Effect of nicotinamide on early graft failure following intraportal islet transplantation

Intraportal islet transplantation (IPIT) may potentially cure Type 1 diabetes mellitus; however, graft failure in the early post-transplantation period presents a major obstacle. In this study, we tested the ability of nicotinamide to prevent early islet destruction in a syngeneic mouse model. Mice (C57BL/6) with chemically-induced diabetes received intraportal transplants of syngeneic islet tissue in various doses. Islets were cultured for 24 h in medium with or without 10 mM nicotinamide supplementation. Following IPIT, islet function was confirmed by an intraperitoneal glucose tolerance test (IPGTT) and hepatectomy. The effects of nicotinamide were evaluated by blood glucose concentration, serum monocyte chemoattractant protein-1 (MCP-1) concentration, and immunohistology at 3 h and 24 h after IPIT. Among the various islet doses, an infusion of 300 syngeneic islets treated with nicotinamide exhibited the greatest differences in glucose tolerance between recipients of treated and untreated (i.e., control) islets. One day after 300 islet equivalent (IEQ) transplantation, islets treated with nicotinamide were better granulated than the untreated islets (P = 0.01), and the recipients displayed a slight decrease in serum MCP-1 concentration, as compared to controls. After 15 days, recipients of nicotinamide-pretreated islets showed higher levels of graft function (as measured by IPGTT) than controls. The pretreatment also prolonged graft survival (> 100 days) and function; these were confirmed by partial hepatectomy, which led to the recurrence of diabetes. Pretreatment of islet grafts with nicotinamide may prevent their deterioration on the early period following IPIT in a syngeneic mouse model.

Prolonged survival of islet allografts in mice treated with rosmarinic acid and anti-CD154 antibody

Pancreatic islet transplantation can correct the abnormal glucose metabolism of Type 1 diabetes. Although immunosuppressants greatly reduce the acute rejection rate in transplant patients, the long-term side effects can be debilitating. Therefore, researchers are seeking to develop new immunosuppressive regimens that induce maximal levels of immunosuppression with minor side effects. Rosmarinic acid (Ros A) is a secondary metabolite of certain herbs and has multiple biological activities, including anti-inflammatory effects. Here, we have investigated whether treatment of mice with a combination of Ros A and anti-CD154 monoclonal antibody (MR1) improves islet allograft survival in a murine model. After transplantation, the mice were treated with either Ros A, MR1, or both (the "double" treatment). Allograft survival was prolonged in the double-treated animals compared to animals that received only Ros A or MR1. As is the case with the single-treated animals at 15 days after transplantation, the double-treated recipients did not display a significant decrease in the expression of cytokines or the population of activated T cells. Infiltrating CD3+ T cells were reduced in the MR1- or double therapy relative to control or RosA group. However, at the same time point, double-treated graft showed fewer apoptotic cells and increased expression of insulin and glucagons, compared to the single-treatment groups. Furthermore, long-term (>150 days) allografts that were received with double therapy exhibited larger islet clusters and contained more insulin- and glucagon-positive cells, relative to the MR1-treated grafts. In conclusion, treatment with both Ros A and MR1 has a synergistic effect in murine islet allotransplantation.

Reconstitution of Human Immune Cells with Co-transplantation of Fetal Liver/Thymus Tissues and Cultured Umbilical Cord Blood-derived Hematopoietic Stem Cells in Rag2(-/-)gamma(c)(-/-) Mice

PURPOSE: Many researchers have tried to develop animal models that mimic the human immune system, e.g. a humanized mouse model, to improve the engraftment of hematopoietic stem cells and develop human immune cells in an animal model. This study evaluated the feasibility of the cultured human umbilical cord blood (hUCB)-derived CD34(+) cells for cell expansion, in Rag2(-/-)gamma(c)(-/-) mice, and establish co-transplantation with human fetal thymus/liver tissue (Thy/Liv) under the kidney capsule. METHODS: Co-transplantation of hUCB-derived CD34(+) cells with Thy/Liv was performed. The hUCB-derived CD34(+) cells were prepared by freshly thawing (G1) and culturing for 7 days with two types of cytokine combinations (G2, G3). The CD45(+) cell populations were measured at 6, 8, 10 and 16 weeks in the peripheral blood. The splenocytes were cultured with mitogenic stimuli (PHA -L or IL-2) at 20 weeks post- transplantation, and the proliferation of human immune cells was evaluated. RESULTS: There were no significant differences in the human CD45(+) cell populations at 6, 8, 10 and 16 weeks post-transplantation between the groups. In the cultured splenocytes at 20 weeks post-transplant with PHA-L or IL-2, there was remarkable expansion of CD3(+) cells in the three groups. Although no CD19(+) cells were detected in the spleen, human Ig G was detected in the sera of these mice. CONCLUSION: The cultured and expanded hUCB-derived cells with cytokine combinations might be a feasible cell source in humanized mouse modeling. In addition, human immune cells can be reconstituted from the co-transplantation of Thy/Liv and cultured hUCB-derived CD34(+) cells.

Two-signal blockade with anti-CD45RB and anti-CD154 monoclonal antibodies inhibits graft rejection via CD4-dependent mechanisms in allogeneic skin transplantation

Blockade of signal 1 or 2 for T-cell activation by the use of anti-CD45RB and anti-CD154 monoclonal antibodies (mAb) (two-signal blockade) has been proven effective in preventing or delaying graft rejection. However, the mechanisms of its immunomodulatory effects are clearly unknown and the present studies were performed to determine how the two-signal blockade modulate allogeneic immune responses, especially T-cell mediated cellular immunity, in a murine skin allograft model. We now report on the profound inhibition of alloreactive T cells by two-signal blockade via CD4-dependent mechanisms. C57BL/6 mice of BALB/c skin allograft were treated with anti-CD45RB, anti-CD154, CTLA4-Ig, or their combinations. For depletion of CD4 or CD8 T cells, the recipients received CD4-depleting or CD8-depleting mAb. We confirmed that survival of skin allograft was markedly prolongated in the two-signal blockade-treated group. In depletion study, anti-CD45RB, anti-CD154 and CD4-depleting mAb-treated group showed acute rejection of skin allograft in contrast to CD8-depleting group treated with the two-signal blockade. In the group treated with the two-signal blockade, the proportions of CD4+CD45RB(low)and CD8+CTLA-4 regulatory T cells were increased while effector CD8+ T cells, including IFN-gamma-secreting and CD8+CD62L(low)T cells, were decreased when compared with non-treated group. In contrast, the CD4-depleted group treated with the two-signal blockade resulted in recovery from immunoregulatory effects of two-signal blockade. In addition, results of IL-4 and IL-10 production were also showed CD4-dependence. Therefore, the two-signal blockade is accompanied by CD4-dependent mechanisms in allogeneic skin transplantation.