RESUMEN
The activation of the endothelial surface in xenografts is still a poorly understood process and the consequences are unpredictable. The role of Ca2+ -messaging during the activation of endothelial cells is well recognized and routinely measured by synthetic Ca2+ -sensitive fluorophors. However, these compounds require fresh loading immediately before each experiment and in particular when grown in state-of-the-art 3D cell culture systems, endothelial cells are difficult to access with such sensors. Therefore, we developed transgenic pigs expressing a Ca2+ -sensitive protein and examined its principal characteristics. Primary transgenic endothelial cells stimulated by ATP showed a definite and short influx of Ca2+ into the cytosol, whereas exposure to human serum resulted in a more intense and sustained response. Surprisingly, not all endothelial cells reacted identically to a stimulus, rather activation took place in adjacent cells in a timely decelerated way and with distinct intensities. This effect was again more pronounced when cells were stimulated with human serum. Finally, we show clear evidence that antibody binding alone significantly activated endothelial cells, whereas antibody depletion dramatically reduced the stimulatory potential of serum. Transgenic porcine endothelial cells expressing a Ca2+ -sensor represent an interesting tool to dissect factors inducing activation of porcine endothelial cells after exposure to human blood or serum.
Asunto(s)
Señalización del Calcio , Células Endoteliales , Suero , Animales , Animales Modificados Genéticamente , Calcio , Células Cultivadas , Células Endoteliales/citología , Humanos , Porcinos , Trasplante HeterólogoRESUMEN
BACKGROUND: Beta-toxin (CPB) is the major virulence factor of Clostridium perfringens type C, causing hemorrhagic enteritis in newborn pigs but also other animals and humans. Vaccines containing inactivated CPB are known to induce protective antibody titers in sow colostrum and neutralization of the CPB activity is thought to be essential for protective immunity in newborn piglets. However, no method is available to quantify the neutralizing effect of vaccine-induced antibody titers in pigs. (2) Methods: We developed a novel assay for the quantification of neutralizing anti-CPB antibodies. Sera and colostrum of sows immunized with a commercial C. perfringens type A and C vaccine was used to determine neutralizing effects on CPB induced cytotoxicity in endothelial cells. Antibody titers of sows and their piglets were determined and compared to results obtained by an ELISA. (3) Results: Vaccinated sows developed neutralizing antibodies against CPB in serum and colostrum. Multiparous sows developed higher serum and colostrum antibody titers after booster vaccinations than uniparous sows. The antibody titers of sows and those of their piglets correlated highly. Piglets from vaccinated sows were protected against intraperitoneal challenge with C. perfringens type C supernatant. (4) Conclusions: The test based on primary porcine endothelial cells quantifies neutralizing antibody activity in serum and colostrum of vaccinated sows and could be used to reduce and refine animal experimentation during vaccine development.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Toxinas Bacterianas/inmunología , Vacunas Bacterianas/administración & dosificación , Calostro/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Toxinas Bacterianas/genética , Bioensayo , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Femenino , Proteínas Recombinantes/farmacología , Porcinos , VacunaciónRESUMEN
PURPOSE: Direct recognition of porcine MHC proteins by human T cells is an impediment to successful xenotransplantation. Therefore, reducing human T cell response initiated by the interaction between TCR/CD8 cell and MHC class I on pig endothelial cell may be beneficial in successful pig- to-human xenotransplantation. METHODS: We examined MHC expression on porcine endothelial cell line, MYP30 cells in the absence or presence of IFN-g by FACS analysis. We introduced human cytomegalovirus (hCMV) US genes, which are known to be able to reduce MHC class I expression on the cell surface after infection, into MYP30 cells in order to test the feasibility of modifying these cells to reduced MHC class I antigens by the introduction of hCMV US genes such as US2, 3, 6 or 11. RESULTS: MHC class I expressions in MYP30 cells were dramatically induced by IFN-gamma treatment. FACS analysis showed that cells transfected with the hCMV US2, 3, 6 or 11 genes exhibited 30~40% of MHC class I expression compared with mock-transfected cells. We next established stable cell lines expressing US6 gene, which had been found to exert best down-regulation effect on MHC class I expression. Stable cell line expressing US6 gene products exhibited more than 10% reduced expression level of the MHC class I compared with transiently transfected cells. CONCLUSION: Although the further analysis of the cytotoxicities of T and NK cells on the hCMV US gene transfected cells are needed to clarify the feasibility of their application, these results suggest that virus stealth technology can be exploited for xenotransplantation.