RESUMEN
BACKGROUND: αGal-deficient xenografts are protected from hyperacute rejection during xenotransplantation but are still rejected more rapidly than allografts. Despite studies showing the roles of non-Gal antibodies and αß T cells in xenograft rejection, the involvement of γδ T cells in xenograft rejection has been limitedly investigated. METHODS: Six male cynomolgus monkeys were transplanted with porcine vessel xenografts from wild-type (n = 3) or GGTA1 knockout (n = 3) pigs. We measured the proportions and T cell receptor (TCR) repertoires of blood γδ T cells before and after xenotransplant. Grafted porcine vessel-infiltrating immune cells were visualized at the end of experiments. RESULTS: Blood γδ T cells expanded and infiltrated into the graft vessel adventitia following xenotransplantation of α-Gal-deficient pig blood vessels. Pre- and post-transplant analysis of γδ TCR repertoire revealed a transition in δ chain usage post-transplantation, with the expansion of several clonotypes of δ1, δ3, or δ7 chains. Furthermore, the distinctions between pre- and post-transplant δ chain usages were more prominent than those observed for γ chain usages. CONCLUSION: γδ TCR repertoire was significantly altered by xenotransplantation, suggesting the role of γδ T cells in sustained xenoreactive immune responses.
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Primates , Subgrupos de Linfocitos T , Animales , Masculino , Xenoinjertos , Receptores de Antígenos de Linfocitos T , Porcinos , Trasplante Heterólogo , Macaca fascicularisRESUMEN
The purpose of this study was to compare the efficiency of the production of cloned transgenic Yucatan miniature pigs (YMPs) using two recipient breeds, i.e., YMPs and domestic pigs (DPs), under various embryo transfer conditions. We initially assessed the in vitro developmental competence of embryos obtained via somatic cell nuclear transfer (SCNT) from three different transgenic donor cells. No difference was observed among the three groups regarding developmental competence. Furthermore, the cloning efficiency remained consistent among the three groups after the transfer of the SCNT embryos to each surrogate mother. Subsequently, to compare the efficiency of the production of cloned transgenic YMPs between the two recipient breeds using varying parameters, including ovulation status (preovulation and postovulation), duration of in vitro culture (IVC) (incubated within 24 h and 24-48 h), and the number of transferred SCNT embryos (less than and more than 300), we assessed the pregnancy rates, delivery rates, mean offspring counts, and cloning efficiency. Regarding the ovulation status, YMPs exhibited higher pregnancy rates, delivery rates, and cloning efficiency compared with DPs in both statuses. Moreover, the pregnancy rates, delivery rates, and cloning efficiency were affected by the ovulation status in DPs, but not in YMPs. The comparison of IVC duration between groups revealed that YMPs had higher pregnancy rates vs. DPs in both conditions. SCNT embryos cultured for 24-48 h in YMPs yielded higher delivery rates and cloning efficiency compared with those cultured for less than 24 h in DPs. Finally, the analysis based on the number of transferred SCNT embryos showed that both the pregnancy and delivery rates were higher in YMPs vs. DPs. However, the highest average number of offspring was obtained when more than 300 SCNT embryos were transferred into DPs, whereas the cloning efficiency was higher in YMPs vs. DPs. Our results suggest that YMPs are more suitable recipients than are DPs under various conditions for the production of cloned transgenic YMPs.
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Clonación de Organismos , Técnicas de Transferencia Nuclear , Embarazo , Femenino , Porcinos/genética , Animales , Porcinos Enanos/genética , Animales Modificados Genéticamente , Clonación de Organismos/veterinaria , Clonación de Organismos/métodos , Técnicas de Transferencia Nuclear/veterinaria , Transferencia de Embrión/veterinaria , Transferencia de Embrión/métodosRESUMEN
Although supplementation with docosahexaenoic acid (DHA) during porcine oocyte IVM is well-established, the available data are limited due to the lack of consistency. Moreover, to our knowledge, the anti-oxidant effects of DHA on porcine oocytes have not been reported. Hence, this study aimed to examine the effects of DHA supplementation on the regulation of energy metabolism during porcine oocyte maturation to improve oocyte maturation and embryonic development. By supplementing the IVM medium with various DHA concentrations, 25 µM DHA was identified as the optimal concentration which improved intraoocyte glutathione content and enhanced embryonic development after parthenogenesis. Compared to embryos derived from the control group, those derived from SCNT or IVF showed significantly improved blastocyst formation upon DHA supplementation during IVM. In addition, various transcription factors associated with oocyte development and apoptosis in mature oocytes were beneficially regulated in the DHA-treated oocytes. Moreover, DHA improved the AMP-activated protein kinase (AMPK)-regulatory ability of porcine oocytes and ameliorated nuclear maturation and embryonic development, which were decreased by artificially downregulating AMPK. To our knowledge, this is the first study to examine the effects of DHA as an AMPK regulator on oocyte maturation and embryo development in pigs. Furthermore, DHA addition to the IVM medium upregulated the relative expression of genes associated with mitochondrial potential and lipid metabolism. Therefore, the membrane potential of mitochondria (evaluated based on the JC-1 aggregate/JC-1 monomer ratio) and the levels of fatty acids and lipid droplets in matured oocytes increased, resulting in increased ATP synthesis. In conclusion, the DHA treatment of porcine oocytes with 25 µM DHA during IVM enhances the homeostasis of energy metabolism by improving mitochondrial function and lipid metabolism, leading to improved quality of matured oocytes and enhanced embryonic developmental potential of in vitro produced (IVP) embryos. Thus, 25 µM DHA supplementation could serve as a tool for improving the quality of IVP embryos. The study findings provide a basis for further research on improving the production efficiency of cloned animals by securing high-quality matured oocytes and enhancing energy metabolism in mammalian oocytes, including those of pigs.
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Ácidos Docosahexaenoicos , Desarrollo Embrionario , Metabolismo Energético , Técnicas de Maduración In Vitro de los Oocitos , Oocitos , Animales , Ácidos Docosahexaenoicos/farmacología , Ácidos Docosahexaenoicos/administración & dosificación , Oocitos/efectos de los fármacos , Porcinos/embriología , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Metabolismo Energético/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Homeostasis/efectos de los fármacos , FemeninoRESUMEN
Introduction: Decreasing rates of blood donation and close margins between blood supply and demand pose challenges in healthcare. Genetically engineered pig red blood cells (pRBCs) have been explored as alternatives to human RBCs for transfusion, and triple-gene knockout (TKO) modification improves the compatibility of pRBCs with human blood in vitro. In this study, we assessed the efficacy and risks of transfusing wild-type (WT)- and TKO-pRBCs into nonhuman primates (NHPs). Methods: Blood from O-type WT and TKO pigs was processed to produce pRBCs for transfusion, which were transfused or not into NHPs (n=4 per group: WT, TKO, and control) after 25% total blood volume withdrawal: their biological responses were compared. Hematological, biochemical, and immunological parameters were measured before, immediately after, and at intervals following transfusion. Two months later, a second transfusion was performed in three NHPs of the transfusion group. Results: Transfusion of both WT- and TKO-pRBCs significantly improved RBC counts, hematocrit, and hemoglobin levels up to the first day post-transfusion, compared to the controls. The transfusion groups showed instant complement activation and rapid elicitation of anti-pig antibodies, as well as elevated liver enzyme and bilirubin levels post-transfusion. Despite the higher agglutination titers with WT-pRBCs in the pre-transfusion crossmatch, the differences between the WT and TKO groups were not remarkable except for less impairment of liver function in the TKO group. After the second transfusion, more pronounced adverse responses without any hematological gain were observed. Conclusions: WT- and TKO-pRBC transfusions effectively increased hematologic parameters on the first day, with rapid clearance from circulation thereafter. However, pRBC transfusion triggers strong antibody responses, limiting the benefits of the pRBC transfusion and increasing the risk of adverse reactions.
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Transfusión de Eritrocitos , Eritrocitos , Técnicas de Inactivación de Genes , Animales , Transfusión de Eritrocitos/efectos adversos , Transfusión de Eritrocitos/métodos , Porcinos , Eritrocitos/inmunología , Eritrocitos/metabolismo , Animales Modificados Genéticamente , Hemoglobinas/metabolismo , Galactosiltransferasas/genética , Galactosiltransferasas/deficiencia , Hematócrito , Femenino , Masculino , PrimatesRESUMEN
BACKGROUND: Xenotransplantation, particularly when involving pig donors, presents challenges related to the transmission of porcine cytomegalovirus (pCMV) and its potential impact on recipient outcomes. This study aimed to investigate the relationship between pCMV positivity in both donors and recipients and the survival time of cynomolgus monkey recipients after xenogeneic kidney transplantation. METHODS: We conducted 20 cynomolgus xenotransplants using 18 transgenic pigs. On the surgery day, donor pig blood was sampled, and DNA was extracted from serum and peripheral blood mononuclear cells. Recipient DNA extraction followed the same protocol from pre-transplantation to post-transplantation. Porcine cytomegalovirus detection used real-time polymerase chain reaction (real-time PCR) with the ViroReal kit, achieving a sensitivity of 50 copies/reaction. A Ct value of 37.0 was the pCMV positivity threshold. RESULTS: Of 20 cynomolgus recipients, when donors tested negative for pCMV, recipients also showed negative results in 9 cases. In 4 cases where donors were negative, recipients tested positive. All 5 cases with pCMV-positive donors resulted in positive assessments for recipients. Detection of donor pCMV correlated with shorter recipient survival. Continuous recipient positivity during observation correlated with shorter survival, whereas transient detection showed no significant change in survival rates. However, donor pig phenotypes and transplantation protocols did not significantly impact survival. CONCLUSION: The detection of pCMV in both donors and recipients plays a crucial role in xenotransplantation outcomes. These findings suggest the importance of monitoring and managing pCMV in xenotransplantation to enhance long-term outcomes.