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Sample records for chimeric mice provide

  1. Steroid metabolism in chimeric mice with humanized liver.

    PubMed

    Lootens, Leen; Van Eenoo, Peter; Meuleman, Philip; Pozo, Oscar J; Van Renterghem, Pieter; Leroux-Roels, Geert; Delbeke, Frans T

    2009-11-01

    Anabolic androgenic steroids are considered to be doping agents and are prohibited in sports. Their metabolism needs to be elucidated to allow for urinary detection by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Steroid metabolism was assessed using uPA(+/+) SCID mice with humanized livers (chimeric mice). This study presents the results of 19-norandrost-4-ene-3,17-dione (19-norAD) administration to these in vivo mice. As in humans, 19-norandrosterone and 19-noretiocholanolone are the major detectable metabolites of 19-norAD in the urine of chimeric mice.A summary is given of the metabolic pathways found in chimeric mice after administration of three model steroid compounds (methandienone, androst-4-ene-3,17-dione and 19-norandrost-4-ene-3,17-dione). From these studies we can conclude that all major metabolic pathways for anabolic steroids in humans are present in the chimeric mouse. It is hoped that, in future, this promising chimeric mouse model might assist the discovery of new and possible longer detectable metabolites of (designer) steroids. PMID:20355169

  2. Chimeric elk/mouse prion proteins in transgenic mice

    PubMed Central

    Tamgüney, Gültekin; Giles, Kurt; Oehler, Abby; Johnson, Natrina L.; DeArmond, Stephen J.

    2013-01-01

    Chronic wasting disease (CWD) of deer and elk is a highly communicable neurodegenerative disorder caused by prions. Investigations of CWD are hampered by slow bioassays in transgenic (Tg) mice. Towards the development of Tg mice that will be more susceptible to CWD prions, we created a series of chimeric elk/mouse transgenes that encode the N terminus of elk PrP (ElkPrP) up to residue Y168 and the C terminus of mouse PrP (MoPrP) beyond residue 169 (mouse numbering), designated Elk3M(SNIVVK). Between codons 169 and 219, six residues distinguish ElkPrP from MoPrP: N169S, T173N, V183I, I202V, I214V and R219K. Using chimeric elk/mouse PrP constructs, we generated 12 Tg mouse lines and determined incubation times after intracerebral inoculation with the mouse-passaged RML scrapie or Elk1P CWD prions. Unexpectedly, one Tg mouse line expressing Elk3M(SNIVVK) exhibited incubation times of <70 days when inoculated with RML prions; a second line had incubation times of <90 days. In contrast, mice expressing full-length ElkPrP had incubation periods of >250 days for RML prions. Tg(Elk3M,SNIVVK) mice were less susceptible to CWD prions than Tg(ElkPrP) mice. Changing three C-terminal mouse residues (202, 214 and 219) to those of elk doubled the incubation time for mouse RML prions and rendered the mice resistant to Elk1P CWD prions. Mutating an additional two residues from mouse to elk at codons 169 and 173 increased the incubation times for mouse prions to >300 days, but made the mice susceptible to CWD prions. Our findings highlight the role of C-terminal residues in PrP that control the susceptibility and replication of prions. PMID:23100369

  3. Using chimeric mice with humanized livers to predict human drug metabolism and a drug-drug interaction.

    PubMed

    Nishimura, Toshihiko; Nishimura, Toshiko; Hu, Yajing; Wu, Manhong; Pham, Edward; Suemizu, Hiroshi; Elazar, Menashe; Liu, Michael; Idilman, Ramazan; Yurdaydin, Cihan; Angus, Peter; Stedman, Catherine; Murphy, Brian; Glenn, Jeffrey; Nakamura, Masato; Nomura, Tatsuji; Chen, Yuan; Zheng, Ming; Fitch, William L; Peltz, Gary

    2013-02-01

    Interspecies differences in drug metabolism have made it difficult to use preclinical animal testing data to predict the drug metabolites or potential drug-drug interactions (DDIs) that will occur in humans. Although chimeric mice with humanized livers can produce known human metabolites for test substrates, we do not know whether chimeric mice can be used to prospectively predict human drug metabolism or a possible DDI. Therefore, we investigated whether they could provide a more predictive assessment for clemizole, a drug in clinical development for the treatment of hepatitis C virus (HCV) infection. Our results demonstrate, for the first time, that analyses performed in chimeric mice can correctly identify the predominant human drug metabolite before human testing. The differences in the rodent and human pathways for clemizole metabolism were of importance, because the predominant human metabolite was found to have synergistic anti-HCV activity. Moreover, studies in chimeric mice also correctly predicted that a DDI would occur in humans when clemizole was coadministered with a CYP3A4 inhibitor. These results demonstrate that using chimeric mice can improve the quality of preclinical drug assessment. PMID:23143674

  4. Temporally chimeric mice reveal flexibility of circadian period-setting in the suprachiasmatic nucleus

    PubMed Central

    Smyllie, Nicola J.; Chesham, Johanna E.; Hamnett, Ryan; Maywood, Elizabeth S.; Hastings, Michael H.

    2016-01-01

    The suprachiasmatic nucleus (SCN) is the master circadian clock controlling daily behavior in mammals. It consists of a heterogeneous network of neurons, in which cell-autonomous molecular feedback loops determine the period and amplitude of circadian oscillations of individual cells. In contrast, circuit-level properties of coherence, synchrony, and ensemble period are determined by intercellular signals and are embodied in a circadian wave of gene expression that progresses daily across the SCN. How cell-autonomous and circuit-level mechanisms interact in timekeeping is poorly understood. To explore this interaction, we used intersectional genetics to create temporally chimeric mice with SCN containing dopamine 1a receptor (Drd1a) cells with an intrinsic period of 24 h alongside non-Drd1a cells with 20-h clocks. Recording of circadian behavior in vivo alongside cellular molecular pacemaking in SCN slices in vitro demonstrated that such chimeric circuits form robust and resilient circadian clocks. It also showed that the computation of ensemble period is nonlinear. Moreover, the chimeric circuit sustained a wave of gene expression comparable to that of nonchimeric SCN, demonstrating that this circuit-level property is independent of differences in cell-intrinsic periods. The relative dominance of 24-h Drd1a and 20-h non-Drd1a neurons in setting ensemble period could be switched by exposure to resonant or nonresonant 24-h or 20-h lighting cycles. The chimeric circuit therefore reveals unanticipated principles of circuit-level operation underlying the emergent plasticity, resilience, and robustness of the SCN clock. The spontaneous and light-driven flexibility of period observed in chimeric mice provides a new perspective on the concept of SCN pacemaker cells. PMID:26966234

  5. Temporally chimeric mice reveal flexibility of circadian period-setting in the suprachiasmatic nucleus.

    PubMed

    Smyllie, Nicola J; Chesham, Johanna E; Hamnett, Ryan; Maywood, Elizabeth S; Hastings, Michael H

    2016-03-29

    The suprachiasmatic nucleus (SCN) is the master circadian clock controlling daily behavior in mammals. It consists of a heterogeneous network of neurons, in which cell-autonomous molecular feedback loops determine the period and amplitude of circadian oscillations of individual cells. In contrast, circuit-level properties of coherence, synchrony, and ensemble period are determined by intercellular signals and are embodied in a circadian wave of gene expression that progresses daily across the SCN. How cell-autonomous and circuit-level mechanisms interact in timekeeping is poorly understood. To explore this interaction, we used intersectional genetics to create temporally chimeric mice with SCN containing dopamine 1a receptor (Drd1a) cells with an intrinsic period of 24 h alongside non-Drd1a cells with 20-h clocks. Recording of circadian behavior in vivo alongside cellular molecular pacemaking in SCN slices in vitro demonstrated that such chimeric circuits form robust and resilient circadian clocks. It also showed that the computation of ensemble period is nonlinear. Moreover, the chimeric circuit sustained a wave of gene expression comparable to that of nonchimeric SCN, demonstrating that this circuit-level property is independent of differences in cell-intrinsic periods. The relative dominance of 24-h Drd1a and 20-h non-Drd1a neurons in setting ensemble period could be switched by exposure to resonant or nonresonant 24-h or 20-h lighting cycles. The chimeric circuit therefore reveals unanticipated principles of circuit-level operation underlying the emergent plasticity, resilience, and robustness of the SCN clock. The spontaneous and light-driven flexibility of period observed in chimeric mice provides a new perspective on the concept of SCN pacemaker cells. PMID:26966234

  6. The Macrophage-depleting Agent Clodronate Promotes Durable Hematopoietic Chimerism and Donor-specific Skin Allograft Tolerance in Mice

    PubMed Central

    Li, Zhanzhuo; Xu, Xin; Feng, Xingmin; Murphy, Philip M.

    2016-01-01

    Hematopoietic chimerism is known to promote donor-specific organ allograft tolerance; however, clinical translation has been impeded by the requirement for toxic immunosuppression and large doses of donor bone marrow (BM) cells. Here, we investigated in mice whether durable chimerism might be enhanced by pre-treatment of the recipient with liposomal clodronate, a macrophage depleting agent, with the goal of vacating BM niches for preferential reoccupation by donor hematopoietic stem cells (HSC). We found that liposomal clodronate pretreatment of C57BL/6 mice permitted establishment of durable hematopoietic chimerism when the mice were given a low dose of donor BM cells and transient immunosuppression. Moreover, clodronate pre-treatment increased durable donor-specific BALB/c skin allograft tolerance. These results provide proof-of-principle that clodronate is effective at sparing the number of donor BM cells required to achieve durable hematopoietic chimerism and donor-specific skin allograft tolerance and justify further development of a tolerance protocol based on this principle. PMID:26917238

  7. An optimization of protocol for mixed chimerism induction in mice model.

    PubMed

    Baśkiewicz-Masiuk, M; Grymuła, K; Pius, E; Hałasa, M; Dziedziejko, V; Schmidt, Ch; Walczak, M; Machaliński, B

    2009-01-01

    Studies on mixed chimerism are currently focused primarily on obtaining less toxic conditioning protocols. With these issues in mind, we have undertaken the attempt to optimize the procedure of mixed chimerism induction in mice. In order to reduce toxicity, we used decreasing doses of total body irradiation (TBI) together with combination of blocking antibodies. We also tried to eliminate immunosuppression (cyclophosphamide - CP) treatment after bone marrow transplantation. B6.SJL-PtprcaPep3b mice were injected with 20-30 x 106 bone marrow cells from Balb C mice. Mice were treated with TBI (3 - 1.5 - 0 Gy) on "-1" day of the experiment and blocking antibodies against CD40L ("0", and "4" days) and additionally anti-CD8 ("-2" day) and/or anti-NK1.1 ("-3" day). Mice in certain groups also received CP (175 mg/kg) on "2" day. Presence of mixed chimerism was assessed in peripheral blood cells by flow cytometry on the 1st, 2nd, 3rd, 4th, 6th and 8th weeks of the experiment by detecting of CD45.1 (characteristic for B6.SJL-PtprcaPep3b strain) and CD45.2 (characteristic for Balb C strain) antigens expression. We also analyzed the percentage of peripheral blood CD8 T-cells (CD3e/CD8a) and NK cells (Ly-49D/NK1.1). We found that reduction of TBI dose and elimination of CP decrease the rate of mixed chimerism formation. The highest percentage of donor cells was obtained in the group of animals treated with 3 Gy of TBI, CP and combination of anti-CD40L, anti-CD8, and anti-NK1.1 antibodies. The 3 Gy TBI was necessary to induce stable mixed chimerism, but it could be obtained without the CP use. The percentage of CD3e/CD8a and Ly-49D/NK1.1 cells was significantly lower in the groups of mice treated by corresponding antibodies. Moreover, we observed the lowest number of peripheral blood Ly-49D/NK1.1 cells in the group of animals with highest mixed chimerism. Our experiments in mice model can help in better understanding of mixed chimerism phenomenon and in selecting the method of

  8. CHIMERIC SINDBIS/EASTERN EQUINE ENCEPHALITIS VACCINE CANDIDATES ARE HIGHLY ATTENUATED AND IMMUNOGENIC IN MICE

    PubMed Central

    Wang, Eryu; Petrakova, Olga; Adams, A. Paige; Aguilar, Patricia V.; Kang, Wenli; Paessler, Slobodan; Volk, Sara M.; Frolov, Ilya; Weaver, Scott C.

    2007-01-01

    We developed chimeric Sindbis (SINV)/Eastern equine encephalitis (EEEV) viruses and investigated their potential for use as live virus vaccines against EEEV. One vaccine candidate contained structural protein genes from a typical North American EEEV strain, while the other had structural proteins from a naturally attenuated Brazilian isolate. Both chimeric viruses replicated efficiently in mammalian and mosquito cell cultures and were highly attenuated in mice. Vaccinated mice did not develop detectable disease or viremia, but developed high titers of neutralizing antibodies. Upon challenge with EEEV, mice vaccinated with >104PFU of the chimeric viruses were completely protected from disease. These findings support the potential use of these SIN/EEEV chimeras as safe and effective vaccines. PMID:17904699

  9. Expression of a Chimeric Antigen Receptor in Multiple Leukocyte Lineages in Transgenic Mice.

    PubMed

    Yong, Carmen S M; Westwood, Jennifer A; Schröder, Jan; Papenfuss, Anthony T; von Scheidt, Bianca; Moeller, Maria; Devaud, Christel; Darcy, Phillip K; Kershaw, Michael H

    2015-01-01

    Genetically modified CD8+ T lymphocytes have shown significant anti-tumor effects in the adoptive immunotherapy of cancer, with recent studies highlighting a potential role for a combination of other immune subsets to enhance these results. However, limitations in present genetic modification techniques impose difficulties in our ability to fully explore the potential of various T cell subsets and assess the potential of other leukocytes armed with chimeric antigen receptors (CARs). To address this issue, we generated a transgenic mouse model using a pan-hematopoietic promoter (vav) to drive the expression of a CAR specific for a tumor antigen. Here we present a characterization of the immune cell compartment in two unique vav-CAR transgenic mice models, Founder 9 (F9) and Founder 38 (F38). We demonstrate the vav promoter is indeed capable of driving the expression of a CAR in cells from both myeloid and lymphoid lineage, however the highest level of expression was observed in T lymphocytes from F38 mice. Lymphoid organs in vav-CAR mice were smaller and had reduced cell numbers compared to the wild type (WT) controls. Furthermore, the immune composition of F9 mice differed greatly with a significant reduction in lymphocytes found in the thymus, lymph node and spleen of these mice. To gain insight into the altered immune phenotype of F9 mice, we determined the chromosomal integration site of the transgene in both mouse strains using whole genome sequencing (WGS). We demonstrated that compared to the 7 copies found in F38 mice, F9 mice harbored almost 270 copies. These novel vav-CAR models provide a ready source of CAR expressing myeloid and lymphoid cells and will aid in facilitating future experiments to delineate the role for other leukocytes for adoptive immunotherapy against cancer. PMID:26505904

  10. Expression of a Chimeric Antigen Receptor in Multiple Leukocyte Lineages in Transgenic Mice

    PubMed Central

    Yong, Carmen S. M.; Westwood, Jennifer A.; Schröder, Jan; Papenfuss, Anthony T.; von Scheidt, Bianca; Moeller, Maria; Devaud, Christel

    2015-01-01

    Genetically modified CD8+ T lymphocytes have shown significant anti-tumor effects in the adoptive immunotherapy of cancer, with recent studies highlighting a potential role for a combination of other immune subsets to enhance these results. However, limitations in present genetic modification techniques impose difficulties in our ability to fully explore the potential of various T cell subsets and assess the potential of other leukocytes armed with chimeric antigen receptors (CARs). To address this issue, we generated a transgenic mouse model using a pan-hematopoietic promoter (vav) to drive the expression of a CAR specific for a tumor antigen. Here we present a characterization of the immune cell compartment in two unique vav-CAR transgenic mice models, Founder 9 (F9) and Founder 38 (F38). We demonstrate the vav promoter is indeed capable of driving the expression of a CAR in cells from both myeloid and lymphoid lineage, however the highest level of expression was observed in T lymphocytes from F38 mice. Lymphoid organs in vav-CAR mice were smaller and had reduced cell numbers compared to the wild type (WT) controls. Furthermore, the immune composition of F9 mice differed greatly with a significant reduction in lymphocytes found in the thymus, lymph node and spleen of these mice. To gain insight into the altered immune phenotype of F9 mice, we determined the chromosomal integration site of the transgene in both mouse strains using whole genome sequencing (WGS). We demonstrated that compared to the 7 copies found in F38 mice, F9 mice harbored almost 270 copies. These novel vav-CAR models provide a ready source of CAR expressing myeloid and lymphoid cells and will aid in facilitating future experiments to delineate the role for other leukocytes for adoptive immunotherapy against cancer. PMID:26505904

  11. Fialuridine Induces Acute Liver Failure in Chimeric TK-NOG Mice: A Model for Detecting Hepatic Drug Toxicity Prior to Human Testing

    PubMed Central

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

    2014-01-01

    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 of chimeric mice in

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

    PubMed Central

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

    2015-01-01

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

  13. Recombinant chimeric Japanese encephalitis virus/tick-borne encephalitis virus is attenuated and protective in mice.

    PubMed

    Wang, Hong-Jiang; Li, Xiao-Feng; Ye, Qing; Li, Shi-Hua; Deng, Yong-Qiang; Zhao, Hui; Xu, Yan-Peng; Ma, Jie; Qin, E-De; Qin, Cheng-Feng

    2014-02-12

    Tick-borne encephalitis virus (TBEV) represents one of the most dangerous human pathogens circulating in Europe and East Asia. No effective treatment for TBEV infection currently exists, and vaccination is the primary preventive measure. Although several inactivated vaccines have been licensed, the development of novel vaccines against TBEV remains a high priority in disease-endemic countries. In the present study, a live chimeric recombinant TBEV (ChinTBEV) was created by substituting the major structural genes of TBEV for the corresponding regions of Japanese encephalitis virus (JEV) live vaccine strain SA14-14-2. The resulting chimera had a small-plaque phenotype, replicated efficiently in both mammalian and mosquito cells. The preliminary data from in vitro passaging indicated the potential for stability of ChinTBEV. ChinTBEV also exhibited significantly attenuated neuroinvasiveness in mice upon either intraperitoneal or subcutaneous inoculation in comparison with its parental TBEV. Importantly, a single immunisation with ChinTBEV elicited TBEV-specific IgG and neutralising antibody responses in a dose-dependent manner, providing significant protection against lethal TBEV challenge in mice. Taken together, the results of this proof-of-concept study indicate that ChinTBEV can be further developed as a potential vaccine candidate against TBEV infection. Moreover, the construction of this type of flavivirus chimera using a JEV vaccine strain as the genetic backbone represents a universal vaccine approach. PMID:24394443

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

    PubMed

    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. PMID:24700822

  15. Mice with Chimeric Livers Are an Improved Model for Human Lipoprotein Metabolism

    PubMed Central

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

    2013-01-01

    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 [Fah(−/−)Rag2(−/−)Il2rg(−/−)]) 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. PMID:24223822

  16. Tissue-specific and developmentally regulated expression of a chimeric actin-globin gene in transgenic mice.

    PubMed Central

    Shani, M

    1986-01-01

    A chimeric plasmid containing about 2/3 of the rat skeletal muscle actin gene plus 730 base pairs of its 5' flanking sequences fused to the 3' end of a human embryonic globin gene (D. Melloul, B. Aloni, J. Calvo, D. Yaffe, and U. Nudel, EMBO J. 3:983-990, 1984) was inserted into mice by microinjection into fertilized eggs. Eleven transgenic mice carrying the chimeric gene with or without plasmid pBR322 DNA sequences were identified. The majority of these mice transmitted the injected DNA to about 50% of their progeny. However, in transgenic mouse CV1, transmission to progeny was associated with amplification or deletion of the injected DNA sequences, while in transgenic mouse CV4 transmission was distorted, probably as a result of insertional mutagenesis. Tissue-specific expression was dependent on the removal of the vector DNA sequences from the chimeric gene sequences prior to microinjection. None of the transgenic mice carrying the chimeric gene together with plasmid pBR322 sequences expressed the introduced gene in striated muscles. In contrast, the six transgenic mice carrying the chimeric gene sequences alone expressed the inserted gene specifically in skeletal and cardiac muscles. Moreover, expression of the chimeric gene was not only tissue specific, but also developmentally regulated. Similar to the endogenous skeletal muscle actin gene, the chimeric gene was expressed at a relatively high level in cardiac muscle of neonatal mice and at a significantly lower level in adult cardiac muscle. These results indicate that the injected DNA included sufficient cis-acting control elements for its tissue-specific and developmentally regulated expression in transgenic mice. Images PMID:3023942

  17. CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice

    PubMed Central

    Oji, Asami; Noda, Taichi; Fujihara, Yoshitaka; Miyata, Haruhiko; Kim, Yeon Joo; Muto, Masanaga; Nozawa, Kaori; Matsumura, Takafumi; Isotani, Ayako; Ikawa, Masahito

    2016-01-01

    Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenotypic analyses in these animals. Here we show that large deletions with two sgRNAs as well as dsDNA-mediated point mutations are efficient in mouse embryonic stem cells (ESCs). The dsDNA-mediated gene knockins are also feasible in ESCs. Finally, we generated chimeric mice with biallelic mutant ESCs for a lethal gene, Dnajb13, and analyzed their phenotypes. Not only was the lethal phenotype of hydrocephalus suppressed, but we also found that Dnajb13 is required for sperm cilia formation. The combination of biallelic genome editing in ESCs and subsequent chimeric analysis provides a useful tool for rapid gene function analysis in the whole organism. PMID:27530713

  18. CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice.

    PubMed

    Oji, Asami; Noda, Taichi; Fujihara, Yoshitaka; Miyata, Haruhiko; Kim, Yeon Joo; Muto, Masanaga; Nozawa, Kaori; Matsumura, Takafumi; Isotani, Ayako; Ikawa, Masahito

    2016-01-01

    Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenotypic analyses in these animals. Here we show that large deletions with two sgRNAs as well as dsDNA-mediated point mutations are efficient in mouse embryonic stem cells (ESCs). The dsDNA-mediated gene knockins are also feasible in ESCs. Finally, we generated chimeric mice with biallelic mutant ESCs for a lethal gene, Dnajb13, and analyzed their phenotypes. Not only was the lethal phenotype of hydrocephalus suppressed, but we also found that Dnajb13 is required for sperm cilia formation. The combination of biallelic genome editing in ESCs and subsequent chimeric analysis provides a useful tool for rapid gene function analysis in the whole organism. PMID:27530713

  19. Chimeric Alphavirus Vaccine Candidates Protect Mice from Intranasal Challenge with Western Equine Encephalitis Virus

    PubMed Central

    Atasheva, Svetlana; Wang, Eryu; Adams, A. Paige; Plante, Kenneth S.; Ni, Sai; Taylor, Katherine; Miller, Mary E.; Frolov, Ilya; Weaver, Scott C.

    2011-01-01

    We developed two types of chimeric Sindbis virus (SINV)/western equine encephalitis virus (WEEV) alphaviruses to investigate their potential use as live virus vaccines against WEE. The first-generation vaccine candidate, SIN/CO92, was derived from structural protein genes of WEEV strain CO92-1356, and two second-generation candidates were derived from WEEV strain McMillan. For both first- and second-generation vaccine candidates, the nonstructural protein genes were derived from SINV strain AR339. Second-generation vaccine candidates SIN/SIN/McM and SIN/EEE/McM included the envelope glycoprotein genes from WEEV strain McMillan; however, the amino-terminal half of the capsid, which encodes the RNA-binding domain, was derived from either SINV or eastern equine encephalitis virus (EEEV) strain FL93-939. All chimeric viruses replicated efficiently in mammalian and mosquito cell cultures and were highly attenuated in 6-week-old mice. Vaccinated mice developed little or no detectable disease and showed little or no evidence of challenge virus replication; however, all developed high titers of neutralizing antibodies. Upon intranasal challenge with high doses of virulent WEEV strains, mice vaccinated with ≥105 PFU of SIN/CO92 or ≥104 PFU of SIN/SIN/McM or SIN/EEE/McM were completely protected from disease. These findings support the potential use of these live-attenuated vaccine candidates as safe and effective vaccines against WEE. PMID:19446595

  20. Hematopoietic mixed chimerism derived from allogeneic embryonic stem cells prevents autoimmune diabetes mellitus in NOD mice.

    PubMed

    Verda, Larissa; Kim, Duck An; Ikehara, Susumu; Statkute, Laisvyde; Bronesky, Delphine; Petrenko, Yevgeniya; Oyama, Yu; He, Xiang; Link, Charles; Vahanian, Nicholas N; Burt, Richard K

    2008-02-01

    Embryonic stem cell (ESC)-derived hematopoietic stem cells (HSC), unlike HSC harvested from the blood or marrow, are not contaminated by lymphocytes. We therefore evaluated whether ESC-derived HSC could produce islet cell tolerance, a phenomenon termed graft versus autoimmunity (GVA), without causing the usual allogeneic hematopoietic stem cell transplant complication, graft-versus-host disease (GVHD). Herein, we demonstrate that ESC-derived HSC may be used to prevent autoimmune diabetes mellitus in NOD mice without GVHD or other adverse side effects. ESC were cultured in vitro to induce differentiation toward HSC, selected for c-kit expression, and injected either i.v. or intra-bone marrow (IBM) into sublethally irradiated NOD/LtJ mice. Nine of 10 mice from the IBM group and 5 of 8 from the i.v. group did not become hyperglycemic, in contrast to the control group, in which 8 of 9 mice developed end-stage diabetes. All mice with >5% donor chimerism remained free of diabetes and insulitis, which was confirmed by histology. Splenocytes from transplanted mice were unresponsive to glutamic acid decarboxylase isoform 65, a diabetic-specific autoantigen, but responded normally to third-party antigens. ESC-derived HSC can induce an islet cell tolerizing GVA effect without GVHD. This study represents the first instance, to our knowledge, of ESC-derived HSC cells treating disease in an animal model. PMID:17975228

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2014-12-01

    Annual health threats and economic damages caused by influenza virus are still a main concern of the World Health Organization and other health departments all over the world. An influenza virosome is a highly efficient immunomodulating carrier mimicking the natural antigen presentation pathway and has shown an excellent tolerability profile due to its biocompatibility and purity. The major purpose of this study was to construct a new chimeric virosome influenza vaccine containing hemagglutinin (HA) and neuraminidase (NA) proteins derived from the A/PR/8/1934 (H1N1) (PR8) and A/X/47 (H3N2) (X47) viruses, and to evaluate its efficacy as a vaccine candidate in mice. A single intramuscular vaccination with the chimeric virosomes provided complete protection against lethal challenge with the PR8 and X47 viruses. The chimeric virosomes induced high IgG antibody responses as well as hemagglutination inhibition (HAI) titers. HAI titers following the chimeric virosome vaccination were at the same level as the whole inactivated influenza vaccine. Mice immunized with the chimeric virosomes displayed considerably less weight loss and exhibited significantly reduced viral load in their lungs compared with the controls. The chimeric virosomes can be used as an innovative vaccine formulation to confer protection against a broad range of influenza viruses. PMID:25066138

  4. Mouse retrovirus mediates porcine endogenous retrovirus transmission into human cells in long-term human-porcine chimeric mice

    PubMed Central

    Yang, Yong-Guang; Wood, James C.; Lan, Ping; Wilkinson, Robert A.; Sykes, Megan; Fishman, Jay A.; Patience, Clive

    2004-01-01

    Porcine endogenous retrovirus (PERV) is a potential pathogen in clinical xenotransplantation; transmission of PERV in vivo has been suggested in murine xenotransplantation models. We analyzed the transmission of PERV to human cells in vivo using a model in which immunodeficient NOD/SCID transgenic mice were transplanted with porcine and human lymphohematopoietic tissues. Our results demonstrate, we believe for the first time, that human and pig cells can coexist long-term (up to 25 weeks) without direct PERV infection of human cells. Despite the transplantation of porcine cells that did not produce human-tropic PERV, human cells from the chimeric mice were frequently found to contain PERV sequences. However, this transmission was due to the pseudotyping of PERV-C (a virus without human tropism) by xenotropic murine leukemia virus, rather than to de novo generation of human-tropic PERV. Thus, pseudotyping might account for the PERV transmission previously observed in mice. The absence of direct human cell infection following long-term in vivo coexistence with large numbers of porcine cells provides encouragement regarding the potential safety of using pigs that do not produce human-tropic PERV as source animals for transplantation to humans. PMID:15343388

  5. Chimeric tick-borne encephalitis and dengue type 4 viruses: effects of mutations on neurovirulence in mice.

    PubMed Central

    Pletnev, A G; Bray, M; Lai, C J

    1993-01-01

    Two new chimeric flaviviruses were constructed from full-length cDNAs that contained tick-borne encephalitis virus (TBEV) CME or ME structural protein genes and the remaining genes derived from dengue type 4 virus (DEN4). Studies involving mice inoculated intracerebrally with the ME chimeric virus indicated that it retained the neurovirulence of its TBEV parent from which its pre-M and E genes were derived. However, unlike parental TBEV, the chimeric virus did not produce encephalitis when mice were inoculated peripherally, indicating a loss of neuroinvasiveness. In the present study, the ME chimeric virus (vME) was subjected to mutational analysis in an attempt to reduce or ablate neurovirulence measured by direct inoculation of virus into the brain. We identified three distinct mutations that were each associated independently with a significant reduction of mouse neurovirulence of vME. These mutations ablated (i) the TBEV pre-M cleavage site, (ii) the TBEV E glycosylation site, or (iii) the first DEN4 NS1 glycosylation site. In contrast, ablation of the second DEN4 NS1 glycosylation site or the TBE pre-M glycosylation site or amino acid substitution at two positions in the TBEV E protein increased neurovirulence. The only conserved feature of the three attenuated mutants was restriction of virus yield in both simian and mosquito cells. Following parenteral inoculation, these attenuated mutants induced complete resistance in mice to fatal encephalitis caused by the highly neurovirulent vME. Images PMID:8331735

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-29

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    2016-01-01

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

  11. Novel recombinant chimeric virus-like particle is immunogenic and protective against both enterovirus 71 and coxsackievirus A16 in mice.

    PubMed

    Zhao, Hui; Li, Hao-Yang; Han, Jian-Feng; Deng, Yong-Qiang; Zhu, Shun-Ya; Li, Xiao-Feng; Yang, Hui-Qin; Li, Yue-Xiang; Zhang, Yu; Qin, E-De; Chen, Rong; Qin, Cheng-Feng

    2015-01-01

    Hand-foot-and-mouth disease (HFMD) has been recognized as an important global public health issue, which is predominantly caused by enterovirus 71 (EV-A71) and coxsackievirus A16 (CVA16). There is no available vaccine against HFMD. An ideal HFMD vaccine should be bivalent against both EV-A71 and CVA16. Here, a novel strategy to produce bivalent HFMD vaccine based on chimeric EV-A71 virus-like particles (ChiEV-A71 VLPs) was proposed and illustrated. The neutralizing epitope SP70 within the capsid protein VP1 of EV-A71 was replaced with that of CVA16 in ChiEV-A71 VLPs. Structural modeling revealed that the replaced CVA16-SP70 epitope is well exposed on the surface of ChiEV-A71 VLPs. These VLPs produced in Saccharomyces cerevisiae exhibited similarity in both protein composition and morphology as naive EV-A71 VLPs. Immunization with ChiEV-A71 VLPs in mice elicited robust Th1/Th2 dependent immune responses against EV-A71 and CVA16. Furthermore, passive immunization with anti-ChiEV-A71 VLPs sera conferred full protection against lethal challenge of both EV-A71 and CVA16 infection in neonatal mice. These results suggested that this chimeric vaccine, ChiEV-A71 might have the potential to be further developed as a bivalent HFMD vaccine in the near future. Such chimeric enterovirus VLPs provide an alternative platform for bivalent HFMD vaccine development. PMID:25597595

  12. Generating Chimeric Mice by Using Embryos from Nonsuperovulated BALB/c Mice Compared with Superovulated BALB/c and Albino C57BL/6 Mice.

    PubMed

    Esmail, Michael Y; Qi, Peimin; Connor, Aurora Burds; Fox, James G; García, Alexis

    2016-01-01

    The reliable generation of high-percentage chimeras from gene-targeted C57BL/6 embryonic stem cells has proven challenging, despite optimization of cell culture and microinjection techniques. To improve the efficiency of this procedure, we compared the generation of chimeras by using 3 different inbred, albino host, embryo-generating protocols: BALB/cAnNTac (BALB/c) donor mice superovulated at 4 wk of age, 12-wk-old BALB/c donor mice without superovulation, and C57BL/6NTac-Tyr(tm1Arte) (albino B6) mice superovulated at 4 wk of age. Key parameters measured included the average number of injectable embryos per donor, the percentage of live pups born from the total number of embryos transferred to recipients, and the number of chimeric pups with high embryonic-stem-cell contribution by coat color. Although albino B6 donors produced significantly more injectable embryos than did BALB/c donors, 12-wk-old BALB/c donor produced high-percentage (at least 70%) chimeras more than 2.5 times as often as did albino B6 mice and 20 times more efficiently than did 4-wk-old BALB/c donors. These findings clearly suggest that 12-wk-old BALB/c mice be used as blastocyst donors to reduce the number of mice used to generate each chimera, reduce the production of low-percentage chimeras, and maximize the generation of high-percentage chimeras from C57BL/6 embryonic stem cells. PMID:27423145

  13. Endogenous and xenobiotic metabolite profiling of liver extracts from SCID and chimeric humanized mice following repeated oral administration of troglitazone.

    PubMed

    Barnes, Alan J; Baker, David R; Hobby, Kirsten; Ashton, Simon; Michopoulos, Filippos; Spagou, Konstantina; Loftus, Neil J; Wilson, Ian D

    2014-01-01

    1. Metabonomic analysis, via a combination of untargeted and targeted liquid chromatography-mass spectrometry (LC-MS) and untargeted (1)H NMR spectroscopy-based metabolite profiling, was performed on aqueous (AQ) and organic liver extracts from control (SCID) and chimeric humanized (PXB) mice dosed with troglitazone at 0, 300 and 600 mg/kg/day for seven days. 2. LC-MS analysis of AQ liver extracts showed a more "human-like" profile for troglitazone metabolites for PXB, compared with SCID, mice. 3. LC-MS detected differences in endogenous metabolites, particularly lipid species in dosed mice, including elevated triacylglycerols and 1-alkyl,2-acylglycerophosphates as well as lowered diacylglycerophosphocholines and 1-alkyl,2-acylglycerophosphocholines for PXB compared with SCID mouse liver extracts. Following drug administration changes in the relative proportions of the ions for various unsaturated fatty acids were observed for both types of mouse, some of which were specific to PXB or SCID mice. 4.  (1)H NMR spectroscopy revealed that AQ PXB mouse liver extracts had elevated amounts of inosine, fumarate, creatine, aspartate, trimethylamine N-oxide, glycerophosphocholine, phosphocholine, choline, glutamine, glutamate, acetate, alanine and lactate relative to SCID mice and decreased histidine, glycogen, α- and β-glucose, taurine, and glutathione. Increased uracil and tyrosine concentrations were detected for PXB mice on troglitazone administration. 5. Metabonomic profiling thus showed clear differences between humanized and SCID mice, including after administration of troglitazone. PMID:24350779

  14. Intravitreal injection of a chimeric phage endolysin Ply187 protects mice from Staphylococcus aureus endophthalmitis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  15. Prevention of Birch Pollen-Related Food Allergy by Mucosal Treatment with Multi-Allergen-Chimers in Mice

    PubMed Central

    Hoflehner, Elisabeth; Hufnagl, Karin; Schabussova, Irma; Jasinska, Joanna; Hoffmann-Sommergruber, Karin; Bohle, Barbara; Maizels, Rick M.; Wiedermann, Ursula

    2012-01-01

    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. PMID:22768077

  16. Improvement of the skeletal and dental hypophosphatasia phenotype in Alpl−/− mice by administration of soluble (non-targeted) chimeric alkaline phosphatase

    PubMed Central

    Gasque, Kellen; Foster, Brian L.; Kuss, Pia; Yadav, Manisha C.; Liu, Jin; Kiffer-Moreira, Tina; van Elsas, Andrea; Hatch, Nan; Somerman, Martha J.; Millán, JoséLuis

    2014-01-01

    Hypophosphatasia (HPP) results from ALPL gene mutations, which lead to a deficiency of tissue-nonspecific alkaline phosphatase (TNAP), and accumulation of inorganic pyrophosphate, a potent inhibitor of mineralization that is also a natural substrate of TNAP, in the extracellular space. HPP causes mineralization disorders including soft bones (rickets or osteomalacia) and defects in teeth and periodontal tissues. Enzyme replacement therapy using mineral-targeting recombinant TNAP has proven effective in preventing skeletal and dental defects in TNAP knockout (Alpl−/−) mice, a model for life-threatening HPP. Here, we show that the administration of a soluble, intestinal-like chimeric alkaline phosphatase (ChimAP) improves the manifestations of HPP in Alpl−/− mice. Mice received daily subcutaneous injections of ChimAP at doses of 1, 8 or 16 mg/kg, from birth for up to 53 days. Lifespan and body weight of Alpl−/− mice were normalized, and vitamin B6-associated seizures were absent with 16 mg/kg/day of ChimAP. Radiographs, μCT and histological analyses documented improved mineralization in cortical and trabecular bone and secondary ossification centers in long bones of ChimAP16-treated mice. There was no evidence of craniosynostosis in the ChimAP16-treated mice and we did not detect ectopic calcification by radiography and histology in the aortas, stomachs, kidneys or lungs in any of the treatment groups. Molar tooth development and function improved with the highest ChimAP dose, including enamel, dentin, and tooth morphology. Cementum remained deficient and alveolar bone mineralization was reduced compared to controls, though ChimAP-treated Alpl−/− mice featured periodontal attachment and retained teeth. This study provides the first evidence for the pharmacological efficacy of ChimAP for use in the treatment of skeletal and dental manifestations of HPP. PMID:25433339

  17. Improvement of the skeletal and dental hypophosphatasia phenotype in Alpl-/- mice by administration of soluble (non-targeted) chimeric alkaline phosphatase.

    PubMed

    Gasque, Kellen C S; Foster, Brian L; Kuss, Pia; Yadav, Manisha C; Liu, Jin; Kiffer-Moreira, Tina; van Elsas, Andrea; Hatch, Nan; Somerman, Martha J; Millán, José Luis

    2015-03-01

    Hypophosphatasia (HPP) results from ALPL gene mutations, which lead to a deficiency of tissue-nonspecific alkaline phosphatase (TNAP), and accumulation of inorganic pyrophosphate, a potent inhibitor of mineralization that is also a natural substrate of TNAP, in the extracellular space. HPP causes mineralization disorders including soft bones (rickets or osteomalacia) and defects in teeth and periodontal tissues. Enzyme replacement therapy using mineral-targeting recombinant TNAP has proven effective in preventing skeletal and dental defects in TNAP knockout (Alpl(-/-)) mice, a model for life-threatening HPP. Here, we show that the administration of a soluble, intestinal-like chimeric alkaline phosphatase (ChimAP) improves the manifestations of HPP in Alpl(-/-) mice. Mice received daily subcutaneous injections of ChimAP at doses of 1, 8 or 16 mg/kg, from birth for up to 53 days. Lifespan and body weight of Alpl(-/-) mice were normalized, and vitamin B6-associated seizures were absent with 16 mg/kg/day of ChimAP. Radiographs, μCT and histological analyses documented improved mineralization in cortical and trabecular bone and secondary ossification centers in long bones of ChimAP16-treated mice. There was no evidence of craniosynostosis in the ChimAP16-treated mice and we did not detect ectopic calcification by radiography and histology in the aortas, stomachs, kidneys or lungs in any of the treatment groups. Molar tooth development and function improved with the highest ChimAP dose, including enamel, dentin, and tooth morphology. Cementum remained deficient and alveolar bone mineralization was reduced compared to controls, though ChimAP-treated Alpl(-/-) mice featured periodontal attachment and retained teeth. This study provides the first evidence for the pharmacological efficacy of ChimAP for use in the treatment of skeletal and dental manifestations of HPP. PMID:25433339

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

    PubMed Central

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

    1992-01-01

    We have developed a method for attenuating vaccinia virus recombinants by expressing a fusion protein of a lymphokine and an immunogen. Chimeric genes were constructed that coded for gamma interferon (IFN-gamma) and structural proteins of the human immunodeficiency virus type 1 (HIV-1). In this study, we describe the biological and immunological properties of vaccinia virus recombinants expressing chimeric genes of murine or human IFN-gamma with glycoprotein gp120, gag, and a fragment of gp41. All fusion proteins retained the antigenic characteristics of both IFN-gamma and HIV as shown by immunoblot analysis. However, the antiviral activity of IFN-gamma could be demonstrated only for the IFN-gamma-gag fusion protein. In contrast, the attenuating activity of IFN-gamma for nude mice was retained by all of the recombinants, albeit at various rates. Unlike the antiviral activity, the attenuating activity of IFN-gamma was not species specific. Implications for the development of attenuated live recombinant vaccines for AIDS are discussed. Images PMID:1565633

  19. A Competitive Advantage by Neonatally Engrafted Human Glial Progenitors Yields Mice Whose Brains Are Chimeric for Human Glia

    PubMed Central

    Schanz, Steven J.; Morrow, Carolyn; Munir, Jared; Chandler-Militello, Devin; Wang, Su

    2014-01-01

    Neonatally transplanted human glial progenitor cells (hGPCs) densely engraft and myelinate the hypomyelinated shiverer mouse. We found that, in hGPC-xenografted mice, the human donor cells continue to expand throughout the forebrain, systematically replacing the host murine glia. The differentiation of the donor cells is influenced by the host environment, such that more donor cells differentiated as oligodendrocytes in the hypomyelinated shiverer brain than in myelin wild-types, in which hGPCs were more likely to remain as progenitors. Yet in each recipient, both the number and relative proportion of mouse GPCs fell as a function of time, concomitant with the mitotic expansion and spread of donor hGPCs. By a year after neonatal xenograft, the forebrain GPC populations of implanted mice were largely, and often entirely, of human origin. Thus, neonatally implanted hGPCs outcompeted and ultimately replaced the host population of mouse GPCs, ultimately generating mice with a humanized glial progenitor population. These human glial chimeric mice should permit us to define the specific contributions of glia to a broad variety of neurological disorders, using human cells in vivo. PMID:25429155

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

    PubMed Central

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

    2015-01-01

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

  1. TLR9 bone marrow chimeric mice define a role for cerebral TNF in neuroprotection induced by CpG preconditioning.

    PubMed

    Packard, Amy E B; Leung, Philberta Y; Vartanian, Keri B; Stevens, Susan L; Bahjat, Frances R; Stenzel-Poore, Mary P

    2012-12-01

    Systemic preconditioning with the TLR9 ligand CpG induces neuroprotection against brain ischemic injury through a tumor necrosis factor (TNF)-dependent mechanism. It is unclear how systemic administration of CpG engages the brain to induce the protective phenotype. To address this, we created TLR9-deficient reciprocal bone marrow chimeric mice lacking TLR9 on either hematopoietic cells or radiation-resistant cells of nonhematopoietic origin. We report that wild-type mice reconstituted with TLR9-deficient hematopoietic cells failed to show neuroprotection after systemic CpG preconditioning. Further, while hematopoietic expression of TLR9 is required for CpG-induced neuroprotection it is not sufficient to restore protection to TLR9-deficient mice that are reconstituted with hematopoietic cells bearing TLR9. To determine whether the absence of protection was associated with TNF, we examined TNF levels in the systemic circulation and the brain. We found that although TNF is required for CpG preconditioning, systemic TNF levels did not correlate with the protective phenotype. However, induction of cerebral TNF mRNA required expression of TLR9 on both hematopoietic and nonhematopoietic cells and correlated with neuroprotection. In accordance with these results, we show the therapeutic potential of intranasal CpG preconditioning, which induces brain TNF mRNA and robust neuroprotection with no concomitant increase in systemic levels of TNF. PMID:23010947

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

    PubMed

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

    2016-07-28

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

  3. Comparative biodistributions of indium-111-labelled macrocycle chimeric B72.3 antibody conjugates in tumour-bearing mice.

    PubMed Central

    Turner, A.; King, D. J.; Farnsworth, A. P.; Rhind, S. K.; Pedley, R. B.; Boden, J.; Boden, R.; Millican, T. A.; Millar, K.; Boyce, B.

    1994-01-01

    A novel 111In ligand (a C-functionalised derivative of 1,4,7-triazacyclononanetriacetic acid), termed 9N3, was covalently attached to chimeric B72.3, labelled with 111In and compared with 111In-labelled chimeric B72.3 diethylenetriaminepentaacetic acid (DTPA) cyclic anhydride conjugate (cDTPA) and a C-linked derivative of DTPA (CT-DTPA) in athymic mice bearing human colon carcinoma xenografts. Significant differences in biodistribution were observed between 9N3 and cDTPA conjugates especially in the tumour uptake and blood, liver, femur and colon levels at 24, 48 and 144 h. Significantly higher tumour uptake was observed for 111In-cB72.3-9N3 compared with 111In-cB72.3-cDTPA at all time points. Radiolocalisation (RI) indices increased with time for the 9N3 conjugate but remained constant for the cDTPA conjugate. The biodistribution of 111In-labelled cB72.3-CT-DTPA was similar to that of 111In-labelled cB72.3-9N3 except for elevated kidney levels. A 12N4 macrocycle (a C-functionalised derivative of 1,4,7,10-tetraazacyclododecanetetraacetic acid) was also tested for its ability to chelate 111In and its biodistribution examined. Labelled conjugates with this macrocycle were more difficult to prepare in a stable form but gave a very similar biodistribution to the 9N3 macrocycle conjugate. Macrocycle-antibody conjugates of this type offer considerable promise for tumour imaging in patients. PMID:8018538

  4. T Cells Engineered With Chimeric Antigen Receptors Targeting NKG2D Ligands Display Lethal Toxicity in Mice.

    PubMed

    VanSeggelen, Heather; Hammill, Joanne A; Dvorkin-Gheva, Anna; Tantalo, Daniela G M; Kwiecien, Jacek M; Denisova, Galina F; Rabinovich, Brian; Wan, Yonghong; Bramson, Jonathan L

    2015-10-01

    Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy targets. To assess the tumoricidal properties of T cells directed to attack NKG2D ligands, we engineered murine T cells with two distinct NKG2D-based chimeric antigen receptors (CARs): (i) a fusion between the NKG2D receptor and the CD3ζ chain and (ii) a conventional second-generation CAR, where the extracellular domain of NKG2D was fused to CD28 and CD3ζ. To enhance the CAR surface expression, we also engineered T cells to coexpress DAP10. In vitro functionality and surface expression levels of all three CARs was greater in BALB/c T cells than C57BL/6 T cells, indicating strain-specific differences. Upon adoptive transfer of NKG2D-CAR-T cells into syngeneic animals, we observed significant clinical toxicity resulting in morbidity and mortality. The severity of these toxicities varied between the CAR configurations and paralleled their in vitro NKG2D surface expression. BALB/c mice were more sensitive to these toxicities than C57BL/6 mice, consistent with the higher in vitro functionality of BALB/c T cells. Treatment with cyclophosphamide prior to adoptive transfer exacerbated the toxicity. We conclude that while NKG2D ligands may be useful targets for immunotherapy, the pursuit of NKG2D-based CAR-T cell therapies should be undertaken with caution. PMID:26122933

  5. The distribution of saponins in vivo affects their synergy with chimeric toxins against tumours expressing human epidermal growth factor receptors in mice

    PubMed Central

    Bachran, C; Weng, A; Bachran, D; Riese, SB; Schellmann, N; Melzig, MF; Fuchs, H

    2010-01-01

    Background and purpose: Certain saponins synergize with antitumour drugs to enhance their efficacy, but the mechanisms underlying this synergy in vivo are not well studied. Here, we describe the distribution of Saponinum album (Spn) from Gypsophila paniculata L. in mice after subcutaneous injection. Experimental approach: The [3H]-labelled Spn used for in vivo experiments was biologically active, as it still increased the cytotoxicity of a chimeric toxin in vitro. Distribution of [3H]-Spn was measured in BALB/c mice, with or without subcutaneous tumours in the flank. Labelled Spn was subcutaneously injected in the neck, and samples of organs, blood, urine and tumour tissue were analysed for radioactivity, 5–240 min after the injection. Key results: The majority of [3H]-Spn distributed within 10 min throughout the entire animal, with high levels of radioactivity in the urine by 30 min. No preferential accumulation in tumour tissue or other organs was observed. In tumour-bearing mice, using a sequential combination of Spn (given first) and a chimeric toxin against the epidermal growth factor receptor, ErbB1, we tested two different pretreatment times for Spn. There was high antitumour efficacy (66% inhibition of tumour growth) after 60 min pre treatment with Spn, but no significant inhibition after 10 min pre treatment with Spn. Conclusions and implications: [3H]-Spn was rapidly cleared from the mice after s.c. injection, and antitumour synergy with chimeric toxins was correlated with the removal of excess Spn from tissues. Disposition of Spn in vivo may critically determine antitumour synergy with chimeric toxins. PMID:20015087

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

    PubMed

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

    2016-09-01

    The present study focused on the interactive pain regulation of endokinin A/B (EKA/B, the common C-terminal decapeptide in EKA and EKB) or endokinin C/D (EKC/D, the common C-terminal duodecapeptide in EKC and EKD) on chimeric peptide MCRT (YPFPFRTic-NH2, based on YPFP-NH2 and PFRTic-NH2) at the supraspinal level in mice. Results demonstrated that the co-injection of nanomolar EKA/B and MCRT showed moderate regulation, whereas 30 pmol EKA/B had no effect on MCRT. The combination of EKC/D and MCRT produced enhanced antinociception, which was nearly equal to the sum of the mathematical values of single EKC/D and MCRT. Mechanism studies revealed that pre-injected naloxone attenuated the combination significantly compared with the equivalent analgesic effects of EKC/D alone, suggesting that EKC/D and MCRT might act on two totally independent pathways. Moreover, based on the above results and previous reports, we made two reasonable hypotheses to explain the cocktail-induced analgesia, which may potentially pave the way to explore the respective regulatory mechanisms of EKA/B, EKC/D, and MCRT and to better understand the complicated pain regulation of NK1 and μ opioid receptors, as follows: (1) MCRT and endomorphin-1 possibly activated different μ subtypes; and (2) picomolar EKA/B might motivate the endogenous NPFF system after NK1 activation. PMID:27285300

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

    PubMed Central

    Lo, Agnes Shuk-Yee; Xu, Chen; Murakami, Akikazu; Marasco, Wayne A

    2014-01-01

    Carbonic anhydrase IX (CAIX) is a tumor-associated antigen and marker of hypoxia that is overexpressed on > 90% of clear-cell type renal cell carcinoma (RCC) but not on neighboring normal kidney tissue. Here, we report on the construction of two chimeric antigen receptors (CARs) that utilize a carbonic anhydrase (CA) domain mapped, human single chain antibody (scFv G36) as a targeting moiety but differ in their capacity to provide costimulatory signaling for optimal T cell proliferation and tumor cell killing. The resulting anti-CAIX CARs were expressed on human primary T cells via lentivirus transduction. CAR-transduced T cells (CART cells) expressing second-generation G36-CD28-TCRζ exhibited more potent in vitro antitumor effects on CAIX+ RCC cells than first-generation G36-CD8-TCRζ including cytotoxicity, cytokine secretion, proliferation, and clonal expansion. Adoptive G36-CD28-TCRζ CART cell therapy combined with high-dose interleukin (IL)-2 injection also lead to superior regression of established RCC in nude mice with evidence of tumor cell apoptosis and tissue necrosis. These results suggest that the fully human G36-CD28-TCRζ CARs should provide substantial improvements over first-generation mouse anti-CAIX CARs in clinical use through reduced human anti-mouse antibody responses against the targeting scFv and administration of lower doses of T cells during CART cell therapy of CAIX+ RCC. PMID:27119093

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

    PubMed

    Lo, Agnes Shuk-Yee; Xu, Chen; Murakami, Akikazu; Marasco, Wayne A

    2014-01-01

    Carbonic anhydrase IX (CAIX) is a tumor-associated antigen and marker of hypoxia that is overexpressed on > 90% of clear-cell type renal cell carcinoma (RCC) but not on neighboring normal kidney tissue. Here, we report on the construction of two chimeric antigen receptors (CARs) that utilize a carbonic anhydrase (CA) domain mapped, human single chain antibody (scFv G36) as a targeting moiety but differ in their capacity to provide costimulatory signaling for optimal T cell proliferation and tumor cell killing. The resulting anti-CAIX CARs were expressed on human primary T cells via lentivirus transduction. CAR-transduced T cells (CART cells) expressing second-generation G36-CD28-TCRζ exhibited more potent in vitro antitumor effects on CAIX(+) RCC cells than first-generation G36-CD8-TCRζ including cytotoxicity, cytokine secretion, proliferation, and clonal expansion. Adoptive G36-CD28-TCRζ CART cell therapy combined with high-dose interleukin (IL)-2 injection also lead to superior regression of established RCC in nude mice with evidence of tumor cell apoptosis and tissue necrosis. These results suggest that the fully human G36-CD28-TCRζ CARs should provide substantial improvements over first-generation mouse anti-CAIX CARs in clinical use through reduced human anti-mouse antibody responses against the targeting scFv and administration of lower doses of T cells during CART cell therapy of CAIX(+) RCC. PMID:27119093

  9. Pharmacokinetics of chimeric L6 conjugated to indium-111- and yttrium-90-DOTA-peptide in tumor-bearing mice

    SciTech Connect

    DeNardo, S.J.; Zhong, G.R.; Salako, Q.

    1995-05-01

    A bifunctional chelating agent, DOTA-Gly{sub 3}-L-(p-isothiocyanato)-phenylalanine amide (DOTA-peptide-NCS), was studied in nude mice bearing human breast cancer xenografts (HBT 3477) to determine its potential for radioimmunoconjugate therapy. Indium-111 and yttrium-90 were attached to an anti-adenocarcinoma chimeric L6 (ChL6) monoclonal antibody (MAb) after pre-chelation to the DOTA-peptide-NCS and the desired neutral radiochelates were obtained by purification. The unique characteristic of the DOTA-peptide-NCS to form neutral complexes with trivalent metals was utilized to separate the resulting {sup 111}In and {sup 90}Y radiochelates from excess chelating agent and other anionic by-products resulting from metal impurities. The purified radiochelates were then conjugated to ChL6. The paramacokinetics of {sup 111}In- and {sup 90}Y-DOTA-peptide-ChL6 were obtained for 5 days after injection in nude mice bearing HBT 3477 xenographs. The results were compared with the pharmacokinetics of {sup 125}I-ChL6 obtained in the same mouse model. The whole-body clearance of {sup 125}I-ChL6, {sup 90}Y-and {sup 111}In-DOTA-peptide-ChL6 was monoexponential with biologic half-times of 92, 104 and 160 hr, respectively. Blood clearances of the three radiopharmaceuticals were biphasic. The radiometal immunoconjugates had greater tumor uptake and slower clearances. Indium-111- and {sup 90}Y-DOTA-peptide-ChL6 can be produced at high specific activity with fewer than one chelate per MAb by using a pre-labeling method that permits radiochelate purification by charge selection. Studies in mouse xenografts indicate that tumor uptake in enhanced and a favorable therapeutic index is achieved using these agents. 29 refs., 7 figs., 2 tabs.

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

    PubMed

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

    2015-05-01

    Di(2-ethylhexyl)phthalate (DEHP) is a reproductive toxicant in male rodents. The aim of the current study was to extrapolate the pharmacokinetics and toxicokinetics of mono(2-ethylhexyl)phthalate (MEHP, a primary metabolite of DEHP) in humans by using data from oral administration of DEHP to chimeric mice transplanted with human hepatocytes. MEHP and its glucuronide were detected in plasma from control mice and chimeric mice after single oral doses of 250mg DEHP/kg body weight. Biphasic plasma concentration-time curves of MEHP and its glucuronide were seen only in control mice. MEHP and its glucuronide were extensively excreted in urine within 24h in mice with humanized liver. In contrast, fecal excretion levels of MEHP glucuronide were high in control mice compared with those with humanized liver. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated urine MEHP concentrations in humans were consistent with reported concentrations. This research illustrates how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of pharmacokinetics or toxicokinetics of the primary or secondary metabolites of DEHP. PMID:25867688

  11. Partial rescue of a lethal phenotype of fragile bones in transgenic mice with a chimeric antisense gene directed against a mutated collagen gene.

    PubMed Central

    Khillan, J S; Li, S W; Prockop, D J

    1994-01-01

    Previously, transgenic mice were prepared that developed a lethal phenotype of fragile bones because they expressed an internally deleted mini-gene for the pro alpha 1(I) chain of human type I procollagen. The shortened pro alpha 1(I) chains synthesized from the human transgene bound to and produced degradation of normal pro alpha 1(I) chains synthesized from the normal mouse alleles. Here we assembled an antisense gene that was similar to the internally deleted COL1A1 minigene but the 3' half of the gene was inverted so as to code for an antisense RNA. Transgenic mice expressing the antisense gene had a normal phenotype, apparently because the antisense gene contained human sequences instead of mouse sequences. Two lines of mice expressing the antisense gene were bred to two lines of transgenic mice expressing the mini-gene. In mice that inherited both genes, the incidence of the lethal fragile bone phenotype was reduced from 92% to 27%. The effects of the antisense gene were directly demonstrated by an increase in the ratio of normal mouse pro alpha 1(I) chains to human mini-pro alpha 1(I) chains in tissues from mice that inherited both genes and had a normal phenotype. The results raise the possibility that chimeric gene constructs that contain intron sequences and in which only the second half of a gene is inverted may be particularly effective as antisense genes. Images PMID:8022775

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2013-08-29

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

  14. Survival of host mast cells after establishment of hematopoietic chimerism by graft-versus-host reaction in nonirradiated F1 hybrid mice

    SciTech Connect

    Tsuyama, K.; Sonoda, T.; Kitamura, Y.; Inoue, R.; Ochi, T.; Ono, K.

    1982-10-01

    Since the tissue mast cell has been shown to be progeny of the multipotential hematopoietic stem cell (CFU-S), and the CFU-S is a sensitive target of graft-versus-host (GVH) reaction, we examined whether or not the mast cell is also the target of GVH reaction. Giant granules of C57BL/6-bgJ/bgJ mice were used as a marker of donor cells. When 10(8) spleen cells of C57BL/6-bgJ/bgJ mice were injected into nonirradiated (C57BL/6 X CBA)F1 hybrid mice, erythrocytes and neutrophils became of donor type in about one-half of the recipient mice. In the bone marrow and spleen of the chimeric mice, the CFU-S was of donor type as well. In contrast, mast cells of host type remained in many tissues of the chimeras. Moreover, mast cell precursors with capabilities of proliferation and differentiation were preserved in the skin of chimeras. The present results suggest that the effect of systemic GVH reaction on mature mast cells and the mast cell precursor fixed in the skin is significantly less severe than that on the CFU-S itself.

  15. Young T Cells Age During a Redirected Anti-Tumor Attack: Chimeric Antigen Receptor-Provided Dual Costimulation is Half the Battle

    PubMed Central

    Hombach, Andreas A.; Abken, Hinrich

    2013-01-01

    Adoptive therapy with chimeric antigen receptor (CAR)-redirected T cells showed spectacular efficacy in the treatment of leukemia in recent early phase trials. Patient’s T cells were ex vivo genetically engineered with a CAR, amplified and re-administered to the patient. While T cells mediating the primary response were predominantly of young effector and central memory phenotype, repetitive antigen engagement irreversible triggers T cell maturation leaving late memory cells with the KLRG1+ CD57+ CD7− CCR7− phenotype in the long-term. These cells preferentially accumulate in the periphery, are hypo-responsive upon TCR engagement and prone to activation-induced cell death. A recent report indicates that those T cells can be rescued by CAR provided CD28 and OX40 (CD134) stimulation. We discuss the strategy with respect to prolong the anti-tumor response and to improve the over-all efficacy of adoptive cell therapy. PMID:23761793

  16. Young T Cells Age During a Redirected Anti-Tumor Attack: Chimeric Antigen Receptor-Provided Dual Costimulation is Half the Battle.

    PubMed

    Hombach, Andreas A; Abken, Hinrich

    2013-01-01

    Adoptive therapy with chimeric antigen receptor (CAR)-redirected T cells showed spectacular efficacy in the treatment of leukemia in recent early phase trials. Patient's T cells were ex vivo genetically engineered with a CAR, amplified and re-administered to the patient. While T cells mediating the primary response were predominantly of young effector and central memory phenotype, repetitive antigen engagement irreversible triggers T cell maturation leaving late memory cells with the KLRG1(+) CD57(+) CD7(-) CCR7(-) phenotype in the long-term. These cells preferentially accumulate in the periphery, are hypo-responsive upon TCR engagement and prone to activation-induced cell death. A recent report indicates that those T cells can be rescued by CAR provided CD28 and OX40 (CD134) stimulation. We discuss the strategy with respect to prolong the anti-tumor response and to improve the over-all efficacy of adoptive cell therapy. PMID:23761793

  17. Boosting BCG-primed mice with chimeric DNA vaccine HG856A induces potent multifunctional T cell responses and enhanced protection against Mycobacterium tuberculosis.

    PubMed

    Ji, Ping; Hu, Zhi-Dong; Kang, Han; Yuan, Qin; Ma, Hui; Wen, Han-Li; Wu, Juan; Li, Zhong-Ming; Lowrie, Douglas B; Fan, Xiao-Yong

    2016-02-01

    The tuberculosis pandemic continues to rampage despite widespread use of the current Bacillus Calmette-Guerin (BCG) vaccine. Because DNA vaccines can elicit effective antigen-specific immune responses, including potent T cell-mediated immunity, they are promising vehicles for antigen delivery. In a prime-boost approach, they can supplement the inadequate anti-TB immunological memory induced by BCG. Based on this, a chimeric DNA vaccine HG856A encoding Mycobacterium tuberculosis (M. tuberculosis) immunodominant antigen Ag85A plus two copies of ESAT-6 was constructed. Potent humoral immune responses, as well as therapeutic effects induced by this DNA vaccine, were observed previously in M. tuberculosis-infected mice. In this study, we further evaluated the antigen-specific T cell immune responses and showed that repeated immunization with HG856A gave modest protection against M. tuberculosis challenge infection and significantly boosted the immune protection primed by BCG vaccination. Enhanced protection was accompanied by increased multifunctional Th1 CD4(+) T cell responses, most notably by an elevated frequency of M. tuberculosis antigen-specific IL-2-producing CD4(+) T cells post-vaccination. These data confirm the potential of chimeric DNA vaccine HG856A as an anti-TB vaccine candidate. PMID:26111521

  18. Assessment of bacterial contamination of drinking water provided to mice.

    PubMed

    Haist, Carrie; Cadillac, Joan; Dysko, Robert

    2004-11-01

    The objective of this study was to evaluate whether an 240-ml water bottle provided to individually housed mice would remain potable for a 2-week interval (based on absence of coliforms). The study used inbred C57BL/6 mice and CB6F1 x C3D2F1 hybrid mice. Test groups were assigned to minimize the variables of strain, caging type (non-ventilated static versus ventilated) and building location. A 3-cc sample of drinking water was removed aseptically from the bottles and vacuum-filtered using a 250-ml filter funnel with a 0.45-mum pore size. The membrane filter was removed using sterile forceps and placed on a blood agar plate for 10 min. The plate was streaked and incubated at 37 degrees C for 5 days. The plates were observed daily, and if growth had occurred, further testing was done to determine specific organisms. Of the 148 samples only 23 had any bacterial growth. Typical bacteria were unspeciated gram-positive bacilli and Staphylococcus, Micrococcus, Streptococcus, and Pantoea species. The absence of coliforms and low percentage of bacterial contamination suggest that drinking water will remain potable for 2 weeks when supplied to an individual mouse. PMID:15636548

  19. A Tripartite Cocktail of Chimeric Monoclonal Antibodies Passively Protects Mice against Ricin, Staphylococcal Enterotoxin B and Clostridium perfringens Epsilon Toxin

    PubMed Central

    Sully, Erin K.; Whaley, Kevin; Bohorova, Natasha; Bohorov, Ognian; Goodman, Charles; Kim, Do; Pauly, Michael; Velasco, Jesus; Holtsberg, Frederick W.; Stavale, Eric; Aman, M. Javad; Tangudu, Chandra; Uzal, Francisco A.; Mantis, Nicholas J.; Zeitlin, Larry

    2014-01-01

    Due to the fast-acting nature of ricin, staphylococcal enterotoxin (SEB), and Clostridium perfringens epsilon toxin (ETX), it is necessary that therapeutic interventions following a bioterrorism incident by one of these toxins occur as soon as possible after intoxication. Moreover, because the clinical manifestations of intoxication by these toxins are likely to be indistinguishable from each other, especially following aerosol exposure, we have developed a cocktail of chimeric monoclonal antibodies that is capable of neutralizing all three toxins. The efficacy of this cocktail was demonstrated in mouse models of lethal dose toxin challenge. PMID:25260254

  20. Bleomycin-Treated Chimeric Thy1-Deficient Mice with Thy1-Deficient Myofibroblasts and Thy-Positive Lymphocytes Resolve Inflammation without Affecting the Fibrotic Response

    PubMed Central

    Cohen, Pazit Y.; Breuer, Raphael; Zisman, Philip; Wallach-Dayan, Shulamit B.

    2015-01-01

    Lung fibrosis is characterized by abnormal accumulation of fibroblasts in the interstitium of the alveolar space. Two populations of myofibroblasts, distinguished by Thy1 expression, are detected in human and murine lungs. Accumulation of Thy1-negative (Thy1−) myofibroblasts was shown in the lungs of humans with idiopathic pulmonary fibrosis (IPF) and of bleomycin-treated mice. We aimed to identify genetic changes in lung myofibroblasts following Thy1 crosslinking and assess the impact of specific lung myofibroblast Thy1-deficiency, in vivo, in bleomycin-injured mouse lungs. Thy1 increased in mouse lung lymphocytes following bleomycin injury but decreased in myofibroblasts when fibrosis was at the highest point (14 days), as assessed by immunohistochemistry. Using gene chip analysis, we detected that myofibroblast Thy1 crosslinking mediates downregulation of genes promoting cell proliferation, survival, and differentiation, and reduces production of extracellular matrix (ECM) components, while concurrently mediating the upregulation of genes known to foster inflammation and immunological functions. Chimeric Thy1-deficient mice with Thy1+ lymphocytes and Thy1− myofibroblasts showed fibrosis similar to wild-type mice and an increased number of CD4/CD25 regulatory T cells, with a concomitant decrease in inflammation. Lung myofibroblasts downregulate Thy1 expression to increase their proliferation but to diminish the in vivo inflammatory milieu. Inflammation is not essential for evolution of fibrosis as was previously stated. PMID:26300593

  1. Hepatitis C virus dynamics and cellular gene expression in uPA-SCID chimeric mice with humanized livers during intravenous silibinin monotherapy.

    PubMed

    DebRoy, S; Hiraga, N; Imamura, M; Hayes, C N; Akamatsu, S; Canini, L; Perelson, A S; Pohl, R T; Persiani, S; Uprichard, S L; Tateno, C; Dahari, H; Chayama, K

    2016-09-01

    Legalon SIL (SIL) is a chemically hydrophilized version of silibinin, an extract of milk thistle (Silybum marianum) seeds that has exhibited hepatoprotective and antiviral effectiveness against hepatitis C virus (HCV) in patients leading to viral clearance in combination with ribavirin. To elucidate the incompletely understood mode of action of SIL against HCV, mathematical modelling of HCV kinetics and human hepatocyte gene expression studies were performed in uPA-SCID-chimeric mice with humanized livers. Chronically HCV-infected mice (n = 15) were treated for 14 days with daily intravenous SIL at 469, 265 or 61.5 mg/kg. Serum HCV and human albumin (hAlb) were measured frequently, and liver HCV RNA was analysed at days 3 and 14. Microarray analysis of human hepatocyte gene expression was performed at days 0, 3 and 14 of treatment. While hAlb remained constant, a biphasic viral decline in serum was observed consisting of a rapid 1st phase followed by a second slower phase (or plateau with the two lower SIL dosings). SIL effectiveness in blocking viral production was similar among dosing groups (median ε = 77%). However, the rate of HCV-infected hepatocyte decline, δ, was dose-dependent. Intracellular HCV RNA levels correlated (r = 0.66, P = 0.01) with serum HCV RNA. Pathway analysis revealed increased anti-inflammatory and antiproliferative gene expression in human hepatocytes in SIL-treated mice. The results suggest that SIL could lead to a continuous second-phase viral decline, that is potentially viral clearance, in the absence of adaptive immune response along with increased anti-inflammatory and antiproliferative gene expression in human hepatocytes. PMID:27272497

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

    SciTech Connect

    Perkins, S.; Fleischman, R.A.

    1988-04-01

    Studies of bone marrow transplant patients have suggested that the stromal cells of the in vitro hematopoietic microenvironment are transplantable into conditioned recipients. Moreover, in patients with myeloproliferative disorders, all of the stromal cells, which include presumptive endothelial cells, appear to be derived from hematopoietic precursors. To confirm these findings, we have constructed two chimeric mouse models: (a) traditional radiation chimeras, and (b) fetal chimeras, produced by placental injection of bone marrow into genetically anemic Wx/Wv fetuses, a technique that essentially precludes engraftment of nonhematopoietic cells. Using two-color indirect immunofluorescence, the stromal cells in long-term bone marrow culture derived from these chimeras were analyzed for donor or host origin by strain-specific H-2 antigens, and for cell lineage by a variety of other specific markers. 75-95% of the stromal cells were shown to be hematopoietic cells of the monocyte-macrophage lineage, based upon donor origin, phagocytosis, and expression of specific hematopoietic surface antigens. The remaining 5-25% of the stromal cells were exclusively host in origin. Apart from occasional fat cells, these cells uniformly expressed collagen type IV, laminin, and a surface antigen associated with endothelial cells. Since these endothelial-like cells are not transplantable into radiation or fetal chimeras, they are not derived from hematopoietic stem cells. The contrast between our findings and human studies suggests either unexpected species differences in the origin of stromal lineages or limitations in the previous methodology used to detect nonhematopoietic stromal cells.

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

    PubMed Central

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

    2013-01-01

    SUMMARY 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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-08-01

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

  6. Regimen-Specific Effects of RNA-Modified Chimeric Antigen Receptor T Cells in Mice with Advanced Leukemia

    PubMed Central

    Barrett, David M.; Liu, Xiaojun; Jiang, Shuguang; June, Carl H.; Zhao, Yangbing

    2013-01-01

    Abstract Cytotoxic T lymphocytes modified with chimeric antigen receptors (CARs) for adoptive immunotherapy of hematologic malignancies have demonstrated activity in early phase clinical trials. While T cells bearing stably expressed CARs are efficacious and have potential long-term persistence, temporary expression of a CAR via RNA electroporation is also potentially efficacious in preclinical models. Temporary CAR expression using RNA presents a method of testing CARs clinically with additional safety where there may be concerns about possible chronic “on-target, off-tumor” toxic effects, as the degradation of RNA ensures complete removal of the CAR over time without relying on suicide induction systems. CD19-directed RNA CAR T cells were tested in vivo for efficacy and comparison to lentiviral vector (LV)-generated stable CAR T cells. We tested the hypothesis that multiple infusions of RNA CAR T cells preceded by lymphodepleting chemotherapy could mediate improved survival and sustained antitumor responses in a robust leukemia xenograft model. The saturation strategy using rationally designed multiple infusions of RNA CARs based on multiple model iterations approached the efficacy of a stable LV expression method. Two-color imaging revealed that relapse was a locoregional phenomenon in both the temporary and the stable expression models. In marked contrast to stably expressed CARs with retroviral or LV technology, the efficacy of RNA CARs appears independent of the costimulatory signaling endodomains likely because they more influence proliferation and persistence rather than short-term efficacy. The efficacy of the RNA CAR infusions may approach that of stably expressed CARs, offer theoretically safer initial clinical testing in addition to suicide systems, and allow for rapid and effective iterative preclinical modeling for the testing of new targets. PMID:23883116

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

    PubMed

    Hong, Hao; Brown, Christine E; Ostberg, Julie R; Priceman, Saul J; Chang, Wen-Chung; Weng, Lihong; Lin, Paul; Wakabayashi, Mark T; Jensen, Michael C; Forman, Stephen J

    2016-01-01

    New therapeutic modalities are needed for ovarian cancer, the most lethal gynecologic malignancy. Recent clinical trials have demonstrated the impressive therapeutic potential of adoptive therapy using chimeric antigen receptor (CAR)-redirected T cells to target hematological cancers, and emerging studies suggest a similar impact may be achieved for solid cancers. We sought determine whether genetically-modified T cells targeting the CE7-epitope of L1-CAM, a cell adhesion molecule aberrantly expressed in several cancers, have promise as an immunotherapy for ovarian cancer, first demonstrating that L1-CAM was highly over-expressed on a panel of ovarian cancer cell lines, primary ovarian tumor tissue specimens, and ascites-derived primary cancer cells. Human central memory derived T cells (TCM) were then genetically modified to express an anti-L1-CAM CAR (CE7R), which directed effector function upon tumor antigen stimulation as assessed by in vitro cytokine secretion and cytotoxicity assays. We also found that CE7R+ T cells were able to target primary ovarian cancer cells. Intraperitoneal (i.p.) administration of CE7R+ TCM induced a significant regression of i.p. established SK-OV-3 xenograft tumors in mice, inhibited ascites formation, and conferred a significant survival advantage compared with control-treated animals. Taken together, these studies indicate that adoptive transfer of L1-CAM-specific CE7R+ T cells may offer a novel and effective immunotherapy strategy for advanced ovarian cancer. PMID:26761817

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

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

    Many studies have shown that calcium phosphate ceramics (CP) have osteoconductive and osteoinductive properties; however, the exact mechanism of bone induction has not yet been reported. This study was performed to investigate if destroying immunological function will influence osteogenesis, to explain the mechanism which is unclear. In this study, twenty C57BL/6 mice were divided into two groups (n = 10), in group 1, a hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ceramic was implanted into both the left and right leg muscles of each mouse; in group 2, ten mice experienced lethal irradiation, then were injected bone marrow (BM) cells from green fluorescent protein (GFP) transgenic mice by tail veil, after bone marrow transplantation (BMT), heart, liver, spleen, lung, kidney, and muscle were harvested for biological analysis, after the GFP chimera model was established successfully, the same HA/β-TCP ceramic was implanted into both leg muscles of each mouse immediately after irradiation. 45 and 90 days after implantation, the ceramics of the two groups were harvested to perform with hematoxylin and eosin (HE) and immunohistochemistry (IHC) staining; the results showed that there was no bone formation in group 2, while new bone tissues were detected in group 1. Our findings suggest that the BM cell from GFP transgenic mice is a good biomarker and it could set a good platform for chimera model; it also shows that BM cell is one of cell resources of bone induction, and destruction of immune function will impede osteoinduction by CP. Overall, our results may shed light on clear mechanism study of bone induction in the future.

  9. A Chimeric Dengue Virus Vaccine using Japanese Encephalitis Virus Vaccine Strain SA14-14-2 as Backbone Is Immunogenic and Protective against Either Parental Virus in Mice and Nonhuman Primates

    PubMed Central

    Li, Xiao-Feng; Deng, Yong-Qiang; Yang, Hui-Qiang; Zhao, Hui; Jiang, Tao; Yu, Xue-Dong; Li, Shi-Hua; Ye, Qing; Zhu, Shun-Ya; Wang, Hong-Jiang; Zhang, Yu; Ma, Jie; Yu, Yong-Xin; Liu, Zhong-Yu; Qin, E-De; Shi, Pei-Yong

    2013-01-01

    The development of a safe and efficient dengue vaccine represents a global challenge in public health. Chimeric dengue viruses (DENV) based on an attenuated flavivirus have been well developed as vaccine candidates by using reverse genetics. In this study, based on the full-length infectious cDNA clone of the well-known Japanese encephalitis virus live vaccine strain SA14-14-2 as a backbone, a novel chimeric dengue virus (named ChinDENV) was rationally designed and constructed by replacement with the premembrane and envelope genes of dengue 2 virus. The recovered chimeric virus showed growth and plaque properties similar to those of the parental DENV in mammalian and mosquito cells. ChinDENV was highly attenuated in mice, and no viremia was induced in rhesus monkeys upon subcutaneous inoculation. ChinDENV retained its genetic stability and attenuation phenotype after serial 15 passages in cultured cells. A single immunization with various doses of ChinDENV elicited strong neutralizing antibodies in a dose-dependent manner. When vaccinated monkeys were challenged with wild-type DENV, all animals except one that received the lower dose were protected against the development of viremia. Furthermore, immunization with ChinDENV conferred efficient cross protection against lethal JEV challenge in mice in association with robust cellular immunity induced by the replicating nonstructural proteins. Taken together, the results of this preclinical study well demonstrate the great potential of ChinDENV for further development as a dengue vaccine candidate, and this kind of chimeric flavivirus based on JE vaccine virus represents a powerful tool to deliver foreign antigens. PMID:24109223

  10. Inhibition of Hepatitis C Virus in Chimeric Mice by Short Synthetic Hairpin RNAs: Sequence Analysis of Surviving Virus Shows Added Selective Pressure of Combination Therapy

    PubMed Central

    Dallas, Anne; Ilves, Heini; Ma, Han; Chin, Daniel J.; MacLachlan, Ian; Klumpp, Klaus

    2014-01-01

    ABSTRACT We have recently shown that a cocktail of two short synthetic hairpin RNAs (sshRNAs), targeting the internal ribosome entry site of hepatitis C virus (HCV) formulated with lipid nanoparticles, was able to suppress viral replication in chimeric mice infected with HCV GT1a by up to 2.5 log10 (H. Ma et al., Gastroenterology 146:63–66.e5, http://dx.doi.org/10.1053/j.gastro.2013.09.049) Viral load remained about 1 log10 below pretreatment levels 21 days after the end of dosing. We have now sequenced the HCV viral RNA amplified from serum of treated mice after the 21-day follow-up period. Viral RNA from the HCV sshRNA-treated groups was altered in sequences complementary to the sshRNAs and nowhere else in the 500-nucleotide sequenced region, while the viruses from the control group that received an irrelevant sshRNA had no mutations in that region. The ability of the most commonly selected mutations to confer resistance to the sshRNAs was confirmed in vitro by introducing those mutations into HCV-luciferase reporters. The mutations most frequently selected by sshRNA treatment within the sshRNA target sequence occurred at the most polymorphic residues, as identified from an analysis of available clinical isolates. These results demonstrate a direct antiviral activity with effective HCV suppression, demonstrate the added selective pressure of combination therapy, and confirm an RNA interference (RNAi) mechanism of action. IMPORTANCE This study presents a detailed analysis of the impact of treating a hepatitis C virus (HCV)-infected animal with synthetic hairpin-shaped RNAs that can degrade the virus's RNA genome. These RNAs can reduce the viral load in these animals by over 99% after 1 to 2 injections. The study results confirm that the viral rebound that often occurred a few weeks after treatment is due to emergence of a virus whose genome is mutated in the sequences targeted by the RNAs. The use of two RNA inhibitors, which is more effective than use of either

  11. Analyses of acute graft-versus-host-like reaction in (MRL/lpr----MRL/+) chimeric mice using MRL/lpr-Thy-1. 1 congenic mice

    SciTech Connect

    Nakagawa, T.; Nagata, N.; Hosaka, N.; Inaba, M.; Yasumizu, R.; Ogawa, R.; Ikehara, S. )

    1991-10-01

    When MRL/Mp(-)+/+(MRL/+) mice are lethally irradiated and then reconstituted with MRL/Mp-lpr/lpr (MRL/lpr) bone marrow and/or spleen cells, these MRL/+ mice develop lpr-GVHD which is similar to acute graft-versus-host disease (GVHD). Using a Thy-1 congenic strain of MRL/lpr mice (MRL/lpr-Thy-1.1), the authors analyzed T cell subpopulations in the thymus and spleen of MRL/+ mice suffering from lpr-GVHD. lpr-GVHD was induced in MRL/+ mice by transplantation of bone marrow cells (BMC) from MRL/lpr-Thy-1.1 mice; severe lymphocyte depletion associated with fibrosis was observed in the spleens after 7 weeks of bone marrow transplantation (BMT). Thymocytes of the host MRL/+ thymus were replaced with donor-derived cells from the early stage of lpr-GVHD, whereas in the spleen, a small number of host T cells (Thy-1.2+) (4-5%) were retained until the late stage of lpr-GVHD. Donor-type (Thy-1.1+) T cell subsets were not different from those of nontreated MRL/+ mice in the thymus, whereas in the spleen. CD8+ T cells (Thy-1.1+) reached a peak at 5 weeks after BMT, and CD4+ T cells (Thy-1.1+), a peak at 6 weeks. The elimination of T cells from MRL/lpr BMC had no evident effect on the prevention of lpr-GVHD. T cell subpopulations showed a similar pattern to GVHD elicited by MHC differences. Analyses of autoreactive T cells expressing V beta 5 or V beta 11 revealed that autoreactive T cells were deleted from the peripheral lymph nodes. Interestingly, the levels of IgG anti-ssDNA antibodies markedly increased, and both IgM and IgG rheumatoid factors slightly increased 5 to 7 weeks after BMT. These findings are discussed in relation to not only GVHD elicited by MHC differences but also autoimmune diseases.

  12. Crystal Structures of Yeast-Produced Enterovirus 71 and Enterovirus 71/Coxsackievirus A16 Chimeric Virus-Like Particles Provide the Structural Basis for Novel Vaccine Design against Hand-Foot-and-Mouth Disease

    PubMed Central

    Lyu, Ke; He, Ya-Ling; Li, Hao-Yang

    2015-01-01

    ABSTRACT Human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two major causative agents for hand-foot-and-mouth disease (HFMD). Previously, we demonstrated that a virus-like particle (VLP) for EV71 produced from Saccharomyces cerevisiae is a potential vaccine candidate against EV71 infection, and an EV71/CVA16 chimeric VLP can elicit protective immune responses against both virus infections. Here, we presented the crystal structures of both VLPs, showing that both the linear and conformational neutralization epitopes identified in EV71 are mostly preserved on both VLPs. The replacement of only 4 residues in the VP1 GH loop converted strongly negatively charged surface patches formed by portions of the SP70 epitope in EV71 VLP into a relatively neutral surface in the chimeric VLP, which likely accounted for the additional neutralization capability of the chimeric VLP against CVA16 infection. Such local variations in the amino acid sequences and the surface charge potential are also present in different types of polioviruses. In comparison to EV71 VLP, the chimeric VLP exhibits structural changes at the local site of amino acid replacement and the surface loops of all capsid proteins. This is consistent with the observation that the VP1 GH loop located near the pseudo-3-fold junction is involved in extensive interactions with other capsid regions. Furthermore, portions of VP0 and VP1 in EV71 VLP are at least transiently exposed, revealing the structural flexibility of the VLP. Together, our structural analysis provided insights into the structural basis of enterovirus neutralization and novel vaccine design against HFMD and other enterovirus-associated diseases. IMPORTANCE Our previous studies demonstrated that the enterovirus 71 (EV71) virus-like particle (VLP) produced from yeast is a vaccine candidate against EV71 infection and that a chimeric EV71/coxsackievirus A16 (CVA16) VLP with the replacement of 4 amino acids in the VP1 GH loop can confer

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

    SciTech Connect

    Ye Ling; Sun Yuliang; Lin Jianguo; Bu Zhigao; Wu Qingyang; Jiang, Shibo; Steinhauer, David A.; Compans, Richard W.; Yang Chinglai . E-mail: chyang@emory.edu

    2006-08-15

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

  14. Generation of monoclonal antibodies of desired specificity using chimeric polyomavirus-derived virus-like particles.

    PubMed

    Zvirbliene, A; Samonskyte, L; Gedvilaite, A; Voronkova, T; Ulrich, R; Sasnauskas, K

    2006-04-20

    Foreign protein sequences presented on hamster polyomavirus (HaPyV) major capsid protein VP1-derived virus-like particles (VLPs) have been demonstrated to be highly immunogenic. The current study was aimed to evaluate VP1-derived chimeric VLPs as tools for hybridoma technology to generate monoclonal antibodies (mAbs) of desired specificity. Chimeric VLPs containing inserts of different size and origin were used as immunogens. Chimeric VLPs carrying a 9 amino acid (aa)-long cytotoxic T-cell epitope (STAPPVHNV) of human mucin 1 (MUC1) elicited a strong epitope-specific humoral immune response in mice and promoted the production of MUC1-specific mAbs. From a total of seven mAbs of IgG isotype generated against the chimeric VLPs, two mAbs were directed against the MUC1 epitope and five mAbs against the VP1-carrier. Two out of five anti-VP1 mAbs recognized epitopes located at the previously defined insertion site #2 (aa 223/224), which confirms its surface-exposed localization. Chimeric VLPs carrying a 120-aa long sequence of Puumala hantavirus (PUUV) nucleocapsid protein (NP) promoted the generation of five mAbs of IgG isotype specific to PUUV NP. All mAbs recognized the full-length NP of different PUUV strains. In contrast, no VP1-specific mAbs were obtained. The ability of chimeric VLPs to activate antigen-presenting cells was evaluated by studying the uptake of chimeric VLPs by murine spleen cell-derived dendritic cells (DCs). Efficient uptake of VLPs and activation of murine DCs were demonstrated, which may represent the basis of the strong immunogenicity of chimeric VLPs. In conclusion, chimeric VLPs effectively stimulated the production of IgG antibodies specific for foreign epitopes presented at surface-exposed regions. Thus, chimeric HaPyV VP1-derived VLPs represent efficient immunogens for hybridoma technology and provide a promising alternative to chemical coupling of synthetic peptides to carrier proteins. PMID:16516908

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  17. Generation of Chimeric Rhesus Monkeys

    PubMed Central

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

    2011-01-01

    Summary 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 4-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. PMID:22225614

  18. Investigation of drug-drug interactions caused by human pregnane X receptor-mediated induction of CYP3A4 and CYP2C subfamilies in chimeric mice with a humanized liver.

    PubMed

    Hasegawa, Maki; Tahara, Harunobu; Inoue, Ryo; Kakuni, Masakazu; Tateno, Chise; Ushiki, Junko

    2012-03-01

    The induction of cytochrome P450 (P450) enzymes is one of the risk factors for drug-drug interactions (DDIs). To date, the human pregnane X receptor (PXR)-mediated CYP3A4 induction has been well studied. In addition to CYP3A4, the expression of CYP2C subfamily is also regulated by PXR, and the DDIs caused by the induction of CYP2C enzymes have been reported to have a major clinical impact. The purpose of the present study was to investigate whether chimeric mice with a humanized liver (PXB mice) can be a suitable animal model for investigating the PXR-mediated induction of CYP2C subfamily, together with CYP3A4. We evaluated the inductive effect of rifampicin (RIF), a typical human PXR ligand, on the plasma exposure to the four P450 substrate drugs (triazolam/CYP3A4, pioglitazone/CYP2C8, (S)-warfarin/CYP2C9, and (S)-(-)-mephenytoin/CYP2C19) by cassette dosing in PXB mice. The induction of several drug-metabolizing enzymes and transporters in the liver was also examined by measuring the enzyme activity and mRNA expression levels. Significant reductions in the exposure to triazolam, pioglitazone, and (S)-(-)-mephenytoin, but not to (S)-warfarin, were observed. In contrast to the in vivo results, all the four P450 isoforms, including CYP2C9, were elevated by RIF treatment. The discrepancy in the (S)-warfarin results between in vivo and in vitro studies may be attributed to the relatively small contribution of CYP2C9 to (S)-warfarin elimination in the PXB mice used in this study. In summary, PXB mice are a useful animal model to examine DDIs caused by PXR-mediated induction of CYP2C and CYP3A4. PMID:22126990

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

    PubMed Central

    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

  20. Affinity-tuned ErbB2 or EGFR chimeric antigen receptor T cells exhibit an increased therapeutic index against tumors in mice

    PubMed Central

    Liu, Xiaojun; Jiang, Shuguang; Fang, Chongyun; Yang, Shiyu; Olalere, Devvora; Pequignot, Edward C.; Cogdill, Alexandria P.; Li, Na; Ramones, Melissa; Granda, Brian; Zhou, Li; Loew, Andreas; Young, Regina M.; June, Carl H.; Zhao, Yangbing

    2015-01-01

    Target-mediated toxicity is a major limitation in the development of chimeric antigen T cell receptors (CAR) for adoptive cell therapy of solid tumors. In this study, we developed a strategy to adjust the affinities of the scFv component of CAR to discriminate tumors overexpressing the target from normal tissues which express it at physiologic levels. A CAR-expressing T cell panel was generated with target antigen affinities varying over three orders of magnitude. High-affinity cells recognized target expressed at any level, including at levels in normal cells that were undetectable by flow cytometry. Affinity-tuned cells exhibited robust antitumor efficacy similar to high-affinity cells, but spared normal cells expressing physiologic target levels. The use of affinity-tuned scFvs offers a strategy to empower wider use of CAR T cells against validated targets widely overexpressed on solid tumors, including those considered undruggable by this approach. PMID:26330166

  1. Affinity-Tuned ErbB2 or EGFR Chimeric Antigen Receptor T Cells Exhibit an Increased Therapeutic Index against Tumors in Mice.

    PubMed

    Liu, Xiaojun; Jiang, Shuguang; Fang, Chongyun; Yang, Shiyu; Olalere, Devvora; Pequignot, Edward C; Cogdill, Alexandria P; Li, Na; Ramones, Melissa; Granda, Brian; Zhou, Li; Loew, Andreas; Young, Regina M; June, Carl H; Zhao, Yangbing

    2015-09-01

    Target-mediated toxicity is a major limitation in the development of chimeric antigen T-cell receptors (CAR) for adoptive cell therapy of solid tumors. In this study, we developed a strategy to adjust the affinities of the scFv component of CAR to discriminate tumors overexpressing the target from normal tissues that express it at physiologic levels. A CAR-expressing T-cell panel was generated with target antigen affinities varying over three orders of magnitude. High-affinity cells recognized target expressed at any level, including at levels in normal cells that were undetectable by flow cytometry. Affinity-tuned cells exhibited robust antitumor efficacy similar to high-affinity cells, but spared normal cells expressing physiologic target levels. The use of affinity-tuned scFvs offers a strategy to empower wider use of CAR T cells against validated targets widely overexpressed on solid tumors, including those considered undruggable by this approach. PMID:26330166

  2. Improved mouse cage provides versatility and ease in handling laboratory mice

    NASA Technical Reports Server (NTRS)

    Jones, N. D.

    1969-01-01

    Mouse cage system provides versatility and ease in handling laboratory mice, cleaning their cages, and collecting uncontaminated metabolic test specimens. The cage, compact and free standing, contains a screened bottom and funnel channel to collect waste. The feed is in the cage top and thereby separates the food and waste.

  3. Increased gastrin gene expression provides a physiological advantage to mice under hypoxic conditions.

    PubMed

    Laval, Marie; Baldwin, Graham S; Shulkes, Arthur; Marshall, Kathryn M

    2015-01-15

    Hypoxia, or a low concentration of O2, is encountered in humans undertaking activities such as mountain climbing and scuba diving and is important pathophysiologically as a limiting factor in tumor growth. Although data on the interplay between hypoxia and gastrins are limited, gastrin expression is upregulated by hypoxia in gastrointestinal cancer cell lines, and gastrins counterbalance hypoxia by stimulating angiogenesis in vitro and in vivo. The aim of this study was to determine if higher concentrations of the gastrin precursor progastrin are protective against hypoxia in vivo. hGAS mice, which overexpress progastrin in the liver, and mice of the corresponding wild-type FVB/N strain were exposed to normoxia or hypoxia. Iron status was assessed by measurement of serum iron parameters, real-time PCR for mRNAs encoding critical iron regulatory proteins, and Perls' stain and atomic absorption spectrometry for tissue iron concentrations. FVB/N mice lost weight at a faster rate and had higher sickness scores than hGAS mice exposed to hypoxia. Serum iron levels were lower in hGAS than FVB/N mice and decreased further when the animals were exposed to hypoxia. The concentration of iron in the liver was strikingly lower in hGAS than FVB/N mice. We conclude that increased circulating concentrations of progastrin provide a physiological advantage against systemic hypoxia in mice, possibly by increasing the availability of iron stores. This is the first report of an association between progastrin overexpression, hypoxia, and iron homeostasis. PMID:25394662

  4. Electroejaculation of chimeric rats

    PubMed Central

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

    2014-01-01

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

  5. Electroejaculation of chimeric rats.

    PubMed

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

    2013-06-01

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

  6. Chimeric Pestivirus Experimental Vaccines.

    PubMed

    Reimann, Ilona; Blome, Sandra; Beer, Martin

    2016-01-01

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

  7. Insulin-regulated aminopeptidase deficiency provides protection against ischemic stroke in mice.

    PubMed

    Pham, Vi; Albiston, Anthony L; Downes, Catherine E; Wong, Connie H Y; Diwakarla, Shanti; Ng, Leelee; Lee, Seyoung; Crack, Peter J; Chai, Siew Yeen

    2012-04-10

    Recent studies have demonstrated that angiotensin IV (Ang IV) provides protection against brain injury caused by cerebral ischemia. Ang IV is a potent inhibitor of insulin-regulated aminopeptidase (IRAP). Therefore, we examined the effect of IRAP gene inactivation on neuroprotection following transient middle cerebral artery occlusion (MCAo) in mice. IRAP knockout mice and wild-type controls were subjected to 2 h of transient MCAo using the intraluminal filament technique. Twenty-four hours after reperfusion, neurological deficits of the stroke-induced mice were assessed and infarct volumes were measured by TTC staining. The cerebral infarct volume was significantly reduced in the IRAP knockout mice compared to wild-type littermates with corresponding improvement in neurological performance at 24 h post-ischemia. An increase in compensatory cerebral blood flow during MCAo was observed in the IRAP knockout animals with no differences in cerebral vascular anatomy detected. The current study demonstrates that deletion of the IRAP gene protects the brain from ischemic damage analogous to the effect of the IRAP inhibitor, Ang IV. This study indicates that IRAP is potentially a new therapeutic target for the development of treatment for ischemic stroke. PMID:21895534

  8. A Human-Mouse Chimeric Model of Obliterative Bronchiolitis after Lung Transplantation

    PubMed Central

    Xue, Jianmin; Zhu, Xuehai; George, M. Patricia; Myerburg, Michael M.; Stoner, Michael W.; Pilewski, Joseph W.; Duncan, Steven R.

    2011-01-01

    Obliterative bronchiolitis is a frequent, morbid, and usually refractory complication of lung transplantation. Mechanistic study of obliterative bronchiolitis would be aided by development of a relevant model that uses human immune effector cells and airway targets. Our objective was to develop a murine chimera model that mimics obliterative bronchiolitis of lung allograft recipients in human airways in vivo. Human peripheral blood mononuclear cells were adoptively transferred to immunodeficient mice lacking activity of T, B, and NK cells, with and without concurrent transplantations of human small airways dissected from allogeneic cadaveric lungs. Chimerism with human T cells occurred in the majority of recipient animals. The chimeric T cells became highly activated, rapidly infiltrated into the small human airway grafts, and caused obliterative bronchiolitis. In contrast, airways implanted into control mice that did not also receive human peripheral blood mononuclear cell transfers remained intact. In vitro proliferation assays indicated that the chimeric T cells had enhanced specific proliferative responses to donor airway alloantigens. This model confirms the critical role of T cells in development of obliterative bronchiolitis among human lung allograft recipients and provides a novel and easily implemented mechanism for detailed, reductionist in vivo studies of human T-cell responses to allogeneic human small airways. PMID:21801868

  9. Acquisition of an anti-idiotypic cytotoxic T lymphocyte repertoire in B cell-transferred or tetraparental bone marrow chimeric mice

    SciTech Connect

    Yamamoto, H.; Bitoh, S.; Fujimoto, S.

    1987-10-01

    In previous studies we showed that major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) specific for the cross-reactive idiotype (CRI) of MOPC-104E myeloma protein could only be induced in BALB/c or BAB-14 mice which have the ability to produce the CRI, but not in C.AL-20 or C.B-20 mice which have no ability to produce the CRI. The strong correlation between CRI-specific CTL responder strains and CRI producers supports the idea that the VH gene products are intrinsic primary antigenic stimuli for the generation of the anti-idiotypic CTL. To investigate the role of B lymphocytes in the selection of T lymphocyte repertoire, the purified B cells of CRI producer strains were repeatedly injected into anti-CRI CTL nonresponder neonatal mice. CRI-specific CTL activity was successfully induced in the CRI nonproducer mice only when they were exposed to CRI producer strain B lymphocytes from neonatal life. When the CTL nonresponder adult mice received CRI producer B lymphocytes, the nonresponder phenotype was not changed into the responder phenotype. Inducibility of CRI-specific CTL was also analyzed in tetraparental bone marrow chimeras. When CRI nonproducer bone marrow cells repopulated along with CRI producer bone marrow cells, the anti-CRI CTL of CRI nonproducer origin were generated. Adaptive differentiation of haplotype preference was also observed. When these observations are taken collectively, we see that the anti-idiotypic T lymphocyte repertoire is not a genetically determined one, but rather that the repertoire of T lymphocytes strongly depends on the postnatal selection process through the intrinsic idiotypic repertoire of B lymphocytes, i.e., internal images.

  10. Tests in mice of a dengue vaccine candidate made of chimeric Junin virus-like particles and conserved dengue virus envelope sequences.

    PubMed

    Mareze, Vania Aparecida; Borio, Cristina Silvia; Bilen, Marcos F; Fleith, Renata; Mirazo, Santiago; Mansur, Daniel Santos; Arbiza, Juan; Lozano, Mario Enrique; Bruña-Romero, Oscar

    2016-01-01

    Two new vaccine candidates against dengue virus (DENV) infection were generated by fusing the coding sequences of the self-budding Z protein from Junin virus (Z-JUNV) to those of two cryptic peptides (Z/DENV-P1 and Z/DENV-P2) conserved on the envelope protein of all serotypes of DENV. The capacity of these chimeras to generate virus-like particles (VLPs) and to induce virus-neutralizing antibodies in mice was determined. First, recombinant proteins that displayed reactivity with a Z-JUNV-specific serum by immunofluorescence were detected in HEK-293 cells transfected with each of the two plasmids and VLP formation was also observed by transmission electron microscopy. Next, we determined the presence of antibodies against the envelope peptides of DENV in the sera of immunized C57BL/6 mice. Results showed that those animals that received Z/DENV-P2 DNA coding sequences followed by a boost with DENV-P2 synthetic peptides elicited significant specific antibody titers (≥6.400). Finally, DENV plaque-reduction neutralization tests (PRNT) were performed. Although no significant protective effect was observed when using sera of Z/DENV-P1-immunized animals, antibodies raised against vaccine candidate Z/DENV-P2 (diluted 1:320) were able to reduce in over 50 % the number of viral plaques generated by infectious DENV particles. This reduction was comparable to that of the 4G2 DENV-specific monoclonal cross-reactive (all serotypes) neutralizing antibody. We conclude that Z-JUNV-VLP is a valid carrier to induce antibody-mediated immune responses in mice and that Z/DENV-P2 is not only immunogenic but also protective in vitro against infection of cells with DENV, deserving further studies. On the other side, DENV's fusion peptide-derived chimera Z/DENV-P1 did not display similar protective properties. PMID:26386688

  11. Chimeric vapA/groEL2 DNA vaccines enhance clearance of Rhodococcus equi in aerosol challenged C3H/He mice.

    PubMed

    Phumoonna, Tongted; Barton, Mary D; Vanniasinkam, Thiru; Heuzenroeder, Michael W

    2008-05-12

    Rhodococcus equi remains a significant bacterial pathogen, causing severe pyogranulomatous pneumonia in foals aged 1-3 months. There is no effective vaccine currently available for the prevention of R. equi pneumonia. DNA vaccines are known to offer specific advantages over conventional vaccines. The aim of this study was to demonstrate efficacy of our recombinant DNA vaccine candidates, namely pcDNA3-Re1, pcDNA3-Re3 and pcDNA3-Re5 by combining a heat shock protein GroEL2 to a virulence-associated protein A (VapA) from R. equi to protect C3H/He mice against the R. equi infection. VapA was shown to be strongly recognised by sera from pneumonic foals. All vaccines elicited at least a doubling of the IgG2a/IgG1 ratio in comparison to the controls, indicating a bias to the Th1 response, which is postulated to be crucial for bacterial clearance and protective immunity against intracellular pathogens including R. equi. In addition, the immunised mice showed a significant reduction in R. equi in their lungs at 7 days after the aerosol challenge in comparison to PBS treated mice. However, examination of lung pathology 14 days after the challenge showed no gross differences in pathological changes between the unvaccinated and vaccinated animals. The lack of significant pathological changes suggests that the precise level of protection against R. equi pneumonia in the murine model of infection may not represent a true effectiveness of the potential vaccine candidates, indicating the mouse may not be the ideal non-equine model for vaccine studies and (or) the incomplete immunogenic antigen of vapA-based DNA vaccine constructs that mount an inadequate cell-mediated immune response against the R. equi infection. PMID:18423949

  12. Construction and characterization of chimeric tick-borne encephalitis/dengue type 4 viruses.

    PubMed Central

    Pletnev, A G; Bray, M; Huggins, J; Lai, C J

    1992-01-01

    Dengue type 4 virus (DEN4) cDNA was used as a vector to express genes of the distantly related tick-borne encephalitis virus (TBEV). Full-length chimeric TBEV/DEN4 cDNAs were constructed by substituting TBEV genes coding for proteins such as capsid (C); pre-membrane, which is the precursor of membrane (M); envelope (E); or nonstructural protein NS1 for the corresponding DEN4 sequences. RNA transcripts prepared from cDNAs were used to transfect permissive simian cells. Two viable chimeric viruses that contained TBEV CME or ME genes were recovered. Compared with DEN4, chimeric TBE(ME)/DEN4 virus [designated vTBE(ME)/DEN4] produced larger plaques and grew to higher titer in simian cells. In contrast, vTBE(ME)/DEN4 produced smaller plaques on mosquito cells and grew to lower titer than DEN4. Analysis of viral RNA and proteins produced in vTBE(ME)/DEN4- and DEN4-infected mosquito or simian cells revealed that the chimera was restricted in its ability to enter and replicate in mosquito cells. In contrast, vTBE(ME)/DEN4 entered simian cells efficiently and its RNA was replicated more rapidly in these cells than was parental DEN4 RNA. Following intracerebral inoculation, vTBE(ME)/DEN4 caused fatal encephalitis in both suckling and adult mice, while nearly all mice inoculated by the same route with DEN4 did not develop disease. Unlike wild-type TBEV, vTBE(ME)/DEN4 did not cause encephalitis when adult mice were inoculated by a peripheral route. Adult mice previously inoculated with the chimera by a peripheral route were completely resistant to subsequent intraperitoneal challenge with 10(3) times the median lethal dose of TBEV, whereas mice previously inoculated with DEN4 were not protected. These findings indicate that (i) the TBEV M and E genes of the chimeric virus are major protective antigens and induce resistance to lethal TBEV challenge and (ii) other regions of the TBEV genome are essential for the ability of this virus to spread from a peripheral site to the brain

  13. A novel M2e-multiple antigenic peptide providing heterologous protection in mice

    PubMed Central

    Wen, Feng; Ma, Ji-Hong; Yang, Fu-Ru; Huang, Meng; Zhou, Yan-Jun; Li, Ze-Jun; Wang, Xiu-Hui; Li, Guo-Xin; Jiang, Yi-Feng; Tong, Wu

    2016-01-01

    Swine influenza viruses (SwIVs) cause considerable morbidity and mortality in domestic pigs, resulting in a significant economic burden. Moreover, pigs have been considered to be a possible mixing vessel in which novel strains loom. Here, we developed and evaluated a novel M2e-multiple antigenic peptide (M2e-MAP) as a supplemental antigen for inactivated H3N2 vaccine to provide cross-protection against two main subtypes of SwIVs, H1N1 and H3N2. The novel tetra-branched MAP was constructed by fusing four copies of M2e to one copy of foreign T helper cell epitopes. A high-yield reassortant H3N2 virus was generated by plasmid based reverse genetics. The efficacy of the novel H3N2 inactivated vaccines with or without M2e-MAP supplementation was evaluated in a mouse model. M2e-MAP conjugated vaccine induced strong antibody responses in mice. Complete protection against the heterologous swine H1N1 virus was observed in mice vaccinated with M2e-MAP combined vaccine. Moreover, this novel peptide confers protection against lethal challenge of A/Puerto Rico/8/34 (H1N1). Taken together, our results suggest the combined immunization of reassortant inactivated H3N2 vaccine and the novel M2e-MAP provided cross-protection against swine and human viruses and may serve as a promising approach for influenza vaccine development. PMID:27051342

  14. H-2 restriction as a consequence of intentional priming: T cells of fully allogeneic chimeric mice as well as of normal mice respond to foreign antigens in the context of H-2 determinants not encountered on thymic epithelial cells.

    PubMed

    Stockinger, H; Pfizenmaier, K; Hardt, C; Rodt, H; Röllinghoff, M; Wagner, H

    1980-12-01

    Fully allogeneic chimeras were able to develop in vitro alloantigen-specific, as well as H-2-restricted, Sendai virus-specific cytotoxic T-lymphocyte (CTL) response. Depending on the immunization regimen used, Sendai virus-specific CTL responses were restricted to the H-2 antigens of either the stem cell donor or the thymus. Similarly, unprimed splenic T cells of normal mice were found to contain CTL-precursor cells that specifically reacted against Sendai virus or trinitrophenyl derivatives in the context of allogeneic major histocompatibility complex determinants that had not been encountered during their thymic differentiation. A frequency analysis of allogeneically versus syngeneically restricted virus-specific CTL precursors present in splenic T cells showed a ratio of about 1 to 6. These results provide evidence that H-2 restriction of trinitrophenyl- or Sendai virus-specific T cells is dictated by the complex type of the antigen-presenting cell and thus appears to be independent of the type of thymus in which the T cells have undergone maturation. PMID:6261255

  15. Mecp2-Null Mice Provide New Neuronal Targets for Rett Syndrome

    PubMed Central

    Urdinguio, Rocio G.; Lopez-Serra, Lidia; Lopez-Nieva, Pilar; Alaminos, Miguel; Diaz-Uriarte, Ramon; Fernandez, Agustin F.; Esteller, Manel

    2008-01-01

    Background Rett syndrome (RTT) is a complex neurological disorder that is one of the most frequent causes of mental retardation in women. A great landmark in research in this field was the discovery of a relationship between the disease and the presence of mutations in the gene that codes for the methyl-CpG binding protein 2 (MeCP2). Currently, MeCP2 is thought to act as a transcriptional repressor that couples DNA methylation and transcriptional silencing. The present study aimed to identify new target genes regulated by Mecp2 in a mouse model of RTT. Methodology/Principal Findings We have compared the gene expression profiles of wild type (WT) and Mecp2-null (KO) mice in three regions of the brain (cortex, midbrain, and cerebellum) by using cDNA microarrays. The results obtained were confirmed by quantitative real-time PCR. Subsequent chromatin immunoprecipitation assays revealed seven direct target genes of Mecp2 bound in vivo (Fkbp5, Mobp, Plagl1, Ddc, Mllt2h, Eya2, and S100a9), and three overexpressed genes due to an indirect effect of a lack of Mecp2 (Irak1, Prodh and Dlk1). The regions bound by Mecp2 were always methylated, suggesting the involvement of the methyl-CpG binding domain of the protein in the mechanism of interaction. Conclusions We identified new genes that are overexpressed in Mecp2-KO mice and are excellent candidate genes for involvement in various features of the neurological disease. Our results demonstrate new targets of MeCP2 and provide us with a better understanding of the underlying mechanisms of RTT. PMID:18989361

  16. BAC transgenic mice provide evidence that p53 expression is highly regulated in vivo

    PubMed Central

    Chen, L; Zhang, G X; Zhou, Y; Zhang, C X; Xie, Y Y; Xiang, C; He, X Y; Zhang, Q; Liu, G

    2015-01-01

    p53 is an important tumor suppressor and stress response mediator. Proper control of p53 level and activity is tightly associated with its function. Posttranslational modifications and the interactions with Mdm2 and Mdm4 are major mechanisms controlling p53 activity and stability. As p53 protein is short-lived and hardly detectable in unstressed situations, less is known on its basal level expression and the corresponding controlling mechanisms in vivo. In addition, it also remains obscure how p53 expression might contribute to its functional regulation. In this study, we established bacterial artificial chromosome transgenic E.coli β-galactosidase Z gene reporter mice to monitor p53 expression in mouse tissues and identify important regulatory elements critical for the expression in vivo. We revealed preferentially high level of p53 reporter expressions in the proliferating, but not the differentiated compartments of the majority of tissues during development and tissue homeostasis. In addition, tumors as well as regenerating tissues in the p53 reporter mice also expressed high level of β-gal. Furthermore, both the enhancer box sequence (CANNTG) in the p53 promoter and the 3′ terminal untranslated region element were critical in mediating the high-level expression of the reporter. We also provided evidence that cellular myelocytomatosis oncogene was a critical player regulating p53 mRNA expression in proliferating cells and tissues. Finally, we found robust p53 activation preferentially in the proliferating compartment of mouse tissues upon DNA damage and the proliferating cells exhibited an enhanced p53 response as compared with cells in a quiescent state. Together, these results suggested a highly regulated expression pattern of p53 in the proliferating compartment controlled by both transcriptional and posttranscriptional mechanisms, and such regulated p53 expression may impose functional significance upon stress by setting up a precautionary mode in

  17. Enhanced humoral and mucosal immune responses after intranasal immunization with chimeric multiple antigen peptide of LcrV antigen epitopes of Yersinia pestis coupled to palmitate in mice.

    PubMed

    Uppada, Srijayaprakash Babu; Bhat, Ajaz Ahmed; Sah, Anil; Donthamshetty, Rao Nageswara

    2011-11-21

    Yersinia pestis is the causative agent of the most deadly disease plague. F1 and V antigens are the major vaccine candidates. Six protective epitopes of V antigen of varying length (15-25aa) were assembled on a lysine backbone as multiple antigen peptide (MAP) using standard Fmoc chemistry. Palmitate was coupled at amino terminus end. Amino acid analysis, SDS-PAGE, immunoblot and immunoreactivity proved the authenticity of MAP. MAP was immunized intranasally encapsulated in PLGA (polylactide-co-glycolide) microspheres and with/without/adjuvants murabutide and CpG ODN 1826 (CpG), in three strains of mice. Humoral and mucosal immune responses were studied till day 120 and memory response was checked after immunization with native V antigen on day 120. Epitope specific serum and mucosal washes IgG, IgA, IgG subclasses and specific activity were measured by indirect ELISA and sandwich ELISA, respectively. IgG and IgA peak antibody titers of all the MAP construct formulations in sera were ranging from 71,944 to 360,578 and 4493 to 28,644, respectively. MAP with CpG showed significantly high (p<0.0001) antibody titers ranging from 101,690 to 360,578 for IgG and 28,644 for IgA. Mucosal peak IgG and IgA titers were ranging from 1425 to 8072 and 1425 to 7183, respectively in intestinal washes and 799-4528 and 566-4027, respectively in lung washes. MAP with CpG showed significantly high (p<0.001) SIgA titers of 8000 in lung and 16,000 in intestinal washes. IgG isotyping revealed IgG2a/IgG1 ratio>1 with CpG. Serum and mucosal antipeptide IgG and IgA specific activities correlated well with antibody titers. All the constituent peptides contributed towards immune response. Structural analysis of MAP revealed little or no interaction between the peptides. Present study showed MAP to be highly immunogenic with high and long lasting antibody titers in serum and mucosal washes with good recall response with/without CpG as an adjuvant which can be used for vaccine development for

  18. Ectopic brown adipose tissue in muscle provides a mechanism for differences in risk of metabolic syndrome in mice

    PubMed Central

    Almind, Katrine; Manieri, Monia; Sivitz, William I.; Cinti, Saverio; Kahn, C. Ronald

    2007-01-01

    C57BL/6 (B6) mice subjected to a high-fat diet develop metabolic syndrome with obesity, hyperglycemia, and insulin resistance, whereas 129S6/SvEvTac (129) mice are relatively protected from this disorder because of differences in higher basal energy expenditure in 129 mice, leading to lower weight gain. At a molecular level, this difference correlates with a marked higher expression of uncoupling protein 1 (UCP1) and a higher degree of uncoupling in vitro in mitochondria isolated from muscle of 129 versus B6 mice. Detailed histological examination, however, reveals that this UCP1 is in mitochondria of brown adipocytes interspersed between muscle bundles. Indeed, the number of UCP1-positive brown fat cells in intermuscular fat in 129 mice is >700-fold higher than in B6 mice. These brown fat cells are subject to further up-regulation of UCP1 after stimulation with a β3-adrenergic receptor agonist. Thus, ectopic deposits of brown adipose tissue in intermuscular depots with regulatable expression of UCP1 provide a genetically based mechanism of protection from weight gain and metabolic syndrome between strains of mice. PMID:17283342

  19. Analyzing cell fusion events within the central nervous system using bone marrow chimerism.

    PubMed

    Kemp, Kevin; Hares, Kelly

    2015-01-01

    It has emerged that cells which typically reside in the bone marrow have the capacity to cross the blood brain barrier and contribute genetic material to a range of neuronal cell types within the central nervous system. One such mechanism to account for this phenomenon is cellular fusion, occurring between migrating bone marrow-derived stem cells and neuronal cells in-situ. Biologically, the significance as to why cells from distinct lineages fuse with cells of the central nervous system is, as yet, unclear. Growing evidence however suggests that these cell fusion events could provide an efficient means of rescuing the highly complex and differentiated neuronal cell types that cannot be replaced in adulthood. To facilitate further understanding of cell fusion within the central nervous system, we describe here a technique to establish chimeric mice that are stably reconstituted with green fluorescent protein expressing sex-mismatched bone marrow. These chimeric mice are known to represent an excellent model for studying bone marrow cell migration and infiltration throughout the body, while in parallel, as will be described here, also provide a means to neatly analyze both bone marrow-derived cell fusion and trans-differentiation events within the central nervous system. PMID:25947664

  20. Electromagnetic Treatment to Old Alzheimer's Mice Reverses β-Amyloid Deposition, Modifies Cerebral Blood Flow, and Provides Selected Cognitive Benefit

    PubMed Central

    Arendash, Gary W.; Mori, Takashi; Dorsey, Maggie; Gonzalez, Rich; Tajiri, Naoki; Borlongan, Cesar

    2012-01-01

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

  1. Chimeric Lyssavirus Glycoproteins with Increased Immunological Potential

    PubMed Central

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

    1999-01-01

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

  2. Mixed chimerism and permanent specific transplantation tolerance induced by a nonlethal preparative regimen

    SciTech Connect

    Sharabi, Y.; Sachs, D.H.

    1989-02-01

    The use of allogeneic bone marrow transplantation as a means of inducing donor-specific tolerance across MHC barriers could provide an immunologically specific conditioning regimen for organ transplantation. However, a major limitation to this approach is the toxicity of whole body irradiation as currently used to abrogate host resistance and permit marrow engraftment. The present study describes methodology for abrogating host resistance and permitting marrow engraftment without lethal irradiation. Our preparative protocol involves administration of anti-CD4 and anti-CD8 mAbs in vivo, 300-rad WBI, 700-rad thymic irradiation, and unmanipulated fully MHC-disparate bone marrow. B10 mice prepared by this regimen developed stable mixed lymphohematopoetic chimerism without any clinical evidence of graft-vs.-host disease. Engraftment was accompanied by induction of specific tolerance to donor skin grafts (B10.D2), while third-party skin grafts (B10.BR) were promptly rejected. Mice treated with the complete regimen without bone marrow transplantation appeared healthy and enjoyed long-term survival. This study therefore demonstrates that stable mixed chimerism with donor-specific tolerance can be induced across an MHC barrier after a nonlethal preparative regimen, without clinical GVHD and without the risk of aplasia.

  3. Anti-idiotypic immunization provides protection against lethal endotoxaemia in BALB/c mice.

    PubMed Central

    Cornelissen, J J; Maassen, K; van Emst, L; Weers, P M; Harmsen, M; Benaissa-Trouw, B J; Oosterlaken, T A; Kraaijeveld, C A; Verhoef, J

    1993-01-01

    Against lipid A (the conserved moiety of lipopolysaccharides from Gram-negative bacteria) neutralizing IgM monoclonal antibodies (mAb) 8-2 and 26-20 anti-idiotypic (Ab2) mAb were produced: Ab2 mAb KM-04 (IgG1) against mAb 8-2, and Ab2 mAb PW-1 (IgG2a) and PW-2 (IgG1) against mAb 26-20. The binding of Ab2 mAb KM-04 to 8-2 (Ab1) was strongly inhibited by a lipopolysaccharide (LPS) extract from either Salmonella minnesota R595 (Re LPS) or Escherichia coli J5 (Rc LPS), whereas the binding of Ab2 mAb PW-1 and PW-2 to 26-20 (Ab1) was only marginally inhibited by both Re LPS and Rc LPS. The results indicated that Ab2 mAb KM-04 recognizes a lipid A-binding site related idiotope on mAb 8-2 and therefore KM-04 might bear the internal image of a neutralization determining epitope of lipid A. Consequently Ab2 KM-04 might induce antibodies to lipid A. Indeed anti-idiotypic immunization of syngeneic BALB/c mice with Ab2 mAb KM-04 resulted in development of lipid A-binding anti-anti-idiotypic (Ab3) antibodies in serum. Similar immunizations with Ab2 mAb PW-1 and PW-2 were unsuccessful. However, induction of lipid A-binding Ab3 by mAb KM-04 proved to be genetically restricted to BALB/c mice. DBA/2 mice, Swiss mice and rabbits did not develop lipid A-binding antibodies upon immunization with mAb KM-04. In protection experiments, it was shown that BALB/c mice vaccinated with mAb KM-04 showed significantly enhanced survival from challenge with either rough (Re) LPS from Salmonella minnesota or smooth LPS from E. coli 0111:B4 when compared to BALB/c mice immunized with a non-relevant Ab2 mAb. The results suggest that mAb KM-04 constitutes a non-internal image vaccine to the lethal effect of lipid A in BALB/c mice. Furthermore an Ab3 mAb was prepared against Ab2 mAb KM-04 that showed reactivity with Re LPS. This Ab3 mAb, designated LE-21 (IgG2a) protected mice against an otherwise lethal challenge of Re LPS. PMID:8406595

  4. Adenosine monophosphate deaminase 3 activation shortens erythrocyte half-life and provides malaria resistance in mice.

    PubMed

    Hortle, Elinor; Nijagal, Brunda; Bauer, Denis C; Jensen, Lora M; Ahn, Seong Beom; Cockburn, Ian A; Lampkin, Shelley; Tull, Dedreia; McConville, Malcolm J; McMorran, Brendan J; Foote, Simon J; Burgio, Gaetan

    2016-09-01

    The factors that determine red blood cell (RBC) lifespan and the rate of RBC aging have not been fully elucidated. In several genetic conditions, including sickle cell disease, thalassemia, and G6PD deficiency, erythrocyte lifespan is significantly shortened. Many of these diseases are also associated with protection from severe malaria, suggesting a role for accelerated RBC senescence and clearance in malaria resistance. Here, we report a novel, N-ethyl-N-nitrosourea-induced mutation that causes a gain of function in adenosine 5'-monophosphate deaminase (AMPD3). Mice carrying the mutation exhibit rapid RBC turnover, with increased erythropoiesis, dramatically shortened RBC lifespan, and signs of increased RBC senescence/eryptosis, suggesting a key role for AMPD3 in determining RBC half-life. Mice were also found to be resistant to infection with the rodent malaria Plasmodium chabaudi. We propose that resistance to P. chabaudi is mediated by increased RBC turnover and higher rates of erythropoiesis during infection. PMID:27465915

  5. Chimeric enzymes with improved cellulase activities

    SciTech Connect

    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.

  6. Chimeric autologous/allogeneic constructs for skin regeneration.

    PubMed

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

    2014-08-01

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

  7. Virus-Neutralizing Monoclonal Antibody Expressed in Milk of Transgenic Mice Provides Full Protection against Virus-Induced Encephalitis

    PubMed Central

    Kolb, Andreas F.; Pewe, Lecia; Webster, John; Perlman, Stanley; Whitelaw, C. Bruce A.; Siddell, Stuart G.

    2001-01-01

    Neutralizing antibodies represent a major host defense mechanism against viral infections. In mammals, passive immunity is provided by neutralizing antibodies passed to the offspring via the placenta or the milk as immunoglobulin G and secreted immunoglobulin A. With the long-term goal of producing virus-resistant livestock, we have generated mice carrying transgenes that encode the light and heavy chains of an antibody that is able to neutralize the neurotropic JHM strain of murine hepatitis virus (MHV-JHM). MHV-JHM causes acute encephalitis and acute and chronic demyelination in susceptible strains of mice and rats. Transgene expression was targeted to the lactating mammary gland by using the ovine β-lactoglobulin promoter. Milk from these transgenic mice contained up to 0.7 mg of recombinant antibody/ml. In vitro analysis of milk derived from different transgenic lines revealed a linear correlation between antibody expression and virus-neutralizing activity, indicating that the recombinant antibody is the major determinant of MHV-JHM neutralization in murine milk. Offspring of transgenic and control mice were challenged with a lethal dose of MHV-JHM. Litters suckling nontransgenic dams succumbed to fatal encephalitis, whereas litters suckling transgenic dams were fully protected against challenge, irrespective of whether they were transgenic. This demonstrates that a single neutralizing antibody expressed in the milk of transgenic mice is sufficient to completely protect suckling offspring against MHV-JHM-induced encephalitis. PMID:11222704

  8. DELETION OF iNOS PROVIDES CARDIOPROTECTION IN MICE WITH 2-KIDNEY, 1-CLIP HYPERTENSION

    PubMed Central

    Sun, Ying; Carretero, Oscar A.; Xu, Jiang; Rhaleb, Nour-Eddine; Yang, James J.; Pagano, Patrick J.; Yang, Xiao-Ping

    2009-01-01

    Inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of hypertension and target organ damage. We hypothesized that induction of iNOS contributes to left ventricular (LV) hypertrophy and dysfunction in mice with 2-kidney, 1-clip hypertension (2K1C). Deletion of iNOS diminishes oxidative stress, thereby attenuating LV hypertrophy and enhancing cardiac performance. 2K1C was induced in mice lacking iNOS (iNOS−/−) and wild-type controls (WT, C57BL/6J). Sham-clipped mice served as controls. Systolic blood pressure (SBP) was measured weekly by tail cuff. Left ventricular (LV) ejection fraction (EF, by echocardiography) and cardiac response (dP/dtmax, dP/dt/ip and dP/dtmin) to isoproterenol (ISO: 50 ng/mouse, i.v.) were studied at the end of the experiment. 4-hydroxy-2-nonenal (4-HNE, a byproduct of lipid peroxidation and an indicator of oxidative stress) was measured by immunohistochemical staining. Nox2, eNOS and iNOS protein expression were determined by Western blot. We found that SBP, LV weight (LVW), myocyte cross-sectional area (MCSA), interstitial collagen fraction (ICF), EF and cardiac response to ISO did not differ between strains with sham clipping. 2K1C increased SBP, LVW, MCSA and ICF similarly in both strains. However, in iNOS−/− dP/dtmax, dP/dt/ip and dP/dtmin markedly increased in response to ISO, associated with decreased cardiac 4-HNE expression and urinary nitrate/nitrite. We concluded that deletion of iNOS does not seem to play a significant role in preventing 2K1C-induced hypertension and cardiac hypertrophy; however, it does enhance preservation of cardiac function, probably due to reduction of iNOS-induced oxidative stress. PMID:19001185

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

    SciTech Connect

    Tomioka, Yukiko; Morimatsu, Masami; Nishijima, Ken-ichi; Usui, Tatsufumi; Yamamoto, Sayo; Suyama, Haruka; Ozaki, Kinuyo; Ito, Toshihiro; and others

    2014-07-18

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

  10. Opportunistic reproduction by white-footed mice (Peromyscus leucopus) provided with supplemental food

    SciTech Connect

    Ormiston, B.G.

    1984-07-01

    The role of food supply in determining P. leucopus habitat suitability was investigated in xeric upland (oak-pine) forest habitat, which previously has been shown to be relatively unfavorable for white-footed mice in summer. Food (Purina 5001 lab chow) was available to mice that visited feeders placed at 40 m intervals on two of four one-ha trapping grids. Grids were censused by trapping for three consecutive days prior to and following the treatment period, which occurred for about 30 days in July 1981. Population density increased on food supplied relative to control grids following treatment, but not significantly so. Adult sex ratio was nearer to unity in food augmented areas compared with control areas, which tended to yield more males than females. Significantly greater proportions of adult males and females were reproductive in food supplied relative to control areas after the treatment period. The results suggest that upland habitat is relatively food deficient in summer, and that the habitat occurrence of breeding P. leucopus depends in part on food supply. The significance of these findings for population organization and regulation in this species is discussed. 26 references, 1 figure, 2 tables.

  11. Surfactant-associated protein A provides critical immunoprotection in neonatal mice.

    PubMed

    George, Caroline L S; Goss, Kelli L; Meyerholz, David K; Lamb, Fred S; Snyder, Jeanne M

    2008-01-01

    The collectins surfactant-associated protein A (SP-A) and SP-D are components of innate immunity that are present before birth. Both proteins bind pathogens and assist in clearing infection. The significance of SP-A and SP-D as components of the neonatal immune system has not been investigated. To determine the role of SP-A and SP-D in neonatal immunity, wild-type, SP-A null, and SP-D null mice were bred in a bacterium-laden environment (corn dust bedding) or in a semisterile environment (cellulose fiber bedding). When reared in the corn dust bedding, SP-A null pups had significant mortality (P < 0.001) compared to both wild-type and SP-D null pups exposed to the same environment. The mortality of the SP-A null pups was associated with significant gastrointestinal tract pathology but little lung pathology. Moribund SP-A null newborn mice exhibited Bacillus sp. and Enterococcus sp. peritonitis. When the mother or newborn produced SP-A, newborn survival was significantly improved (P < 0.05) compared to the results when there was a complete absence of SP-A in both the mother and the pup. Significant sources of SP-A likely to protect a newborn include the neonatal lung and gastrointestinal tract but not the lactating mammary tissue of the mother. Furthermore, exogenous SP-A delivered by mouth to newborn SP-A null pups with SP-A null mothers improved newborn survival in the corn dust environment. Therefore, a lack of SP-D did not affect newborn survival, while SP-A produced by either the mother or the pup or oral exogenous SP-A significantly reduced newborn mortality associated with environmentally induced infection in SP-A null newborns. PMID:17967856

  12. Effect of IL-2-Bax, a novel interleukin-2-receptor-targeted chimeric protein, on bleomycin lung injury1

    PubMed Central

    Segel, Michael J; Aqeilan, Rami; Zilka, Keren; Lorberboum-Galski, Haya; Wallach-Dayan, Shulamit B; Conner, Michael W; Christensen, Thomas G; Breuer, Raphael

    2005-01-01

    The role of lymphocytes in the pathogenesis of lung fibrosis is not clear, but the weight of the evidence supports a pro-fibrotic effect for lymphocytes. The high-affinity interleukin-2 receptor (haIL-2R) is expressed on activated, but not quiescent, T lymphocytes. This selective expression of haIL-2R provides the basis for therapeutic strategies that target IL-2R-expressing cells. We hypothesized that elimination of activated lymphocytes by IL-2R-targeted chimeric proteins might ameliorate lung fibrosis. We investigated the effects of IL-2-Bax, a novel apoptosis-inducing IL-2R-targeted chimeric protein, on bleomycin-induced lung injury in mice. Treatment groups included (i) a single intratracheal instillation of bleomycin and twice-daily intraperitoneal injections of IL-2-Bax; (ii) intratracheal bleomycin and intraperitoneal IL-2-PE664Glu, an older-generation chimeric protein; (iii) intratracheal bleomycin/intraperitoneal PBS; (iv) intratracheal saline/intraperitoneal PBS. Lung injury was evaluated 14 days after intratracheal instillation by cell count in bronchoalveolar lavage (BAL) fluid, semi-quantitative and quantitative histomorphological measurements and by biochemical analysis of lung hydroxyproline. Bleomycin induced a BAL lymphocytosis that was significantly attenuated by IL-2-Bax and IL-2-PE664Glu. However, morphometric parameters and lung hydroxyproline were unaffected by the chimeric proteins. These results show that IL-2-Bax reduces the lymphocytic infiltration of the lungs in response to bleomycin, but this effect is not accompanied by a decrease in lung fibrosis. PMID:16191100

  13. A Novel Self-Replicating Chimeric Lentivirus-Like Particle

    PubMed Central

    Young, Kelly R.; Madden, Victoria J.; Johnson, Philip R.; Johnston, Robert E.

    2012-01-01

    Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4+ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation. PMID:22013035

  14. A chimeric protein composed of the binding domains of Clostridium perfringens phospholipase C and Trueperella pyogenes pyolysin induces partial immunoprotection in a mouse model.

    PubMed

    Hu, Yunhao; Zhang, Wenlong; Bao, Jun; Wu, Yuhong; Yan, Minghui; Xiao, Ya; Yang, Lingxiao; Zhang, Yue; Wang, Junwei

    2016-08-01

    Trueperella pyogenes and Clostridium perfringens are two kinds of conditional pathogens frequently associated with wound infections and succeeding lethal complications in various economic livestock. Pyolysin (PLO) and phospholipase C (PLC) are the key virulence factors of these two pathogens, respectively. In our study, a chimeric protein called rPC-PD4, which is composed of the binding regions of PLO and PLC, was synthesized. The toxicity of rPC-PD4 was evaluated. Results revealed that rPC-PD4 is a safe chimeric molecule that can be used to develop vaccines. Immunizing BALB/c mice with rPC-PD4 induced high titers of serum antibodies that could efficiently neutralize the hemolytic activity of recombinant PLO and PLC. After the challenge with T. pyogenes or C. perfringens was performed through the intraperitoneal route, we observed that rPC-PD4 immunization could provide partial immunoprotection and reduce lung, intestine, and liver tissue damage to mice. This work demonstrated the efficacy of the rationally designed rPC-PD4 chimeric protein as a potential vaccine candidate against C. perfringens and T. pyogenes. PMID:27473983

  15. CHIMERIC ALPHAVIRUS VACCINE CANDIDATES FOR CHIKUNGUNYA

    PubMed Central

    Wang, Eryu; Volkova, Eugenia; Adams, A. Paige; Forrester, Naomi; Xiao, Shu-Yuan; Frolov, Ilya; Weaver, Scott C.

    2008-01-01

    Chikungunya virus (CHIKV) is an emerging alphavirus that has caused major epidemics in India and islands off the east coast of Africa since 2005. Importations into Europe and the Americas, including one that led to epidemic transmission in Italy during 2007, underscore the risk of endemic establishment elsewhere. Because there is no licensed human vaccine, and an attenuated Investigational New Drug product developed by the U.S. Army causes mild arthritis in some vaccinees, we developed chimeric alphavirus vaccine candidates using either Venezuelan equine encephalitis attenuated vaccine strain TC-83, a naturally attenuated strain of eastern equine encephalitis virus (EEEV), or Sindbis virus as a backbone and the structural protein genes of CHIKV. All vaccine candidates replicated efficiently in cell cultures, and were highly attenuated in mice. All of the chimeras also produced robust neutralizing antibody responses, although the TC-83 and EEEV backbones appeared to offer greater immunogenicity. Vaccinated mice were fully protected against disease and viremia after CHIKV challenge. PMID:18692107

  16. RNAi-Mediated CCR5 Knockdown Provides HIV-1 Resistance to Memory T Cells in Humanized BLT Mice

    PubMed Central

    Shimizu, Saki; Ringpis, Gene-Errol; Marsden, Matthew D; Cortado, Ruth V; Wilhalme, Holly M; Elashoff, David; Zack, Jerome A; Chen, Irvin S Y; An, Dong Sung

    2015-01-01

    Transplantation of hematopoietic stem/progenitor cells (HSPC) modified with a lentiviral vector bearing a potent nontoxic short hairpin RNA (sh1005) directed to the HIV coreceptor CCR5 is capable of continuously producing CCR5 downregulated CD4+ T lymphocytes. Here, we characterized HIV-1 resistance of the sh1005-modified CD4+ T lymphocytes in vivo in humanized bone marrow/liver/thymus (hu BLT) mice. The sh1005-modified CD4+ T lymphocytes were positively selected in CCR5-tropic HIV-1–challenged mice. The sh1005-modified memory CD4+ T lymphocytes (the primary target of CCR5-tropic HIV-1) expressing sh1005 were maintained in lymphoid tissues in CCR5-tropic HIV-1–challenged mice. Frequencies of HIV-1 p24 expressing cells were significantly reduced in the sh1005-modified splenocytes by ex vivo cell stimulation confirming that CCR5 downregulated sh1005 modified cells are protected from viral infection. These results demonstrate that stable CCR5 downregulation through genetic modification of human HSPC by lentivirally delivered sh1005 is highly effective in providing HIV-1 resistance. Our results provide in vivo evidence in a relevant small animal model that sh1005 is a potent early-step anti-HIV reagent that has potential as a novel anti-HIV-1 HSPC gene therapeutic reagent for human applications. PMID:25689223

  17. A measure of glucocorticoid load provided by DNA methylation of Fkbp5 in mice

    PubMed Central

    Lee, R. S.; Tamashiro, K. L. K.; Purcell, R. H.; Huo, Y.; Rongione, M.; Potash, J. B.; Yang, X.; Wand, G. S.

    2014-01-01

    Rationale Given the contribution of cortisol dysregulation to neuropsychiatric and metabolic disorders, it is important to be able to accurately compute glucocorticoid burden, a measure of allostatic load. One major problem in calculating cortisol burden is that existing measures reflect cortisol exposure over a short duration and have not been proven to reliably quantify cortisol burden over weeks or months. Method We treated two cohorts of mice with corticosterone in the drinking water and determined the relationship between serial plasma corticosterone levels drawn over 4 weeks and the whole-blood DNA methylation (DNAm) changes in a specific glucocorticoid-sensitive gene, Fkbp5, determined at the end of the treatment period. Results We observed that the percent reduction in DNAm in the intron 1 region of Fkbp5 determined from a single blood draw strongly reflected average glucocorticoid burden generated weekly during the prior month of glucocorticoid exposure. There were also strong correlations in DNAm with glucocorticoid-induced end organ changes in spleen weight and visceral fat. We tested a subset of these animals for anxiety-like behavior in the elevated plus maze and found that DNAm in the blood also has predictive value in determining the behavioral consequences of glucocorticoid exposure. Conclusion A whole-blood assessment of Fkbp5 gene methylation is a biomarker that integrates 4 weeks of glucocorticoid exposure and may be a useful measure in states of excess exposure. It will be important to determine if Fkbp5 DNAm changes can also be a biomarker of glucocorticoid burden during chronic social stress. PMID:21509501

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

    SciTech Connect

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

    1990-06-01

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

  19. Dre - Cre Sequential Recombination Provides New Tools for Retinal Ganglion Cell Labeling and Manipulation in Mice

    PubMed Central

    Shi, Melody; Liu, Pinghu; Dong, Lijin; Parmhans, Nadia; Popescu, Octavian; Badea, Tudor Constantin

    2014-01-01

    Background Genetic targeting methods have greatly advanced our understanding of many of the 20 Retinal Ganglion Cell (RGC) types conveying visual information from the eyes to the brain. However, the complexity and partial overlap of gene expression patterns in RGCs call for genetic intersectional or sparse labeling strategies. Loci carrying the Cre recombinase in conjunction with conditional knock-out, reporter or other genetic tools can be used for targeted cell type ablation and functional manipulation of specific cell populations. The three members of the Pou4f family of transcription factors, Brn3a, Brn3b and Brn3c, expressed early during RGC development and in combinatorial pattern amongst RGC types are excellent candidates for such gene manipulations. Methods and Findings We generated conditional Cre knock-in alleles at the Brn3a and Brn3b loci, Brn3aCKOCre and Brn3bCKOCre. When crossed to mice expressing the Dre recombinase, the endogenous Brn3 gene expressed by Brn3aCKOCre or Brn3bCKOCre is removed and replaced with a Cre recombinase, generating Brn3aCre and Brn3bCre knock-in alleles. Surprisingly both Brn3aCre and Brn3bCre knock-in alleles induce early ubiquitous recombination, consistent with germline expression. However in later stages of development, their expression is limited to the expected endogenous pattern of the Brn3a and Brn3b genes. We use the Brn3aCre and Brn3bCre alleles to target a Cre dependent Adeno Associated Virus (AAV) reporter to RGCs and demonstrate its use in morphological characterization, early postnatal gene delivery and tracing the expression of Brn3 genes in RGCs. Conclusions Dre recombinase effectively recombines the Brn3aCKOCre and Brn3bCKOCre alleles containing its roxP target sites. Sequential Dre to Cre recombination reveals Brn3a and Brn3b expression in early mouse development. The generated Brn3aCre and Brn3bCre alleles are useful tools that can target exogenously delivered Cre dependent reagents to RGCs in early

  20. Expression of a recombinant chimeric protein of hepatitis A virus VP1-Fc using a replicating vector based on Beet curly top virus in tobacco leaves and its immunogenicity in mice.

    PubMed

    Chung, Ho Yong; Lee, Hyun Ho; Kim, Kyung Il; Chung, Ha Young; Hwang-Bo, Jeon; Park, Jong Hwa; Sunter, Garry; Kim, Jong Bum; Shon, Dong Hwa; Kim, Wonyong; Chung, In Sik

    2011-08-01

    We describe the expression and immunogenicity of a recombinant chimeric protein (HAV VP1-Fc) consisting of human hepatitis A virus VP1 and an Fc antibody fragment using a replicating vector based on Beet curly top virus (BCTV) in Agrobacterium-infiltrated Nicotiana benthamiana leaves. Recombinant HAV VP1-Fc was expressed with a molecular mass of approximately 68 kDa. Recombinant HAV VP1-Fc, purified using Protein A Sepharose affinity chromatography, elicited production of specific IgG antibodies in the serum after intraperitoneal immunization. Following vaccination with recombinant HAV VP1-Fc protein, expressions of IFN-γ and IL-4 were increased in splenocytes at the time of sacrifice. Recombinant VP1-Fc from infiltrated tobacco plants can be used as an effective experimental immunogen for research into vaccine development. PMID:21442402

  1. A Chimeric Virus-Mouse Model System for Evaluating the Function and Inhibition of Papain-Like Proteases of Emerging Coronaviruses

    PubMed Central

    Deng, Xufang; Agnihothram, Sudhakar; Mielech, Anna M.; Nichols, Daniel B.; Wilson, Michael W.; StJohn, Sarah E.; Larsen, Scott D.; Mesecar, Andrew D.; Lenschow, Deborah J.; Baric, Ralph S.

    2014-01-01

    ABSTRACT To combat emerging coronaviruses, developing safe and efficient platforms to evaluate viral protease activities and the efficacy of protease inhibitors is a high priority. Here, we exploit a biosafety level 2 (BSL-2) chimeric Sindbis virus system to evaluate protease activities and the efficacy of inhibitors directed against the papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV), a biosafety level 3 (BSL-3) pathogen. We engineered Sindbis virus to coexpress PLpro and a substrate, murine interferon-stimulated gene 15 (ISG15), and found that PLpro mediates removal of ISG15 (deISGylation) from cellular proteins. Mutation of the catalytic cysteine residue of PLpro or addition of a PLpro inhibitor blocked deISGylation in virus-infected cells. Thus, deISGylation is a marker of PLpro activity. Infection of alpha/beta interferon receptor knockout (IFNAR−/−) mice with these chimeric viruses revealed that PLpro deISGylation activity removed ISG15-mediated protection during viral infection. Importantly, administration of a PLpro inhibitor protected these mice from lethal infection, demonstrating the efficacy of a coronavirus protease inhibitor in a mouse model. However, this PLpro inhibitor was not sufficient to protect the mice from lethal infection with SARS-CoV MA15, suggesting that further optimization of the delivery and stability of PLpro inhibitors is needed. We extended the chimeric-virus platform to evaluate the papain-like protease/deISGylating activity of Middle East respiratory syndrome coronavirus (MERS-CoV) to provide a small-animal model to evaluate PLpro inhibitors of this recently emerged pathogen. This platform has the potential to be universally adaptable to other viral and cellular enzymes that have deISGylating activities. IMPORTANCE Evaluating viral protease inhibitors in a small-animal model is a critical step in the path toward antiviral drug development. We modified a biosafety level 2 chimeric virus

  2. Production and characterization of a recombinant chimeric antigen consisting botulinum neurotoxin serotypes A, B and E binding subdomains.

    PubMed

    Ebrahimi, Firouz; Rasaee, Mohammad Javad; Mousavi, Seyed Latif; Babaeipour, Valiollah

    2010-02-01

    Botulinum neurotoxins (BoNTs) are potent toxicant proteins composed of a heavy chain (100 kDa) and a light chain (50 kDa) of seven (A-G) serotypes that is responsible for botulism syndrome. In this study, polypeptides from C-terminal heavy chain of BoNTs serotypes A, B and E to the length of 54, 45 and 48 amino acid respectively were selected, linked together using a hydrophobic linker and expressed in E. coli. The expression efficiency of the chimeric protein was found to be 51%. The chimeric protein was produced in the form of inclusion body (IB) both at two studied temperatures, 30 degrees C and 37 degrees C. This IB was extracted by ultracentrifugation and followed for chimeric protein solubilization and purification using of ultrafiltration and preparative electrophoresis. The purified chimeric protein was characterized using blotting and ELISA. To evaluate the protection ability of this chimeric antigen against their active toxins, it was injected to mice and the antibody titer as well as the extent of protectivity were determined. Mice given three injections (10 microg/mice) of the antigen were protected against an intra-peritoneal administration of 10 LD(50 )of serotypes A and E, but 100 LD(50) of serotype B. We conclude that a significant correlation exists between the antigenic characteristics and protection capability of the chimeric protein prepared in this study. PMID:20118620

  3. Enhanced efficacy of an AAV vector encoding chimeric, highly secreted acid alpha-glucosidase in glycogen storage disease type II.

    PubMed

    Sun, Baodong; Zhang, Haoyue; Benjamin, Daniel K; Brown, Talmage; Bird, Andrew; Young, Sarah P; McVie-Wylie, Alison; Chen, Y-T; Koeberl, Dwight D

    2006-12-01

    Glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) is an inherited muscular dystrophy caused by deficiency in the activity of the lysosomal enzyme acid alpha-glucosidase (GAA). We hypothesized that chimeric GAA containing an alternative signal peptide could increase the secretion of GAA from transduced cells and enhance the receptor-mediated uptake of GAA in striated muscle. The relative secretion of chimeric GAA from transfected 293 cells increased up to 26-fold. Receptor-mediated uptake of secreted, chimeric GAA corrected cultured GSD-II patient cells. High-level hGAA was sustained in the plasma of GSD-II mice for 24 weeks following administration of an AAV2/8 vector encoding chimeric GAA; furthermore, GAA activity was increased and glycogen content was significantly reduced in striated muscle and in the brain. Administration of only 1 x 10(10) vector particles increased GAA activity in the heart and diaphragm for >18 weeks, whereas 3 x 10(10) vector particles increased GAA activity and reduced glycogen content in the heart, diaphragm, and quadriceps. Furthermore, an AAV2/2 vector encoding chimeric GAA produced secreted hGAA for >12 weeks in the majority of treated GSD-II mice. Thus, chimeric, highly secreted GAA enhanced the efficacy of AAV vector-mediated gene therapy in GSD-II mice. PMID:16987711

  4. Chimeric NK-receptor–bearing T cells mediate antitumor immunotherapy

    PubMed Central

    Zhang, Tong; Lemoi, Bethany A.; Sentman, Charles L.

    2005-01-01

    NKG2D is an activating cell-surface receptor expressed on natural killer (NK) cells and some T-cell subsets. Its ligands are primarily expressed on tumor cells. The aim of this study was to determine whether chimeric NK-receptor—bearing T cells would directly kill tumor cells and lead to induction of host immunity against tumors. Chimeric NK receptors were produced by linking NKG2D or DNAX activating protein of 10 kDa (Dap10) to the cytoplasmic portion of the CD3ζ chain. Our results showed that chimeric (ch) NKG2D-bearing T cells responded to NKG2D-ligand–bearing tumor cells (RMA/Rae-1β, EG7) but not to wild-type tumor cells (RMA). This response was dependent upon ligand expression on the target cells but not on expression of major histocompatibility complex (MHC) molecules, and the response could be blocked by anti-NKG2D antibodies. These T cells produced large amounts of T-helper 1 (Th1) cytokines and proinflammatory chemokines and killed ligand–expressing tumor cells. Adoptive transfer of chNKG2D-bearing T cells inhibited RMA/Rae-1β tumor growth in vivo. Moreover, mice that had remained tumor-free were resistant to subsequent challenge with the wild-type RMA tumor cells, suggesting the generation of immunity against other tumor antigens. Taken together, our findings indicate that modification of T cells with chimeric NKG2D receptors represents a promising approach for immunotherapy against cancer. PMID:15890688

  5. Can Humanized Mice Predict Drug "Behavior" in Humans?

    PubMed

    Xu, Dan; Peltz, Gary

    2016-01-01

    Most of what we know about a drug prior to human clinical studies is derived from animal testing. Because animals and humans have substantial differences in their physiology and in their drug metabolism pathways, we do not know very much about the pharmacokinetic and pharmacodynamic behavior of a drug in humans until after it is administered to many people. Hence, drug-induced liver injury has become a significant public health problem, and we have a very inefficient drug development process with a high failure rate. Because the human liver is at the heart of these problems, chimeric mice with humanized livers could be used to address these issues. We examine recent evidence indicating that drug testing in chimeric mice could provide better information about a drug's metabolism, disposition, and toxicity (i.e., its "behavior") in humans and could aid in developing personalized medicine strategies, which would improve drug efficacy and safety. PMID:26514208

  6. Multi-Stage Tuberculosis Subunit Vaccine Candidate LT69 Provides High Protection against Mycobacterium tuberculosis Infection in Mice

    PubMed Central

    Niu, Hongxia; Peng, Jinxiu; Bai, Chunxiang; Liu, Xun; Hu, Lina; Luo, Yanping; Wang, Bingxiang; Zhang, Ying; Chen, Jianzhu; Yu, Hongjuan; Xian, Qiaoyang; Zhu, Bingdong

    2015-01-01

    Effective tuberculosis (TB) vaccine should target tubercle bacilli with various metabolic states and confer long-term protective immunity. In this study, we constructed a novel multi-stage TB subunit vaccine based on fusion protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-HspX (LT69 for short) which combined early expressed antigens and latency-associated antigen. The fusion protein was mixed with an adjuvant being composed of N, N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA) and polyriboinosinic polyribocytidylic acid (PolyI:C) to construct subunit vaccine, whose immunogenicity and protective ability were evaluated in C57BL/6 mice. The results showed that LT69 had strong immunogenicity and high protective effect against Mycobacterium tuberculosis (M. tuberculosis) H37Rv aerosol challenge. Low-dose (2 μg) of LT69 generated long-term immune memory responses and provided effective protection, which was even higher than traditional vaccine BCG did at 30 weeks post the last vaccination. In conclusion, multistage subunit vaccine LT69 showed high and long-term protection against M. tuberculosis infection in mice, whose effect could be enhanced by using a relative low dosage of antigen. PMID:26098302

  7. Mice overexpressing CD97 in intestinal epithelial cells provide a unique model for mammalian postnatal intestinal cylindrical growth.

    PubMed

    Aust, Gabriela; Kerner, Christiane; Gonsior, Susann; Sittig, Doreen; Schneider, Hartmut; Buske, Peter; Scholz, Markus; Dietrich, Norman; Oldenburg, Sindy; Karpus, Olga N; Galle, Jörg; Amasheh, Salah; Hamann, Jörg

    2013-07-01

    Postnatal enlargement of the mammalian intestine comprises cylindrical and luminal growth, associated with crypt fission and crypt/villus hyperplasia, respectively, which subsequently predominate before and after weaning. The bipartite adhesion G protein-coupled receptor CD97 shows an expression gradient along the crypt-villus axis in the normal human intestine. We here report that transgenic mice overexpressing CD97 in intestinal epithelial cells develop an upper megaintestine. Intestinal enlargement involves an increase in length and diameter but does not affect microscopic morphology, as typical for cylindrical growth. The megaintestine is acquired after birth and before weaning, independent of the genotype of the mother, excluding altered availability of milk constituents as driving factor. CD97 overexpression does not regulate intestinal growth factors, stem cell markers, and Wnt signaling, which contribute to epithelial differentiation and renewal, nor does it affect suckling-to-weaning transition. Consistent with augmented cylindrical growth, suckling but not adult transgenic mice show enlarged crypts and thus more crypt fissions caused by a transient increase of the crypt transit-amplifying zone. Intestinal enlargement by CD97 requires its seven-span transmembrane/cytoplasmic C-terminal fragment but not the N-terminal fragment binding partner CD55. In summary, ectopic expression of CD97 in intestinal epithelial cells provides a unique model for intestinal cylindrical growth occurring in breast-fed infants. PMID:23676664

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

    SciTech Connect

    Kawano, Masaaki; Morikawa, Katsuma; Suda, Tatsuya; Ohno, Naohito; Matsushita, Sho; Akatsuka, Toshitaka; Handa, Hiroshi; Matsui, Masanori

    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.

  9. 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

    NASA Astrophysics Data System (ADS)

    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.

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

    SciTech Connect

    Zhang Fanglin; Wu Xingan; Luo Wen; Bai Wentao; Liu Yong; Yan Yan; Wang Haitao; Xu Zhikai . E-mail: zhikaixu@fmmu.edu.cn

    2007-03-23

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

  11. Novel Protective Antigens Expressed by Trypanosoma cruzi Amastigotes Provide Immunity to Mice Highly Susceptible to Chagas' Disease▿

    PubMed Central

    Silveira, Eduardo L. V.; Claser, Carla; Haolla, Filipe A. B.; Zanella, Luiz G.; Rodrigues, Mauricio M.

    2008-01-01

    Earlier studies have demonstrated in A/Sn mice highly susceptible to Chagas' disease protective immunity against lethal Trypanosoma cruzi infection elicited by vaccination with an open reading frame (ORF) expressed by amastigotes. In our experiments, we used this mouse model to search for other amastigote-expressed ORFs with a similar property. Fourteen ORFs previously determined to be expressed in this developmental stage were individually inserted into a eukaryotic expression vector containing a nucleotide sequence that encoded a mammalian secretory signal peptide. Immunization with 13 of the 14 ORFs induced specific antibodies which recognized the amastigotes. Three of those immune sera also reacted with trypomastigotes and epimastigotes. After a lethal challenge with Y strain trypomastigotes, the vast majority of plasmid-injected mice succumbed to infection. In some cases, a significant delay in mortality was observed. Only two of these ORFs provided protective immunity against the otherwise lethal infection caused by trypomastigotes of the Y or Colombia strain. These ORFs encode members of the trans-sialidase family of surface antigens related to the previously described protective antigen amastigote surface protein 2 (ASP-2). Nevertheless, at the level of antibody recognition, no cross-reactivity was observed between the ORFs and the previously described ASP-2 from the Y strain. In immunofluorescence analyses, we observed the presence of epitopes related to both proteins expressed by amastigotes of seven different strains. In conclusion, our approach allowed us to successfully identify two novel protective ORFs which we consider interesting for future studies on the immune response to Chagas' disease. PMID:18579696

  12. Novel protective antigens expressed by Trypanosoma cruzi amastigotes provide immunity to mice highly susceptible to Chagas' disease.

    PubMed

    Silveira, Eduardo L V; Claser, Carla; Haolla, Filipe A B; Zanella, Luiz G; Rodrigues, Mauricio M

    2008-08-01

    Earlier studies have demonstrated in A/Sn mice highly susceptible to Chagas' disease protective immunity against lethal Trypanosoma cruzi infection elicited by vaccination with an open reading frame (ORF) expressed by amastigotes. In our experiments, we used this mouse model to search for other amastigote-expressed ORFs with a similar property. Fourteen ORFs previously determined to be expressed in this developmental stage were individually inserted into a eukaryotic expression vector containing a nucleotide sequence that encoded a mammalian secretory signal peptide. Immunization with 13 of the 14 ORFs induced specific antibodies which recognized the amastigotes. Three of those immune sera also reacted with trypomastigotes and epimastigotes. After a lethal challenge with Y strain trypomastigotes, the vast majority of plasmid-injected mice succumbed to infection. In some cases, a significant delay in mortality was observed. Only two of these ORFs provided protective immunity against the otherwise lethal infection caused by trypomastigotes of the Y or Colombia strain. These ORFs encode members of the trans-sialidase family of surface antigens related to the previously described protective antigen amastigote surface protein 2 (ASP-2). Nevertheless, at the level of antibody recognition, no cross-reactivity was observed between the ORFs and the previously described ASP-2 from the Y strain. In immunofluorescence analyses, we observed the presence of epitopes related to both proteins expressed by amastigotes of seven different strains. In conclusion, our approach allowed us to successfully identify two novel protective ORFs which we consider interesting for future studies on the immune response to Chagas' disease. PMID:18579696

  13. Yellow Fever/Japanese Encephalitis Chimeric Viruses: Construction and Biological Properties

    PubMed Central

    Chambers, Thomas J.; Nestorowicz, Ann; Mason, Peter W.; Rice, Charles M.

    1999-01-01

    A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA14-14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA14-14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 106 PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein. PMID:10074160

  14. Low dose busulfan facilitates chimerism and tolerance in a murine model.

    PubMed

    Anam, Khairul; Black, Alfred T; Hale, Douglas A

    2006-01-01

    T cell depletion, sirolimus and "mega" dose donor specific bone marrow (DSBM) infusion promotes stable multilineage chimerism and indefinite survival of skin allografts in completely mismatched mice. The purpose of this study is to determine whether the addition of low dose busulfan can reduce the amount of DSBM required while preserving efficacy. C57BL/6 recipients of BALB/c skin allografts were treated with alphaCD4 and alphaCD8 monoclonal antibodies, DSBM, sirolimus and various doses of busulfan. The kinetics and phenotype of chimerism and the presence of clonal deletion of alloreactive T-cells were defined using flow cytometry. In vitro reactivity was determined using mixed lymphocyte culture. Second skin grafts confirmed the presence of tolerance. All doses of busulfan resulted in engraftment when combined with this regimen using a reduced dose of donor marrow. The level, kinetics and character of chimerism observed were dose related. Chimerism was associated with indefinite allograft acceptance (>200 days). Tolerance was documented both in vitro/in vivo and was associated with clonal deletion. Addition of a single low dose of busulfan to an established tolerance protocol reduced the required DSBM dose by over 80% while still promoting comparable levels of donor chimerism and donor-specific tolerance. PMID:16431286

  15. Thrombopoietin treatment of one graft in a double cord blood transplant provides early platelet recovery while contributing to long-term engraftment in NSG mice.

    PubMed

    van der Garde, Mark; van Hensbergen, Yvette; Brand, Anneke; Slot, Manon C; de Graaf-Dijkstra, Alice; Mulder, Arend; Watt, Suzanne M; Zwaginga, Jaap Jan

    2015-01-01

    Human cord blood (CB) hematopoietic stem cell (HSC) transplants demonstrate delayed early neutrophil and platelet recovery and delayed longer term immune reconstitution compared to bone marrow and mobilized peripheral blood transplants. Despite advances in enhancing early neutrophil engraftment, platelet recovery after CB transplantation is not significantly altered when compared to contemporaneous controls. Recent studies have identified a platelet-biased murine HSC subset, maintained by thrombopoietin (TPO), which has enhanced capacity for short- and long-term platelet reconstitution, can self-renew, and can give rise to myeloid- and lymphoid-biased HSCs. In previous studies, we have shown that transplantation of human CB CD34(+) cells precultured in TPO as a single graft accelerates early platelet recovery as well as yielding long-term repopulation in immune-deficient mice. In this study, using a double CB murine transplant model, we investigated whether TPO cultured human CB CD34(+) cells have a competitive advantage or disadvantage over untreated human CB CD34(+) cells in terms of (1) short-term and longer term platelet recovery and (2) longer term hematological recovery. Our studies demonstrate that the TPO treated graft shows accelerated early platelet recovery without impairing the platelet engraftment of untreated CD34(+) cells. Notably, this was followed by a dominant contribution to platelet production through the untreated CD34(+) cell graft over the intermediate to longer term. Furthermore, although the contribution of the TPO treated graft to long-term hematological engraftment was reduced, the TPO treated and untreated grafts both contributed significantly to long-term chimerism in vivo. PMID:25137252

  16. Haematoporphyrin based photodynamic therapy combined with hyperthermia provided effective therapeutic vaccine effect against colon cancer growth in mice.

    PubMed

    He, Yaoming; Ge, Haiyan; Li, Shuping

    2012-01-01

    Photodynamic therapy (PDT) has become an attractive option used in tumor treatment via its direct tumoricidal activities or its immune-boosting activities. On the other hand, heat shock protein 70 has been found to be largely associated with the establishment of anti-tumor activities offered by hyperthermia treated tumor cells. In the present study, we found that injection of tumor-bearing mice with colon cancer cell line CT-26 treated with haematoporphyrin based photodynamic therapy (hematoporphyrin monomethyl ether based PDT, HMME-PDT) together with hyperthermia demonstrated the most effective therapeutic effects against tumor growth, followed by cells treated by hyperthermia alone. CT-26 cells treated only with HMME-PDT failed to provide any therapeutic effects, although significant cell death was induced by HMME-PDT. Compared to hyperthermia treatment, HMME-PDT induced more efficient surface localization of HSP70 on CT-26 cells which correlated with efficient activation of cytolytic CD8 T cells and with effective anti-tumor responses. Thus, our study demonstrated that the surface expression of HSP70 may play a more important role than the total expression or release of this molecule in the activation of immune responses. And our study offered a novel modified PDT approach to the treatment of tumor cells intrinsically low on HSP70 expression. PMID:23055814

  17. Chimeric rabies SADB19-VSVg-pseudotyped lentiviral vectors mediate long-range retrograde transduction from the mouse spinal cord.

    PubMed

    Schoderboeck, L; Riad, S; Bokor, A M; Wicky, H E; Strauss, M; Bostina, M; Oswald, M J; Empson, R M; Hughes, S M

    2015-05-01

    Lentiviral vectors have proved an effective method to deliver transgenes into the brain; however, they are often hampered by a lack of spread from the site of injection. Modifying the viral envelope with a portion of a rabies envelope glycoprotein can enhance spread in the brain by using long-range axon projections to facilitate retrograde transport. In this study, we generated two chimeric envelopes containing the extra-virion and transmembrane domain of rabies SADB19 or CVS-N2c with the intra-virion domain of vesicular stomatitis virus. Viral particles were packaged containing a green fluorescent protein reporter construct under the control of the phosphoglycerokinase promoter. Both vectors produced high-titer particles with successful integration of the glycoproteins into the particle envelope and significant transduction of neurons in vitro. Injection of the SADB19 chimeric viral vector into the lumbar spinal cord of adult mice mediated a strong preference for gene transfer to local neurons and axonal terminals, with retrograde transport to neurons in the brainstem, hypothalamus and cerebral cortex. Development of this vector provides a useful means to reliably target select populations of neurons by retrograde targeting. PMID:25630949

  18. Deletional and regulatory mechanisms coalesce to drive transplantation tolerance through mixed chimerism.

    PubMed

    Hock, Karin; Mahr, Benedikt; Schwarz, Christoph; Wekerle, Thomas

    2015-09-01

    Establishing donor-specific immunological tolerance could improve long-term outcome by obviating the need for immunosuppressive drug therapy, which is currently required to control alloreactivity after organ transplantation. Mixed chimerism is defined as the engraftment of donor hematopoietic stem cells in the recipient, leading to viable coexistence of both donor and recipient leukocytes. In numerous experimental models, cotransplantation of donor bone marrow (BM) into preconditioned (e.g., through irradiation or cytotoxic drugs) recipients leads to transplantation tolerance through (mixed) chimerism. Mixed chimerism offers immunological advantages for clinical translation; pilot trials have established proof of concept by deliberately inducing tolerance in humans. Widespread clinical application is prevented, however, by the harsh preconditioning currently necessary for permitting BM engraftment. Recently, the immunological mechanisms inducing and maintaining tolerance in experimental mixed chimerism have been defined, revealing a more prominent role for regulation than historically assumed. The evidence from murine models suggests that both deletional and regulatory mechanisms are critical in promoting complete tolerance, encompassing also the minor histocompatibility antigens. Here, we review the current understanding of tolerance through mixed chimerism and provide an outlook on how to realize widespread clinical translation based on mechanistic insights gained from chimerism protocols, including cell therapy with polyclonal regulatory T cells. PMID:26200095

  19. Chimeric Antigens of Toxoplasma gondii: Toward Standardization of Toxoplasmosis Serodiagnosis Using Recombinant Products

    PubMed Central

    Beghetto, Elisa; Spadoni, Andrea; Bruno, Luca; Buffolano, Wilma; Gargano, Nicola

    2006-01-01

    We have evaluated the diagnostic utility of six antigenic regions of the Toxoplasma gondii MIC2, MIC3, M2AP, GRA3, GRA7, and SAG1 gene products, assembled in recombinant chimeric antigens by genetic engineering, in order to replace the soluble, whole-cell tachyzoite extract in serological assays. Serum samples from 100 adults with acquired T. gondii infection and from 30 infants born to mothers with primary toxoplasmosis contracted during pregnancy, of whom 20 were congenitally infected, were included. Immunoglobulin G (IgG) and IgM antibodies against epitopes carried by chimeric antigens were measured by performing parallel enzyme immunoassays (recombinant enzyme-linked immunosorbent assays [Rec-ELISAs]), and the results obtained by standard commercial assays with the whole-cell Toxoplasma antigen and assays with the chimeric antigens were compared. Our results demonstrate that IgG and IgM Rec-ELISAs with individual chimeric antigens have performance characteristics comparable to those of the corresponding commercial assays. Furthermore, we show that IgM-capture assays based on chimeric antigens improve the ability to diagnose congenital toxoplasmosis postnatally compared with the ability to diagnose congenital toxoplasmosis by the use of standard assays. The use of recombinant chimeric antigens is effective in distinguishing T. gondii-infected individuals from T. gondii-uninfected individuals and shows that immunoassays based on recombinant products could provide the basis for standardized commercial tests for the serodiagnosis of toxoplasmosis. PMID:16757610

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

    PubMed Central

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

    2009-01-01

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

  1. Autoantibodies induced by chimeric cytokine - HIV envelope glycoprotein immunogens

    PubMed Central

    Isik, Gözde; van Montfort, Thijs; Chung, Nancy P.Y.; Moore, John P.; Sanders, Rogier W.

    2014-01-01

    Cytokines are often used as adjuvants to increase the immunogenicity of vaccines as they can improve the immune response and/or direct it into a desired direction. As an alternative to co-delivering antigens and cytokines separately they can be fused into a composite protein, with the advantage that both moieties act on the same immune cells. The HIV-1 envelope glycoprotein (Env) spike, located on the outside of virus particles and the only relevant protein for the induction of neutralizing antibodies (NAbs), is poorly immunogenic. The induction of anti-Env Abs can be improved by coupling Env proteins to co-stimulatory molecules such as a proliferation inducing ligand (APRIL). Here, we evaluated the immunogenicity of chimeric molecules containing uncleaved Env gp140 fused to the species-matched cytokines IL-21 or GM-CSF in rabbits and mice. Each cytokine was either fused to the C-terminus of Env or embedded within Env at the position of the variable loops 1 and 2 (V1V2). The cytokine components of the chimeric Env-GM-CSF and Env-IL-21 molecules were functional in vitro, but none of the Env-cytokine fusion proteins resulted in improved Ab responses in vivo. Both the Env-GM-CSF and the Env-IL-21 molecules induced strong anti-cytokine Ab responses, in both test species. These autoimmune responses were independent of the location of the cytokine in the chimeric Env molecules; in that they were induced by cytokines inserted within the V1V2 of Env or fused to its Ct. The induction of undesired autoimmune responses should be considered when using cytokines as co-stimulatory molecules in fusion proteins. PMID:24729614

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

    PubMed

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

    2011-07-01

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

  3. Chimeric Antisense Oligonucleotide Conjugated to α-Tocopherol

    PubMed Central

    Nishina, Tomoko; Numata, Junna; Nishina, Kazutaka; Yoshida-Tanaka, Kie; Nitta, Keiko; Piao, Wenying; Iwata, Rintaro; Ito, Shingo; Kuwahara, Hiroya; Wada, Takeshi; Mizusawa, Hidehiro; Yokota, Takanori

    2015-01-01

    We developed an efficient system for delivering short interfering RNA (siRNA) to the liver by using α-tocopherol conjugation. The α-tocopherol–conjugated siRNA was effective and safe for RNA interference–mediated gene silencing in vivo. In contrast, when the 13-mer LNA (locked nucleic acid)-DNA gapmer antisense oligonucleotide (ASO) was directly conjugated with α-tocopherol it showed markedly reduced silencing activity in mouse liver. Here, therefore, we tried to extend the 5′-end of the ASO sequence by using 5′-α-tocopherol–conjugated 4- to 7-mers of unlocked nucleic acid (UNA) as a “second wing.” Intravenous injection of mice with this α-tocopherol–conjugated chimeric ASO achieved more potent silencing than ASO alone in the liver, suggesting increased delivery of the ASO to the liver. Within the cells, the UNA wing was cleaved or degraded and α-tocopherol was released from the 13-mer gapmer ASO, resulting in activation of the gapmer. The α-tocopherol–conjugated chimeric ASO showed high efficacy, with hepatic tropism, and was effective and safe for gene silencing in vivo. We have thus identified a new, effective LNA-DNA gapmer structure in which drug delivery system (DDS) molecules are bound to ASO with UNA sequences. PMID:25584900

  4. Mixed chimerism to induce tolerance for solid organ transplantation

    SciTech Connect

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

    1991-04-01

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

  5. Blood chimerism in a dizygotic dichorionic pregnancy.

    PubMed

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

    2010-10-01

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

  6. Enhanced Efficacy of an AAV Vector Encoding Chimeric, Highly-Secreted Acid α-glucosidase in Glycogen Storage Disease Type II

    PubMed Central

    Sun, Baodong; Zhang, Haoyue; Benjamin, Daniel K.; Brown, Talmage; Bird, Andrew; Young, Sarah P.; McVie-Wylie, Alison; Chen, Y-T; Koeberl, Dwight D.

    2009-01-01

    Glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) is an inherited muscular dystrophy caused by deficiency in the activity of the lysosomal enzyme acid α-glucosidase (GAA). We hypothesized that chimeric GAA containing an alternative signal peptide could increase the secretion of GAA from transduced cells and enhance the receptor-mediated uptake of GAA in striated muscle. The relative secretion of chimeric GAA from transfected 293 cells increased up to 26-fold. Receptor-mediated uptake of secreted, chimeric GAA corrected cultured GSD-II patient cells. High-level hGAA was sustained in the plasma of GSD-II mice for 24 weeks following administration of an AAV2/8 vector encoding chimeric GAA; furthermore, GAA activity was increased and glycogen content was significantly reduced in striated muscle and in the brain. Administration of only 1×1010 vector particles increased GAA activity in the heart and diaphragm for >18 weeks, whereas 3×1010 vector particles increased GAA activity and reduced glycogen content in the heart, diaphragm, and quadriceps. Furthermore, an AAV2/2 vector encoding chimeric GAA produced secreted hGAA for >12 weeks in the majority of treated GSD-II mice. Thus, chimeric, highly secreted GAA enhanced the efficacy of AAV vector-mediated gene therapy in GSD-II mice. PMID:16987711

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

    PubMed Central

    2011-01-01

    Background To develop a plant-based vaccine against Plasmodium vivax, two P. vivax candidate proteins were chosen. First, the merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen that is currently considered a strong vaccine candidate. Second, the circumsporozoite protein (CSP), a component of sporozoites that contains a B-cell epitope. Methods A synthetic chimeric recombinant 516 bp gene encoding containing PvMSP-1, a Pro-Gly linker motif, and PvCSP was synthesized; the gene, named MLC, encoded a total of 172 amino acids. The recombinant gene was modified with regard to codon usage to optimize gene expression in Brassica napus. The Ti plasmid inducible gene transfer system was used for MLC chimeric recombinant gene expression in B. napus. Gene expression was confirmed by polymerase chain reaction (PCR), beta-glucuronidase reporter gene (GUS) assay, and Western blot. Results The MLC chimeric recombinant protein expressed in B. napus had a molecular weight of approximately 25 kDa. It exhibited a clinical sensitivity of 84.21% (n = 38) and a clinical specificity of 100% (n = 24) as assessed by enzyme-linked immunosorbent assay (ELISA). Oral immunization of BALB/c mice with MLC chimeric recombinant protein successfully induced antigen-specific IgG1 production. Additionally, the Th1-related cytokines IL-12 (p40), TNF, and IFN-γ were significantly increased in the spleens of the BALB/c mice. Conclusions The chimeric MLC recombinant protein produced in B. napus has potential as both as an antigen for diagnosis and as a valuable vaccine candidate for oral immunization against vivax malaria. PMID:21529346

  8. A synthetic cGMP-sensitive gene switch providing Viagra(®)-controlled gene expression in mammalian cells and mice.

    PubMed

    Kim, Taeuk; Folcher, Marc; Charpin-El Hamri, Ghislaine; Fussenegger, Martin

    2015-05-01

    Cyclic guanosine monophosphate (cGMP) is a universal second messenger that is synthesized from guanosine triphosphate (GTP) by guanylyl cyclases (GCs) and hydrolyzed into guanosine monophosphate (GMP) by phosphodiesterases (PDEs). Small-molecule drugs that induce high cGMP levels in specialized tissues by boosting GC activity or inhibiting PDE activity have become the predominant treatment strategy for a wide range of medical conditions, including congestive heart failure, pulmonary hypertension, atherosclerosis-based claudication and erectile dysfunction. By fusing the cGMP receptor protein (CRP) of Rhodospirillum centenum to the Herpes simplex-derived transactivation domain VP16, we created a novel synthetic mammalian cGMP-sensing transcription factor (GTA) that activates synthetic promoters (PGTA) containing newly identified GTA-specific operator sites in a concentration-dependent manner. In cell lines expressing endogenous natriuretic peptide receptor A (NPR-A) (HeLa), GTA/PGTA-driven transgene expression was induced by B-type natriuretic peptide (BNP; Nesiritide(®)) in a concentration-dependent manner, which activated NPR-A׳s intracellular GC domain and triggered a corresponding cGMP surge. Ectopic expression of NPR-A in NPR-A-negative cell lines (HEK-293T) produced high cGMP levels and mediated maximum GTA/PGTA-driven transgene expression, which was suppressed by co-expression of PDEs (PDE-3A, PDE-5A and PDE-9A) and was re-triggered by the corresponding PDE inhibitor drugs (Pletal(®), Perfan(®), Primacor(®) (PDE-3A), Viagra(®), Levitra(®), Cialis(®) (PDE-5A) and BAY73-6691 (PDE-9A)). Mice implanted with microencapsulated designer cells co-expressing the GTA/PGTA device with NPR-A and PDE-5A showed control of blood SEAP levels through administration of sildenafil (Viagra(®)). Designer cells engineered for PDE inhibitor-modulated transgene expression may provide a cell-based PDE-targeting drug discovery platform and enable drug-adjusted gene- and cell

  9. Hepatitis C Virus Infection Suppresses the Interferon Response in the Liver of the Human Hepatocyte Chimeric Mouse

    PubMed Central

    Tsuge, Masataka; Fujimoto, Yoshifumi; Hiraga, Nobuhiko; Zhang, Yizhou; Ohnishi, Mayu; Kohno, Tomohiko; Abe, Hiromi; Miki, Daiki; Imamura, Michio; Takahashi, Shoichi; Ochi, Hidenori; Hayes, C. Nelson; Miya, Fuyuki; Tsunoda, Tatsuhiko; Chayama, Kazuaki

    2011-01-01

    Background and Aims Recent studies indicate that hepatitis C virus (HCV) can modulate the expression of various genes including those involved in interferon signaling, and up-regulation of interferon-stimulated genes by HCV was reported to be strongly associated with treatment outcome. To expand our understanding of the molecular mechanism underlying treatment resistance, we analyzed the direct effects of interferon and/or HCV infection under immunodeficient conditions using cDNA microarray analysis of human hepatocyte chimeric mice. Methods Human serum containing HCV genotype 1b was injected into human hepatocyte chimeric mice. IFN-α was administered 8 weeks after inoculation, and 6 hours later human hepatocytes in the mouse livers were collected for microarray analysis. Results HCV infection induced a more than 3-fold change in the expression of 181 genes, especially genes related to Organismal Injury and Abnormalities, such as fibrosis or injury of the liver (P = 5.90E-16 ∼ 3.66E-03). IFN administration induced more than 3-fold up-regulation in the expression of 152 genes. Marked induction was observed in the anti-fibrotic chemokines such as CXCL9, suggesting that IFN treatment might lead not only to HCV eradication but also prevention and repair of liver fibrosis. HCV infection appeared to suppress interferon signaling via significant reduction in interferon-induced gene expression in several genes of the IFN signaling pathway, including Mx1, STAT1, and several members of the CXCL and IFI families (P = 6.0E-12). Genes associated with Antimicrobial Response and Inflammatory Response were also significantly repressed (P = 5.22×10−10 ∼ 1.95×10−2). Conclusions These results provide molecular insights into possible mechanisms used by HCV to evade innate immune responses, as well as novel therapeutic targets and a potential new indication for interferon therapy. PMID:21886832

  10. Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae

    PubMed Central

    Pionnier, Nicolas; Brotin, Emilie; Karadjian, Gregory; Hemon, Patrice; Gaudin-Nomé, Françoise; Vallarino-Lhermitte, Nathaly; Nieguitsila, Adélaïde; Fercoq, Frédéric; Aknin, Marie-Laure; Marin-Esteban, Viviana; Chollet-Martin, Sylvie; Schlecht-Louf, Géraldine

    2016-01-01

    Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4+/1013). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4+/1013 mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4+/1013 mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis. PMID:27111140

  11. Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae.

    PubMed

    Pionnier, Nicolas; Brotin, Emilie; Karadjian, Gregory; Hemon, Patrice; Gaudin-Nomé, Françoise; Vallarino-Lhermitte, Nathaly; Nieguitsila, Adélaïde; Fercoq, Frédéric; Aknin, Marie-Laure; Marin-Esteban, Viviana; Chollet-Martin, Sylvie; Schlecht-Louf, Géraldine; Bachelerie, Françoise; Martin, Coralie

    2016-04-01

    Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4(+/1013)). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4(+/1013) mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4(+/1013) mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis. PMID:27111140

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

    PubMed

    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. PMID:24293125

  13. Alloreactive Regulatory T Cells Allow the Generation of Mixed Chimerism and Transplant Tolerance

    PubMed Central

    Ruiz, Paulina; Maldonado, Paula; Hidalgo, Yessia; Sauma, Daniela; Rosemblatt, Mario; Bono, Maria Rosa

    2015-01-01

    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 (NM-BMT) protocol using retinoic acid (RA)-induced alloantigen-specific Tregs, clinically available immunosuppressive drugs, and lower doses of irradiation. We demonstrate that RA-induced alloantigen-specific Tregs in addition to a NM-BMT protocol generates stable mixed chimerism and induces 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. PMID:26635810

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

    PubMed Central

    Mishina, Yukiko; Mutoh, Hiroki; Song, Chenchen; Knöpfel, Thomas

    2014-01-01

    Deciphering how the brain generates cognitive function from patterns of electrical signals is one of the ultimate challenges in neuroscience. To this end, it would be highly desirable to monitor the activities of very large numbers of neurons while an animal engages in complex behaviors. Optical imaging of electrical activity using genetically encoded voltage indicators (GEVIs) has the potential to meet this challenge. Currently prevalent GEVIs are based on the voltage-sensitive fluorescent protein (VSFP) prototypical design or on the voltage-dependent state transitions of microbial opsins. We recently introduced a new VSFP design in which the voltage-sensing domain (VSD) is sandwiched between a fluorescence resonance energy transfer pair of fluorescent proteins (termed VSFP-Butterflies) and also demonstrated a series of chimeric VSD in which portions of the VSD of Ciona intestinalis voltage-sensitive phosphatase are substituted by homologous portions of a voltage-gated potassium channel subunit. These chimeric VSD had faster sensing kinetics than that of the native Ci-VSD. Here, we describe a new set of VSFPs that combine chimeric VSD with the Butterfly structure. We show that these chimeric VSFP-Butterflies can report membrane voltage oscillations of up to 200 Hz in cultured cells and report sensory evoked cortical population responses in living mice. This class of GEVIs may be suitable for imaging of brain rhythms in behaving mammalians. PMID:25324718

  15. Preparation and characterization of a novel chimeric protein VEGI-CTT in Escherichia coli.

    PubMed

    Cai, Jiping; Wei, Ruili; Cheng, Jinwei

    2008-01-01

    Vascular endothelial cell growth inhibitor (VEGI) is a recently identified antiangiogenic cytokine that belongs to the TNF superfamily, and could effectively inhibit endothelial cell proliferation and angiogenesis. Synthetic peptide CTT (CTTHWGFTLC) has been found to suppress invasion and migration of both tumor and endothelial cells by potent and selective inhibition of MMP-2 and MMP-9. To prepare chimeric protein VEGI-CTT for more potent antitumor therapy, the recombinant expression vector pET-VEGI-CTT was constructed. This fusion protein was expressed in inclusion bodies in E. coli BL21 (DE3), and was refolded and purified by immobilized metal affinity chromatography using His-tag. Purified VEGI-CTT protein was characterized by proliferation assays of the endothelial cells and casein degradation assay in vitro. The results demonstrated that chimeric protein VEGI-CTT had a potent activity of antiangiogenesis through inhibiting the proliferation of endothelial cells, and could effectively reduce the activity of MMP-2 and MMP-9. The preliminarily in vivo study demonstrated that chimeric protein VEGI-CTT had more potent antitumor activity than VEGI and/or CTT peptide against CA46 human lymphoma xenografts in nude mice. Thus, these facts that are derived from the present study suggest that the chimeric protein VEGI-CTT may be used for tumor therapy in the future. PMID:18769489

  16. CCR2 antagonist CCX140-B provides renal and glycemic benefits in diabetic transgenic human CCR2 knockin mice

    PubMed Central

    Sullivan, Timothy; Miao, Zhenhua; Dairaghi, Daniel J.; Krasinski, Antoni; Wang, Yu; Zhao, Bin N.; Baumgart, Trageen; Ertl, Linda S.; Pennell, Andrew; Seitz, Lisa; Powers, Jay; Zhao, Ruiping; Ungashe, Solomon; Wei, Zheng; Boring, Landin; Tsou, Chia-Lin; Charo, Israel; Schall, Thomas J.; Jaen, Juan C.

    2013-01-01

    Chemokine (C-C motif) receptor 2 (CCR2) is central for the migration of monocytes into inflamed tissues. The novel CCR2 antagonist CCX140-B, which is currently in two separate phase 2 clinical trials in diabetic nephropathy, has recently been shown to reduce hemoglobin A1c and fasting blood glucose levels in type 2 diabetics. In this report, we describe the effects of this compound on glycemic and renal function parameters in diabetic mice. Since CCX140-B has a low affinity for mouse CCR2, transgenic human CCR2 knockin mice were generated and rendered diabetic with either a high-fat diet (diet-induced obesity) or by deletion of the leptin receptor gene (db/db). CCX140-B treatment in both models resulted in decreased albuminuria, which was associated with decreased glomerular hypertrophy and increased podocyte density. Moreover, treatment of diet-induced obese mice with CCX140-B resulted in decreased levels of fasting blood glucose and insulin, normalization of homeostatic model assessment of insulin resistance values, and decreased numbers of adipose tissue inflammatory macrophages. Unlike other CCR2 antagonists, CCX140-B had no effect on plasma levels of the CCR2 ligand CCL2 or on the numbers of blood monocytes. These results support the ongoing evaluation of this molecule in diabetic subjects with impaired renal function. PMID:23986513

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

    PubMed

    Wu, Jun; Izpisua Belmonte, Juan Carlos

    2016-06-01

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

  18. The basic principles of chimeric antigen receptor (CAR) design

    PubMed Central

    Sadelain, Michel; Brentjens, Renier; Riviere, Isabelle

    2013-01-01

    CARs are recombinant receptors that provide both antigen-binding and T cell activating functions. A multitude of CARs has been reported over the past decade, targeting an array of cell surface tumor antigens. Their biological functions have dramatically changed following the introduction of tri-partite receptors comprising a costimulatory domain, termed second generation CARs. These have recently demonstrated clinical benefit in patients treated with CD19-targeted autologous T cells. CARs may be combined with costimulatory ligands, chimeric costimulatory receptors or cytokines to further enhance T cell potency, specificity and safety. CARs represent a new class of drugs with exciting potential for cancer immunotherapy. PMID:23550147

  19. Bioengineered Chimeric Spider Silk-Uranium Binding Proteins

    PubMed Central

    Krishnaji, Sreevidhya Tarakkad; Kaplan, David L.

    2014-01-01

    Heavy metals constitute a source of environmental pollution. Here, novel functional hybrid biomaterials for specific interactions with heavy metals are designed by bioengineering consensus sequence repeats from spider silk of Nephila clavipes with repeats of a uranium peptide recognition motif from a mutated 33-residue of calmodulin protein from Paramecium tetraurelia. The self-assembly features of the silk to control nanoscale organic/inorganic material interfaces provides new biomaterials for uranium recovery. With subsequent enzymatic digestion of the silk to concentrate the sequestered metals, options can be envisaged to use these new chimeric protein systems in environmental engineering, including to remediate environments contaminated by uranium. PMID:23212989

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2016-03-01

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

  2. Enhancement of the in vivo persistence and antitumor efficacy of CD19 chimeric antigen receptor T cells through the delivery of modified TERT mRNA

    PubMed Central

    Bai, Yun; Kan, Shifeng; Zhou, Shixin; Wang, Yuting; Xu, Jun; Cooke, John P; Wen, Jinhua; Deng, Hongkui

    2015-01-01

    Chimeric antigen receptor T cell immunotherapy is a promising therapeutic strategy for treating tumors, demonstrating its efficiency in eliminating several hematological malignancies in recent years. However, a major obstacle associated with current chimeric antigen receptor T cell immunotherapy is that the limited replicative lifespan of chimeric antigen receptor T cells prohibits the long-term persistence and expansion of these cells in vivo, potentially hindering the long-term therapeutic effects of chimeric antigen receptor T cell immunotherapy. Here we showed that the transient delivery of modified mRNA encoding telomerase reverse transcriptase to human chimeric antigen receptor T cells targeting the CD19 antigen (CD19 chimeric antigen receptor T cells) would transiently elevate the telomerase activity in these cells, leading to increased proliferation and delayed replicative senescence without risk of insertion mutagenesis or immortalization. Importantly, compared to conventional CD19 chimeric antigen receptor T cells, after the transient delivery of telomerase reverse transcriptase mRNA, these CD19 chimeric antigen receptor T cells showed improved persistence and proliferation in mouse xenograft tumor models of human B-cell malignancies. Furthermore, the transfer of CD19 chimeric antigen receptor T cells after the transient delivery of telomerase reverse transcriptase mRNA enhanced long-term antitumor effects in mouse xenograft tumor models compared with conventional CD19 chimeric antigen receptor T cell transfer. The results of the present study provide an effective and safe method to improve the therapeutic potential of chimeric antigen receptor T cells, which might be beneficial for treating other types of cancer, particularly solid tumors. PMID:27462436

  3. Japanese encephalitis virus vaccine candidates generated by chimerization with dengue virus type 4.

    PubMed

    Gromowski, Gregory D; Firestone, Cai-Yen; Hanson, Christopher T; Whitehead, Stephen S

    2014-05-23

    Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis worldwide and vaccination is one of the most effective ways to prevent disease. A suitable live-attenuated JEV vaccine could be formulated with a live-attenuated tetravalent dengue vaccine for the control of these viruses in endemic areas. Toward this goal, we generated chimeric virus vaccine candidates by replacing the precursor membrane (prM) and envelope (E) protein structural genes of recombinant dengue virus type 4 (rDEN4) or attenuated vaccine candidate rDEN4Δ30 with those of wild-type JEV strain India/78. Mutations were engineered in E, NS3 and NS4B protein genes to improve replication in Vero cells. The chimeric viruses were attenuated in mice and some elicited modest but protective levels of immunity after a single dose. One particular chimeric virus, bearing E protein mutation Q264H, replicated to higher titer in tissue culture and was significantly more immunogenic in mice. The results are compared with live-attenuated JEV vaccine strain SA14-14-2. PMID:24699473

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

    PubMed Central

    Bello-Rivero, Iraldo; Torrez-Ruiz, Yeny; Blanco-Garcés, Elizabeth; Pentón-Rol, Giselle; Fernández-Batista, Osmani; Javier-González, Luís; Gerónimo-Perez, Haydee; López-Saura, Pedro

    2006-01-01

    Background TH1 immune response antagonism is a desirable approach to mitigate some autoimmune and inflammatory reactions during the course of several diseases where IL-2 and IFN-γ are two central players. Therefore, the neutralization of both cytokines could provide beneficial effects in patients suffering from autoimmune or inflammatory illnesses. Results A chimeric antagonist that can antagonize the action of TH1 immunity mediators, IFN-γ and IL-2, was designed, engineered, expressed in E. coli, purified and evaluated for its in vitro biological activities. The TH1 antagonist molecule consists of the extracellular region for the human IFNγ receptor chain 1 fused by a four-aminoacid linker peptide to human 60 N-terminal aminoacid residues of IL-2. The corresponding gene fragments were isolated by RT-PCR and cloned in the pTPV-1 vector. E. coli (W3110 strain) was transformed with this vector. The chimeric protein was expressed at high level as inclusion bodies. The protein was partially purified by pelleting and washing. It was then solubilized with strong denaturant and finally refolded by gel filtration. In vitro biological activity of chimera was demonstrated by inhibition of IFN-γ-dependent HLA-DR expression in Colo 205 cells, inhibition of IFN-γ antiproliferative effect on HEp-2 cells, and by a bidirectional effect in assays for IL-2 T-cell dependent proliferation: agonism in the absence versus inhibition in the presence of IL-2. Conclusion TH1 antagonist is a chimeric protein that inhibits the in vitro biological activities of human IFN-γ, and is a partial agonist/antagonist of human IL-2. With these attributes, the chimera has the potential to offer a new opportunity for the treatment of autoimmune and inflammatory diseases. PMID:16716222

  5. Repetitive behavior and increased activity in mice with Purkinje cell loss: a model for understanding the role of cerebellar pathology in autism.

    PubMed

    Martin, Loren A; Goldowitz, Dan; Mittleman, Guy

    2010-02-01

    Repetitive behaviors and hyperactivity are common features of developmental disorders, including autism. Neuropathology of the cerebellum is also a frequent occurrence in autism and other developmental disorders. Recent studies have indicated that cerebellar pathology may play a causal role in the generation of repetitive and hyperactive behaviors. In this study, we examined the relationship between cerebellar pathology and these behaviors in a mouse model of Purkinje cell loss. Specifically, we made aggregation chimeras between Lc/+ mutant embryos and +/+ embryos. Lc/+ mice lose 100% of their Purkinje cells postnatally due to a cell-intrinsic gain-of-function mutation. Through our histological examination, we demonstrated that Lc/+<-->+/+ chimeric mice have Purkinje cells ranging from zero to normal numbers. Our analysis of these chimeric cerebella confirmed previous studies on Purkinje cell lineage. The results of both open-field activity and hole-board exploration testing indicated negative relationships between Purkinje cell number and measures of activity and investigatory nose-poking. Additionally, in a progressive-ratio operant paradigm, we found that Lc/+ mice lever-pressed significantly less than +/+ controls, which led to significantly lower breakpoints in this group. In contrast, chimeric mice lever-pressed significantly more than controls and this repetitive lever-pressing behavior was significantly and negatively correlated with total Purkinje cell numbers. Although the performance of Lc/+ mice is probably related to their motor deficits, the significant relationships between Purkinje cell number and repetitive lever-pressing behavior as well as open-field activity measures provide support for a role of cerebellar pathology in generating repetitive behavior and increased activity in chimeric mice. PMID:20105240

  6. Engineering of chimeric class II polyhydroxyalkanoate synthases.

    PubMed

    Niamsiri, Nuttawee; Delamarre, Soazig C; Kim, Young-Rok; Batt, Carl A

    2004-11-01

    PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB(-)4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic

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

    PubMed

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

    2016-03-01

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

  8. Immunization of mice with formalin-inactivated spores from avirulent Bacillus cereus strains provides significant protection from challenge with Bacillus anthracis Ames.

    PubMed

    Vergis, James M; Cote, Christopher K; Bozue, Joel; Alem, Farhang; Ventura, Christy L; Welkos, Susan L; O'Brien, Alison D

    2013-01-01

    Bacillus anthracis spores are the infectious form of the organism for humans and animals. However, the approved human vaccine in the United States is derived from a vegetative culture filtrate of a toxigenic, nonencapsulated B. anthracis strain that primarily contains protective antigen (PA). Immunization of mice with purified spore proteins and formalin-inactivated spores (FIS) from a nonencapsulated, nontoxigenic B. anthracis strain confers protection against B. anthracis challenge when PA is also administered. To investigate the capacity of the spore particle to act as a vaccine without PA, we immunized mice subcutaneously with FIS from nontoxigenic, nonencapsulated B. cereus strain G9241 pBCXO1(-)/pBC210(-) (dcG9241), dcG9241 ΔbclA, or 569-UM20 or with exosporium isolated from dcG9241. FIS vaccination provided significant protection of mice from intraperitoneal or intranasal challenge with spores of the virulent B. anthracis Ames or Ames ΔbclA strain. Immunization with dcG9241 ΔbclA FIS, which are devoid of the immunodominant spore protein BclA, provided greater protection from challenge with either Ames strain than did immunization with FIS from BclA-producing strains. In addition, we used prechallenge immune antisera to probe a panel of recombinant B. anthracis Sterne spore proteins to identify novel immunogenic vaccine candidates. The antisera were variably reactive with BclA and with 10 other proteins, four of which were previously tested as vaccine candidates. Overall our data show that immunization with FIS from nontoxigenic, nonencapsulated B. cereus strains provides moderate to high levels of protection of mice from B. anthracis Ames challenge and that neither PA nor BclA is required for this protection. PMID:23114705

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

    PubMed

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

    2014-09-17

    A multi-antigen chimera composed of three antigens of Mycoplasma hyopneumoniae (R1, P42, and NrdF) and the mucosal adjuvant Escherichia coli heat-labile enterotoxin B subunit (LTB) was constructed, and its antigenic and immunogenic properties were evaluated in mice and pigs. In addition, we compared the effect of the fusion and co-administration of these proteins in mice. Antibodies against each subunit recognized the chimeric protein. Intranasal and intramuscular immunization of mice with the chimeric protein significantly increased IgG and IgA levels in the serum and tracheobronchial lavages, respectively, against some of the antigens present in the chimeric. Swine immunized with the chimeric protein developed an immune response against all M. hyopneumoniae antigens present in the fusion with a statistically significant difference (P<0.05). The adjuvant rLTB enhanced the immune response in both fused and co-administered antigens; however, better results were obtained with the chimeric protein. This multi-antigen is a promising vaccine candidate that may help control M. hyopneumoniae infection. PMID:25091529

  10. Principles of Bone Marrow Transplantation (BMT): Providing Optimal Veterinary and Husbandry Care to Irradiated Mice in BMT Studies

    PubMed Central

    Duran-Struuck, Raimon; Dysko, Robert C

    2009-01-01

    Bone marrow transplantation (BMT) is the treatment of choice for many leukemias, solid tumors, and metabolic diseases. The field of bone marrow research is highly dependent on in vivo experimentation, because in vitro techniques do not mimic these complicated in vivo systems. Therefore, understanding the medical and husbandry care needs of these transiently immunodeficient bone marrow recipient animals is crucial for researchers, veterinary and animal care personnel. Here we discuss the principles of bone marrow transplantation, mouse pathogens that can interfere with transplantation research, and important husbandry and veterinary practices for mice that may help to minimize unnecessary infections during the transplantation process. Whole-body irradiation is one of the most common tools for myeloablation of the recipient's bone marrow. We discuss the crucial role of the irradiator for BMT research and the importance of aseptic husbandry practices to lessen the possibility of the irradiator for being a source for disease transmission. Finally, we discuss some important guidelines for Institutional Animal Use and Care Committees reviewing irradiation and BMT protocols. PMID:19245745

  11. Molecular analyses provide insight into mechanisms underlying sarcopenia and myofibre denervation in old skeletal muscles of mice.

    PubMed

    Barns, Mitchell; Gondro, Cedric; Tellam, Ross L; Radley-Crabb, Hannah G; Grounds, Miranda D; Shavlakadze, Tea

    2014-08-01

    Molecular mechanisms that are associated with age-related denervation and loss of skeletal muscle mass and function (sarcopenia) are described for female C57Bl/6J mice aged 3, 15, 24, 27 and 29 months (m). Changes in mRNAs and proteins associated with myofibre denervation and protein metabolism in ageing muscles are reported, across the transition from healthy adult myofibres to sarcopenia that occurs between 15 and 24 m. This onset of sarcopenia at 24 m, corresponded with increased expression of genes associated with neuromuscular junction denervation including Chnrg, Chrnd, Ncam1, Runx1, Gadd45a and Myog. Sarcopenia in quadriceps muscles also coincided with increased protein levels for Igf1 receptor, Akt and ribosomal protein S6 (Rps6) with increased phosphorylation of Rps6 (Ser235/236) and elevated Murf1 mRNA and protein, but not Fbxo32: many of these changes are also linked to denervation. Global transcription profiling via microarray analysis confirmed these functional themes and highlighted additional themes that may be a consequence of pathology associated with sarcopenia, including changes in fatty acid metabolism, extracellular matrix structure and protein catabolism. Ageing was also associated with increased global gene expression variance, consistent with decreased control of gene regulation. PMID:24836906

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

    PubMed

    Kikuchi, Yosuke; Kunitoh-Asari, Ayami; Hayakawa, Katsuyuki; Imai, Shinjiro; Kasuya, Kenji; Abe, Kimio; Adachi, Yu; Fukudome, Shin-Ichi; Takahashi, Yoshimasa; Hachimura, Satoshi

    2014-01-01

    The mucosal immune system provides the first line of defense against inhaled and ingested pathogenic microbacteria and viruses. This defense system, to a large extent, is mediated by the actions of secretory IgA. In this study, we screened 140 strains of lactic acid bacteria for induction of IgA production by murine Peyer's patch cells. We selected one strain and named it Lactobacillus plantarum AYA. We found that L. plantarum AYA-induced production of IL-6 in Peyer's patch dendritic cells, with this production promoting IgA(+) B cells to differentiate into IgA-secreting plasma cells. We also observed that oral administration of L. plantarum AYA in mice caused an increase in IgA production in the small intestine and lung. This production of IgA correlated strongly with protective ability, with the treated mice surviving longer than the control mice after lethal influenza virus infection. Our data therefore reveals a novel immunoregulatory role of the L. plantarum AYA strain which enhances mucosal IgA production and provides protection against respiratory influenza virus infection. PMID:24466081

  13. Structure-Function Analysis of Peroxisomal ATP-binding Cassette Transporters Using Chimeric Dimers*

    PubMed Central

    Geillon, Flore; Gondcaille, Catherine; Charbonnier, Soëli; Van Roermund, Carlo W.; Lopez, Tatiana E.; Dias, Alexandre M. M.; Pais de Barros, Jean-Paul; Arnould, Christine; Wanders, Ronald J.; Trompier, Doriane; Savary, Stéphane

    2014-01-01

    ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned in this respect as well as in their capability to form functional heterodimers. Using a cell model expressing an ABCD2-EGFP fusion protein, we first demonstrated by proximity ligation assay and co-immunoprecipitation assay that ABCD1 interacts with ABCD2. Next, we tested in the pxa1/pxa2Δ yeast mutant the functionality of ABCD1/ABCD2 dimers by expressing chimeric proteins mimicking homo- or heterodimers. For further structure-function analysis of ABCD1/ABCD2 dimers, we expressed chimeric dimers fused to enhanced GFP in human skin fibroblasts of X-linked adrenoleukodystrophy patients. These cells are devoid of ABCD1 and accumulate very long-chain fatty acids (C26:0 and C26:1). We checked that the chimeric proteins were correctly expressed and targeted to the peroxisomes. Very long-chain fatty acid levels were partially restored in transfected X-linked adrenoleukodystrophy fibroblasts regardless of the chimeric construct used, thus demonstrating functionality of both homo- and heterodimers. Interestingly, the level of C24:6 n-3, the immediate precursor of docosahexaenoic acid, was decreased in cells expressing chimeric proteins containing at least one ABCD2 moiety. Our data demonstrate for the first time that both homo- and heterodimers of ABCD1 and ABCD2 are functionally active. Interestingly, the role of ABCD2 (in homo- and heterodimeric forms) in the metabolism of polyunsaturated fatty acids is clearly evidenced, and the chimeric dimers provide a novel tool to study substrate specificity of peroxisomal ATP-binding cassette transporters. PMID:25043761

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  15. Development of chimeric laccases by directed evolution.

    PubMed

    Pardo, Isabel; Vicente, Ana Isabel; Mate, Diana M; Alcalde, Miguel; Camarero, Susana

    2012-12-01

    DNA recombination methods are useful tools to generate diversity in directed evolution protein engineering studies. We have designed an array of chimeric laccases with high-redox potential by in vitro and in vivo DNA recombination of two fungal laccases (from Pycnoporus cinnabarinus and PM1 basidiomycete), which were previously tailored by laboratory evolution for functional expression in Saccharomyces cerevisiae. The laccase fusion genes (including the evolved α-factor prepro-leaders for secretion in yeast) were subjected to a round of family shuffling to construct chimeric libraries and the best laccase hybrids were identified in dual high-throughput screening (HTS) assays. Using this approach, we identified chimeras with up to six crossover events in the whole sequence, and we obtained active hybrid laccases with combined characteristics in terms of pH activity and thermostability. PMID:22729887

  16. In vitro and in vivo characterization of chimeric duck Tembusu virus based on Japanese encephalitis live vaccine strain SA14-14-2.

    PubMed

    Wang, Hong-Jiang; Liu, Long; Li, Xiao-Feng; Ye, Qing; Deng, Yong-Qiang; Qin, E-De; Qin, Cheng-Feng

    2016-07-01

    Duck Tembusu virus (DTMUV), a newly identified flavivirus, has rapidly spread to China, Malaysia and Thailand. The potential threats to public health have been well-highlighted; however its virulence and pathogenesis remain largely unknown. Here, by using reverse genetics, a recombinant chimeric DTMUV based on Japanese encephalitis live vaccine strain SA14-14-2 was obtained by substituting the corresponding prM and E genes (named ChinDTMUV). In vitro characterization demonstrated that ChinDTMUV replicated efficiently in mammalian cells with small-plaque phenotype in comparison with its parental viruses. Mouse tests showed ChinDTMUV exhibited avirulent phenotype in terms of neuroinvasiveness, while it retained neurovirulence from its parental virus DTMUV. Furthermore, immunization with ChinDTMUV was evidenced to elicit robust IgG and neutralizing antibody responses in mice. Overall, we successfully developed a viable chimeric DTMUV, and these results provide a useful platform for further investigation of the pathogenesis of DTMUV and development of a live attenuated DTMUV vaccine candidate. PMID:27100268

  17. Transcriptome analysis revealed chimeric RNAs, single nucleotide polymorphisms and allele-specific expression in porcine prenatal skeletal muscle

    PubMed Central

    Yang, Yalan; Tang, Zhonglin; Fan, Xinhao; Xu, Kui; Mu, Yulian; Zhou, Rong; Li, Kui

    2016-01-01

    Prenatal skeletal muscle development genetically determines postnatal muscle characteristics such as growth and meat quality in pigs. However, the molecular mechanisms underlying prenatal skeletal muscle development remain unclear. Here, we performed the first genome-wide analysis of chimeric RNAs, single nuclear polymorphisms (SNPs) and allele-specific expression (ASE) in prenatal skeletal muscle in pigs. We identified 14,810 protein coding genes and 163 high-confidence chimeric RNAs expressed in prenatal skeletal muscle. More than 94.5% of the chimeric RNAs obeyed the canonical GT/AG splice rule and were trans-splicing events. Ten and two RNAs were aligned to human and mouse chimeric transcripts, respectively. We detected 106,457 high-quality SNPs (6,955 novel), which were mostly (89.09%) located within QTLs for production traits. The high proportion of non-exonic SNPs revealed the incomplete annotation status of the current swine reference genome. ASE analysis revealed that 11,300 heterozygous SNPs showed allelic imbalance, whereas 131 ASE variants were located in the chimeric RNAs. Moreover, 4 ASE variants were associated with various economically relevant traits of pigs. Taken together, our data provide a source for studies of chimeric RNAs and biomarkers for pig breeding, while illuminating the complex transcriptional events underlying prenatal skeletal muscle development in mammals. PMID:27352850

  18. Transcriptome analysis revealed chimeric RNAs, single nucleotide polymorphisms and allele-specific expression in porcine prenatal skeletal muscle.

    PubMed

    Yang, Yalan; Tang, Zhonglin; Fan, Xinhao; Xu, Kui; Mu, Yulian; Zhou, Rong; Li, Kui

    2016-01-01

    Prenatal skeletal muscle development genetically determines postnatal muscle characteristics such as growth and meat quality in pigs. However, the molecular mechanisms underlying prenatal skeletal muscle development remain unclear. Here, we performed the first genome-wide analysis of chimeric RNAs, single nuclear polymorphisms (SNPs) and allele-specific expression (ASE) in prenatal skeletal muscle in pigs. We identified 14,810 protein coding genes and 163 high-confidence chimeric RNAs expressed in prenatal skeletal muscle. More than 94.5% of the chimeric RNAs obeyed the canonical GT/AG splice rule and were trans-splicing events. Ten and two RNAs were aligned to human and mouse chimeric transcripts, respectively. We detected 106,457 high-quality SNPs (6,955 novel), which were mostly (89.09%) located within QTLs for production traits. The high proportion of non-exonic SNPs revealed the incomplete annotation status of the current swine reference genome. ASE analysis revealed that 11,300 heterozygous SNPs showed allelic imbalance, whereas 131 ASE variants were located in the chimeric RNAs. Moreover, 4 ASE variants were associated with various economically relevant traits of pigs. Taken together, our data provide a source for studies of chimeric RNAs and biomarkers for pig breeding, while illuminating the complex transcriptional events underlying prenatal skeletal muscle development in mammals. PMID:27352850

  19. A recombinant chimeric protein containing B chains of ricin and abrin is an effective vaccine candidate

    PubMed Central

    Wang, Junhong; Gao, Shan; Zhang, Tao; Kang, Lin; Cao, Wuchun; Xu, Na; Liu, Wensen; Wang, Jinglin

    2014-01-01

    Both ricin toxin (RT) and abrin toxin (AT) are 2 important toxin agents as potantial bioweapons. A dual subunit vaccine against RT and AT exposure is a promising option for developing prophylactic vaccination. In this study, we constructed a dual vaccine with RT B chain and AT B chain named RTB-ATB. The RTB-ATB chimeric protein was expressed in Escherichia coli (E. coli), and the purified protein was used to evaluate the immune response by a 2 × 2 × 2 × 2 factorial design. The main effects included dose of RTB-ATB, route of immunization injection, immunization time interval, and dose of native toxins challenge. For 2 × LD50 challenge of RT or AT, 100% of the RTB-ATB immunized mice survived and regained or exceeded their initial weights within 10 days. For 4 × LD50 challenge, different routes of immunization injection caused significant difference (P < 0.05), intraperitoneal (i.p.) administration of immunogen protected mice better than the subcutaneous (s.c.) administration. In conclusion, when administered i.p. to mice with 25 μg per mouse and immunization time interval Π in the absence of adjuvant, the chimeric protein elicited a stronger immune response and protected the animals from a dose of native toxins which was 4 times higher than their LD50 in unvaccinated mice. Besides, the RTB-ATB chimeric protein could induce specific neutralizing antibodies against these 2 toxins. We anticipate that this study will open new possibilities in the preparation of RTB-ATB dual subunit vaccine against the exposure to deadly RT and AT. PMID:24509607

  20. Metabolic studies of prostanozol with the uPA-SCID chimeric mouse model and human liver microsomes.

    PubMed

    Geldof, Lore; Lootens, Leen; Decroix, Lieselot; Botrè, Francesco; Meuleman, Philip; Leroux-Roels, Geert; Deventer, Koen; Van Eenoo, Peter

    2016-03-01

    Anabolic androgenic steroids are prohibited by the World Anti-Doping Agency because of their adverse health and performance enhancing effects. Effective control of their misuse by detection in urine requires knowledge about their metabolism. In case of designer steroids, ethical objections limit the use of human volunteers to perform excretion studies. Therefore the suitability of alternative models needs to be investigated. In this study pooled human liver microsomes (HLM) and an uPA(+/+)-SCID chimeric mouse model were used to examine the metabolism of the designer steroid prostanozol as a reference standard. Metabolites were detected by GC-MS (full scan) and LC-MS/MS (precursor ion scan). In total twenty-four prostanozol metabolites were detected with the in vitro and in vivo metabolism studies, which could be grouped into two broad classes, those with a 17-hydroxy- and those with a 17-keto-substituent. Major first phase metabolic sites were tentatively identified as C-3'; C-4 and C-16. Moreover, 3'- and 16β-hydroxy-17-ketoprostanozol could be unequivocally identified, since authentic reference material was available, in both models. Comparison with published data from humans showed a good correlation, except for phase II metabolism. As metabolites were in contrast to the human studies predominantly present in the free fraction. Two types of metabolites ((di)hydroxylated prostanozol metabolites) that have not been described before could be confirmed in a real positive doping control sample. Hence, the results provide further evidence for the applicability of chimeric mice and HLM to perform metabolism studies of designer steroids. PMID:26774429

  1. Uncoupling of Molecular Maturation from Peripheral Target Innervation in Nociceptors Expressing a Chimeric TrkA/TrkC Receptor

    PubMed Central

    Gorokhova, Svetlana; Gaillard, Stéphane; Urien, Louise; Malapert, Pascale; Legha, Wassim; Baronian, Grégory; Desvignes, Jean-Pierre; Alonso, Serge; Moqrich, Aziz

    2014-01-01

    Neurotrophins and their receptors control a number of cellular processes, such as survival, gene expression and axonal growth, by activating multiple signalling pathways in peripheral neurons. Whether each of these pathways controls a distinct developmental process remains unknown. Here we describe a novel knock-in mouse model expressing a chimeric TrkA/TrkC (TrkAC) receptor from TrkA locus. In these mice, prospective nociceptors survived, segregated into appropriate peptidergic and nonpeptidergic subsets, projected normally to distinct laminae of the dorsal spinal cord, but displayed aberrant peripheral target innervation. This study provides the first in vivo evidence that intracellular parts of different Trk receptors are interchangeable to promote survival and maturation of nociceptors and shows that these developmental processes can be uncoupled from peripheral target innervation. Moreover, adult homozygous TrkAC knock-in mice displayed severe deficits in acute and tissue injury-induced pain, representing the first viable adult Trk mouse mutant with a pain phenotype. PMID:24516396

  2. Detection and quantification of chimerism by droplet digital PCR.

    PubMed

    George, David; Czech, Juliann; John, Bobby; Yu, Min; Jennings, Lawrence J

    2013-01-01

    Accurate quantification of chimerism and microchimerism is proving to be increasingly valuable for hematopoietic cell transplantation as well as non-transplant conditions. However, methods that are available to quantify low-level chimerism lack accuracy. Therefore, we developed and validated a method for quantifying chimerism based on digital PCR technology. We demonstrate accurate quantification that far exceeds what is possible with analog qPCR down to 0.01% with the potential to go even lower. Also, this method is inherently more informative than qPCR. We expect the advantages of digital PCR will make it the preferred method for chimerism analysis. PMID:23974275

  3. Mixed chimerism to induce tolerance: lessons learned from nonhuman primates

    PubMed Central

    Murakami, Toru; Cosimi, A. Benedict; Kawai, Tatsuo

    2013-01-01

    The mixed chimerism approach has been demonstrated to be an effective means of inducing allograft tolerance. Based on our rodent studies on mixed chimerism, we previously developed a clinically relevant nonmyeloablative preparative regimen that permits the induction of mixed chimerism and renal allograft tolerance following donor bone marrow transplantation in major histocompatibility complex fully mismatched cynomolgus monkeys. This approach has been successfully extended to HLA matched or mismatched kidney transplant recipients. In the manuscript, we summarize some of the important conclusions made in our laboratories regarding induction of mixed chimerism and allograft tolerance in a nonhuman primate model. PMID:19027614

  4. Construction and Evaluation of a Maize Chimeric Promoter with Activity in Kernel Endosperm and Embryo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chimeric promoters contain DNA sequences from different promoters. Chimeric promoters are developed to increase the level of recombinant protein expression, precisely control transgene activity, or to escape homology-based gene silencing. Sets of chimeric promoters, each containing different lengt...

  5. Recombinant BCG prime and PPE protein boost provides potent protection against acute Mycobacterium tuberculosis infection in mice.

    PubMed

    Yang, Enzhuo; Gu, Jin; Wang, Feifei; Wang, Honghai; Shen, Hongbo; Chen, Zheng W

    2016-04-01

    Since BCG, the only vaccine widely used against tuberculosis (TB) in the world, provides varied protective efficacy and may not be effective for inducing long-term cellular immunity, it is in an urgent need to develop more effective vaccines and more potent immune strategies against TB. Prime-boost is proven to be a good strategy by inducing long-term protection. In this study, we tested the protective effect against Mycobacterium tuberculosis (Mtb) challenge of prime-boost strategy by recombinant BCG (rBCG) expressing PPE protein Rv3425 fused with Ag85B and Rv3425. Results showed that the prime-boost strategy could significantly increase the protective efficiency against Mtb infection, characterized by reduction of bacterial load in lung and spleen, attenuation of tuberculosis lesions in lung tissues. Importantly, we found that Rv3425 boost, superior to Ag85B boost, provided better protection against Mtb infection. Further research proved that rBCG prime-Rv3425 boost could obviously increase the expansion of lymphocytes, significantly induce IL-2 production by lymphocytes upon PPD stimulation, and inhibit IL-6 production at an early stage. It implied that rBCG prime-Rv3425 boost opted to induce Th1 immune response and provided a long-term protection against TB. These results implicated that rBCG prime-Rv3425 boost is a potent and promising strategy to prevent acute Mtb infection. PMID:26792673

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

    SciTech Connect

    Datta, T.; Doermer, P.

    1987-12-01

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

  7. Novel synthetic plasmid and Doggybone™ DNA vaccines induce neutralizing antibodies and provide protection from lethal influenza challenge in mice

    PubMed Central

    Scott, Veronica L; Patel, Ami; Villarreal, Daniel O; Hensley, Scott E; Ragwan, Edwin; Yan, Jian; Sardesai, Niranjan Y; Rothwell, Paul J; Extance, Jonathan P; Caproni, Lisa J; Weiner, David B

    2015-01-01

    Nucleic acid-based vaccines (NAVs) are a promising alternative to conventional influenza vaccines with the potential to increase influenza vaccine availability due to their simplicity in design and rapid speed of production. NAVs can also target multiple influenza antigens and control flu variants. Traditionally NAVs have been DNA plasmids however, we are continuing to explore new methods that may enhance vaccine efficacy. Recently new focus has been on RNA cassettes as NAVs. RNA vaccines combine conceptual advantages in that they focus on delivery of only the coding cassette. However, RNA vaccines have a short half-life and cause interferon-induced fevers. Here we describe a new NAV approach where we study delivery of a linear DNA cassette [Doggybone™ linear closed DNA [(dbDNA™)] produced by an enzymatic process that yields an antigen expression cassette comprising a promoter, DNA antigen, poly A tail, and telomeric ends. This focused approach has many of the advantages of plasmid DNA as well as a minimal cassette size similar to RNA strategies. For this study, we characterized the specific CD4+ and CD8+ T cell responses and determined the hemagglutination inhibition (HI) titers induced by dbDNA™ and compared the responses with those of an optimized plasmid DNA (pDNA) vaccine encoding the same H1N1 influenza A/PR/8/34 HA gene. Immunizations with the constructs resulted in similar humoral and cellular immune responses. Both constructs induced high-titer HI antibodies and fully protected animals from lethal viral challenge. The data obtained from this study provides important validation for further development of novel vector approaches. PMID:26091432

  8. An inactivated recombinant rabies CVS-11 virus expressing two copies of the glycoprotein elicits a higher level of neutralizing antibodies and provides better protection in mice.

    PubMed

    Xue, Xiang-Hong; Zheng, Xue-Xing; Wang, Hua-Lei; Ma, Jin-Zhu; Li, Ling; Gai, Wei-Wei; Wang, Tie-Cheng; Yang, Song-Tao; Xia, Xian-Zhu

    2014-06-01

    The rabies virus (RABV) G protein is the primary contributor to the pathogenicity and protective immunity of RABV. In this study, we generated a recombinant rCVS-11-G strain containing two copies of the G protein derived from the pathogenic wild-type (wt) CVS-11 strain and based on its infectious clone. Compared with the wtCVS-11 strain, the rCVS-11-G strain possessed a larger virion and 1.4-fold more G protein, but it exhibited a similar growth property to the rCVS-11 strain, including passaging stability in vitro. qPCR results showed that the two G genes were over-expressed in BHK-21 cells infected with the rCVS-11-G strain. However, the rCVS-11-G strain presented an 80 % lower LD50 than the wtCVS-11 strain when intracranially (i.c.) inoculated in adult mice. Adult mice that were either intracranially (i.c.) or intramuscularly (i.m.) inoculated with rCVS-11-G strain developed more acute neurological symptoms and greater mortality than those inoculated with the wtCVS-11 strain. Furthermore, the rCVS-11-G strain was more easily and rapidly taken up by neuroblastoma cells. These data indicated that the rCVS-11-G strain might have increased neurotropism because of the over-expression of the pathogenic G protein. The inactivated rCVS-11-G strain induced significantly higher levels of virus neutralization antibodies and provided better protection from street rabies virus challenge in mice. Therefore, the rCVS-11-G strain may be a promising inactivated vaccine strain due to its better immunogenicity. PMID:24535572

  9. Overexpression of β-Klotho in Adipose Tissue Sensitizes Male Mice to Endogenous FGF21 and Provides Protection From Diet-Induced Obesity.

    PubMed

    Samms, Ricardo J; Cheng, Christine C; Kharitonenkov, Alexei; Gimeno, Ruth E; Adams, Andrew C

    2016-04-01

    The endocrine hormone fibroblast growth factor 21 (FGF21) is induced in the adaptive response to nutrient deprivation, where it serves to regulate the integrated response to fasting via its primary receptor complex, FGF receptor 1 coupled with the cofactor β-klotho (KLB) in target tissues. Curiously, endogenous FGF21 levels are also elevated in preclinical models of obesity and in obese/diabetic individuals. In addition to higher FGF21 levels, reduced KLB expression in liver and adipose tissue has been noted in these same individuals, suggesting that obesity may represent an FGF21 resistant state. To explore the contribution of tissue-specific KLB levels to endogenous FGF21 activity, in both fasting and high-fat diet feeding conditions, we generated animals overexpressing KLB in liver (LKLBOE) or adipose (ATKLBOE). Supportive of tissue-specific partitioning of FGF21 action, after chronic high-fat feeding, ATKLBOE mice gained significantly less weight than WT. Reduced weight gain was associated with elevated caloric expenditure, accompanied by a reduced respiratory exchange ratio and lower plasma free fatty acids levels, suggestive of augmented lipid metabolism. In contrast, LKLBOE had no effect on body weight but did reduce plasma cholesterol. The metabolic response to fasting was enhanced in LKLBOE mice, evidenced by increased ketone production, whereas no changes in this were noted in ATKLBOE mice. Taken together, these data provide further support that specific effects of FGF21 are mediated via engagement of distinct target organs. Furthermore, enhancing KLB expression in adipose may sensitize to endogenous FGF21, thus representing a novel strategy to combat metabolic disease. PMID:26901091

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

    PubMed Central

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

    2009-01-01

    Background Mixed chimerism induces donor-specific tolerance to composite tissue allotransplants (CTA). In the present studies, we used a nonmyeloablative conditioning approach to establish chimerism and promote CTA acceptance. Methods WF (RT1Au) rats were conditioned with 600-300 cGy total body irradiation (TBI, day-1), 100 × 106 T cell-depleted ACI (RT1Aabl) bone marrow cells were transplanted day 0, followed by a 11-day course of tacrolimus and one dose of anti-lymphocyte serum (day 10). Heterotopic osteomyocutaneous flap transplantation was performed 4-6 weeks after bone marrow transplantation. Results Mixed chimerism was initially achieved in almost all recipients, but long-term acceptance of CTA was only achieved in rats treated with 600 cGy TBI. When anti-αβ-TCR mAb (day-3) was added into the regimens, donor chimerism was similar to recipients preconditioned without anti-αβ-TCR mAb. However, the long-term CTA survival was significantly improved in chimeras receiving ≥ 300 cGy TBI plus anti-αβ-TCR mAb. Higher levels of donor chimerism were associated with CTA acceptance. The majority of flap-acceptors lost peripheral blood (PB) chimerism within 6 months. However, donor chimerism persisted in transplanted bone at significantly higher levels compared to other hematopoietic compartments. The compartment donor chimerism may be responsible for the maintenance of tolerance to CTA. Long-term acceptors were tolerant to a donor skin graft challenge even in the absence of PB chimerism. Conclusions Mixed chimerism established by nonmyeloablative conditioning induces long-term acceptance of CTA which is associated with persistent chimerism preferentially in transplanted donor bone. PMID:19920776

  11. How Chimeric Antigen Receptor Design Affects Adoptive T Cell Therapy.

    PubMed

    Gacerez, Albert T; Arellano, Benjamine; Sentman, Charles L

    2016-12-01

    Chimeric antigen receptor (CAR) T cells have been developed to treat tumors and have shown great success against B cell malignancies. Exploiting modular designs and swappable domains, CARs can target an array of cell surface antigens and, upon receptor-ligand interactions, direct signaling cascades, thereby driving T cell effector functions. CARs have been designed using receptors, ligands, or scFv binding domains. Different regions of a CAR have each been found to play a role in determining the overall efficacy of CAR T cells. Therefore, this review provides an overview of CAR construction and common designs. Each CAR region is discussed in the context of its importance to a CAR's function. Additionally, the review explores how various engineering strategies have been applied to CAR T cells in order to regulate CAR T cell function and activity. J. Cell. Physiol. 231: 2590-2598, 2016. © 2016 Wiley Periodicals, Inc. PMID:27163336

  12. Tolerance Induction in HLA Disparate Living Donor Kidney Transplantation by Donor Stem Cell Infusion: durable chimerism predicts outcome1

    PubMed Central

    Leventhal, Joseph; Abecassis, Michael; Miller, Joshua; Gallon, Lorenzo; Tollerud, David; Elliott, Mary Jane; Bozulic, Larry D.; Houston, Chris; Sustento-Reodica, Nedjema; Ildstad, Suzanne T.

    2012-01-01

    Background We recently reported that durable chimerism can be safely established in mismatched kidney recipients through nonmyeloablative conditioning followed by infusion of a facilitating cell (FC)-based hematopoietic stem cell transplant termed FCRx. Here we provide intermediate-term follow-up on this phase 2 trial. Methods Fifteen HLA mismatched living donor renal transplant recipients underwent low intensity conditioning (fludarabine, cyclophosphamide, 200cGyTBI), received a living donor kidney transplant on day 0, then infusion of cryopreserved FCRx on day +1. Maintenance immunosuppression(IS),consisting of tacrolimus and mycophenolate, was weaned over one year. Results All but one patient demonstrated peripheral blood macrochimerism post-transplantation. Engraftment failure occurred in a highly sensitized (PRA of52%) recipient. Chimerism was lost in 3patients at 2, 3, and 6 months post transplantation. Two of these subjects had received either a reduced cell dose or incomplete conditioning; the other 2 had PRA >20%. All demonstrated donor-specific hyporesponsiveness and were weaned from full dose immunosuppression. Complete immunosuppression withdrawal at one year post-transplant was successful in all patients with durable chimerism. There has been no GVHD or engraftment syndrome. Renal transplant loss occurred in 1 patient who developed sepsis following an atypical viral infection. Two subjects with only transient chimerism demonstrated subclinical rejection on protocol biopsy despite donor-specific hyporesponsiveness. Conclusions Low intensity conditioning plus FCRx safely achieved durable chimerism in mismatched allograft recipients. Sensitization represents an obstacle to successful induction of chimerism. Sustained T cell chimerism is a more robust biomarker of tolerance than donor-specific hyporeactivity. PMID:23222893

  13. 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.

    PubMed

    Russo, Lisa M; Melton-Celsa, Angela R; Smith, Michael J; O'Brien, Alison D

    2014-01-01

    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. PMID:24671194

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

    PubMed Central

    Bounds, Callie E.; Kwilas, Steven A.; Kuehne, Ana I.; Brannan, Jennifer M.; Bakken, Russell R.; Dye, John M.; Hooper, Jay W.; Dupuy, Lesley C.; Ellefsen, Barry; Hannaman, Drew; Wu, Hua; Jiao, Jin-an; Sullivan, Eddie J.; Schmaljohn, Connie S.

    2015-01-01

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

  15. Network analysis of gene expression in mice provides new evidence of involvement of the mTOR pathway in antipsychotic-induced extrapyramidal symptoms.

    PubMed

    Mas, S; Gassó, P; Boloc, D; Rodriguez, N; Mármol, F; Sánchez, J; Bernardo, M; Lafuente, A

    2016-06-01

    To identify potential candidate genes for future pharmacogenetic studies of antipsychotic (AP)-induced extrapyramidal symptoms (EPS), we used gene expression arrays to analyze changes induced by risperidone in mice strains with different susceptibility to EPS. We proposed a systems biology analytical approach that combined the identification of gene co-expression modules related to AP treatment, the construction of protein-protein interaction networks with genes included in identified modules and finally, gene set enrichment analysis of constructed networks. In response to risperidone, mice strain with susceptibility to develop EPS showed downregulation of genes involved in the mammalian target of rapamycin (mTOR) pathway and biological processes related to this pathway. Moreover, we also showed differences in the phosphorylation pattern of the ribosomal protein S6 (rpS6), which is a major downstream effector of mTOR. The present study provides new evidence of the involvement of the mTOR pathway in AP-induced EPS and offers new and valuable markers for pharmacogenetic studies. PMID:26122020

  16. Human prion strain selection in transgenic mice

    PubMed Central

    Giles, Kurt; Glidden, David V.; Patel, Smita; Korth, Carsten; Groth, Darlene; Lemus, Azucena; DeArmond, Stephen J.; Prusiner, Stanley B.

    2010-01-01

    Transgenic (Tg) mice expressing chimeras of mouse and human prion proteins (PrP) have shorter incubation periods for Creutzfeldt-Jakob disease (CJD) prions than mice expressing full-length human PrP. Increasing the sequence similarity of the chimeric PrP to mouse PrP, by reverting human residues to mouse, resulted in a Tg line, denoted Tg22372, which was susceptible to sporadic (s) CJD prions in ~110 days 1. Reversion of one additional residue (M111V) resulted in a new Tg line, termed Tg1014, susceptible to sCJD prions in ~75 days. Tg1014 mice also has shorter incubation periods for variant (v) CJD prions, providing a more tractable model for studying this prion strain. Transmission of vCJD prions to Tg1014 mice resulted in two different strains, determined by neuropathology and biochemical analysis, which correlated with the length of the incubation time. One strain had the biochemical, neuropathological, and transmission characteristics including longer incubation times of the inoculated vCJD strain; the second strain produced a phenotype resembling that of sCJD prions including relatively shorter incubation periods. Mice with intermediate incubation periods for vCJD prions had a mixture of the two strains. Both strains were serially transmitted in Tg1014 mice, which led to further reduction in incubation periods. Conversion of vCJD-like to sCJD-like strains was favored in Tg1014 mice more than in the Tg22372 line. The single amino acid difference therefore appears to offer selective pressure for propagation of the sCJD-like strain. These two Tg mouse lines provide relatively rapid models to study human prion diseases as well as the evolution of human prion strains. PMID:20695008

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

    PubMed

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

    2006-12-01

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

  18. ChiTaRS: a database of human, mouse and fruit fly chimeric transcripts and RNA-sequencing data.

    PubMed

    Frenkel-Morgenstern, Milana; Gorohovski, Alessandro; Lacroix, Vincent; Rogers, Mark; Ibanez, Kristina; Boullosa, Cesar; Andres Leon, Eduardo; Ben-Hur, Asa; Valencia, Alfonso

    2013-01-01

    Chimeric RNAs that comprise two or more different transcripts have been identified in many cancers and among the Expressed Sequence Tags (ESTs) isolated from different organisms; they might represent functional proteins and produce different disease phenotypes. The ChiTaRS database of Chimeric Transcripts and RNA-Sequencing data (http://chitars.bioinfo.cnio.es/) collects more than 16 000 chimeric RNAs from humans, mice and fruit flies, 233 chimeras confirmed by RNA-seq reads and ∼2000 cancer breakpoints. The database indicates the expression and tissue specificity of these chimeras, as confirmed by RNA-seq data, and it includes mass spectrometry results for some human entries at their junctions. Moreover, the database has advanced features to analyze junction consistency and to rank chimeras based on the evidence of repeated junction sites. Finally, 'Junction Search' screens through the RNA-seq reads found at the chimeras' junction sites to identify putative junctions in novel sequences entered by users. Thus, ChiTaRS is an extensive catalog of human, mouse and fruit fly chimeras that will extend our understanding of the evolution of chimeric transcripts in eukaryotes and can be advantageous in the analysis of human cancer breakpoints. PMID:23143107

  19. Dasatinib Targets B-Lineage Cells but Does Not Provide an Effective Therapy for Myeloproliferative Disease in c-Cbl RING Finger Mutant Mice

    PubMed Central

    Duyvestyn, Johanna M.; Taylor, Samuel J.; Dagger, Samantha A.; Orandle, Marlene; Morse, Herbert C.; Thien, Christine B. F.; Langdon, Wallace Y.

    2014-01-01

    This study aimed to determine whether the multi-kinase inhibitor dasatinib would provide an effective therapy for myeloproliferative diseases (MPDs) involving c-Cbl mutations. These mutations, which occur in the RING finger and linker domains, abolish the ability of c-Cbl to function as an E3 ubiquitin ligase and downregulate activated protein tyrosine kinases. Here we analyzed the effects of dasatinib in a c-Cbl RING finger mutant mouse that develops an MPD with a phenotype similar to the human MPDs. The mice are characterized by enhanced tyrosine kinase signaling resulting in an expansion of hematopoietic stem cells, multipotent progenitors and cells within the myeloid lineage. Since c-Cbl is a negative regulator of c-Kit and Src signaling we reasoned that dasatinib, which targets these kinases, would be an effective therapy. Furthermore, two recent studies showed dasatinib to be effective in inhibiting the in vitro growth of cells from leukemia patients with c-Cbl RING finger and linker domain mutations. Surprisingly we found that dasatinib did not provide an effective therapy for c-Cbl RING finger mutant mice since it did not suppress any of the hematopoietic lineages that promote MPD development. Thus we conclude that dasatinib may not be an appropriate therapy for leukemia patients with c-Cbl mutations. We did however find that dasatinib caused a marked reduction of pre-B cells and immature B cells which correlated with a loss of Src activity. This study is therefore the first to provide a detailed characterization of in vivo effects of dasatinib in a hematopoietic disorder that is driven by protein tyrosine kinases other than BCR-ABL. PMID:24718698

  20. The Pathogenesis of Saffold Virus in AG129 Mice and the Effects of Its Truncated L Protein in the Central Nervous System

    PubMed Central

    Tan, Shawn Zheng Kai; Chua, Kaw Bing; Xu, Yishi; Prabakaran, Mookkan

    2016-01-01

    Saffold Virus (SAFV) is a human cardiovirus that has been suggested to cause severe infection of the central nervous system (CNS). Compared to a similar virus, Theiler’s murine encephalomyelitis virus (TMEV), SAFV has a truncated Leader (L) protein, a protein essential in the establishment of persistent CNS infections. In this study, we generated a chimeric SAFV by replacing the L protein of SAFV with that of TMEV. We then compared the replication in cell cultures and pathogenesis in a mouse model. We showed that both SAFV and chimeric SAFV are able to infect Vero and Neuro2a cells well, but only chimeric SAFV was able to infect RAW264.7. We then showed that mice lacking IFN-α/β and IFN-γ receptors provide a good animal model for SAFV infection, and further identified the locality of the infection to the ventral horn of the spine and several locations in the brain. Lastly, we showed that neither SAFV nor chimeric SAFV causes persistence in this model. Overall, our results provide a strong basis on which the mechanisms underlying Saffold virus induced neuropathogenesis can be further studied and, hence, facilitating new information about its pathogenesis. PMID:26901216

  1. A chimeric platelet-targeted urokinase prodrug selectively blocks new thrombus formation

    PubMed Central

    Fuentes, Rudy E.; Zaitsev, Sergei; Ahn, Hyun Sook; Hayes, Vincent; Kowalska, M. Anna; Lambert, Michele P.; Wang, Yuhuan; Siegel, Donald L.; Bougie, Daniel W.; Aster, Richard H.; Myers, Daniel D.; Stepanova, Victoria; Cines, Douglas B.; Muzykantov, Vladimir R.; Poncz, Mortimer

    2015-01-01

    The use of fibrinolytic agents to prevent new thrombus formation is limited by an increased risk of bleeding due to lysis of hemostatic clots that prevent hemorrhage in damaged blood vessels. We sought to develop an agent that provides thromboprophylaxis without carrying a significant risk of causing systemic fibrinolysis or disrupting hemostatic clots. We previously showed that platelet (PLT) α granule–delivered urokinase plasminogen activator (uPA) is highly effective in preventing thrombosis, while being associated with little systemic fibrinolysis or bleeding. Here, we generated a chimeric prodrug composed of a single-chain version of the variable region of an anti-αIIbβ3 mAb fused to a thrombin-activatable, low-molecular-weight pro-uPA (PLT/uPA-T). PLT/uPA-T recognizes human αIIbβ3 on both quiescent and activated platelets and is enzymatically activated specifically by thrombin. We found that this prodrug binds tightly to human platelets even after gel filtration, has a prolonged half-life in mice transgenic for human αIIb compared with that of uPA-T, and prevents clot formation in a microfluidic system. Importantly, in two murine injury models, PLT/uPA-T did not lyse preexisting clots, even when administration was delayed by as little as 10 minutes, while it concurrently prevented the development of nascent thrombi. Thus, PLT/uPA-T represents the prototype of a platelet-targeted thromboprophylactic agent that selectively targets nascent over preexisting thrombi. PMID:26690701

  2. Chimerism of buccal membrane cells in a monochorionic dizygotic twin.

    PubMed

    Fumoto, Seiko; Hosoi, Kenichiro; Ohnishi, Hiroaki; Hoshina, Hiroaki; Yan, Kunimasa; Saji, Hiroh; Oka, Akira

    2014-04-01

    No monochorionic dizygotic twins (MCDZTs) with cellular chimerism involving cells other than blood cells have been reported in the literature to date. Here we report a probable first case of MCDZTs with buccal cell chimerism. A 32-year-old woman conceived twins by in vitro fertilization by using 2 cryopreserved blastocysts that were transferred into her uterus. An ultrasound scan at 8 weeks' gestation showed signs indicative of monochorionic twins. A healthy boy and a healthy girl were born, showing no sexual ambiguity. Cytogenetic analyses and microsatellite studies demonstrated chimerism in blood cells of both twins. Notably, repeated fluorescence in situ hybridization and microsatellite studies revealed chimerism in buccal cells obtained from 1 of the twins. Although the mechanism through which buccal cell chimerism was generated remains to be elucidated, ectopic differentiation of chimeric hematopoietic cells that migrated to the buccal membrane or the cellular transfer between the 2 embryos at the early stage of development might be responsible for the phenomenon. This hypothesis raises an interesting issue regarding embryonic development and cellular differentiation into organs during fetal development. Given the possibility of cryptic chimerism in various organs including gonadal tissues in MCDZTs, close observation will be required to determine whether complications develop in the course of the patients' growth. PMID:24685957

  3. Envelope-chimeric Entry-targeted Measles Virus Escapes Neutralization and Achieves Oncolysis

    PubMed Central

    Miest, Tanner S; Yaiw, Koon-Chu; Frenzke, Marie; Lampe, Johanna; Hudacek, Andrew W; Springfeld, Christoph; von Messling, Veronika; Ungerechts, Guy; Cattaneo, Roberto

    2011-01-01

    Measles virus (MV) is a promising vector for cancer therapy and multivalent vaccination, but high prevalence of pre-existing neutralizing antibodies may reduce therapeutic efficacy, particularly following systemic administration. MV has only one serotype, but here we show that its envelope glycoproteins can be exchanged with those of the closely related canine distemper virus (CDV), generating a chimeric virus capable of escaping neutralization. To target its entry, we displayed on the CDV attachment protein a single-chain antibody specific for a designated receptor. To enhance oncolytic efficacy we armed the virus with a prodrug convertase gene capable of locally activating chemotherapeutic prodrugs. The new virus achieved high titers, was genetically stable, and was resistant to neutralization by sera from both MV-immunized mice and MV-immune humans. The new virus targeted syngeneic murine tumor cells expressing the designated receptor implanted in immunocompetent mice, and synergized with a chemotherapeutic prodrug in a model of oncolysis. Importantly, the chimeric MV remained oncolytic when administered systemically even in the presence of anti-MV antibodies capable of abrogating the therapeutic efficacy of the parental, nonshielded MV. This work shows that targeting, arming, and shielding can be combined to generate a tumor-specific, neutralization-resistant virus that can synergize with chemotherapeutics. PMID:21610701

  4. A PLGA-encapsulated chimeric protein protects against adherence and toxicity of enterotoxigenic Escherichia coli.

    PubMed

    Nazarian, Shahram; Gargari, Seyed Latif Mousavi; Rasooli, Iraj; Hasannia, Sadegh; Pirooznia, Nazanin

    2014-01-01

    Enterotoxigenic Escherichia coli (ETEC) are the most common cause of diarrhea among children. Colonization factors and enterotoxins are the major ETEC candidate vaccines. Since protection against ETEC mostly occurs by induction of IgA antibodies, much effort is focused on the development of oral vaccines. In this study oral immunogenicity of a poly(lactic-co-glycolic acid) (PLGA) encapsulated chimeric protein containing CfaB, CstH, CotA and LTB (Heat-labile B subunit) was investigated. The protein was encapsulated in PLGA by double emulsion method and nanoparticles were characterized physicochemically. Immunogenicity was assessed by evaluating IgG1, IgG2 and IgA titers after BALB/c mice vaccination. Non aggregated nanoparticles had a spherical shape with an average particle size of 252.7±23 nm and 91.96±4.4% of encapsulation efficiency. Western blotting showed maintenance of the molecular weight and antigenicity of the released protein. Oral immunization of mice induced serum IgG and fecal IgA antibody responses. Immunization induced protection against ETEC binding to Caco-2 cells. The effect of LT toxin on fluid accumulation in ileal loops was neutralized by inhibition of enterotoxin binding to GM1-ganglosides. Delivery of the chimeric protein in PLGA elicited both systemic and mucosal immune responses. The findings could be exploited to development of oral multi-component ETEC prophylactic measures. PMID:23906742

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

    SciTech Connect

    Taguchi, Kazuhiro . E-mail: s3061@nms.ac.jp; Ogawa, Rei; Migita, Makoto; Hanawa, Hideki; Ito, Hiromoto; Orimo, Hideo

    2005-05-27

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

  6. Generation of Gene Knockout Mice by ES Cell Microinjection

    PubMed Central

    Longenecker, Glenn; Kulkarni, Ashok B

    2009-01-01

    This unit lists and describes protocols used in the production of chimeric mice leading to the generation of gene knockout mice. These protocols include the collection of blastocyst embryos, ES cell injection, and uterine transfer of injected blastocysts. Support protocols in the superovulation of blastocyst donor mice, generation of pseudopregnant recipients, fabrication of glass pipettes, and generation of germline mice are also included. Practical tips and solutions are mentioned to help troubleshoot problems that may occur. PMID:19731226

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

    PubMed

    Gedvilaite, Alma; Kucinskaite-Kodze, Indre; Lasickiene, Rita; Timinskas, Albertas; Vaitiekaite, Ausra; Ziogiene, Danguole; Zvirbliene, Aurelija

    2015-08-01

    Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes. PMID:26230706

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

    PubMed Central

    Gedvilaite, Alma; Kucinskaite-Kodze, Indre; Lasickiene, Rita; Timinskas, Albertas; Vaitiekaite, Ausra; Ziogiene, Danguole; Zvirbliene, Aurelija

    2015-01-01

    Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes. PMID:26230706

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

    PubMed

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

    2016-07-01

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

  10. Vaginal transmission of chimeric simian/human immunodeficiency viruses in rhesus macaques.

    PubMed Central

    Lu, Y; Brosio, P; Lafaile, M; Li, J; Collman, R G; Sodroski, J; Miller, C J

    1996-01-01

    Chimeric simian/human immunodeficiency viruses (SHIVs) that express the env genes derived from distinct HIV type 1 (HIV-1) isolates were tested for the ability to infect rhesus macaques following intravaginal inoculation. SHIVs containing either the HIV-1 HXBc2 or the HIV-1 89.6 envelope glycoproteins were capable of replicating in intravenously inoculated rhesus macaques. However, intravaginal inoculation of animals with these two SHIVs resulted in infection only with the SHIV containing the HIV-1 89.6 glycoprotein. Thus, properties conferred by the envelope glycoproteins in the chimeric virus affect the ability of particular SHIVs to initiate a systemic infection following vaginal inoculation. These results provide indirect support for the hypothesis that the selection of specific viral variants occurs in the genital tracts of individuals exposed to HIV by sexual contact. PMID:8627782

  11. Engraftment kinetics and hematopoietic chimerism after reduced-intensity conditioning with fludarabine and treosulfan before allogeneic stem cell transplantation.

    PubMed

    Blau, I W; Schmidt-Hieber, Martin; Leschinger, N; Göldner, H; Knauf, W; Hopfenmüller, W; Thiel, E; Blau, O

    2007-08-01

    Reduced-intensity conditioning with fludarabine and treosulfan before allogeneic stem cell transplantation (SCT) was introduced several years ago. Although its feasibility has recently been proven, only limited data are available on myelotoxicity, engraftment kinetics, and the significance of hematopoietic chimerism using this novel conditioning regimen. To clarify these open questions, we analyzed 27 patients with various hematological diseases, who received allogeneic SCT preceded by fludarabine/treosulfan conditioning. Further assessment endpoints included graft-vs-host disease (GvHD), mortality, and overall survival (OS). Allogeneic SCT was followed by neutropenia (absolute neutrophil count < or = 0.5 x 10(9)/l) and thrombocytopenia (platelets < or = 20 x 10(9)/l) in all patients. All patients showed stable neutrophil engraftment, and all except one had stable platelet engraftment. Grades II-IV acute GvHD was found in 48% of patients, whereas 52% developed chronic GvHD. The treatment-related mortality on day +100, 1 year after SCT, and at the last follow-up was 11, 26, and 33%, respectively. We found complete chimerism rates of 46, 57, and 72% on days +28, +56, and at the last follow-up or before death, respectively. The underlying malignancy tended to relapse more frequently in patients with mixed chimerism than in those with complete chimerism on day +28 as well as on day +56 (not significant). Additionally, no significant association was found between hematopoietic chimerism and donor type, GvHD, or OS, respectively. We conclude that reduced-intensity conditioning with fludarabine and treosulfan before allogeneic SCT is myeloablative, provides stable engraftment, and leads to complete chimerism in the majority of patients. PMID:17468869

  12. Chimerism in piglets developed from aggregated cloned embryos.

    PubMed

    Huang, Yongye; Li, Zhanjun; Wang, Anfeng; Han, Xiaolei; Song, Yuning; Yuan, Lin; Li, Tianye; Wang, Bing; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

    2016-04-01

    Porcine chimeras are valuable in the study of pluripotency, embryogenesis and development. It would be meaningful to generate chimeric piglets from somatic cell nuclear transfer embryos. In this study, two cell lines expressing the fluorescent markers enhanced green fluorescent protein (EGFP) and tdTomato were used as donor cells to produce reconstructed embryos. Chimeric embryos were generated by aggregating two EGFP-cell derived embryos with two tdTomato-cell derived embryos at the 4-cell stage, and embryo transfer was performed when the aggregated embryos developed into blastocysts. Live porcine chimeras were successfully born and chimerism was observed by their skin color, gene integration, microsatellite loci composition and fluorescent protein expression. The chimeric piglets were largely composed of EGFP-expressing cells, and this phenomenon was possibly due to the hyper-methylation of the promoter of the tdTomato gene. In addition, the expression levels of tumorigenicity-related genes were altered after tdTomato transfection in bladder cancer cells. The results show that chimeric pigs can be produced by aggregating cloned embryos and that the developmental capability of the cloned embryo in the subsequent chimeric development could be affected by the growth characteristics of its donor cell. PMID:27239442

  13. Inhibition of myeloid differentiation factor 88(MyD88) by ST2825 provides neuroprotection after experimental traumatic brain injury in mice.

    PubMed

    Zhang, Hua-Sheng; Li, Hua; Zhang, Ding-Ding; Yan, Hui-Ying; Zhang, Zi-Huan; Zhou, Chen-Hui; Ye, Zhen-Nan; Chen, Qiang; Jiang, Tian-Wei; Liu, Jing-Peng; Hang, Chun-Hua

    2016-07-15

    Myeloid differentiation factor 88(MyD88) is an endogenous adaptor protein that plays an important role in coordinating intracellular inflammatory responses induced by agonists of the Toll-like receptor and interleukin-1 receptor families. MyD88 has been reported to be essential for neuronal death in animal models and may represent a therapeutic target for pharmacologic inhibition following traumatic brain injury (TBI). The purpose of the current study was to investigate the neuroprotective effect of MyD88 specific inhibitor ST2825 in an experimental mouse model of TBI. Intracerebroventricular (ICV) injection of high concentration (20μg/μL) ST2825 (15min post TBI) attenuated the development of TBI in mice, markedly improved neurological function and reduced brain edema. Decreased neural apoptosis and increased neuronal survival were also observed. Biochemically, the high concentration of ST2825 significantly reduced the levels of MyD88, further decreased TAK1, p-TAK1, nuclear p65 and increased IκB-α. Additionally, ST2825 significantly reduced the levels of Iba-1 and inflammatory factors TNF-α and IL-1β. These data provide an experimental rationale for evaluation of MyD88 as a drug target and highlight the potential therapeutic implications of ST2825 in TBI. PMID:27155455

  14. Effects of T cell depletion in radiation bone marrow chimeras. I. Evidence for a donor cell population which increases allogeneic chimerism but which lacks the potential to produce GVHD

    SciTech Connect

    Sykes, M.; Sheard, M.; Sachs, D.H.

    1988-10-01

    The opposing problems of graft-vs-host disease (GVHD) and failure of alloengraftment present major obstacles to the application of bone marrow transplantation (BMT) across complete MHC barriers. The addition of syngeneic T-cell-depleted (TCD) bone marrow (BM) to untreated fully allogeneic marrow inocula in lethally irradiated mice has been previously shown to provide protection from GVHD. We have used this model to study the effects of allogeneic T cells on levels of chimerism in recipients of mixed marrow inocula. The results indicate that T cells in allogeneic BM inocula eliminate both coadministered recipient-strain and radioresistant host hematopoietic elements to produce complete allogeneic chimerism without clinical GVHD. To determine the role of GVH reactivity in this phenomenon, we performed similar studies in an F1 into parent combination, in which the genetic potential for GVHD is lacking. The presence of T cells in F1 marrow inocula led to predominant repopulation with F1 lymphocytes in such chimeras, even when coadministered with TCD-recipient-strain BM. These results imply that the ability of allogeneic BM cells removed by T cell depletion to increase levels of allochimerism may be mediated by a population which is distinct from that which produces GVHD. These results may have implications for clinical BM transplantation.

  15. Mouse-human immunoglobulin G1 chimeric antibodies with activities against Cryptococcus neoformans.

    PubMed Central

    Zebedee, S L; Koduri, R K; Mukherjee, J; Mukherjee, S; Lee, S; Sauer, D F; Scharff, M D; Casadevall, A

    1994-01-01

    Passive antibody administration is a potentially useful approach for the therapy of human Cryptococcus neoformans infections. To evaluate the efficacy of the human immunoglobulin G1 (IgG1) constant region against C. neoformans and to construct murine antibody derivatives with reduced immunogenicities and longer half-lives in humans, two mouse-human IgG1 chimeric antibodies were generated from the protective murine monoclonal antibodies 2D10 (IgM) and 18B7 (IgG1). The 2D10 mouse-human IgG1 chimeric antibody (ch2D10) had significantly lower binding affinity than its parent murine antibody (m2D10), presumably because of a loss of avidity contribution on switching from IgM to IgG. The 18B7 mouse-human IgG1 chimeric antibody (ch18B7) had higher affinity for cryptococcal polysaccharide antigen than its parent murine antibody (m18B7). ch18B7 and ch2D10 promoted phagocytosis of C. neoformans by primary human microglial cells and the murine J774.16 macrophage-like cell line. ch18B7 and m18B7 enhanced fungistatic or fungicidal activity of J774.16 cells and prolonged the survival of lethally infected mice. We conclude that the human IgG1 constant chain can be effective in mediating antifungal activity against C. neoformans. ch18B7 or similar antibodies are potential candidates for passive antibody therapy of human cryptococcosis. PMID:7979280

  16. The use of fabricated chimeric flap for reconstruction of extensive foot defects.

    PubMed

    Song, Baoqiang; Chen, Jianwu; Han, Yan; Hu, Yalan; Su, Yingjun; Li, Yang; Zhang, Juan; Guo, Shuzhong

    2016-05-01

    Repair of extensive foot defects requires both adequate tissues for wound coverage and special tissues for functional reconstruction. To maximize its function reconstruction, fabricated chimeric flaps consisting of multiple separate flaps were designed to reconstruct such defects. Five patients suffered extensive foot defects with sizes ranging from 23 × 12 cm to 38 × 14 cm(2) in multiple regions including heel, forefoot, dorsum, ankle, anterior leg, and even toes. Causes included crushing injuries, avulsion injuries, and scar excision. Most areas of the defects except heel were first covered by latissimus dorsi muscle flap or anterolateral thigh flap and their pedicles were anastomosed with recipient vessels. Then free medial plantar flaps were transferred for heel reconstruction and their pedicles were further attached to either side branches of the main source vessel or to its distal continuation. All chimeric flaps survived uneventfully and all patients were able to walk in normal footwear during the 1.5- to 4-years follow-up. None of the flaps developed ulcer and flap breakdown. The assessment by Maryland Foot Score showed that four of the five patients gained a "good" recovery and one patient showed moderate improvement of foot functions. Appearances of reconstructed heels were near-normal. The results indicate that fabricated chimeric flap has good design flexibility and may provide an option for functional reconstruction of extensive foot defects. © 2015 Wiley Periodicals, Inc. Microsurgery 36:303-309, 2016. PMID:25752811

  17. Chimeric Aptamer-Gelatin Hydrogels as an Extracellular Matrix Mimic for Loading Cells and Growth Factors

    PubMed Central

    Zhang, Xiaolong; Battig, Mark R.; Chen, Niancao; Gaddes, Erin R.; Duncan, Katelyn L.; Wang, Yong

    2016-01-01

    It is important to synthesize materials to recapitulate critical functions of biological systems for a variety of applications such as tissue engineering and regenerative medicine. The purpose of this study was to synthesize a chimeric hydrogel as a promising extracellular matrix (ECM) mimic using gelatin, a nucleic acid aptamer and polyethylene glycol (PEG). This hydrogel had a macroporous structure that was highly permeable for fast molecular transport. Despite its high permeability, it could strongly sequester and sustainably release growth factors with high bioactivity. Notably, growth factors retained in the hydrogel could maintain ~50% bioactivity during a 14-day release test. It also provided cells with effective binding sites, which led to high efficiency of cell loading into the macroporous hydrogel matrix. When cells and growth factors were co-loaded into the chimeric hydrogel, living cells could still be observed by day 14 in a static serum-reduced culture condition. Thus, this chimeric aptamer-gelatin hydrogel constitutes a promising biomolecular ECM mimic for loading cells and growth factors. PMID:26791559

  18. Generation of Potent T-cell Immunotherapy for Cancer Using DAP12-Based, Multichain, Chimeric Immunoreceptors.

    PubMed

    Wang, Enxiu; Wang, Liang-Chuan; Tsai, Ching-Yi; Bhoj, Vijay; Gershenson, Zack; Moon, Edmund; Newick, Kheng; Sun, Jing; Lo, Albert; Baradet, Timothy; Feldman, Michael D; Barrett, David; Puré, Ellen; Albelda, Steven; Milone, Michael C

    2015-07-01

    Chimeric antigen receptors (CAR) bearing an antigen-binding domain linked in cis to the cytoplasmic domains of CD3ζ and costimulatory receptors have provided a potent method for engineering T-cell cytotoxicity toward B-cell leukemia and lymphoma. However, resistance to immunotherapy due to loss of T-cell effector function remains a significant barrier, especially in solid malignancies. We describe an alternative chimeric immunoreceptor design in which we have fused a single-chain variable fragment for antigen recognition to the transmembrane and cytoplasmic domains of KIR2DS2, a stimulatory killer immunoglobulin-like receptor (KIR). We show that this simple, KIR-based CAR (KIR-CAR) triggers robust antigen-specific proliferation and effector function in vitro when introduced into human T cells with DAP12, an immunotyrosine-based activation motifs-containing adaptor. T cells modified to express a KIR-CAR and DAP12 exhibit superior antitumor activity compared with standard first- and second-generation CD3ζ-based CARs in a xenograft model of mesothelioma highly resistant to immunotherapy. The enhanced antitumor activity is associated with improved retention of chimeric immunoreceptor expression and improved effector function of isolated tumor-infiltrating lymphocytes. These results support the exploration of KIR-CARs for adoptive T-cell immunotherapy, particularly in immunotherapy-resistant solid tumors. PMID:25941351

  19. Generation of Potent T-cell Immunotherapy for Cancer using DAP12-based, Multichain, Chimeric Immunoreceptors

    PubMed Central

    Wang, Enxiu; Wang, Liang-Chuan; Tsai, Ching-Yi; Bhoj, Vijay; Gershenson, Zack; Moon, Edmund; Newick, Kheng; Sun, Jing; Lo, Albert; Baradet, Timothy; Feldman, Michael D.; Barrett, David; Puré, Ellen; Albelda, Steven; Milone, Michael C.

    2015-01-01

    Chimeric antigen receptors (CAR) bearing an antigen-binding domain linked in cis to the cytoplasmic domains of CD3ζ and costimulatory receptors have provided a potent method for engineering T-cell cytotoxicity towards B-cell leukemia and lymphoma. However, resistance to immunotherapy due to loss of T-cell effector function remains a significant barrier, especially in solid malignancies. We describe an alternative chimeric immunoreceptor design in which we have fused a single-chain variable fragment for antigen recognition to the transmembrane and cytoplasmic domains of KIR2DS2, a stimulatory killer immunoglobulin-like receptor (KIR). We show that this simple, KIR-based CAR (KIR-CAR) triggers robust antigen-specific proliferation and effector function in vitro when introduced into human T cells with DAP12, an immunotyrosine-based activation motifs (ITAM)-containing adaptor. T cells modified to express a KIR-CAR and DAP12 exhibit superior antitumor activity compared to standard first and second generation CD3ζ-based CARs in a xenograft model of mesothelioma highly resistant to immunotherapy. The enhanced antitumor activity is associated with improved retention of chimeric immunoreceptor expression and improved effector function of isolated tumor-infiltrating lymphocytes. These results support the exploration of KIR-CARs for adoptive T-cell immunotherapy, particularly in immunotherapy-resistant solid tumors. PMID:25941351

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

    PubMed Central

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

    2016-01-01

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

  1. Humanization of predicted T-cell epitopes reduces the immunogenicity of chimeric antibodies: new evidence supporting a simple method.

    PubMed

    Roque-Navarro, Lourdes; Mateo, Cristina; Lombardero, Josefa; Mustelier, Geraudis; Fernández, Alicia; Sosa, Katya; Morrison, Sherrie L; Pérez, Rolando

    2003-08-01

    Genetic engineering has provided several approaches to reduce immunogenicity of murine antibodies. We described previously a new method based on the humanization of the linear epitopes presented to T cells. In brief, potential immunogenic epitopes in the variable region were identified and subjected to point mutations to make them human and/or to modify amphipatic motifs. The resulting recombinant antibody retained its antigen binding affinity and was less immunogenic in monkeys than their murine or chimeric predecessors are. The present study provides two new examples of this T-cell epitope humanization approach: ior-t1A murine monoclonal antibody (mMAb), which recognizes the human-CD6 molecule, and ior-C5 mMAb, which recognizes a novel glycoprotein expressed on the surface of malignant colorectal cells. Seven amino acids were substituted in ior-C5 and eleven residues in ior-t1A, by the corresponding residues from the highest homologous human sequences. Surprisingly, the homology between re-shaped chimeric antibody variable region frameworks and human sequences was 80-90%. Experiments in monkeys showed that T1AhT and C5hT "detopes" antibodies were less immunogenic than their chimeric analogues while they retained 30-50% of antigen binding affinities. The proposed method might be of general applicability to reduce immunogenicity of chimeric antibodies with therapeutic potential. PMID:14511570

  2. Designing Chimeric Antigen Receptors to Effectively and Safely Target Tumors

    PubMed Central

    Jensen, Michael C.; Riddell, Stanley R.

    2015-01-01

    The adoptive transfer of T cells engineered to express artificial chimeric antigen receptors (CARs) that target a tumor cell surface molecule has emerged as an exciting new approach for cancer immunotherapy. Clinical trials in patients with advanced B cell malignancies treated with CD19-specific CAR-modified T cells (CAR-T) have shown impressive antitumor efficacy, leading to optimism that this approach will be useful for treating common solid tumors. Because CAR-T cells recognize tumor cells independent of their expression of human leukocyte antigen (HLA) molecules, tumors that escape conventional T cells by downregulating HLA and/or mutating components of the antigen processing machinery can be eliminated. The ability to introduce or delete additional genes in T cells has the potential to provide therapeutic cell products with novel attributes that overcome impediments to immune mediated tumor elimination in immunosuppressive tumor microenvironments. This review will discuss recent concepts in the development of effective and safe synthetic CARs for adoptive T cell therapy (ACT). PMID:25621840

  3. Utilizing Chimeric Antigen Receptors to Direct Natural Killer Cell Activity

    PubMed Central

    Hermanson, David L.; Kaufman, Dan S.

    2015-01-01

    Natural killer (NK) cells represent an attractive lymphocyte population for cancer immunotherapy due to their ability to lyse tumor targets without prior sensitization and without need for human leukocyte antigens-matching. Chimeric antigen receptors (CARs) are able to enhance lymphocyte targeting and activation toward diverse malignancies. CARs consist of an external recognition domain (typically a small chain variable fragment) directed at a specific tumor antigen that is linked with one or more intracellular signaling domains that mediate lymphocyte activation. Most CAR studies have focused on their expression in T cells. However, use of CARs in NK cells is starting to gain traction because they provide a method to redirect these cells more specifically to target refractory cancers. CAR-mediated anti-tumor activity has been demonstrated using NK cell lines, as well as NK cells isolated from peripheral blood, and NK cells produced from human pluripotent stem cells. This review will outline the CAR constructs that have been reported in NK cells with a focus on comparing the use of different signaling domains in combination with other co-activating domains. PMID:25972867

  4. Efficient induction of human T-cell leukemia virus-1-specific CTL by chimeric particle without adjuvant as a prophylactic for adult T-cell leukemia.

    PubMed

    Kozako, Tomohiro; Fukada, Katsuhiko; Hirata, Shinya; White, Yohann; Harao, Michiko; Nishimura, Yasuharu; Kino, Youichiro; Soeda, Shinji; Shimeno, Hiroshi; Lemonnier, François; Sonoda, Shunro; Arima, Naomichi

    2009-12-01

    Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with the human T-cell leukemia virus-1 (HTLV-1). HTLV-1-specific cytotoxic T lymphocytes (CTLs) play an important role in suppressing proliferation of HTLV-1-infected or transformed T-cells in vitro. Efficient induction of antigen-specific CTLs is important for immunologic suppression of oncogenesis, but has evaded strategies utilizing poorly immunogenic free synthetic peptides. In the present study, we examined the efficient induction of HTLV-1-specific CD8+ T-cell response by an HTLV-1/hepatitis B virus core (HBc) chimeric particle incorporating the HLA-A*0201-restricted HTLV-1 Tax-epitope. The immunization of HLA-A*0201-transgenic mice with the chimeric particle induced antigen-specific gamma-interferon reaction, whereas immunization with epitope peptide only induced no reaction as assessed by enzyme-linked immunospot assay. Immunization with the chimeric particle also induced HTLV-1-specific CD8+ T-cells in spleen and inguinal lymph nodes. Furthermore, upon exposure of dendritic cells from HLA-A*0201-transgenic mice to the chimeric particle, the expression of CD86, HLA-A02, TLR4 and MHC class II was increased. Additionally, our results show that HTLV-1-specific CD8+ T-cells can be induced by peptide with HTLV-1/HBc particle from ATL patient, but not by peptide only and these HTLV-1-specific CD8+ T-cells were able to lyse cells presenting the peptide. These results suggest that HTLV-1/HBc chimeric particle is capable of inducing strong cellular immune responses without adjuvants via effective maturation of dendritic cells and is potentially useful as an effective carrier for therapeutic vaccines in tumors, or in infectious diseases by substituting the epitope peptide. PMID:19889459

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

    PubMed

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

    2016-06-01

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

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

    SciTech Connect

    Joseph, Tomy; McAuliffe, Josephine; Lu, Bin; Vogel, Leatrice; Swayne, David; Jin, Hong; Kemble, George; Subbarao, Kanta

    2008-08-15

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

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

    PubMed Central

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

    2016-01-01

    Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders. PMID:26955620

  8. Chimeric antigen receptors: driving immunology towards synthetic biology.

    PubMed

    Sadelain, Michel

    2016-08-01

    The advent of second generation chimeric antigen receptors and the CD19 paradigm have ushered a new therapeutic modality in oncology. In contrast to earlier forms of adoptive cell therapy, which were based on the isolation and expansion of naturally occurring T cells, CAR therapy is based on the design and manufacture of engineered T cells with optimized properties. A new armamentarium, comprising not only CARs but also chimeric costimulatory receptors, chimeric cytokine receptors, inhibitory receptors and synthetic Notch receptors, expressed in naïve, central memory or stem cell-like memory T cells, is being developed for clinical use in a wide range of cancers. Immunological principles are thus finding a new purpose thanks to advances in genetic engineering, synthetic biology and cell manufacturing sciences. PMID:27372731

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

    PubMed Central

    Polejaeva, Irina; Mitalipov, Shoukhrat

    2013-01-01

    Mouse embryonic chimeras are a well-established tool for studying cell lineage commitment and pluripotency. Experimental chimeras were successfully produced by combining of two or more preimplantation embryos or, by introduction into a host embryo cultured pluripotent embryonic stem cells (ESCs). Chimera production using genetically modified ESCs became the method of choice for 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. PMID:23221011

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2013-08-01

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

  12. Combination of liquid-chromatography tandem mass spectrometry in different scan modes with human and chimeric mouse urine for the study of steroid metabolism.

    PubMed

    Pozo, Oscar J; Lootens, Leen; Van Eenoo, Peter; Deventer, Koen; Meuleman, Philip; Leroux-Roels, Geert; Parr, Maria K; Schänzer, Wilhelm; Delbeke, Frans T

    2009-11-01

    Anabolic steroids are among the most frequently detected compounds in doping analysis. They are extensively metabolized and therefore an in-depth knowledge about steroid metabolism is needed. In this study, a liquid chromatography tandem mass spectometry (LC-MS/MS) method based on a precursor ion scan with a uPA-SCID mouse with humanized liver (a chimeric mouse) was explored for the detection of steroid metabolism. Methandienone was used as a model compound. The application of the precursor ion scan method in positive human samples and chimeric mice samples after methandienone administration allowed the detection of most steroid metabolites without any structural restriction. Three hitherto unreported metabolites were found using this approach. These metabolites were characterized using LC-MS/MS and feasible structures were proposed. The structure of one of them, 6-ene-epimethandienone, was confirmed by the synthesis of the reference compound. A selected reaction monitoring (SRM) method for the specific detection of all these metabolites has been developed. The application of this method to several human and chimeric mouse samples confirmed that more than 80% of the steroid metabolites were found in both samples. Only metabolites that are poorly detectable by LC-MS/MS were not detected in some urine samples. The metabolic nature of the unreported metabolites was also confirmed. A global strategy for the detection of steroid metabolites combining both human and chimeric mouse urine is proposed. PMID:20355172

  13. A case of canine chimerism diagnosed using coat color tests.

    PubMed

    Dreger, Dayna L; Schmutz, Sheila M

    2012-12-01

    Through the use of PCR based coat color tests, we were able to diagnose a dog that exhibits an unusual coat color phenotype as an XX/XX chimera. Coat color alleles vary widely among dog breeds, presenting a novel method for detecting chimerism using diagnostic tests for known coat color alleles. PMID:22433982

  14. The Sirt1 activator SRT3025 provides atheroprotection in Apoe−/− mice by reducing hepatic Pcsk9 secretion and enhancing Ldlr expression

    PubMed Central

    Miranda, Melroy X.; van Tits, Lambertus J.; Lohmann, Christine; Arsiwala, Tasneem; Winnik, Stephan; Tailleux, Anne; Stein, Sokrates; Gomes, Ana P.; Suri, Vipin; Ellis, James L.; Lutz, Thomas A.; Hottiger, Michael O.; Sinclair, David A.; Auwerx, Johan; Schoonjans, Kristina; Staels, Bart; Lüscher, Thomas F.; Matter, Christian M.

    2015-01-01

    Aims The deacetylase sirtuin 1 (Sirt1) exerts beneficial effects on lipid metabolism, but its roles in plasma LDL-cholesterol regulation and atherosclerosis are controversial. Thus, we applied the pharmacological Sirt1 activator SRT3025 in a mouse model of atherosclerosis and in hepatocyte culture. Methods and results Apolipoprotein E-deficient (Apoe−/−) mice were fed a high-cholesterol diet (1.25% w/w) supplemented with SRT3025 (3.18 g kg−1 diet) for 12 weeks. In vitro, the drug activated wild-type Sirt1 protein, but not the activation-resistant Sirt1 mutant; in vivo, it increased deacetylation of hepatic p65 and skeletal muscle Foxo1. SRT3025 treatment decreased plasma levels of LDL-cholesterol and total cholesterol and reduced atherosclerosis. Drug treatment did not change mRNA expression of hepatic LDL receptor (Ldlr) and proprotein convertase subtilisin/kexin type 9 (Pcsk9), but increased their protein expression indicating post-translational effects. Consistent with hepatocyte Ldlr and Pcsk9 accumulation, we found reduced plasma levels of Pcsk9 after pharmacological Sirt1 activation. In vitro administration of SRT3025 to cultured AML12 hepatocytes attenuated Pcsk9 secretion and its binding to Ldlr, thereby reducing Pcsk9-mediated Ldlr degradation and increasing Ldlr expression and LDL uptake. Co-administration of exogenous Pcsk9 with SRT3025 blunted these effects. Sirt1 activation with SRT3025 in Ldlr−/− mice reduced neither plasma Pcsk9, nor LDL-cholesterol levels, nor atherosclerosis. Conclusion We identify reduction in Pcsk9 secretion as a novel effect of Sirt1 activity and uncover Ldlr as a prerequisite for Sirt1-mediated atheroprotection in mice. Pharmacological activation of Sirt1 appears promising to be tested in patients for its effects on plasma Pcsk9, LDL-cholesterol, and atherosclerosis. PMID:24603306

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

    PubMed

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

    2016-01-01

    In the present study, the free radical scavenging activities (against 1,1-diphenyl-2-pierylhydrazy (DPPH), 2,2'-Azinobis-(3-ethylbenzthiazoline-6- sulphonate) (ABTS+), Hydrogen peroxide (H2O2)) of dimethylglycine sodium salt (DMG-Na) were measured and compared with those of Trolox (6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid), a commonly used antioxidant. The radical scavenging activities of DMG-Na were found to be the highest at 40 mg/ml. In Experiment 2, gastric intubation in mice with 12 mg DMG-Na/0.3 ml sterile saline solution significantly increased (P < 0.05) the body weight (BW) (28 d), organ proportion (liver and spleen), and antioxidant capacity in serum and the liver (Superoxide dismutase (SOD), Hydrogen peroxidase (CAT), Glutathione peroxidase (GPx), and Total antioxidant capacity (T-AOC)), and significantly decreased (P < 0.05) the activities of serum Glutamic-pyruvic transaminase (ALT) and Glutamic oxalacetic transaminase (AST) and Methane Dicarboxylic Aldehyde (MDA) contents in the serum and liver. Specifically, the effect of 12 mg DMG-Na/0.3 ml sterile saline solution, which showed the highest antioxidant capacity, was further studied using a mice model. In Experiment 3, the mice CL (CON+ lipopolysaccharide (LPS)) group showed a significant decrease (P < 0.05) in the serum ALT and AST content; hepatic mitochondrial antioxidant capacity (Manganese Superoxide dismutase (MnSOD), Glutathione reductase (GR), GPx, Glutathione (GSH)); MDA and Protein carbonyl (PC) content; Reactive oxygen species (ROS) level, Mitochondrial membrane potential (MMP) level, and expression of liver antioxidant genes (Nuclear factor erythroid 2-related factor 2 (Nrf2), Heme oxygenase 1 (HO-1), Manganese superoxide dismutase (MnSOD), Glutathione peroxidase 1 (Gpx1), Sirtuin 1 (Sirt1)) relative to the mice CS (CON+ sterile saline) group. The DL (DMG+LPS) group showed a significant decrease (P < 0.05) in serum ALT and AST content, ROS level, and expression of liver

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

    PubMed Central

    Zhang, Jingfei; Kou, Tao; Niu, Yu; Wan, Xiaoli; Zhang, Lili; Wang, Chao; Wang, Tian

    2016-01-01

    In the present study, the free radical scavenging activities (against 1,1-diphenyl-2-pierylhydrazy (DPPH), 2,2'-Azinobis-(3-ethylbenzthiazoline-6- sulphonate) (ABTS+), Hydrogen peroxide (H2O2)) of dimethylglycine sodium salt (DMG-Na) were measured and compared with those of Trolox (6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid), a commonly used antioxidant. The radical scavenging activities of DMG-Na were found to be the highest at 40 mg/ml. In Experiment 2, gastric intubation in mice with 12 mg DMG-Na/0.3 ml sterile saline solution significantly increased (P < 0.05) the body weight (BW) (28 d), organ proportion (liver and spleen), and antioxidant capacity in serum and the liver (Superoxide dismutase (SOD), Hydrogen peroxidase (CAT), Glutathione peroxidase (GPx), and Total antioxidant capacity (T-AOC)), and significantly decreased (P < 0.05) the activities of serum Glutamic-pyruvic transaminase (ALT) and Glutamic oxalacetic transaminase (AST) and Methane Dicarboxylic Aldehyde (MDA) contents in the serum and liver. Specifically, the effect of 12 mg DMG-Na/0.3 ml sterile saline solution, which showed the highest antioxidant capacity, was further studied using a mice model. In Experiment 3, the mice CL (CON+ lipopolysaccharide (LPS)) group showed a significant decrease (P < 0.05) in the serum ALT and AST content; hepatic mitochondrial antioxidant capacity (Manganese Superoxide dismutase (MnSOD), Glutathione reductase (GR), GPx, Glutathione (GSH)); MDA and Protein carbonyl (PC) content; Reactive oxygen species (ROS) level, Mitochondrial membrane potential (MMP) level, and expression of liver antioxidant genes (Nuclear factor erythroid 2-related factor 2 (Nrf2), Heme oxygenase 1 (HO-1), Manganese superoxide dismutase (MnSOD), Glutathione peroxidase 1 (Gpx1), Sirtuin 1 (Sirt1)) relative to the mice CS (CON+ sterile saline) group. The DL (DMG+LPS) group showed a significant decrease (P < 0.05) in serum ALT and AST content, ROS level, and expression of liver

  17. Advances in chimeric antigen receptor immunotherapy for neuroblastoma.

    PubMed

    Heczey, Andras; Louis, Chrystal U

    2013-12-01

    Neuroblastoma (NBL) is the most common extracranial pediatric solid tumor and has heterogeneous biology and behavior. Patients with high-risk disease have poor prognosis despite complex multimodal therapy; therefore, novel curative approaches are needed. Immunotherapy is a novel therapeutic approach that harnesses the inherent activity of the immune system to control and eliminate malignant cells. One form of immunotherapy uses chimeric antigen receptors (CAR) to target tumor-associated antigens. CARs are derived from the antigen-binding domain of a monoclonal antibody (MAb) coupled with the intracellular signaling portion of the T cell receptor. CARs can combine the specificity and effectiveness of MAbs with the active bio-distribution, direct cytotoxicity, and long-term persistence of T cells. NBL provides an attractive target for CAR immunotherapy as many of its tumor-associated antigens are not expressed at significant levels on normal tissues, thus decreasing potential treatment related toxicity. Two previous clinical trials utilizing L1-cell adhesion molecule (L1-CAM) and disialoganglioside (GD2) specific CARs (GD2-CAR) have demonstrated safety and anti-tumor efficacy in heavily pretreated relapsed/refractory neuroblastoma patients. Based on these promising results and on improved techniques that can further potentiate CAR therapies, two clinical trials are currently investigating the use of GD2-CARs in children with NBL. Several approaches may further enhance anti-tumor activity and persistence of CAR modified cells, and if these can be safely translated into the clinic, CAR-based immunotherapy could become a viable adjunct or potential alternative to conventional treatment options for patients with NBL. PMID:24333408

  18. Chimeric aptamers in cancer cell-targeted drug delivery

    PubMed Central

    Kanwar, Jagat R; Roy, Kislay; Kanwar, Rupinder K

    2011-01-01

    Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets ("apatopes") with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern Pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer-aptamer, aptamer-nonaptamer biomacromolecules (siRNAs, proteins) and aptamer-nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe3O4) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities. PMID:21955150

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-12-01

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

  1. Fluorescent transgenic mice suitable for multi-color aggregation chimera studies.

    PubMed

    Ohtsuka, Masato; Miura, Hiromi; Gurumurthy, Channabasavaiah B; Kimura, Minoru; Inoko, Hidetoshi; Yoshimura, Shinichi; Sato, Masahiro

    2012-11-01

    We recently reported a novel method of mouse transgenesis called Pronuclear Injection-based Targeted Transgenisis (PITT) using which a series of fluorescent transgenic (Tg) mice lines were generated. These lines, unlike those generated using conventional random integration methods, express the transgenes faithfully and reproducibly generation after generation. Because of this superior nature, these lines are ideal for the generation of multi-colored aggregation chimeras that can be used to study cell-cell interactions and lineage analyses in living embryos/organs, where the transgenes can be detected and the clonal origin of a given cell population easily traced by its distinct fluorescence. In this study, to verify if Tg fluorescent mice generated through PITT were suitable for such applications, we sought to generate chimeric blastocysts and chimeric-Tg mice by aggregating two- or three-colored 8-cell embryos. Our analyses using these models led to the following observations. First, we noticed that cell mixing was infrequent during the stages of morula to early blastocyst. Second, chimeric fetuses obtained after aggregation of the two-colored 8-cell embryos exhibited uniform cell mixing. And third, in the organs of adult chimeric mice, the mode of cell distribution could be either clonal or polyclonal, as previously pointed out by others. Implications of our novel and improved Tg-chimeric mice approach for clonal cell lineage and developmental studies are discussed. PMID:22868913

  2. Possible Role of Minor H Antigens in the Persistence of Donor Chimerism after Stem Cell Transplantation; Relevance for Sustained Leukemia Remission

    PubMed Central

    van der Torren, Cornelis R.; van Hensbergen, Yvette; Luther, Susanne; Aghai, Zohara; Rychnavská, Zuzana Stachová; Slot, Manon; Scherjon, Sicco; Kröger, Nicolaus; Ganser, Arnold; Weissinger, Eva M.; Goulmy, Els; Hambach, Lothar

    2015-01-01

    Persistent complete donor chimerism is an important clinical indicator for remissions of hematological malignancies after HLA-matched allogeneic stem cell transplantation (SCT). However, the mechanisms mediating the persistence of complete donor chimerism are poorly understood. The frequent coincidence of complete donor chimerism with graft-versus-leukemia effects and graft-versus-host disease suggests that immune responses against minor histocompatibility antigens (mHags) are playing an important role in suppressing the host hematopoiesis after allogeneic SCT. Here, we investigated a possible relationship between donor immune responses against the hematopoiesis-restricted mHag HA-1 and the long-term kinetics of host hematopoietic chimerism in a cohort of 10 patients after allogeneic HLA-matched, HA-1 mismatched SCT. Functional HA-1 specific CTLs (HA-1 CTLs) were detectable in 6/10 patients lysing host-type hematopoietic cells in vitro. Presence of HA-1 CTLs in the peripheral blood coincided with low host hematopoiesis levels quantified by highly sensitive mHag specific PCR. Additionally, co-incubation of host type CD34+ cells with HA-1 CTLs isolated after allogeneic SCT prevented progenitor and cobblestone area forming cell growth in vitro and human hematopoietic engraftment in immunodeficient mice. Conversely, absence or loss of HA-1 CTLs mostly coincided with high host hematopoiesis levels and/or relapse. In summary, in this first study, presence of HA-1 CTLs paralleled low host hematopoiesis levels. This coincidence might be supported by the capacity of HA-1 CTLs isolated after allogeneic SCT to specifically eliminate host type hematopoietic stem/progenitor cells. Additional studies involving multiple mismatched mHags in more patients are required to confirm this novel characteristic of mHag CTLs as factor for the persistence of complete donor chimerism and leukemia remission after allogeneic SCT. PMID:25774796

  3. Repression of the alpha-fetoprotein gene promoter by progesterone and chimeric receptors in the presence of hormones and antihormones.

    PubMed Central

    Turcotte, B; Meyer, M E; Bocquel, M T; Bélanger, L; Chambon, P

    1990-01-01

    Using transient transfection assays, we showed that repression of the alpha-fetoprotein promoter by intact and deletion mutants of the progesterone receptor and by chimeric progesterone/glucocorticoid-estrogen receptors in the presence of their cognate hormones was closely correlated with their ability to bind to a progesterone/glucocorticoid-responsive element. This negative regulation was also observed in the presence of antihormones, providing evidence that receptor-antihormone complexes can bind to their responsive elements in vivo. Images PMID:1697036

  4. The novel anti-CD19 chimeric antigen receptors with humanized scFv (single-chain variable fragment) trigger leukemia cell killing.

    PubMed

    Qian, Liren; Li, Dan; Ma, Lie; He, Ting; Qi, Feifei; Shen, Jianliang; Lu, Xin-An

    2016-01-01

    The molecular design of CARs (Chimeric Antigen Receptors), especially the scFv, has been a major part to use of CAR-T cells for targeted adoptive immunotherapy. To address this issue, we chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR. Next, we generated a panel of humanized scFvs and tested in vitro for their ability to direct CAR-T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. Furthermore, in a xenograft model of lymphoma, human T cells expressing humanized scFvs exhibited the same anti-tumor efficacy as those expressing murine scFv and prolonged survival compared with cells expressing control CAR. Therefore, we uncovered CARs expressing humanized scFv domain that contribute the similar enhanced antileukemic efficacy and survival in tumor bearing mice. These results provide the basis for the future clinical studies of CAR-T cells transduced with humanized scFv directed to CD19. PMID:26996927

  5. Metaphase I arrest and spontaneous parthenogenetic activation of strain LTXBO oocytes: chimeric reaggregated ovaries establish primary lesion in oocytes.

    PubMed

    Eppig, J J; Wigglesworth, K; Hirao, Y

    2000-08-01

    Oocytes of strain LT mice, and related strains such as LTXBO, exhibit a high incidence of arrest in the progression of meiosis at metaphase I (MI) and in spontaneous parthenogenetic activation. Activation of these oocytes within the ovary leads to the formation of ovarian teratomas. In this study, the role of the oocyte's companion granulosa cells, the cumulus cells, was investigated using fully grown oocytes matured in vitro after isolation from LTXBO mice. Results showed that the role of cumulus cells in MI arrest is dichotomous. Cumulus cells temporarily helped to sustain MI arrest, but they also promoted a delayed progression to metaphase II. Cumulus cells also promoted parthenogenetic activation that occurred in association with the delayed progression to metaphase II. Next, the question of whether the lesion(s) promoting MI arrest and spontaneous activation is due to defects in the somatic cells or is intrinsic to the oocyte was addressed using chimeric reaggregated ovaries. An improved method for completely exchanging the germ cell and the somatic cell compartments of ovaries from newborn mice is described. These chimeric reaggregated ovaries, grafted beneath the renal capsule of SCID mice, allowed the complete development of LTXBO oocytes to occur in association with somatic cells from control (B6SJLF(1)) ovaries and development of control oocytes in association with LTXBO somatic cells. Oocyte growth and follicular development appeared generally normal in reaggregated ovaries. High incidences of MI arrest and spontaneous activation of LTXBO oocytes occurred regardless of the genotype of the somatic cells. Moreover, there was a low incidence of MI arrest and spontaneous activation of control oocytes, even though they underwent complete development and maturation associated with LTXBO somatic cells. It is concluded that the phenotypes of MI arrest and parthenogenetic activation in LTXBO oocytes are defects caused by lesions intrinsic to the oocyte

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

    SciTech Connect

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

    1989-05-15

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

  7. Haplotype and minimum-chimerism consensus determination using short sequence data

    PubMed Central

    2012-01-01

    Background Assembling haplotypes given sequence data derived from a single individual is a well studied problem, but only recently has haplotype assembly been considered for population-sampled data. We discuss a software tool called Hapler, which is designed specifically for low-diversity, low-coverage data such as ecological samples derived from natural populations. Because such data may contain error as well as ambiguous haplotype information, we developed methods that increase confidence in these assemblies. Hapler also reconstructs full consensus sequences while minimizing and identifying possible chimeric points. Results Experiments on simulated data indicate that Hapler is effective at assembling haplotypes from gene-sized alignments of short reads. Further, in our tests Hapler-generated consensus sequences are less chimeric than the alternative consensus approaches of majority vote and viral quasispecies estimation regardless of error rate, read length, or population haplotype bias. Conclusions The analysis of genetically diverse sequence data is increasingly common, particularly in the field of ecoinformatics where transcriptome sequencing of natural populations is a cost effective alternative to genome sequencing. For such studies, it is important to consider and identify haplotype diversity. Hapler provides robust haplotype information and identifies possible phasing errors in consensus sequences, providing valuable information for population studies and downstream usage of resulting assemblies. PMID:22537299

  8. CNX-012-570, a direct AMPK activator provides strong glycemic and lipid control along with significant reduction in body weight; studies from both diet-induced obese mice and db/db mice models

    PubMed Central

    2014-01-01

    Objectives AMP activated protein kinase (AMPK) regulates the coordination of anabolic and catabolic processes and is an attractive therapeutic target for T2DM, obesity and metabolic syndrome. We report the anti-hyperglycemic and anti-hyperlipidemic effects of CNX-012-570 is an orally bioavailable small molecule (molecular weight of 530 Daltons) that directly activates AMPK in DIO and db/db animal models of diabetes. Methods Activity and efficacy of the compound was tested in cell based as well as cell free systems in vitro. Male C57BL/6 mice fed with high fat diet (HFD) were assigned to either vehicle or CNX-012-570 (3 mg/kg, orally once a day) for 8 weeks (n = 8). Genetically diabetic db/db mice on chow diet were dosed with vehicle control or CNX-012-570 (2.5 mg/kg, orally once a day) for 6 weeks (n = 8). Results CNX-012-570 is a highly potent and orally bioavailable compound activating AMPK in both cell and cell free systems. It inhibits lipolysis (33%) and gluconeogenesis (28%) in 3T3L1 cells and rat primary hepatocytes respectively. The efficacy of the molecule was translated to both DIO and db/db animal models of diabetes. CNX-012-570 has reduced fasting blood glucose levels by 14%, body weight by 24% and fasting serum triglycerides (TG) by 24%. CNX-012-570 showed a 22% reduction in fed serum cholesterol levels and 19% increase in HDL levels. In db/db mice model, CNX-012-570 has shown 18% decrease in fed glucose and 32% decrease in fasting glucose with a 2.57% reduction in absolute HbA1c. Decrease in serum insulin and glucose AUC indicates the increased insulin sensitivity. Body weight was reduced by 13% with increased browning of adipose tissue and decreased inguinal and mesenteric fat mass. There was significant reduction in liver TG and liver total cholesterol. Conclusions CNX-012-570 has the potential to control hyperglycemia and hyperlipidemia. It also reduces body weight gain with an additional benefit of minimizing cardiovascular risks in

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

    PubMed

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

    2016-02-01

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

  10. Poly (lactic acid)-poly (ethylene glycol) nanoparticles provide sustained delivery of a Chlamydia trachomatis recombinant MOMP peptide and potentiate systemic adaptive immune responses in mice

    PubMed Central

    Dixit, Saurabh; Singh, Shree R.; Yilma, Abebayehu N.; Agee, Ronald D.; Taha, Murtada; Dennis, Vida A.

    2014-01-01

    PLA-PEG [poly (lactic acid)-poly (ethylene glycol)], a biodegradable copolymer, is underexploited for vaccine delivery although it exhibits enhanced biocompatibility and slow release immune-potentiating properties. We document here successful encapsulation of M278, a Chlamydia trachomatis MOMP (major outer membrane protein) peptide, within PLA-PEG nanoparticles by size (~73–100 nm), zeta potential (−16 mV), smooth morphology, encapsulation efficiency (~60%), slow release pattern, and non-toxicity to macrophages. Immunization of mice with encapsulated-M278 elicited higher M278-specific T-cell cytokines [Th1 (IFN-γ, IL-2), Th17 (IL-17)] and antibodies [Th1 (IgG2a), Th2 (IgG1, IgG2b)] compared to bare M278. Encapsulated-M278 mouse serum inhibited Chlamydia infectivity of macrophages, with a concomitant transcriptional down-regulation of MOMP, its cognate TLR2 and CD80 co-stimulatory molecule. Collectively, encapsulated-M278 potentiated crucial adaptive immune responses, which are required by a vaccine candidate for protective immunity against Chlamydia. Our data highlights PLA-PEG’s potential for vaccines, which resides in its slow release and potentiating effects to bolster immune responses. PMID:24602605