Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience

Hearts from alpha1,3-galactosyltransferase knockout pigs (GalT-KO, n = 8) were transplanted heterotopically into baboons using an anti-CD154 monoclonal antibody-based regimen. The elimination of the galactose-alpha1,3-galactose epitope prevented hyperacute rejection and extended survival of pig hearts in baboons for 2-6 months (median, 78 d); the predominant lesion associated with graft failure was a thrombotic microangiopathy, with resulting ischemic injury. There were no infectious complications directly related to the immunosuppressive regimen. The transplantation of hearts from GalT-KO pigs increased graft survival over previous studies.     

Aberrant methylation of Meg3 in alpha1,3-galactosyltransferase knockout pig induced pluripotent stem cells

Pigs have been used as a good research model for xenotransplantation. Several groups have generated porcine-induced pluripotent stem cells (piPSCs) from differentiated somatic cells. Transgenic pigs with the alpha1,3-galactosyltransferase gene-knockout (GalT-KO) could successfully govern hyper acute rejection of organ transplants into primates. Thus, GalT-KO piPSCs could be a powerful cell resource for agricultural and biomedical applications. This study was performed to generate iPSCs from GalT-KO pigs and characterize their properties. We successfully generated a GalT-KO iPSC from a genetically modified pig using double alpha1,3-galactosyltransferase knockout alterations. Similar to mouse embryonic stem cells, the GalT-KO piPSCs were positive for classical pluripotency markers: POU5F1, NANOG, SOX2 and SSEA1, and were negative for: SSEA3, TRA-1-60 and TRA-1-81. Furthermore, these cells could form an embryoid body that differentiated into three germ layers in vitro, and were highly proliferative under leukemia inhibitory factor culture conditions. However, the methylation status in DMR2 of the Meg3 gene was higher in GalT-KO piPSCs than in porcine ear fibroblast. In conclusion, GalT-KO piPSCs could be successfully generated by six human factors without expression of Gal-epitopes. Although aberrant methylation observed in GalT-KO piPSCs, this cell line maintained pluripotency and had differentiation properties into all three germ layers. Therefore, GalT-KO piPSCs might be a good cell source for biomedical application and basic research.     

Detection of serum antibodies in Ethiopian baboons that cross-react with SIV,HTLV-I,and type D retroviral antigens

Baboons (Papio cynocephalus) imported from Ethiopia were screened for antibodies to various primate retroviruses by immunoblotting. Antibodies that cross-reacted with SIV/Mne or with type D viral antigens were detected in approximately one-third of these animals. In addition, 20% of these baboons had antibodies that cross-reacted with HTLV-I viral antigens. These data suggest that wild-caught baboons are infected with retroviruses only partially related to known primate viral isolates.     

Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates

Cell-based diabetes therapy requires an abundant cell source. Here, we report reversal of diabetes for more than 100 d in cynomolgus macaques after intraportal transplantation of cultured islets from genetically unmodified pigs without Gal-specific antibody manipulation. Immunotherapy with CD25-specific and CD154-specific monoclonal antibodies, FTY720 (or tacrolimus), everolimus and leflunomide suppressed indirect activation of T cells, elicitation of non-Gal pig-specific IgG antibody, intragraft expression of proinflammatory cytokines and invasion of infiltrating mononuclear cells into islets.     

Immunogenicity of B-antigen in experimental plague and pseudotuberculosis

The results of the evaluation of the immunogenic properties of B-antigen, earlier identified in the culture fluid of Yersinia pseudotuberculosis submerged culture, with respect to experimental plague and pseudotuberculosis are presented. B-antigen has been shown to produce protective effect in guinea pigs and, probably, hamadryas baboons, but not in white mice infected with the causative agent of plague. Immunizaton with B-antigen protects guinea pigs from primary pneumonic plague caused by both capsule-forming and noncapsular Y. pestis virulent strains. Passive immunization with antibodies to B-antigen induces limitedly pronounced protective effect in guinea pigs and is not effective for white mice with respect to experimental plague. No active or passive protection of white mice or guinea pigs, infected with Y. pseudotuberculosis cultures, has been achieved by the injection of B-antigen or antibodies to it.     

Immunoluminescent detection of antibodies to testicular antigens in experimental autoimmune orchitis]

Antibodies interacting with spermatid structures in the testis sections and with acrosome and caudal regions of spermatozoa in the smears of sperm were determined in the sera of guinea pigs and mice with experimental autoimmune orchitis by the indirect immunofluorescent method. The antibodies were found in mice from the 14th to the 30th day and in guinea pigs from the 14th to the 60th day after immunization. Circulation time and the level of the fluorescent antibodies were significantly decreased in immunized female guinea pigs and mice. Fluorescent antibodies did not show cytotoxic effect and belonged to the IgG fraction. The fluorescent antibodies of guinea pigs did not react with the mouse testicular antigens in cross reaction. The analogous results were seen in the cross reaction of the mouse antibodies with the guinea pig antigens.     

Molecular cloning of pigGnT-I and I.2: an application to xenotransplantation

Xenotransplantation is one of the most attractive solutions for the current worldwide shortage of organs. The knocking out of alpha1,3-galactosyltransferase in pigs resulted in a drastic reduction in xenoantigenicity. However, more recent studies indicate that other xeno-antigens, so-called non-Gal antigens, will also need to be downregulated. In this study, pig N-acetylglucosaminyltransferase I (GnT-I), a key enzyme that initiates the biosynthesis of hybrid- and complex-type N-linked sugar chains, was isolated and the pigGnT-I.2 specific for the O-linked sugar chain was also isolated. Point mutants, pigGnT-I(123) and pigGnT-I(320), were subsequently constructed. While pigGnT-I(123) shows an indistinct dominant negative effect for endogenous GnT-I in pig cells, pigGnT-I(320) had a drastic effect. In addition, in the case of pig cell transfectants with pigGnT-I(320), cell surface carbohydrate structures were significantly altered and its antigenicity to human serum was reduced. Consequently, pigGnT-I(320) appears to be potentially useful in xenotransplantation by remodeling the carbohydrate structures on pig cells.     

Mammalian mismatches in nucleotide metabolism: implications for xenotransplantation

Acute humoral rejection (AHR) limits the clinical application of animal organs for xenotransplantation. Mammalian disparities in nucleotide metabolism may contribute significantly to the microvascular component in AHR; these, however remain ill-defined. We evaluated the extent of species-specific differences in nucleotide metabolism. HPLC analysis was performed on venous blood samples (nucleotide metabolites) and heart biopsies (purine enzymes) from wild type mice, rats, pigs, baboons, and human donors. Ecto-5′-nucleotidase (E5′N) activities were 4-fold lower in pigs and baboon hearts compared to human and mice hearts while rat activity was highest. Similar differences between pigs and humans were also observed with kidneys and endothelial cells. More than 10-fold differences were observed with other purine enzymes. AMP deaminase (AMPD) activity was exceptionally high in mice but very low in pig and baboon hearts. Adenosine deaminase (ADA) activity was highest in baboons. Adenosine kinase (AK) activity was more consistent across different species. Pig blood had the highest levels of hypoxanthine, inosine and adenine. Human blood uric acid concentration was almost 100 times higher than in other species studied. We conclude that species-specific differences in nucleotide metabolism may affect compatibility of pig organs within a human metabolic environment. Furthermore, nucleotide metabolic mismatches may affect clinical relevance of animal organ transplant models. Supplementation of deficient precursors or application of inhibitors of nucleotide metabolism (e.g., allopurinol) or transgenic upregulation of E5'N may overcome some of these differences.     

Adequate selection of antigens for serologic diagnostics of experimental melioidosis

Sera of Burkholderia pseudomallei-infected golden hamsters, white mice, guinea pigs and white rats were studied. Immunochemical analysis revealed presence of antibodies against antigens 2, 3, 6, d, and g with significant predominance of antibodies to antigens 6 and d. Antigen d was detected irrespectively to strain used for experimental infection and experimental animal species. Antibodies to antigen 6 were detected only when strains belonging to Asian serovar, but not to Australian serovar, were used. On the basis of antigens 6 and d following methods of serologic diagnostics were developed: reaction of indirect hemagglutination, reaction of latex agglutination, and radioimmunological assay. Their sensitivity and specificity reached 100% during experimental melioidosis in golden hamsters, guinea pigs and white rats.     

Antibodies to bacterial and tumor-derived antigens in sera from normal guinea pigs.

Antibodies that react with radiolabeled antigens derived from guinea pig line-10 tumor cells and Mycobacterium bovis (BCG) were detected in sera from normal tumor-free strain-2 guinea pigs (NGPS). Binding by NGPS to the two antigens was inhibited by extracts of either line-10 cells or BCG. Binding by NGPS to the line-10 antigen was inhibited by a number of other bacterial extracts. NGPS was tested after absorption with a variety of cells including line-10, line-1, normal guinea pig spleen, normal adult and fetal liver cells. Results indicated that some of the antibodies in NGPS were directed to line-10-specific determinants. The specific stimulating antigen for these antibodies was not identified but because of the antigenic relationship between BCG, line-10 cells and other bacteria, antibodies to line-10-associated antigens might have been induced by exposure to environmental microorganisms.     

Genetic variability at the pig SLA complex in U.S. breeds of pigs

Lymphocytes from 90 mature pigs and 548 growing pigs from 65 litters were screened with cytotoxic antibodies against swine lymphocyte antigens. Pigs included in the survey were from the Chester White, Duroc, Hampshire, Landrace and Yorkshire breeds. Significant differences between breeds were found in the frequency of the three haplotypes tested. Such differences may provide the genetic variability needed to determine associations between disease susceptibility and the pig's major histocompatibility gene complex.     

Genetically-engineered pig-to-baboon liver xenotransplantation: histopathology of xenografts and native organs

Orthotopic liver transplantation was carried out in baboons using wild-type (WT, n = 1) or genetically-engineered pigs (α1,3-galactosyltransferase gene-knockout, GTKO), n = 1; GTKO pigs transgenic for human CD46, n = 7) and a clinically-acceptable immunosuppressive regimen. Biopsies were obtained from the WT pig liver pre-Tx and at 30 min, 1, 2, 3, 4 and 5 h post-transplantation. Biopsies of genetically-engineered livers were obtained pre-Tx, 2 h after reperfusion and at necropsy (4–7 days after transplantation). Tissues were examined by light, confocal, and electron microscopy. All major native organs were also examined. The WT pig liver underwent hyperacute rejection. After genetically-engineered pig liver transplantation, hyperacute rejection did not occur. Survival was limited to 4–7 days due to repeated spontaneous bleeding in the liver and native organs (as a result of profound thrombocytopenia) which necessitated euthanasia. At 2 h, graft histology was largely normal. At necropsy, genetically-engineered pig livers showed hemorrhagic necrosis, platelet aggregation, platelet-fibrin thrombi, monocyte/macrophage margination mainly in liver sinusoids, and vascular endothelial cell hypertrophy, confirmed by confocal and electron microscopy. Immunohistochemistry showed minimal deposition of IgM, and almost absence of IgG, C3, C4d, C5b-9, and of a cellular infiltrate, suggesting that neither antibody- nor cell-mediated rejection played a major role.     

Genetic variability at the pig SLA complex in U.S. breeds of pigs1

Lymphocytes from 90 mature pigs and 548 growing pigs from 6.5 litters were screened with cytotoxic antibodies against swine lymphocyte antigens. Pigs included in the survey were from the Chester White, Duroc, Hampshire, Landrace and Yorkshire breeds. Significant differences between breeds were found in the frequency of the three haplotypes tested. Such differences may provide the genetic variability needed to determine associations between disease susceptibility and the pig's major histocompatibility gene complex.     

Schistosoma mansoni: parasitology and immunology of baboons vaccinated with irradiated cryopreserved schistosomula

Young, captivity-born male baboons (Papio cynocephalus) were vaccinated with γ-irradiated (500 Gy) cryopreserved Puerto Rican strain schistosomula of S. mansoni. Protection against heterologous, normal Kenyan Strain S. mansoni challenge infection was erratic and partial; and two putative correlates of immunity, reduced worm fecundity and change in worm location (anterior shift) were not observed. However, immunization of baboons with this vaccine resulted in a stimulated immune system. Both cellular and humoral anamnesis were demonstrable in vaccinated-challenged baboons. Schistosome infection-associated IgM hypergammaglobulinema was also greatly reduced in vaccinated-challenged baboons. On the other hand, IgG antibodies to adult, egg, and cercarial antigens were increased after challenge infection in preimmunized baboons. Vaccination appears to have resulted in a redirection of the immune system into anti-parasite channels, but this more specific immune response was insufficient to confer good protection against challenge infection in this experiment. The dampening effect of the vaccine on the hypergammaglobulinemia of schistosomiasis is another candidate for a possible “anti-pathogenesis” effect of irradiated schistosome larval vaccines, to be added to the reduced granuloma size already reported (Damian, Roberts, Powell, Clark, Lewis &; Stirewalt, 1984) to occur in these vaccinated baboons. Together, they could increase the potential benefit of live, irradiated vaccines for schistosomiasis. Human trials with vaccines only partially protective against challenge infections may therefore ultimately be warranted.     

Characterization of antibodies to the structural polypeptides of HGSAg: evidence for subtype-specific determinants.

Antisera prepared in guinea pigs to the structural polypeptides of HBAAg/adw and HBSAg/ayw were examined by a modified passive hemagglutination assay for antibodies to the subtype-specific d and y determinants. All of the isolated polypeptide fractions stimulated antibodies to both group specific and subtype-specific antigens of the native HBSAg particle from which they were derived. These data indicate that the polypeptides have similarities in their immunochemical structure.     

Trichinella spiralis: immunization of pigs with newborn larval antigens

The potential of crude Trichinella spiralis newborn larval antigens for pig immunization was investigated. A preparation of whole newborn larvae killed by freezing and thawing, and combined with Freund's complete adjuvant, induced a high level of protection against challenge (78%), compared to a 40% resistance level in pigs immunized with excretory secretory antigens of muscle larvae. Sera from pigs immunized with newborn larvae contained antibodies which bound to the surface of the newborn larvae, as determined by immunofluorescence. In a second trial, the freeze thawed newborn larvae preparation was compared with a soluble and insoluble fraction prepared by sonication of whole newborn larvae. Pigs receiving whole newborn larvae or the insoluble fraction developed strong immunity to challenge (88.2 and 85.5%, respectively); the soluble fraction was ineffective. Immunization with all preparations induced antibody to newborn larval antigens, but not to adult or muscle larvae excretory secretory antigens. Polyacrylamide gel electrophoresis of the soluble and insoluble fractions indicated that sonication was ineffective in solubilizing the larger molecular weight components. These results demonstrate that newborn larval antigens are highly protective in pigs, but that their further development as a vaccine will require more efficient procedures for antigen solubilization and large-scale production.     

Detection of noncapsular antigens in pneumococcus

Complex antigenic preparations obtained from noncapsular pneumococcal strains were used for the immunization of rabbits and guinea pigs. The injection of the preparations in complete Freund's adjuvant for 5 weeks led to the appearance of antibodies in their sera. The antibodies were detected by the double immunodiffusion test. The preparations obtained from different strains by extraction (with Triton X-100 or sodium deoxycholate) or by disintegration contain common pneumococcal antigens.     

Rejection of ticks from guinea pigs by anti-hapten-antibody-mediated degranulation of basophils at cutaneous basophil hypersensitivity sites: role of mediators other than histamine

Previous studies have established that recruitment of basophils to sites of tick feeding in guinea pigs is required to effect immune resistance. In the current study, actively sensitized guinea pigs treated three times daily with H-1 (mepyramine) and H-2 (cimetidine) histamine receptor antagonists, during the challenge tick infestation period, expressed normal resistance to Amblyomma americanum larvae. Similarly, naive guinea pigs treated with anti-histamines four times daily, beginning 7 days before transfer of immune serum and tick challenge and continuing through the tick infestation period, also expressed normal antibody-mediated resistance to A. americanum. These results indicated that histamine was not an important basophil mediator of the resistance response. Ticks allowed to feed on tissue rich in basophils that were induced by sensitization and subsequent local challenge with non-tick protein antigen, keyhole limpet hemocyanin (KLH), expressed normal yield. Ticks that fed on similar tissue rich in basophils induced by sensitization and challenge with KLH, in which the basophils expressed anti-picryl specificity due to systemic passive transfer of anti-picryl antibodies, were rejected when basophils were induced to degranulate by i.v. challenge with picryl antigen at 6 hr (29% rejection), 12 hr (18% rejection), 24 hr (22% rejection), and 48 hr (37% rejection) post-tick attachment. However, basophil degranulation at 18, 72 and 96 hr post-tick attachment had no adverse effect on tick feeding. These hosts were protected from systemic anaphylaxis by treatment with the anti-histamine mepyramine. Release of histamine occurred at tick feeding sites, but vasoactive effects were blocked by mepyramine treatment as evidenced by a lack of increased vascular permeability (bluing) at these sites compared with non-tick-infested tissues, or to cutaneous basophil hypersensitivity (CBH) sites of animals not protected with mepyramine. These results indicate that local recruitment and subsequent degranulation of basophils via immune mechanisms dependent on non-tick antigens can lead to tick rejection, and that basophil-derived mediators other than histamine are involved in this immune resistance response to A. americanum ticks. The identity of the crucial basophil mediator(s) is not known. The significant susceptibility of ticks to basophil-mediator release at 6 to 12 hr and 24 to 48 hr post-attachment coincides with the tick attaching and fast-feeding phases, respectively, suggesting that these phases of tick parasitism are particularly susceptible to the effect of basophil mediators other than histamine.     

Use of microcapsular leptospiral antigens for detecting specific antibodies

The sensitivity of microcapsular leptospiral antigens, produced by Japan Lyophilization Laboratory and intended for use in tests for the detection of antibodies to leptospires in the sera of experimentally immunized laboratory animals, were studied. The comparative study of the microcapsular agglutination (MCA) test and other serological tests, such as the microagglutination (MA) test and the indirect enzyme immunoassay (EIA), was made. The leptospiral antigens under study were found to actively react with serospecific and group-specific antibodies. In infected guinea pigs and rabbits specific antibodies could be detected from days 3-4 in the MCA test and only from days 5-7 in the MA test. The average antibody level determined by titration in the MCA test was 3.3 times higher and in indirect EIA, 4.3 times higher than that determined by titration in the MA test. These data make it possible to recommend the use of microcapsular leptospiral antigens for the early diagnosis of leptospirosis.     

组织相容性Ⅰ类相关链A位点基因多态性在预判肾移植物排斥中的意义

目的 研究组织相容性Ⅰ类相关链A位点(MICA)基因多态性和抗MICA特异性抗体在肾移植排斥反应发生中的意义.方法 采用免疫磁珠液相芯片技术对40例肾移植患者在移植术前和移植术后1个月、3个月、6个月、1年和2年动态检测抗MICA抗体的特异性和阳性分值的变化,同时采用SSOP方法分析16对肾移植供受者的MICA基因分型...