Effect of zinc on early graft failure following intraportal islet transplantation in rat recipients.

BACKGROUND: Zinc (Zn) is related to insulin synthesis, storage and secretion. Zn status in patients with Type 1 diabetes is significantly lower than in healthy controls. Intraportal islet transplantation (IPIT) is a radical treatment for diabetes, the success of which depends largely on the survival of the transplanted islets. This study demonstrates the impact of a Zn-rich environment on transplanted islet survival. MATERIAL/METHODS: Diabetic Wistar rats were transplanted with syngeneic islets. Rats in the high-Zn-diet group were fed a standard pelleted diet containing ZnSO4 at 1000 ppm, whereas those in the control group were fed an ordinary diet alone (ZnSO4 at 50 ppm) for two weeks prior to islet transplantation. We examined Zn level of plasma, the blood glucose levels, and histological findings, etc., after intraportal islet transplantation. RESULTS: The high-Zn-diet group showed excellent blood glucose control compared with the control group on observation days 3, (237.1±120.6 mg/dl vs. 164.2±69.1 mg/dl; p<0.05) and 14, (273.7±160.9 mg/dl vs. 179.2±114.3 mg/dl; p<0.05). Early graft failure was found to be suppressed in the high-Zn-diet group on day 3 after transplantation (6.7% vs. 33.3%: p<0.05). As for the percentage of granulated islets, the high-Zn-diet group was improved (65.1% vs. 41.8%: p<0.05). CONCLUSIONS: These results indicated that a zinc-rich environment is advantageous for the recipient in intraportal islet transplantation. Zn is harmless in humans; thus, we consider that Zn supplementation could be a simple way to improve clinical results of intraportal islet transplantation.

Magnetic resonance imaging of pancreatic islets transplanted into the right liver lobes of diabetic mice.

INTRODUCTION: Pancreatic islets (PI) labeled with Feridex can be visualized using magnetic resonance imaging (MRI) after transplantation into the liver. However, there is still no accurate method of quantifying the signal loss caused by the iron contrast agent within transplanted tissue. The aim of this study was to test a new method for quantifying signal loss during the early posttransplantation period. METHODS: Isolated mouse PI (C57BL/6 and BALB/c) were labeled in CMRL-1066 culture media supplemented with Feridex. Two hundred twenty PI were injected directly into the right liver lobes of BALB/c diabetic (streptozotocine 220 mg/kg) recipients (isografts, n = 3; allografts, n = 3). Animals were scanned at 3 T, on a whole body scanner equipped with a high-performance gradient insert and using the 3D FIESTA sequence, on days 1, 7, and 14. Signal loss was quantified by comparison of liver tissue with and without labeled PI. Signal loss detected on the first scan was rated as 100% and subsequent measurements were recalculated as relative numbers. RESULTS: While the function of the isografts remained stable throughout the study, the allografts failed on days 5 and 10. A decrease in the amount of signal loss was observed in all animals and was comparable after the first week in both groups. However, there was a difference between groups after the second week (mean +/- SD; isografts, 100% --> 61.8 +/- 6.74% --> 47.18 +/- 7.14%; allografts, 100% --> 59.39 +/- 8.54% --> 38.16 +/- 6.81%). CONCLUSION: Disappearance of signal loss is comparable in all animals during the first week and seems to be independent of acute rejection.

Culture medium modulates proinflammatory conditions of human pancreatic islets before transplantation.

A portion of transplanted islets is lost during engraftment as a result of stressful events, involving hypoxia and production of proinflammatory molecules by islets. Two of these molecules (monocyte chemoattractant protein-1, CCL2/MCP-1 and tissue factor, TF) are directly correlated with reduced graft function. We evaluated which factors reduce islet proinflammatory conditions. In particular the effects of different culture media supplemented with proteins or antioxidant agents on CCL2/MCP-1 and TF human islet release were evaluated. We observed that human islets after culture in final wash culture medium (FW) significantly decreased CCL2/MCP-1 release and TF production compared with CMRL and M199. These effects were independent from the type of protein added to the media (human serum, human albumin, fetal calf serum). Glutathione in FW further decreased CCL2/MCP-1 in a dose-dependent manner. Culture conditions can modulate the proinflammatory state of islets, and could be used in clinical islet transplantation to reduce inflammation during engraftment.

An antagonist IL-15/Fc protein prevents costimulation blockade-resistant rejection.

IL-15 is a powerful T cell growth factor (TCGF) with particular importance for the maintenance of CD8(+) T cells. Because costimulation blockade does not result in universal tolerance, we hypothesized that "escape" from costimulation blockade might represent a CD8(+) and IL-15/IL-15R(+)-dependent process. For this analysis, we have used an IL-15 mutant/Fcgamma2a protein, a potentially cytolytic protein that is also a high-affinity receptor site specific antagonist for the IL-15Ralpha receptor protein, as a therapeutic agent. The IL-15-related fusion protein was used as monotherapy or in combination with CTLA4/Fc in murine islet allograft models. As monotherapies, CTLA4/Fc and an IL-15 mutant/Fcgamma2a were comparably effective in a semiallogeneic model system, and combined treatment with IL-15 mutant/Fcgamma2a plus CTLA4/Fc produced universal permanent engraftment. In a fully MHC-mismatched strain combination known to be refractory to costimulation blockade treatment, combined treatment with both fusion proteins proved to be highly effective; >70% of recipients were tolerized. The analysis revealed that the IL-15 mutant/Fc treatment confers partial protection from both CD4(+) and CD8(+) T cell graft infiltration. In rejections occurring despite CTLA4/Fc treatment, concomitant treatment with the IL-15 mutant/Fcgamma2a protein blocked a CD8(+) T cell-dominated rejection processes. This protection was linked to a blunted proliferative response of alloreactive T cells as well silencing of CTL-related gene expression events. Hence, we have demonstrated that targeting the IL-15/IL-15R pathway represents a new and potent strategy to prevent costimulation blockade-resistant CD8(+) T cell-driven rejection.

Prevention of pancreatic islet xenograft rejection by dietary vitamin E.

In pancreatic islet transplantation, the adhesion of activated leukocytes to endothelial cells and the loss of microvascular integrity represent the critical microcirculatory events, which promote loss of graft function due to rejection. With the view that oxygen radicals may contribute to graft rejection, we studied the effect of the antioxidant vitamin E on microvascular rejection of islet grafts. Islets were transplanted syngeneically and xenogeneically (rat) into dorsal skin-fold chambers of hamsters, which received a non-vitamin-E-supplemented laboratory chow. Treated animals with xenografts were fed with a diet supplemented with vitamin E in a low (150 mg/kg) and high (8000 mg/kg) concentration. Intravital fluorescence microscopy demonstrated complete vascularization of syngeneic grafts at day 10 after transplantation, intact islet microcirculation at day 20 with a functional capillary density of 653 +/- 6 cm-1, and only few leukocytes adherent to the endothelial lining of the islets' microvasculature (88 +/- 23 mm-2). Xenogeneic islets showed initial signs of rejection at day 6, including adhesion of leukocytes to the microvascular endothelium (610 +/- 110 mm-2) and loss of endothelial integrity. After 20 days, functional capillary density was significantly lower (173 +/- 68 cm-1) when compared with syngeneic grafts, indicating failure of graft acceptance. Supplementation of the diet with low and high concentrations of vitamin E resulted in a significant (P < 0.05) reduction of xenograft leukocyte-endothelium interaction (146 +/- 29 mm-2 and 109 +/- 42 mm-2) at day 6 after transplantation and and adequate development of functional capillary density at day 20 (478 +/- 36 cm-1 and 539 +/- 86 cm-1; P < 0.05), indicating prevention of microvascular rejection. We conclude that dietary supplementation of the lipophilic antioxidant vitamin E attenuates leukocyte-endothelial cell interactions, preserves microvascular integrity, and thus inhibits microvascular rejection in a dose-dependent fashion. Our study underscores the pivotal mediator role of reactive oxygen species in islet xenograft rejection and, furthermore, suggests that dietary vitamin E may act as an adjunct anti-rejection treatment in clinical islet transplantation.

Improved functional survival of human islets of Langerhans in three-dimensional matrix culture.

The current study evaluates functional survival of human islets maintained in tissue culture for up to 4 wk in suspension media (CMRL-1066 with supplements) and contrasts these results with immobilizing three-dimensional matrices (agarose or alginate). The absolute number and volume of islets retrieved from agarose is significantly higher after two and four wk of culture compared to conventional free-floating media. In vitro function of islets, assessed by insulin/DNA content, insulin secretion into the culture media over 24 h and glucose-theophylline stimulated insulin release in a dynamic perifusion system, was not significantly different between free-floating and matrix preserved islets. In vivo islet function was evaluated by the effectiveness for reversal of insulin-dependent diabetes mellitus by transplantation of the islets under the kidney capsule of nude mice. Although adequate insulin responses to glucose were seen after culture in conventional or matrix media, only agarose embedded islets were consistently able to induce normoglycemia in diabetic recipients after 14 days of culture. Additional transplantation experiments defined the threshold level required to reverse diabetes to be between 1,000 and 1,500 agarose preserved islets. Our data suggest improved engraftment of human islets after agarose culture. This culture method may be of benefit for the accumulation of functionally competent human islets, thus facilitating the implementation of clinical protocols that utilize freshly isolated islets from multiple donors without the need for cryopreservation.