Search

Objective: To ascertain the safety and function of the transplantation of neonatal pig islets (NPIs) for diabetic patients., Methods: NPIs were injected into the hepatic artery of 22 patients. After the transplantation, the patients were treated with a multiple drug immunosuppressive regimens. The first 14 patients were treated with cyclosporine (CsA), mycophenolate mofetil (MMF) and prednisolon, and porcine C-peptide was not monitored, the following 2 patients were given cyklosporin and MMF only, while the next 6 patients were given a quadruple drug regimen consisting of OKT3, takrolimus, sirolimus and prednisolon. The blood glucose levels,exogenous insulin requirement,HbA1c, porcine endogenous retrovirus (PERV) and liver function were assessed before and after NPI transplantation. The serum porcine C peptide were monitored in last 8 patients., Results: The first 14 patients required less insulin and the HbA1c dropped after the transplantation. In the 2 subsequent patients, the metabolic parameters remained unchanged and monitor of porcine C-peptide was negative. Insulin requirements were reduced in all 6 patients, and HbA1c was normalized 3 months after the transplantation. Significant levels of porcine C-peptide were detected in the patient serum. Two of the patients were given a second injection of NPIs, and one of them became insulin independent for 7 d. No serious adverse events were noted after the transplantation. There was no evidence of PERV transmission. Six out of the 22 patients were followed up for 4-6 years after the NPIs injection, immunosuppressive treatment was stopped 1 year after the transplantation. The patients started to take insulin at the time of follow up. Four patients restricted the intake of sugar, while the other 2 did not. One patient had ketoacidosis twice and slight diabetic retinopathy, and another patient had ketoacidosis induced by acute gastroenteritis. The remaining 4 patients did not have any complications. Assays for PERV were again negative., Conclusion: Xenogenic islets can survive and function in the human body. No serious adverse events are noted.

Objective: To determine the genetic modification on neonatal porcine islet cell clusters (NICC) by small interfering RNA (siRNA)-mediated tissue factor (TF) knockdown in vitro., Methods: Porcine NICC were transfected with 5 pairs of designed siRNA respectively or in different combinations with lipofectamine 2000. Transfected NICC were analyzed for TF gene by real-time PCR to select the siRNA which worked best. Meanwhile, the viability of NICC after the TF siRNA transfection was examined by FACS. The efficiency of TF gene and protein suppression was measured by real-time PCR and and FACS respectively., Results: Real-time PCR and FACS showed that a 60% reduction in the TF gene expression and a 50% reduction in the protien level of TF on NICC were achieved by transfecting 3 pairs of selected siRNA. The siRNA transfection had no significant effect on the viability of NICC which was analyzed by FACS., Conclusion: The expression of TF on porcine NICC is efficiently suppressed by 3 pairs of designed siRNA in vitro.

In order to investigate the mechanism by which CD4+ T cells and macrophages interact in the xenogeneic immune response, murine CD4+ T cells and macrophages were used as responder cells in culture with irradiated fetal pig spleen cells (FPSC) as pig xenogeneic stimulators. In this in vitro model, murine CD4+ T cells and macrophages were cultured individually, or together with FPSC. In addition, mouse CD4+ T cells were also cultured with autologous macrophages which were previously stimulated by FPSC. The cultured murine cells were analyzed for expression of CD4+ T cell and macrophage activation markers (cell surface markers and cytokines) as well as cytokine production. CD4+ T cells and macrophages cultured alone or together without FPSC showed unchanged low levels of expression of activation markers. Coculture of macrophages with FPSC and in the absence of CD4+ T cells induced increased expression levels of all the activation markers examined except B7.2 and ICAM-1. Addition of CD4+ T cells to the coculture further enhanced this up-regulation. Coculture of CD4+ T cells with FPSC-stimulated macrophages, but not naive macrophages, or FPSC alone, resulted in significantly increased numbers of CD4+ T cells coexpressing their activation markers, especially IFN-gamma and CD40L, and this expression was enhanced further by including FPSC in the coculture. The activation of both CD4+ T cells and macrophages in their coculture with FPSC was suppressed by neutralizing IFN-gamma but not IL-4. Our results demonstrated that interaction of CD4+ T cells and autologous macrophages was required for their optimal activation in response to pig xenogeneic stimulation. The mechanisms involved included cell-cell and/or cytokine interactions, and in particular IFN-gamma mediated communication was involved. Macrophages activated by pig cells in the absence of CD4+ T cells were able to activate naive CD4+ T cells, thus providing an important communication pathway between innate immune activation and a T cell mediated response in xenograft rejection.

Background: In this study, the mechanisms by which CD4+ T cells interact with the innate immune system in xenograft rejection were investigated., Methods: Fetal pig pancreas (FPP) grafts were transplanted into female SCID mice. The FPP recipient SCID mice were reconstituted with exogenous leukocytes obtained from male BALB/c mice., Results: Although nonreconstituted SCID recipients or recipients reconstituted with CD4+ T cell-depleted leukocytes showed indefinite FPP graft survival with very few macrophages infiltrating their grafts, reconstitution of SCID recipients with as few as 2x10(5) CD4+ T cells was sufficient to induce rapid xenograft rejection. CD4+ T cells secreted interferon-gamma but not interleukin-4 and initiated the activation and accumulation of macrophages and natural killer cells, that were responsible for the rapid graft destruction. Suppression of interferon-gamma prolonged graft survival and suppressed the macrophages and natural killer cell accumulation and activation., Conclusions: These results demonstrate that CD4+ T cell-dependent cellular xenograft rejection was a result of macrophage and natural killer cell accumulation and activation, but was not mediated by eosinophils. Consistent with this was the finding that interferon-gamma but not interleukin-4 was in part responsible for mediating this effect.