severely impairs antibody responses suggesting involvement of the classical pathway. In spite of this, normal antibody responses are found in mice lacking several activators of the classical pathway (complement activating natural IgM, serum amyloid P component (SAP), specific intracellular adhesion molecule-grabbing non-integrin R1 (SIGN-R1) or C-reactive protein. Possible explanations to these observations will be discussed.

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

    Institute of Scientific and Technical Information of China (English)

    XUXIN; SUMIOSUGANO; 等

    1999-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2015-08-13

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

  6. Rapid recombination among transfected plasmids, chimeric episome formation and trans gene expression in Plasmodium falciparum.

    Science.gov (United States)

    Kadekoppala, M; Cheresh, P; Catron, D; Ji, D D; Deitsch, K; Wellems, T E; Seifert, H S; Haldar, K

    2001-02-01

    Although recombination is known to be important to generating diversity in the human malaria parasite P. falciparum, the low efficiencies of transfection and the fact that integration of transfected DNA into chromosomes is observed only after long periods (typically 12 weeks or more) have made it difficult to genetically manipulate the blood stages of this major human pathogen. Here we show that co-transfection of a P. falciparum line with two plasmids, one expressing a green fluorescent protein (gfp) reporter and the other expressing a drug resistance marker (Tgdhfr-ts M23), allowed selection of a population in which about approximately 30% of the parasites produce GFP. In these GFP-producing parasites, the transfected plasmids had recombined into chimeric episomes as large as 20 kb and could be maintained under drug pressure for at least 16 weeks. Our data suggest that chimera formation occurs early (detected by 7--14 days) and that it involves homologous recombination favored by presence of the same P. falciparum 5'hrp3 UTR promoting transcription from each plasmid. This indicates the presence of high levels of homologous recombination activity in blood stage parasites that can be used to drive rapid recombination of newly introduced DNA, study mechanisms of recombination, and introduce genes for trans expression in P. falciparum.

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

    Science.gov (United States)

    Yu, Shengnan; Li, Anping; Liu, Qian; Li, Tengfei; Yuan, Xun; Han, Xinwei; Wu, Kongming

    2017-03-29

    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.

  8. Chimeric antigen receptor T cell therapy in AML: How close are we?

    Science.gov (United States)

    Gill, Saar

    2016-12-01

    The majority of patients presenting with acute myeloid leukemia (AML) initially respond to chemotherapy but post-remission therapy is required to consolidate this response and achieve long-term disease-free survival. The most effective form of post-remission therapy relies on T cell immunotherapy in the form of allogeneic hematopoietic cell transplantation (HCT). However, patients with active disease cannot usually expect to be cured with HCT. This inherent dichotomy implies that traditional T cell-based immunotherapy in the form of allogeneic HCT stops being efficacious somewhere between the measurable residual disease (MRD) and the morphologically obvious range. This is in part because the full power of T cells must be restrained in order to avoid lethal graft-versus-host disease (GVHD) and partly because only a sub-population of donor T cells are expected to be able to recognize AML cells via their T cell receptor. Chimeric antigen receptor (CAR) T cell therapy, most advanced in the treatment of patients with B-cell malignancies, may circumvent some of these limitations. However, major challenges remain to be overcome before CAR T cell therapy can be safely applied to AML.

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

    Science.gov (United States)

    Takeshima, Yasuhiro; Yagi, Mariko; Matsuo, Masafumi

    2012-01-01

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

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

    Directory of Open Access Journals (Sweden)

    H.G.P. van Gennip

    2008-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yelei Guo

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    R. Briant

    2014-05-01

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

  14. Diverging catalytic capacities and selectivity profiles with haloalkane substrates of chimeric alpha class glutathione transferases.

    Science.gov (United States)

    Kurtovic, Sanela; Shokeer, Abeer; Mannervik, Bengt

    2008-05-01

    Six homologous Alpha class glutathione transferases of human, bovine, and rat origins were hybridized by means of DNA shuffling. The chimeric mutants were compared with the parental enzymes in their activities with several alkyl iodides. In order to facilitate a multivariate analysis of relationships between substrates and enzyme activities, three descriptors were introduced: 'specific catalytic capacity', 'substrate selectivity', and 'unit-scaled substrate selectivity'. In some cases the purified mutants showed higher specific activity with a certain alkyl iodide than any of the parental enzymes. However, the overriding effect of DNA shuffling was the generation of chimeras with altered substrate selectivity profiles and catalytic capacities. The altered substrate selectivity profiles of some mutants could be rationalized by changes of the substrate-binding residues in the active site of the enzyme. However, in four of the isolated mutants all active-site residues were found identical with those of rat GST A2-2, even though their substrate specificity profiles were significantly different. Clearly, amino acid residues distant from first-sphere interactions with the substrate influence the catalytic activity. These results are relevant both to the understanding how functional properties may develop in natural enzyme evolution and in the tailoring of novel functions in protein engineering.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-08-10

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

  16. Unique chimeric composition of the trehalase gene from brine shrimp, Artemia franciscana.

    Science.gov (United States)

    Tanaka, Shin; Nambu, Fumiko; Nambu, Ziro

    2010-03-01

    To investigate the exon/intron structure of the Artemia trehalase gene, four overlapping clones were isolated from a genome library derived from an inbred strain of crustacean Artemia franciscana, and a 49 kb genetic area was re-constructed. The re-constructed area contained eight exons corresponding to the trehalase cDNA sequence that we had previously reported [1]. Comparative analysis of the Artemia trehalase gene with other animal trehalase genes revealed the existence of conserved exon/intron boundaries among different phyla. Comparison of the 5' UTR region of trehalase mRNA obtained by the 5' RACE method with the trehalase genes indicated the existence of a novel exon/intron boundary in the region designated "Exon I". Surprisingly, a part of a mitochondrial ribosomal protein gene (MRP-S33) was found to be inserted in the 5' UTR region of the trehalase gene. This sequence had the same polyadenylation signal that the Artemia MRP-S33 cDNAs did. Using the 3' RACE method, it was demonstrated that the poly (A) additional signal is still functional and that the chimeric mRNAs composed of the 5' UTR of the trehalase mRNA and of the 3' end derived from the MRP-S33 gene are transcribed.

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

    Directory of Open Access Journals (Sweden)

    Lisa Oestereich

    2016-05-01

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

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

    Science.gov (United States)

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

    2016-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Fu-Nan Cho

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

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

    Science.gov (United States)

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

    2000-04-01

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

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

    Science.gov (United States)

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

    2014-05-06

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

  2. The antibody Hijikata Tatsumi

    Directory of Open Access Journals (Sweden)

    Éden Peretta

    2012-11-01

    Full Text Available Considered one of the most influential modern dance representatives in Japan, Tatsumi Hijikata’s work was a milestone in the Japanese post-war experimental artistic scene. Heretic son of his time, he staged a fertile mix of artistic and cultural influences, overlapping subversive elements of European arts and philosophy with radical references from pre-modern Japanese culture. In this way he built the foundations of its unstable antibody, its political-artistic project of dissolution of a organism, both physical and social.

  3. Use of radiolabeled monoclonal antibodies for diagnostic imaging

    Energy Technology Data Exchange (ETDEWEB)

    Endo, Keigo (Kyoto Univ. (Japan). Faculty of Medicine)

    1990-08-01

    Monoclonal antibodies (MoAbs) are expected to carry radionuclides selectively to target tissues and to offer antigen-specific diagnosis. Indium (In)-111 has many favorable nuclear properties and is efficiently labeled with MoAbs using DAPA as a bifunctional chelating agent. In-111 labeled MoAbs are clinically employed for the diagnosis of malignant melanoma, colorectal cancer and acute myocardial infarction in Japan. Although non-specific deposit of In-111 was seen in liver and bone-marrow, scintigraphy using In-111 labeled MoAbs was encouraging, since it detected about 80% of tumors, tumors missed by conventional diagnostic methods such as CT, and tumors in patients with normal serum CEA values, and acute myocarditis as well as acute myocardial infarction was positive with In-111 labeled Fab fraction of anti-myosin Ab. Acute or subacute toxicity was not observed. Human anti-murine antibody (HAMA) was detected in 53 of 64 (82.8%) patients who were intravenously administered with 20 to 42 mg of anti-melanoma or anti-CEA MoAbs (whole IgG). In contrast, only 5 of 406 (1.2%) patients had detectable levels of HAMA in their serum after receiving 0.5 mg of Fab fraction of MoAb. Recently mouse-human chimeric Ab has been produced by recombinant DNA techniques, which localized well in xenografted tumors and seems to be promising for clinical use. Investigations are under way to increase the tumor to non-tumor ratio by modifying chelating agents for coupling MoAbs with radionuclides. (author).

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

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    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

  5. Cancer imaging with radiolabeled antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Goldenberg, D.M. (Center for Molecular Medicine and Immunology, Newark, NJ (US))

    1990-01-01

    This book presents a perspective of the use of antibodies to target diagnostic isotopes to tumors. Antibodies with reasonable specificity can be developed against almost any substance. If selective targeting to cancer cells can be achieved, the prospects for a selective therapy are equally intriguing. But the development of cancer detection, or imaging, with radiolabeled antibodies has depended upon advances in a number of different areas, including cancer immunology and immunochemistry for identifying suitable antigen targets and antibodies to these targets, tumor biology for model systems, radiochemistry for he attachment of radionuclides to antibodies, molecular biology for reengineering the antibodies for safer and more effective use in humans, and nuclear medicine for providing the best imaging protocols and instrumentation to detect minute amounts of elevated radioactivity against a background of considerable noise. Accordingly, this book has been organized to address the advances that are being made in many of these areas.

  6. VIRAL ANTIBODIES IN PRESCHOOL CHILDREN

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    S. Saidi

    1974-08-01

    Full Text Available One hundred sera from children 1 - 6 years of age, representative of a large serum collection, were tested for the prevalence of antibodies against different viruses. Hemagglutination-inhibition (HI antibodies were found in 68% for measles; 61 % for rubella; 75'% for influenza A2/Hong Kong/68, 16% for influenza B/Md./59, 0% for group A arboviruses, 10% for group B arboviruses, 3% for phlebotomus fever group and 4% for Congo-Crimean hemorrhagic fever (C-CHF group of arboviruses Poliomyelitis-neutralizing antibodies for type 1, 2 and 3 were 90%; 85% and 84%~ respectively. Antibody to EH virus was detected in 84% of the sera by immuno-fluorescence. None of the sera were positive for hepatitis-B antigen or antibody by immuno-precipitation test. The prevalence of some viral antibodies found in this survey are compared with results obtained from surveys in other parts of the country.

  7. Protective efficacy of neutralizing monoclonal antibodies in a nonhuman primate model of Ebola hemorrhagic fever.

    Science.gov (United States)

    Marzi, Andrea; Yoshida, Reiko; Miyamoto, Hiroko; Ishijima, Mari; Suzuki, Yasuhiko; Higuchi, Megumi; Matsuyama, Yukie; Igarashi, Manabu; Nakayama, Eri; Kuroda, Makoto; Saijo, Masayuki; Feldmann, Friederike; Brining, Douglas; Feldmann, Heinz; Takada, Ayato

    2012-01-01

    Ebola virus (EBOV) is the causative agent of severe hemorrhagic fever in primates, with human case fatality rates up to 90%. Today, there is neither a licensed vaccine nor a treatment available for Ebola hemorrhagic fever (EHF). Single monoclonal antibodies (MAbs) specific for Zaire ebolavirus (ZEBOV) have been successfully used in passive immunization experiments in rodent models, but have failed to protect nonhuman primates from lethal disease. In this study, we used two clones of human-mouse chimeric MAbs (ch133 and ch226) with strong neutralizing activity against ZEBOV and evaluated their protective potential in a rhesus macaque model of EHF. Reduced viral loads and partial protection were observed in animals given MAbs ch133 and ch226 combined intravenously at 24 hours before and 24 and 72 hours after challenge. MAbs circulated in the blood of a surviving animal until virus-induced IgG responses were detected. In contrast, serum MAb concentrations decreased to undetectable levels at terminal stages of disease in animals that succumbed to infection, indicating substantial consumption of these antibodies due to virus replication. Accordingly, the rapid decrease of serum MAbs was clearly associated with increased viremia in non-survivors. Our results indicate that EBOV neutralizing antibodies, particularly in combination with other therapeutic strategies, might be beneficial in reducing viral loads and prolonging disease progression during EHF.

  8. Antibodies against antibodies: immunogenicity of adalimumab as a model

    NARCIS (Netherlands)

    van Schouwenburg, P.A.

    2012-01-01

    Upon repeated adalimumab exposure part of the patients start to produce ADA. The antibody response is polyclonal and consists mainly of antibodies of IgG1 and IgG4 isotype. In the majority of ADA positive patients ADA are already produced within the first 28 weeks of treatment and in part of the pat

  9. Neutralizing antibodies respond to a bivalent dengue DNA vaccine or/and a recombinant bivalent antigen.

    Science.gov (United States)

    Zhang, Zhi-Shan; Weng, Yu-Wei; Huang, Hai-Long; Zhang, Jian-Ming; Yan, Yan-Sheng

    2015-02-01

    There is currently no effective vaccine to prevent dengue infection, despite the existence of multiple studies on potential methods of immunization. The aim of the present study was to explore the effect of DNA and/or recombinant protein on levels of neutralizing antibodies. For this purpose, envelope domain IIIs of dengue serotypes 1 and 2 (DEN-1/2)were spliced by a linker (Gly‑Gly‑Ser‑Gly‑Ser)3 and cloned into the prokaryotic expression plasmid pET30a (+) and eukaryotic vector pcDNA3.1 (+). The chimeric bivalent protein was expressed in Escherichia coli, and one‑step purification by high‑performance liquid chromatography was conducted. Protein expression levels of the DNA plasmid were tested in BHK‑21 cells by indirect immunofluorescent assay. In order to explore a more effective immunization strategy and to develop neutralizing antibodies against the two serotypes, mice were inoculated with recombinant bivalent protein, the DNA vaccine, or the two given simultaneously. Presence of the specific antibodies was tested by ELISA and the presence of the neutralizing antibodies was determined by plaque reduction neutralization test. Results of the analysis indicated that the use of a combination of DNA and protein induced significantly higher titers of neutralizing antibodies against either DEN‑1 or DEN‑2 (1:64.0 and 1:76.1, respectively) compared with the DNA (1:24.7 and 1:26.9, DEN‑1 and DEN‑2, respectively) or the recombinant protein (1:34.9 and 1:45.3 in DEN‑1 and DEN‑2, respectively). The present study demonstrated that the combination of recombinant protein and DNA as an immunization strategy may be an effective method for the development of a vaccine to prevent dengue virus infection.

  10. Functional characterization of an scFv-Fc antibody that immunotherapeutically targets the common cancer cell surface proteoglycan CSPG4.

    Science.gov (United States)

    Wang, Xinhui; Katayama, Akihiro; Wang, Yangyang; Yu, Ling; Favoino, Elvira; Sakakura, Koichi; Favole, Alessandra; Tsuchikawa, Takahiro; Silver, Susan; Watkins, Simon C; Kageshita, Toshiro; Ferrone, Soldano

    2011-12-15

    Cell surface chondroitin sulfate proteoglycan 4 (CSPG4) is an attractive target for antibody-based cancer immunotherapy because of its role in tumor cell biology, its high expression on malignant cells including cancer-initiating cells, and its restricted distribution in normal tissues. The clinical use of CSPG4 has been hampered by the lack of a CSPG4-specific chimeric, humanized, or fully human monoclonal antibody. To overcome this limitation, we generated a CSPG4-specific fully human single-chain antibody termed scFv-FcC21 and characterized its specificity and antitumor activity. Viable CSPG4(+) melanoma cells were used in a screen of a human scFv phage display library that included CDR3 engineered to optimize antibody binding sites. The scFv antibody isolated was then recombinantly engineered with a human immunoglobulin G1 Fc region to construct the fully human antibody scFv-FcC21, which recognized tumors of neuroectodermal origin, various types of carcinomas, mesotheliomas, and sarcomas as well as myeloid leukemias. scFv-FcC21 inhibited in vitro growth and migration of tumor cells and in vivo growth of human tumor xenografts. These effects were mediated by inhibition of the activation of extracellular signal-regulated kinase and focal adhesion kinase signaling pathways that are critical for tumor cell growth and migration, respectively. Our findings define the CSPG4-specific fully human scFv-FcC21 antibody as a candidate therapeutic agent to target the many types of tumors that express CSPG4.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-02-01

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

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

    Science.gov (United States)

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

    2001-01-01

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

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

    Science.gov (United States)

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

    2002-01-01

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

  14. 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.

  15. Evidence of a chimeric genome in the cyanobacterial ancestor of plastids

    Directory of Open Access Journals (Sweden)

    Bhattacharya Debashish

    2008-04-01

    Full Text Available Abstract Background Horizontal gene transfer (HGT is a vexing fact of life for microbial phylogeneticists. Given the substantial rates of HGT observed in modern-day bacterial chromosomes, it is envisaged that ancient prokaryotic genomes must have been similarly chimeric. But where can one find an ancient prokaryotic genome that has maintained its ancestral condition to address this issue? An excellent candidate is the cyanobacterial endosymbiont that was harnessed over a billion years ago by a heterotrophic protist, giving rise to the plastid. Genetic remnants of the endosymbiont are still preserved in plastids as a highly reduced chromosome encoding 54 – 264 genes. These data provide an ideal target to assess genome chimericism in an ancient cyanobacterial lineage. Results Here we demonstrate that the origin of the plastid-encoded gene cluster for menaquinone/phylloquinone biosynthesis in the extremophilic red algae Cyanidiales contradicts a cyanobacterial genealogy. These genes are relics of an ancestral cluster related to homologs in Chlorobi/Gammaproteobacteria that we hypothesize was established by HGT in the progenitor of plastids, thus providing a 'footprint' of genome chimericism in ancient cyanobacteria. In addition to menB, four components of the original gene cluster (menF, menD, menC, and menH are now encoded in the nuclear genome of the majority of non-Cyanidiales algae and plants as the unique tetra-gene fusion named PHYLLO. These genes are monophyletic in Plantae and chromalveolates, indicating that loci introduced by HGT into the ancestral cyanobacterium were moved over time into the host nucleus. Conclusion Our study provides unambiguous evidence for the existence of genome chimericism in ancient cyanobacteria. In addition we show genes that originated via HGT in the cyanobacterial ancestor of the plastid made their way to the host nucleus via endosymbiotic gene transfer (EGT.

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

    Science.gov (United States)

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

    1996-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    Binghua Xue

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

  20. Algorithms for joint optimization of stability and diversity in planning combinatorial libraries of chimeric proteins.

    Science.gov (United States)

    Zheng, Wei; Friedman, Alan M; Bailey-Kellogg, Chris

    2009-08-01

    In engineering protein variants by constructing and screening combinatorial libraries of chimeric proteins, two complementary and competing goals are desired: the new proteins must be similar enough to the evolutionarily-selected wild-type proteins to be stably folded, and they must be different enough to display functional variation. We present here the first method, Staversity, to simultaneously optimize stability and diversity in selecting sets of breakpoint locations for site-directed recombination. Our goal is to uncover all "undominated" breakpoint sets, for which no other breakpoint set is better in both factors. Our first algorithm finds the undominated sets serving as the vertices of the lower envelope of the two-dimensional (stability and diversity) convex hull containing all possible breakpoint sets. Our second algorithm identifies additional breakpoint sets in the concavities that are either undominated or dominated only by undiscovered breakpoint sets within a distance bound computed by the algorithm. Both algorithms are efficient, requiring only time polynomial in the numbers of residues and breakpoints, while characterizing a space defined by an exponential number of possible breakpoint sets. We applied Staversity to identify 2-10 breakpoint plans for different sets of parent proteins taken from the purE family, as well as for parent proteins TEM-1 and PSE-4 from the beta-lactamase family. The average normalized distance between our plans and the lower bound for optimal plans is around 2%. Our plans dominate most (60-90% on average for each parent set) of the plans found by other possible approaches, random sampling or explicit optimization for stability with implicit optimization for diversity. The identified breakpoint sets provide a compact representation of good plans, enabling a protein engineer to understand and account for the trade-offs between two key considerations in combinatorial chimeragenesis.

  1. Studies on mu and delta opioid receptor selectivity utilizing chimeric and site-mutagenized receptors.

    Science.gov (United States)

    Wang, W W; Shahrestanifar, M; Jin, J; Howells, R D

    1995-01-01

    Opioid receptors are members of the guanine nucleotide binding protein (G protein)-coupled receptor family. Three types of opioid receptors have been cloned and characterized and are referred to as the delta, kappa and mu types. Analysis of receptor chimeras and site-directed mutant receptors has provided a great deal of information about functionally important amino acid side chains that constitute the ligand-binding domains and G-protein-coupling domains of G-protein-coupled receptors. We have constructed delta/mu opioid receptor chimeras that were express in human embryonic kidney 293 cells in order to define receptor domains that are responsible for receptor type selectivity. All chimeric receptors and wild-type delta and mu opioid receptors displayed high-affinity binding of etorphine (an agonist), naloxone (an antagonist), and bremazocine (a mixed agonist/antagonist). In contrast, chimeras that lacked the putative first extracellular loop of the mu receptor did not bind the mu-selective peptide [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO). Chimeras that lacked the putative third extracellular loop of the delta receptor did not bind the delta-selective peptide, [D-Ser2,D-Leu5]enkephalin-Thr (DSLET). Point mutations in the putative third extracellular loop of the wild-type delta receptor that converted vicinal arginine residues to glutamine abolished DSLET binding while not affecting bremazocine, etorphine, and naltrindole binding. We conclude that amino acids in the putative first extracellular loop of the mu receptor are critical for high-affinity DAMGO binding and that arginine residues in the putative third extracellular loop of the delta receptor are important for high-affinity DSLET binding. Images Fig. 3 PMID:8618916

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

    Institute of Scientific and Technical Information of China (English)

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

    2015-01-01

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

  3. Pathogenic role of antiphospholipid antibodies

    NARCIS (Netherlands)

    Salmon, J. E.; de Groot, P. G.

    2008-01-01

    The antiphospholipid antibody syndrome (APS) is characterized by recurrent arterial and venous thrombosis and/or pregnancy in association with antiphospholipid (aPL) antibodies. The pathogenic mechanisms in APS that lead to in vivo injury are incompletely understood. Recent evidence suggests that AP

  4. Educational paper: Primary antibody deficiencies

    NARCIS (Netherlands)

    G.J.A. Driessen (Gertjan); M. van der Burg (Mirjam)

    2011-01-01

    textabstractPrimary antibody deficiencies (PADs) are the most common primary immunodeficiencies and are characterized by a defect in the production of normal amounts of antigen-specific antibodies. PADs represent a heterogeneous spectrum of conditions, ranging from often asymptomatic selective IgA a

  5. Targeting of Antibodies using Aptamers

    OpenAIRE

    2003-01-01

    The chapter presents a methodology for the rapid selection of aptamers against antibody targets. It is a detailed account of the various methodological steps that describe the selection of aptamers, including PCR steps, buffers to be used, target immobilisation, partitioning and amplification of aptamers, clonning and sequencing, to results in high affinity and specificity ligands for the chosen target antibody.

  6. New engineered antibodies against prions

    Science.gov (United States)

    Škrlj, Nives; Dolinar, Marko

    2014-01-01

    A number of recently developed and approved therapeutic agents based on highly specific and potent antibodies have shown the potential of antibody therapy. As the next step, antibody-based therapeutics will be bioengineered in a way that they not only bind pathogenic targets but also address other issues, including drug targeting and delivery. For antibodies that are expected to act within brain tissue, like those that are directed against the pathogenic prion protein isoform, one of the major obstacles is the blood-brain barrier which prevents efficient transfer of the antibody, even of the engineered single-chain variants. We recently demonstrated that a specific prion-specific antibody construct which was injected into the murine tail vein can be efficiently transported into brain tissue. The novelty of the work was in that the cell penetrating peptide was used as a linker connecting both specificity-determining domains of the antibody peptide, thus eliminating the need for the standard flexible linker, composed of an arrangement of three consecutive (Gly4Ser) repeats. This paves the road toward improved bioengineered antibody variants that target brain antigens. PMID:23941991

  7. Metrics for antibody therapeutics development.

    Science.gov (United States)

    Reichert, Janice M

    2010-01-01

    A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approved in the United States, were derived from analysis of a dataset of over 600 therapeutic mAbs that entered clinical study sponsored, at least in part, by commercial firms. The results presented provide an overview of the field and context for the evaluation of on-going and prospective mAb development programs. The expansion of therapeutic antibody use through supplemental marketing approvals and the increase in the study of therapeutics derived from alternative antibody formats are discussed.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  9. Use of ubiquitous, highly heterozygous copy number variants and digital droplet polymerase chain reaction to monitor chimerism after allogeneic haematopoietic stem cell transplantation.

    Science.gov (United States)

    Whitlam, John B; Ling, Ling; Swain, Michael; Harrington, Tom; Mirochnik, Oksana; Brooks, Ian; Cronin, Sara; Challis, Jackie; Petrovic, Vida; Bruno, Damien L; Mechinaud, Francoise; Conyers, Rachel; Slater, Howard

    2017-01-29

    Chimerism analysis has an important role in the management of allogeneic hematopoietic stem cell transplantation. It informs response to disease relapse, graft rejection, and graft-versus-host disease. We have developed a method for chimerism analysis using ubiquitous copy number variation (CNV), which has the benefit of a "negative background" against which multiple independent informative markers are quantified using digital droplet polymerase chain reaction. A panel of up to 38 CNV markers with homozygous deletion frequencies of approximately 0.4-0.6 were used. Sensitivity, precision, reproducibility, and informativity were assessed. CNV chimerism results were compared against established fluorescence in situ hybridization, single nucleotide polymorphism, and short tandem repeat-based methods with excellent correlation. Using 30 ng of input DNA per well, the limit of detection was 0.05% chimerism and the limit of quantification was 0.5% chimerism. High informativity was seen with a median of four informative markers detectable per individual in 39 recipients and 43 donor genomes studied. The strength of this approach was exemplified in a multiple donor case involving four genomes (three related). The precision, sensitivity, and informativity of this approach recommend it for use in clinical practice.

  10. Recombinant S-layer proteins of Lactobacillus brevis mediating antibody adhesion to calf intestine alleviated neonatal diarrhea syndrome.

    Science.gov (United States)

    Khang, Yong-Ho; Park, Hee-Young; Jeong, Yoo-Seok; Kim, Jung-Ae; Kim, Young-Hwan

    2009-05-01

    A chimeric gene encoding enhanced green fluorescent protein (EGFP) and a S-layer protein from Lactobacillus brevis KCTC3102, and/or two copies of the Fc-binding Z-domain, a synthetic analog of the B-domain of protein A, was constructed and expressed in Escherichia coli BL21(DE3). The S-layer fusion proteins produced in a 500-l fermentor were likely to be stable in the range of pH 5 to 8 and 0 degree to 40 degrees . Their adhesive property enabled an easy and rapid immobilization of enzymes or antibodies on solid materials such as plastics, glass, sol-gel films, and intestinal epithelial cells. Owing to their affinity towards intestinal cells and immunoglobulin G, the Slayer fusion proteins enabled the adhesion of antibodies to human epithelial cells. In addition, feeding a mixture of the S-layer fusion proteins and antibodies against neonatal calf diarrhea (coronavirus, rotavirus, Escherichia coli, and Salmonella typhimurium) to Hanwoo calves resulted in 100% prevention of neonatal calf diarrhea syndrome (p<0.01),whereas feeding antibodies only resulted in 56% prevention.

  11. Comprehensive analysis of antibody recognition in convalescent humans from highly pathogenic avian influenza H5N1 infection.

    Science.gov (United States)

    Zuo, Teng; Sun, Jianfeng; Wang, Guiqin; Jiang, Liwei; Zuo, Yanan; Li, Danyang; Shi, Xuanling; Liu, Xi; Fan, Shilong; Ren, Huanhuan; Hu, Hongxing; Sun, Lina; Zhou, Boping; Liang, Mifang; Zhou, Paul; Wang, Xinquan; Zhang, Linqi

    2015-12-04

    Understanding the mechanism of protective antibody recognition against highly pathogenic avian influenza A virus H5N1 in humans is critical for the development of effective therapies and vaccines. Here we report the crystal structure of three H5-specific human monoclonal antibodies bound to the globular head of hemagglutinin (HA) with distinct epitope specificities, neutralization potencies and breadth. A structural and functional analysis of these epitopes combined with those reported elsewhere identifies four major vulnerable sites on the globular head of H5N1 HA. Chimeric and vulnerable site-specific mutant pseudoviruses are generated to delineate broad neutralization specificities of convalescent sera from two individuals who recovered from the infection with H5N1 virus. Our results show that the four vulnerable sites on the globular head rather than the stem region are the major neutralizing targets, suggesting that during natural H5N1 infection neutralizing antibodies against the globular head work in concert to provide protective antibody-mediated immunity.

  12. Research progress of application of immunotherapy by chimeric antigen receptor-engineered T cells%嵌合抗原受体修饰的T细胞免疫治疗研究进展

    Institute of Scientific and Technical Information of China (English)

    李伟; 于慧杰

    2015-01-01

    The technique of chimeric antigen receptor-engineered T cells (CAR-T) is combined single chain antibody and T-cell activation modification and has specific and durable killing effect on tumors.It had achieved stunning results on clinical trials of B lymphocytic leukemias,B lymphomas and solid tumors.Meanwhile it also had potential risk on the off target effects,cytokine release syndrome,graft versus host disease and so on.This paper briefly reviews the principal of CAR-T cellular mechanism,advantages and clinical applications.%嵌合抗原受体修饰的T细胞(CAR-T细胞)是将单链抗体技术和T细胞活化基序结合应用的一种新的免疫治疗技术,具有特异、持久的肿瘤杀伤效应,在B淋巴细胞白血病、B淋巴瘤及实体瘤的临床试验中取得了令人震惊的效果,但也存在着脱靶效应、细胞因子释放综合征、移植物抗宿主病等潜在的风险.文章就CAR-T细胞原理、优势、临床应用等方面进行简要综述.

  13. Modifications of Visual Field Asymmetries for Face Categorization in Early Deaf Adults: A Study With Chimeric Faces

    Science.gov (United States)

    Dole, Marjorie; Méary, David; Pascalis, Olivier

    2017-01-01

    Right hemisphere lateralization for face processing is well documented in typical populations. At the behavioral level, this right hemisphere bias is often related to a left visual field (LVF) bias. A conventional mean to study this phenomenon consists of using chimeric faces that are composed of the left and right parts of two faces. In this paradigm, participants generally use the left part of the chimeric face, mostly processed through the right optic tract, to determine its identity, gender or age. To assess the impact of early auditory deprivation on face processing abilities, we tested the LVF bias in a group of early deaf participants and hearing controls. In two experiments, deaf and hearing participants performed a gender categorization task with chimeric and normal average faces. Over the two experiments the results confirmed the presence of a LVF bias in participants, which was less frequent in deaf participants. This result suggested modifications of hemispheric lateralization for face processing in deaf participants. In Experiment 2 we also recorded eye movements to examine whether the LVF bias could be related to face scanning behavior. In this second study, participants performed a similar task while we recorded eye movements using an eye tracking system. Using areas of interest analysis we observed that the proportion of fixations on the mouth relatively to the other areas was increased in deaf participants in comparison with the hearing group. This was associated with a decrease of the proportion of fixations on the eyes. In addition these measures were correlated to the LVF bias suggesting a relationship between the LVF bias and the patterns of facial exploration. Taken together, these results suggest that early auditory deprivation results in plasticity phenomenon affecting the perception of static faces through modifications of hemispheric lateralization and of gaze behavior. PMID:28163692

  14. Characterization of NoV P particle-based chimeric protein vaccines developed from two different expression systems.

    Science.gov (United States)

    Fu, Lu; Jin, Hao; Yu, Yongjiao; Yu, Bin; Zhang, Haihong; Wu, Jiaxin; Yin, Yuhe; Yu, Xianghui; Wu, Hui; Kong, Wei

    2017-02-01

    The Norovirus (NoV) P domain, with three surface loops for foreign antigen insertion, has been demonstrated as an excellent platform for antigen presentation and novel vaccine development. The P domain alone can self-assemble into a P dimer, 12-mer small particle or 24-mer P particle, and vaccines based on those particles may elicit different levels of immunogenicity. Currently, P particles are generally produced in soluble expression systems in Escherichia coli, mainly in the 24-mer form. However, the low yield of the soluble protein has hindered further clinical applications of P particle-based protein vaccines. In this study, we inserted the Alzheimer's disease (AD) immunogen Aβ1-6 into the three loops of the P particle to generate an AD protein vaccine. To increase the yield of this chimeric protein, we tested the generation of proteins in a soluble expression system and an inclusion body expression system separately in E. coli. The result showed that the inclusion body expression system could greatly enhance the product yield of the chimeric protein compared with the soluble expression system. The refolded protein from the inclusion bodies was mainly in the 12-mer form, while the protein generated from the soluble supernatant was mainly in the 24-mer form. Moreover, the immunogenicity of soluble proteins was significantly stronger than that of the refolded proteins. Thus, comparisons between the two expression methods suggested that the soluble expression system generated chimeric P particles with better immunogenicity, while inclusion body expression system yielded more P particle proteins.

  15. Antibodies to Phospholipids and Liposomes: Binding of Antibodies to Cells

    Science.gov (United States)

    1987-01-01

    LIPOSOMES: BINDING OF ANTIBODIES TO CELLS 12. PERSONAL AUTHOR(S) W.E. FOGLER , G. M. SWARTZ, AND C.R. ALVING 13a TYPE OF REPORT 13b. TIME COVERED 14. DATE...Elsevier BBA 73693 Antibodies to phospholipids and liposomes: binding of antibodies to cells William E. Fogler *, Glenn M. Swartz, Jr. and Carl R. Alving...Immunol. 21. Research Associateship from the U.S. National 12863-86812Hall. T. and Esser, K. (1984) 3. Immunol. 132. 2059-2063 Research Council. 13 Fogler

  16. Simultaneous expression of displayed and secreted antibodies for antibody screen.

    Directory of Open Access Journals (Sweden)

    Yuanping Zhou

    Full Text Available The display of full-length antibody on the cell surface was achieved by fusing a transmembrane domain of the platelet-derived growth factor receptor (PDGFR to the C-terminus of the heavy chain constant region. We also incorporated a furin cleavage site between the constant region and PDGFR transmembrane domain to obtain secreted antibodies. As a result, antibodies can be expressed simultaneously on the cell surface in a membrane-anchored version for screening and selecting through fluorescence-activated cell sorting (FACS analysis, as well as in conditioned medium in a secreted version for function analysis.

  17. Minor displacements in the insertion site provoke major differences in the induction of antibody responses by chimeric parvovirus-like particles

    DEFF Research Database (Denmark)

    Rueda, P.; Hurtado, A.; del Barrio, M.;

    1999-01-01

    An antigen-delivery system based on hybrid virus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of canine parvovirus (CPV) and expressing foreign peptides was investigated. In this report, we have studied the effects of inserting the poliovirus C3:B epitope in the four...

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

    NARCIS (Netherlands)

    Knipping, Karen; Simons, Peter; 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

    BACKGROUND: 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), sensiti

  19. Growth enhancement in transgenic Atlantic salmon by the use of an "all fish" chimeric growth hormone gene construct.

    Science.gov (United States)

    Du, S J; Gong, Z Y; Fletcher, G L; Shears, M A; King, M J; Idler, D R; Hew, C L

    1992-02-01

    We have developed an "all fish" growth hormone (GH) chimeric gene construct by using an antifreeze protein gene (AFP) promoter from ocean pout linked to a chinook salmon GH cDNA clone. After microinjection into fertilized, nonactivated Atlantic salmon eggs via the micropyle, transgenic Atlantic salmon were generated. The presence of the transgene was detected by polymerase chain reaction (PCR) using specific oligonucleotide primers. A number of these transgenic fish showed dramatic increases in their growth rate. At one year old, the average increase of the transgenic fish was 2 to 6 fold and the largest transgenic fish was 13 times that of the average non-transgenic control.

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

    Science.gov (United States)

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

    2015-11-11

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

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

    Science.gov (United States)

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

    2013-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Eran Hadas

    2013-09-01

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

  3. 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

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

    Directory of Open Access Journals (Sweden)

    Fabrice E. Graf

    2015-08-01

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

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

    Science.gov (United States)

    Graf, Fabrice E; Baker, Nicola; Munday, Jane C; de Koning, Harry P; Horn, David; Mäser, Pascal

    2015-08-01

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

  6. Tabhu: tools for antibody humanization

    DEFF Research Database (Denmark)

    Olimpieri, Pier Paolo; Marcatili, Paolo; Tramontano, Anna

    2015-01-01

    Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can...... elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity...

  7. Crystal structure of a human rhinovirus that displays part of the HIV-1 V3 loop and induces neutralizing antibodies against HIV-1.

    Science.gov (United States)

    Ding, Jianping; Smith, Allen D; Geisler, Sheila C; Ma, Xuejun; Arnold, Gail Ferstandig; Arnold, Eddy

    2002-07-01

    We report the 2.7 A resolution structure of a chimeric rhinovirus, MN-III-2, that displays part of the HIV-1 gp120 V3 loop and elicits HIV-neutralizing antibodies. The V3 loop insert is dominated by two type I beta turns. The structures of two adjacent tripeptides resemble those of analogous segments in three Fab/V3 loop peptide complexes. Although two of the three corresponding antibodies bind and neutralize MN-III-2 well, only one of the three can bind without significant rearrangement. These results suggest that the V3 loop insert: (1) can share some local conformational similarity to V3 loop sequences presented on different structural frameworks; (2) must be able to adopt multiple conformations, even in a relatively constrained environment; and (3) may mimic the conformational variability of the epitope on HIV-1, increasing the likelihood of eliciting appropriate neutralizing immune responses.

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

    Directory of Open Access Journals (Sweden)

    Lisa M Russo

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

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

    Directory of Open Access Journals (Sweden)

    María J Esteva

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

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

    Science.gov (United States)

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

    2015-11-01

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

  11. Comparative insecticidal properties of two nucleopolyhedrovirus vectors encoding a similar toxin gene chimer.

    Science.gov (United States)

    Treacy, M F; Rensner, P E; All, J N

    2000-08-01

    Laboratory, greenhouse and field studies were conducted to characterize the insecticidal properties of genetically altered forms of Autographa californica (Speyer) nucleopolyhedrovirus (AcNPV) and Helicoverpa zea (Boddie) NPV (HzNPV) against selected heliothine species. The altered viruses each contained a chimeric 0.8-kb fragment encoding the insect-specific, sodium channel neurotoxin from the Algerian scorpion Androctonus australis Hector (AaIT, hence recombinant viruses designated Ac-AaIT and Hz-AaIT). Based on LD50 values, results from diet-overlay bioassays showed Ac-AaIT and Hz-AaIT to be equally virulent against larval tobacco budworm, Heliothis virescens (F.), but Hz-AaIT averaged 1,335-fold greater bioactivity than Ac-AaIT against larval cotton bollworm, Helicoverpa zea (Boddie). Hz-AaIT killed larvae of both heliothine species at rates significantly faster than those imparted by HzNPV (viral LT50 values averaged 2.5 and 5.6 d, respectively). In greenhouse studies, foliar sprays of Ac-AaIT and Hz-AaIT were equally effective in controlling H. virescens on cotton; however, Hz-AaIT provided control of H. zea on cotton at a level superior to that of Ac-AaIT. For example, after three weekly sessions of foliar application and H. zea artificial infestation, cotton treated with Ac-AaIT or Hz-AaIT at 10 x 10(11) occulsion bodies (OB)/ha averaged 2.5 and 16.2 nondamaged flower buds per plant, respectively. Another greenhouse study conducted against heliothine species on cotton showed that the quicker killing speed exhibited by Hz-AaIT led to improved plant protection versus HzNPV. Finally, results from three field trials demonstrated that Hz-AaIT at 5-12 x 10(11) OB/ha provided control of the heliothine complex in cotton at levels slightly better than Bacillus thuringiensis, equal to the macrolide, spinosad, and only slightly less than that of selected pyrethroid and carbamate insecticides. Overall, results from these studies indicate that, because of host range

  12. Chimeric Yellow Fever/Dengue Virus as a Candidate Dengue Vaccine: Quantitation of the Dengue Virus-Specific CD8 T-Cell Response

    OpenAIRE

    van der Most, Robbert G.; Murali-Krishna, Kaja; Ahmed, Rafi; Strauss, James H.

    2000-01-01

    We have constructed a chimeric yellow fever/dengue (YF/DEN) virus, which expresses the premembrane (prM) and envelope (E) genes from DEN type 2 (DEN-2) virus in a YF virus (YFV-17D) genetic background. Immunization of BALB/c mice with this chimeric virus induced a CD8 T-cell response specific for the DEN-2 virus prM and E proteins. This response protected YF/DEN virus-immunized mice against lethal dengue encephalitis. Control mice immunized with the parental YFV-17D were not protected against...

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

  14. An intermediate alemtuzumab schedule reduces the incidence of mixed chimerism following reduced-intensity conditioning hematopoietic cell transplantation for hemophagocytic lymphohistiocytosis.

    Science.gov (United States)

    Marsh, Rebecca A; Kim, Mi-Ok; Liu, Chunyan; Bellman, Denise; Hart, Laura; Grimley, Michael; Kumar, Ashish; Jodele, Sonata; Myers, Kasiani C; Chandra, Sharat; Leemhuis, Tom; Mehta, Parinda A; Bleesing, Jack J; Davies, Stella M; Jordan, Michael B; Filipovich, Alexandra H

    2013-11-01

    Reduced-intensity conditioning (RIC) improves the outcomes of hematopoietic cell transplantation (HCT) in patients with hemophagocytic lymphohistiocytosis (HLH). Proximal (ie, close to graft infusion) dosing of alemtuzumab is associated with a high incidence of mixed chimerism, whereas distal (ie, distant from graft infusion) dosing is associated with less mixed chimerism but more acute graft-versus-host disease (GVHD). The alemtuzumab dose per kilogram of body weight also influences these outcomes. We hypothesized that an intermediate alemtuzumab dosing schedule would reduce mixed chimerism and maintain a low incidence of acute GVHD. In this study, 24 consecutive HCTs were performed in patients with HLH or a related disorder using a novel intermediate alemtuzumab schedule of 1 mg/kg starting on day -14. The cumulative incidences (CIs) of mixed chimerism, upfront acute GVHD grades II-IV, and receipt of additional hematopoietic cell products after HCT were compared in patients treated with a distal alemtuzumab schedule (n = 15) and those treated with a proximal alemtuzumab schedule (n = 33). All patients received fludarabine and melphalan. The CI of mixed chimerism was 31% in the intermediate group, 72% in the proximal group (P alemtuzumab (P = .03). The CI of acute GVHD grades II-IV before the development of mixed chimerism was 4% in the intermediate group, 0% in the proximal group, and 13% in the distal group (P = .04, proximal versus distal). The 1-year CI of administration of additional hematopoietic cell products for mixed chimerism (donor lymphocyte infusion ± hematopoietic stem cell boost ± repeat HCT) was 14% in the intermediate group, 53% in the proximal group (P = .01), and 38% in the distal ≥2 mg/kg alemtuzumab group (P = .02). Our findings indicate that intermediate RIC reduces the incidence of mixed chimerism, is associated with a low incidence of upfront acute GVHD, and decreases the need for additional hematopoietic cell

  15. Role of minimal residual disease and chimerism after reduced-intensity and myeloablative allo-transplantation in acute myeloid leukemia and high-risk myelodysplastic syndrome.

    Science.gov (United States)

    Bernal, Teresa; Diez-Campelo, María; Godoy, Vicky; Rojas, Silvia; Colado, Enrique; Alcoceba, Miguel; González, Marcos; Vidriales, Belén; Sánchez-Guijo, Fermín M; López-Corral, Lucía; Luño, Elisa; del Cañizo, Consuelo

    2014-05-01

    We evaluated the impact of detection of minimal residual disease by flow cytometry (FCMRD) and CD3 chimerism in relapse in a cohort of 87 patients with acute myeloid leukemia or myelodysplastic syndrome undergoing stem cell transplantation. Patients with a positive FCMRD at day +100 after transplantation showed higher relapse rates and worse overall survival. In multivariate analysis, a positive FCMRD after transplantation was a significant predictor of relapse. Mixed chimerism showed a trend to statistical signification. We conclude that FCMRD at day 100 after SCT is the best predictor of relapse after SCT in patients with aggressive myeloid malignancies.

  16. Advances in cancer immunotherapy based on chimeric antigen receptor%基于嵌合抗原受体的肿瘤免疫治疗的研究进展

    Institute of Scientific and Technical Information of China (English)

    李玲; 马建波

    2015-01-01

    近来关于嵌合抗原受体( chimeric antigen receptor,CAR)修饰T细胞在治疗包括淋巴瘤、白血病、脑胶质瘤、黑色素瘤、肺癌、前列腺癌、胰腺癌及卵巢癌等肿瘤所取得成绩令人振奋。 CAR是利用基因工程将能够与肿瘤抗原结合的受体与跨细胞膜的部分和细胞内信号转导的部分结合起来形成的一种新型受体,它可脱离主要组织相容性复合体的限制单独执行杀伤细胞的功能。 CAR修饰T细胞因其独特的设计和强效的抗肿瘤作用受到人们的追捧。作者就CAR修饰T细胞在临床治疗中的应用、遇到的问题和对应策略作一综述。%Recently,application of chimeric antigen receptor ( CAR) modified T cells in the treatment of lymphoma, leukemia, brain glioma, melanoma, lung cancer, prostate cancer, panc ̄reatic cancer and ovarian cancer has made very exciting progress. CAR is a kind of genetically engi ̄neered receptor,the common form of these molecules consists of extracellular antigen recognition re ̄gion,spacer,transmembrane domain ( TM ) and intracellular endodomain, the antigen recognition region generally is single ̄chain variable fragments ( scFv) derived from monoclonal antibodies which can recognize target antigen without MHC restriction, and the intracellular endodomain generally is comprised of CD3 zeta and co ̄stimulating molecules. This kind of unique design and strong anti ̄tumor effect make the CAR ̄T more and more widely used. In this review, we highlight recent ad ̄vances of CAR ̄T in clinical applications, discovered problems and possible solving strategies in cancer therapy.

  17. Protection of Macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies.

    Science.gov (United States)

    Mascola, J R; Lewis, M G; Stiegler, G; Harris, D; VanCott, T C; Hayes, D; Louder, M K; Brown, C R; Sapan, C V; Frankel, S S; Lu, Y; Robb, M L; Katinger, H; Birx, D L

    1999-05-01

    The role of antibody in protection against human immunodeficiency virus (HIV-1) has been difficult to study in animal models because most primary HIV-1 strains do not infect nonhuman primates. Using a chimeric simian/human immunodeficiency virus (SHIV) based on the envelope of a primary isolate (HIV-89.6), we performed passive-transfer experiments in rhesus macaques to study the role of anti-envelope antibodies in protection. Based on prior in vitro data showing neutralization synergy by antibody combinations, we evaluated HIV immune globulin (HIVIG), and human monoclonal antibodies (MAbs) 2F5 and 2G12 given alone, compared with the double combination 2F5/2G12 and the triple combination HIVIG/2F5/2G12. Antibodies were administered 24 h prior to intravenous challenge with the pathogenic SHIV-89.6PD. Six control monkeys displayed high plasma viremia, rapid CD4(+)-cell decline, and clinical AIDS within 14 weeks. Of six animals given HIVIG/2F5/2G12, three were completely protected; the remaining three animals became SHIV infected but displayed reduced plasma viremia and near normal CD4(+)-cell counts. One of three monkeys given 2F5/2G12 exhibited only transient evidence of infection; the other two had marked reductions in viral load. All monkeys that received HIVIG, 2F5, or 2G12 alone became infected and developed high-level plasma viremia. However, compared to controls, monkeys that received HIVIG or MAb 2G12 displayed a less profound drop in CD4(+) T cells and a more benign clinical course. These data indicate a general correlation between in vitro neutralization and protection and suggest that a vaccine that elicits neutralizing antibody should have a protective effect against HIV-1 infection or disease.

  18. DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody

    Science.gov (United States)

    2016-03-01

    ECBC-TR-1356 DARPA ANTIBODY TECHNOLOGY PROGRAM STANDARDIZED TEST BED FOR ANTIBODY CHARACTERIZATION...From - To) Oct 2010 – Sep 2012 4. TITLE AND SUBTITLE DARPA Antibody Technology Program Standardized Test Bed for Antibody Characterization...Arlington, VA 22203-2114 10. SPONSOR/MONITOR’S ACRONYM(S) DARPA 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 12. DISTRIBUTION / AVAILABILITY STATEMENT

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

    Institute of Scientific and Technical Information of China (English)

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

    2009-01-01

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

  20. Crystal structure of an FIV/HIV chimeric protease complexed with the broad-based inhibitor, TL-3

    Directory of Open Access Journals (Sweden)

    Elder John H

    2007-01-01

    Full Text Available Abstract We have obtained the 1.7 Å crystal structure of FIV protease (PR in which 12 critical residues around the active site have been substituted with the structurally equivalent residues of HIV PR (12X FIV PR. The chimeric PR was crystallized in complex with the broad-based inhibitor TL-3, which inhibits wild type FIV and HIV PRs, as well as 12X FIV PR and several drug-resistant HIV mutants 1234. Biochemical analyses have demonstrated that TL-3 inhibits these PRs in the order HIV PR > 12X FIV PR > FIV PR, with Ki values of 1.5 nM, 10 nM, and 41 nM, respectively 234. Comparison of the crystal structures of the TL-3 complexes of 12X FIV and wild-typeFIV PR revealed theformation of additinal van der Waals interactions between the enzyme inhibitor in the mutant PR. The 12X FIV PR retained the hydrogen bonding interactions between residues in the flap regions and active site involving the enzyme and the TL-3 inhibitor in comparison to both FIV PR and HIV PR. However, the flap regions of the 12X FIV PR more closely resemble those of HIV PR, having gained several stabilizing intra-flap interactions not present in wild type FIV PR. These findings offer a structural explanation for the observed inhibitor/substrate binding properties of the chimeric PR.

  1. Temporary Ectopic Implantation of a Single Finger Using a Perforator as a Feeding Vessel, and Subsequent Prefabricated Chimeric Flap Transplantation

    Science.gov (United States)

    Takumi, Yamamoto; Hisako, Hara; Yusuke, Yamamoto; Azusa, Oshima; Kazuki, Kikuchi; Harunosuke, Kato; Kumiko, Sata; Kentaro, Doi; Takeshi, Todokoro; Jun, Araki; Makoto, Mihara; Takuya, Higashino; Takuya, Iida; Isao, Koshima

    2012-01-01

    Objective: Ectopic implantation was first reported by Godina in 1986. We herein present 2 cases in which amputated fingers were salvaged and reconstructed by means of temporary ectopic implantation utilizing perforator anastomoses and chimeric flaps. Methods: Case 1. A 30-year-old man injured his right hand. All of the fingers were completely crushed with the exception of the little finger. We performed an ectopic implantation by using the superficial circumflex iliac artery perforator. Three months later, the little finger was transplanted with the superficial circumflex iliac artery perforator flap, vascularized nerve, and the 2nd metacarpal bone. Case 2. A 29-year-old man suffered a degloving injury of the index finger. The digital artery was anastomosed to deep inferior epigastric artery perforator. One month later, a deep inferior epigastric artery perforator flap containing the ectopically transplanted index finger was transplanted, but the index fingertip became pale and necrotized. After debridement, a hemipulp transplantation was performed. Results/Conclusions: As the diameter of perforators is similar to that of digital arteries, and perforators are capable of supplying large areas of tissue, they can be used as recipient vessels for ectopic implantation in finger salvage procedures. Another advantage of perforators as feeding vessels in ectopic implantation is the possibility of forming an ectopic chimera; the finger can be incorporated as a part of the chimeric reconstructive flap. With respect to these advantages, the perforator can be used as a feeder in an ectopic implantation of single finger. PMID:27648114

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

    Science.gov (United States)

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

    2014-10-01

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

  3. PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses.

    Science.gov (United States)

    Fedorov, Victor D; Themeli, Maria; Sadelain, Michel

    2013-12-11

    T cell therapies have demonstrated long-term efficacy and curative potential for the treatment of some cancers. However, their use is limited by damage to bystander tissues, as seen in graft-versus-host disease after donor lymphocyte infusion, or "on-target, off-tumor" toxicities incurred in some engineered T cell therapies. Nonspecific immunosuppression and irreversible T cell elimination are currently the only means to control such deleterious responses, but at the cost of abrogating therapeutic benefits or causing secondary complications. On the basis of the physiological paradigm of immune inhibitory receptors, we designed antigen-specific inhibitory chimeric antigen receptors (iCARs) to preemptively constrain T cell responses. We demonstrate that CTLA-4- or PD-1-based iCARs can selectively limit cytokine secretion, cytotoxicity, and proliferation induced through the endogenous T cell receptor or an activating chimeric receptor. The initial effect of the iCAR is temporary, thus enabling T cells to function upon a subsequent encounter with the antigen recognized by their activating receptor. iCARs thus provide a dynamic, self-regulating safety switch to prevent, rather than treat, the consequences of inadequate T cell specificity.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    1994-01-01

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

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

    Science.gov (United States)

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

    2013-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Huawei Zhang

    2014-12-01

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

  8. Evolution of antiphospholipid antibody syndrome.

    Science.gov (United States)

    Baviskar, Rutuja R; Amonkar, Gayathri P; Chaudhary, Vinod A; Balasubramanian, Meenakshi; Mohite, Shailesh C; Puranik, Gururaj V

    2012-12-01

    Antiphospholipid antibody syndrome is a very important cause of cerebral infarction, myocardial infarction, and repeated pregnancy losses in women. We present an extremely rare case of a 44-year-old man with antiphospholipid syndrome who collapsed and died suddenly. At autopsy, he was found to have both cerebral and myocardial infarction. In all young patients with cerebral infarction, myocardial infarction, pulmonary embolism, recurrent miscarriages, and unexplained low platelet count, one must consider the strong possibility of antiphospholipid antibody syndrome.

  9. Antibodies to watch in 2016

    OpenAIRE

    Reichert, Janice M

    2015-01-01

    The number of novel antibody therapeutics that received first marketing approvals in 2015 met expectations, with 6 (alirocumab (Praluent®), evolocumab (Repatha®), daratumumab (Darzalex®), dinutuximab (Unituxin®), idarucizumab (Praxbind®), mepolizumab (Nucala®)) granted first approvals as of mid-November*. Seven novel antibody therapeutics (begelomab, brodalumab, elotuzumab, ixekizumab, necitumumab, obiltoxaximab, reslizumab) are in regulatory review, and thus a similar number, if not more, ar...

  10. The construction and expression of chimeric urokinase-type plasminogen activator genes containing kringle domains of human plasminogen.

    Science.gov (United States)

    Boutaud, A; Castellino, F J

    1993-06-01

    A series of chimeric urokinase-type plasminogen activator (uPA) genes, which contain combinations of kringle domains of human plasminogen (HPg) in place of the uPA kringle (KuPA), has been constructed and expressed. Some of the resulting recombinant (r) variant uPA chimeras contain modules that potentially mediate the macroscopic binding of HPg to its activation effectors, fibrin(ogen) and 6-aminohexanoic acid (EACA). Such binding sites are not possessed by KuPA, but are present in certain of the HPg kringles, viz., kringle 1 (K1HPg), kringle 4 (K4HPg), and kringle 5 (K5HPg). The recombinant (r) chimeras constructed included molecules with replacements of KuPA with K1HPg (r-[KuPA-->K1HPg]uPA), and with KuPA replaced by double kringle combinations of K1HPgK4HPg (r-[KuPA-->K1HPgK4HPg]uPA), K2HPgK3HPg (r-[KuPA-->K2HPgK3HPg]uPA), and K4HPgK5HPg (r-[KuPA-->K4HPgK5HPg]uPA). All of these variant genes, along with their wild-type (wt) r-uPA counterparts, were expressed in human kidney 293 cells. In cases wherein EACA-binding kringles from HPg have been placed in uPA, this property has been retained in the chimeric molecule and employed as an essential part of the purification procedures for the variants. The steady state amidolytic activity of two-chain (tc) wtr-uPA toward the chromogenic substrate, H-D-pyroglutamyl-Gly-L-Arg-p-nitroanilide (S2444), is characterized by a kcat/KM (pH 7.4, 37 degrees C) of 120 s-1 mM-1. This value ranges from 92 s-1 mM-1 (tcr-[KuPA-->K1HPg]uPA) to 166 s-1 mM-1 (tcr-[KuPA-->K1HPgK4HPg]uPA) for each of the variants, demonstrating that the catalytic efficiency of the active site is altered only in a small way by changes in the noncatalytic domain of uPA. Small differences are also observed in the abilities of these tcr variants to interact with the fast-acting plasma inhibitor of uPA, viz., plasminogen activator inhibitor-1 (PAI-1). The second-order rate constant for the interaction of PAI-1 with tcr-uPA, 0.46 x 10(7) M-1s-1 (pH 7.4, 10 degrees

  11. Tabhu: tools for antibody humanization.

    KAUST Repository

    Olimpieri, Pier Paolo

    2014-10-09

    SUMMARY: Antibodies are rapidly becoming essential tools in the clinical practice, given their ability to recognize their cognate antigens with high specificity and affinity, and a high yield at reasonable costs in model animals. Unfortunately, when administered to human patients, xenogeneic antibodies can elicit unwanted and dangerous immunogenic responses. Antibody humanization methods are designed to produce molecules with a better safety profile still maintaining their ability to bind the antigen. This can be accomplished by grafting the non-human regions determining the antigen specificity into a suitable human template. Unfortunately, this procedure may results in a partial or complete loss of affinity of the grafted molecule that can be restored by back-mutating some of the residues of human origin to the corresponding murine ones. This trial-and-error procedure is hard and involves expensive and time-consuming experiments. Here we present tools for antibody humanization (Tabhu) a web server for antibody humanization. Tabhu includes tools for human template selection, grafting, back-mutation evaluation, antibody modelling and structural analysis, helping the user in all the critical steps of the humanization experiment protocol. AVAILABILITY: http://www.biocomputing.it/tabhu CONTACT: anna.tramontano@uniroma1.it, pierpaolo.olimpieri@uniroma1.it SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

  12. Avian Diagnostic and Therapeutic Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Bradley, David Sherman [UND SMHS

    2012-12-31

    A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.

  13. Migration of antibody secreting cells towards CXCL12 depends on the isotype that forms the BCR

    Science.gov (United States)

    Achatz-Straussberger, Gertrude; Zaborsky, Nadja; Königsberger, Sebastian; Luger, Elke O.; Lamers, Marinus; Crameri, Reto; Achatz, Gernot

    2010-01-01

    Truncation of the cytoplasmic tail of membrane-bound IgE in vivo results in lower serum IgE levels, decreased numbers of IgE-secreting plasma cells and the abrogation of specific secondary immune responses. Here we present mouse strain KN1 that expresses a chimeric ε-γ1 BCR, consisting of the extracellular domains of the ε gene and the trans-membrane and cytoplasmic domains of the γ1 gene. Thus, differences in the IgE immune response of KN1 mice reflect the influence of the “γ1-mediated signalling” of mIgE bearing B cells. KN1 mice show an increased serum IgE level, resulting from an elevated number of IgE-secreting cells. Although the primary IgE immune response in KN1 mice is inconspicuous, the secondary response is far more robust. Most strikingly, IgE-antibody secreting cells with “γ1-signalling history” migrate more efficiently towards the chemokine CXCL12, which guides plasmablasts to plasma cell niches, than IgE-antibody secreting cells with WT “ε-signalling history”. We conclude that IgE plasmablasts have an intrinsic, lower chance to contribute to the long-lived plasma cell pool than IgG1 plasmablasts