Gang Mai

Improved survival of fulminant liver failure by transplantation of microencapsulated cryopreserved porcine hepatocytes in mice.

The aim of this study was to establish hepatocyte isolation in pigs, and to evaluate function of isolated hepatocytes after encapsulation, cryopreservation, and transplantation (Tx) in a mouse model of fulminant liver failure (FLF). After isolation, porcine hepatocytes were microencapsulated with alginate-poly-L-Lysine-alginate membranes and cryopreserved. In vitro, albumin production of free and encapsulated hepatocytes were measured by enzyme linked-immunoadsorbent assay. In vivo, encapsulated hepatocytes were transplanted into different groups of mice with FLF and the following experimental groups were performed: group 1, Tx of empty capsules; group 2, Tx of free primary porcine hepatocytes; group 3, Tx of fresh encapsulated porcine hepatocytes; group 4, Tx of cryopreserved encapsulated porcine hepatocytes. In vitro, fresh or cryopreserved encapsulated porcine hepatocytes showed a continuous decreasing metabolic function over 1 week (albumin and urea synthesis, drug catabolism). In vivo, groups 1 and 2 showed similar survival (18% and 25%, respectively, p > 0.05). In groups 3 and 4, Tx of fresh or cryopreserved encapsulated porcine hepatocytes significantly increased survival rate to 75% and 68%, respectively (p < 0.05). Primary porcine hepatocytes maintained metabolic functions after encapsulation and cryopreservation. In mice with FLF, Tx of encapsulated xenogeneic hepatocytes significantly improved survival. These results indicate that porcine hepatocytes can successfully be isolated, encapsulated, stored using cryopreservation, and transplanted into xenogeneic recipients with liver failure and sustain liver metabolic functions.

Treatment of fulminant liver failure by transplantation of microencapsulated primary or immortalized xenogeneic hepatocytes

Abstract:  Background:  The aim of this study was to evaluate in vitro and in vivo functions of isolated hepatocytes after immortalization, cryopreservation, encapsulation and xenotransplantation into mice with fulminant liver failure (FLF).

Transplantation of Encapsulated Hepatocytes During Acute Liver Failure Improves Survival Without Stimulating Native Liver Regeneration

The aim of this study was to evaluate the effects of intraperitoneal transplantation of encapsulated human hepatocytes on liver metabolism and regeneration of mice with acute liver failure. Primary human hepatocytes were immortalized using lentiviral vectors coding for antiapoptotic genes and microencapsulated using alginate-polylysine polymers. In vitro, immortalized human hepatocytes showed low, but stable, synthetic and catabolitic functions over time, when compared to primary hepatocytes. In vivo, mice with acute liver failure and transplanted with encapsulated immortalized human hepatocytes had a significantly improved survival and biochemical profile, compared to mice transplanted with empty capsules. Serum levels of cytokines implicated in liver regeneration were lower in mice transplanted with hepatocytes compared to mice receiving empty capsules. This decrease was significant for IL-6 and HGF at 3 h. Measurement of liver regeneration showed no significant difference between mice transplanted with hepatocytes compared to control groups. Intraperitoneal transplantation of encapsulated immortalized hepatocytes significantly improved survival of mice with acute liver failure by providing metabolic support and without modifying liver regeneration. The lower levels of cytokines implicated in liver regeneration suggest that the metabolic support provided by the encapsulated hepatocytes reduced the inflammatory stress on the liver and herein decreased the regenerative trigger on residual hepatocytes. These data emphasize that metabolic function and regeneration of hepatocytes are two distinct aspects that need to be studied and approached separately during acute liver failure.

Anti-CD154 mAb and Rapamycin Induce T Regulatory Cell Mediated Tolerance in Rat-to-Mouse Islet Transplantation

Background Anti-CD154 (MR1) monoclonal antibody (mAb) and rapamycin (RAPA) treatment both improve survival of rat-to-mouse islet xenograft. The present study investigated the effect of combined RAPA/MR1 treatment on rat-to-mouse islet xenograft survival and analyzed the role of CD4+CD25+Foxp3+ T regulatory cells (Treg) in the induction and maintenance of the ensuing tolerance. Methodology/Principal Findings C57BL/6 mice were treated with MR1/RAPA and received additional monoclonal anti-IL2 mAb or anti CD25 mAb either early (0–28 d) or late (100–128 d) post-transplantation. Treg were characterised in the blood, spleen, draining lymph nodes and within the graft of tolerant and rejecting mice by flow cytometry and immunohistochemistry. Fourteen days of RAPA/MR1 combination therapy allowed indefinite islet graft survival in >80% of the mice. Additional administration of anti-IL-2 mAb or depleting anti-CD25 mAb at the time of transplantation resulted in rejection (100% and 89% respectively), whereas administration at 100 days post transplantation lead to lower rejection rates (25% and 40% respectively). Tolerant mice showed an increase of Treg within the graft and in draining lymph nodes early post transplantation, whereas 100 days post transplantation no significant increase of Treg was observed. Rejecting mice showed a transient increase of Treg in the xenograft and secondary lymphoid organs, which disappeared within 7 days after rejection. Conclusions/Significances These results suggest a critical role for Treg in the induction phase of tolerance early after islet xenotransplantation. These encouraging data support the need of developing further Treg therapy for overcoming the species barrier in xenotransplantation.

Macrophage depletion prolongs discordant but not concordant islet xenograft survival

Background. T cells and macrophages play a major role in the rejection of xenografted islets. Depending on the phylogenetic disparity, direct or indirect antigen presentation is predominant. The aim of this study was to analyze in vitro the predominance of direct or indirect presentation by depleting antigen-presenting cells in concordant and discordant xenogeneic combinations. In vivo, we analyzed the effect of macrophage depletion on concordant and discordant islet xenograft survival to assess in which combination this strategy can be used as therapeutic tool. Materials and Methods. In vitro, we performed mouse anti-rat and anti-human mixed lymphocyte reactions (MLR) after depletion of responder or stimulator antigen-presenting cells by magnetic sorting. In vivo, streptozotocin-induced diabetic C57BL/6 mice were treated by gadolinium chloride (GdCl) to deplete macrophages, and rat or human islets were transplanted under the kidney capsule. Islet function was followed by glycemia, and xenografts were analyzed at regular intervals for histology and immunohistochemistry. Results. Mouse anti-rat MLR showed a predominant direct antigen presentation pathway, whereas in mouse anti-human MLR, direct and indirect pathways were similarly involved. Survival of rat islets was not modified by GdCl therapy. In contrast, survival of human islets was significantly prolonged in GdCl-treated mice. Macrophage infiltration was decreased in concordant and discordant GdCl-treated xenografts at day 4 compared with controls. At day 15, macrophage, CD4+, and CD8+ cell infiltration was similar in all groups. Conclusions. Our results indicate that direct antigen presentation is dominant in the rejection mechanism of concordant islet xenografts and cannot be influenced by host macrophage depletion. Both direct and indirect antigen presentation are involved in rejection of discordant xenogeneic islets. Macrophage depletion should only be considered as therapeutic tool for discordant islet xenotransplantation.

Anti‐CD154 mAb Treatment But Not Recipient CD154 Deficiency Leads to Long‐Term Survival of Xenogeneic Islet Grafts

The aim of the study was to evaluate the role of recipient CD40 and CD154 in the rejection process of concordant and discordant islets xenotransplantation (Tx). Diabetic C57BL/6 mice, CD40‐ or CD154‐knockout (KO) and complement C3‐deficient (C3−/−) mice were transplanted with either rat or human islets. Group 1, C57BL/6 Tx without therapy; Group 2, C57BL/6 Tx with anti‐CD154 monoclonal antibody (mAb) (MR1) therapy; Group 3, CD40‐KO; and Group 4, CD154‐KO Tx without therapy; Group 5, C3−/− Tx without therapy and Group 6, C3−/−Tx with MR1 therapy. Mixed lymphocyte reactions (MLR) were performed. Compared to Group 1, MR1 induced long‐term survival of xenografts in Group 2, but not in Group 6, survival of islets was not prolonged significantly in Groups 3 and 4. MLR responses in Group 2 were reduced approximately 50% compared to Group 1. In Groups 3, 4 and 6, MLR responses were not modified by the absence of CD40 or CD154 molecules, or MR1 and were similar to Group 1. Improved graft survival and reduced MLR responses in Group 2, but not in Group 6, could be explained by specific targeting of activated T cells with inactivation by complement‐ or cellular‐mediated mechanisms. Rejection of xenografts and strong MLR responses in Groups 3 and 4 are possible through efficient activation of alternate pathways of costimulation.

Blockade of the PD‐1/PD‐1L pathway reverses the protective effect of anti‐CD40L therapy in a rat to mouse concordant islet xenotransplantation model

Abstract: Background: We have previously demonstrated that costimulatory blockade with anti‐CD40L monoclonal antibody (mAb) prolongs the survival of non‐vascularized concordant rat to mouse islet xenografts. Here, we examine whether signaling through the PD‐1/PD‐1L pathway is required for the anti‐CD40L therapy to prolong concordant islet graft survival using a novel anti‐murine PD‐1 mAb (clone 4F10).

Anti-cd25 Mab, Anti-il2 Mab And Il2 Block Tolerance Induction Through Anti-cd154 Mab And Rapamycin In Xenogeneic Islet Transplantation

BACKGROUND We have used anti-CD154 monoclonal antibody (mAb; MR1) and rapamycin (rapa) to induce tolerance to islet xenografts. The aim of this study was to investigate whether classical anergy and/or regulation by interleukin (IL)2-dependent CD25+ T regulatory cells played roles in the induction and maintenance of tolerance in this model. METHODS Streptozotocin-induced diabetic mice were transplanted with rat islets. We performed the following groups: control group, islet transplantation without therapy; rapamycin group, 0.2 mg/kg by oral gavage on days 0, 1, 2, and every other day to day 14; anti-CD154 mAb (MR1) group, 0.5 mg intraperitoneally on days 0, 2, and 4; combination therapy group with rapa and MR1. We then administered in addition to the combination therapy with early (from days 0 to 14 [for IL2] or to 28 [for anti-IL2 mAb and anti-CD25 mAb] post-transplantation) or late (from days 100 to 114 [for IL2] or to 128 [for anti-IL2 mAb and anti-CD25 mAb] posttransplantation) recombinant IL2 (2000 U, intraperitoneally twice a day), a neutralizing anti-IL2 mAb (S4B6-1, 0.3 mg intraperitoneally twice weekly), and a depleting anti-CD25 mAb (PC61, 0.3 mg intraperitoneally twice weekly), respectively. Histology was performed at time of rejection. RESULTS Rapa and MR1 therapy alone significantly prolonged xenograft survival compared to the control group: median graft survival was 34 days versus 17 days (P<.05) and 98 days versus 17 days (P<.05), respectively, but rejection still occurred. Combination therapy with MR1 and rapa allowed indefinite graft survival (median graft survival [MGS]>200 days, P<.001). When exogenous IL2 was administered early with MR1 and rapa, rapid rejection developed in 18 of 18 mice (MGS 7 days), whereas when IL2 was given late, only 3 of 10 developed rejection. Early administration of anti-IL2 mAb led to rejection in 10 of 10 mice (MGS 42 days), whereas late administration led to rejection in only one of four mice. Early administration of anti-CD25 mAb led to rejection in eight of nine mice (MGS 49 days), whereas late administration led to rejection in only three of seven mice. CONCLUSIONS Rapa and MR1 allowed indefinite graft survival of islet xenografts. Classical anergy and regulation by IL2-dependent CD25+ T regulatory cells were critical in the induction of tolerance in the immediate posttransplantation period and less important for maintenance of tolerance.