Intrahepatic Islet Research Articles

A mouse model for studying intrahepatic islet transplantation.

Intrahepatic human islet transplantation has raised hopes for a cure for diabetes mellitus, especially in patients with type 1 diabetes; however, the need for a substantial amount of islets and, in many instances, repeated transplantations demonstrates underlying problems with this procedure, such as failure of angiogenesis and immunologic rejection. Studies using rodent models may be helpful in improving the success of islet transplantation. However, most of the studies using rodents for islet transplantation have been under the kidney capsule rather than the liver. Using islets from transgenic mice expressing green fluorescent protein under the control of mouse insulin I promoter, the authors have developed a method with which to visualize histologic and pathologic changes in intraportally transplanted islets and surrounding hepatic tissue using reflected light confocal imaging. Initial events 24 hr after islet transplantation in the liver include beta-cell loss and hepatic ischemic injuries.

Prevalence of Hepatic Steatosis After Islet Transplantation and Its Relation to Graft Function

Islet allotransplantation can provide insulin independence in selected individuals with type 1 diabetes. The long-term effects of these transplants on the liver are unknown. Recently, two cases of periportal steatosis after islet transplantation have been described. In this study, we performed ultrasound and magnetic resonance imaging (MRI) in 30 C-peptide-positive islet transplant recipients to detect steatosis and to explore the association of the radiological findings with clinical and metabolic factors. Steatosis was observed on MRI in six (20%) subjects. Histological findings of hepatic steatosis concurred with the imaging findings. Steatosis completely resolved in one subject whose graft failed. More subjects with steatosis required supplementary exogenous insulin than not (67 vs. 21%; P < 0.05). The clinical features of subjects with and without steatosis were otherwise similar, although C-peptide levels were higher in insulin-independent subjects with steatosis (0.98 +/- 0.12 vs. 0.70 +/- 0.18 nmol/l; P = 0.05), despite similar blood glucose levels. Serum triglycerides and the use of exogenous insulin were associated with increased odds of steatosis in a logistic regression model (chi(2) [degrees freedom] = 13.6 [2]); P = 0.001). MRI-detected steatosis is a common finding; the steatosis appears to be due to a paracrine action of insulin secreted from intrahepatic islets. Hepatic steatosis may be associated with insulin resistance or graft dysfunction.

Acute and Chronic Imaging Findings in Patients with Intrahepatic Islet Transplants

Acute and Chronic Imaging Findings in Patients with Intrahepatic Islet Transplants

Peripheral mobilization of recipient bone marrow-derived endothelial progenitor cells enhances pancreatic islet revascularization and engraftment after intraportal transplantation

Background. Pancreatic islet transplantation has been validated as a treatment for type 1 diabetes. However, a high number of islets is required to establish euglycemia. Transplantation of islets leads to loss of islet vasculature, which requires revascularization to ensure adequate survival. Islet vascular density in transplanted islets is markedly decreased compared with endogenous islets. The feasibility of revascularization of ischemic tissues by mobilizing endothelial progenitor cells or angioblasts has been demonstrated. Therefore, we investigated the therapeutic potential of angioblast mobilization for stimulation of islet revascularization and therefore engraftment after transplantation. Methods. FVB/NJ mice underwent bone marrow transplantation from transgenic mice constitutively expressing β-galactosidase encoded by LacZ under regulation of the endothelial cell-specific promoter TIE-2 (FEV/NJ-TIE-2-LacZ). Three weeks after reconstitution, animals received an intrahepatic islet syngeneic infusion (FVB/NJ donors). The contribution of angioblasts into sites of islet revascularization was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR), β-galactosidase (β-gal) activity, and immunohistochemistry. Islet vascular density was assessed morphometrically followed by in situ BS-1 lectin staining and functional islet mass after transplantation by metabolic studies. Angioblasts were mobilized with murine granulocyte-macrophage colony-stimulating factor (GM-CSF) (0.5 μg/day/7 days). Results. An islet dose-dependent increase in β-gal was demonstrated after transplantation. These results were confirmed by RT-PCR and immunohistochemistry. GM-CSF increased the number of peripheral angioblasts and their localization into sites of islet revascularization. A significant increase in islet vascular density was observed in animals treated with GM-CSF versus controls. Higher functional islet mass was demonstrated in animals treated with GM-CSF. Conclusions. Augmentation of angioblasts in the peripheral circulation resulted in higher islet vascular density and engraftment. This novel strategy may improve the results in clinical islet transplantation. (Surgery 2003;134:390-8.)

Islet transplantation: where do we stand now?

After many years of limited success in islet transplantation, researchers developing this procedure have made great strides, and several centers have now reported that islet transplantation can result in long-term insulin independence for patients with type 1 diabetes mellitus. The improved quality of life achieved in some islet allograft recipients suggests that this important line of investigation should proceed. Yet, several factors limit the technique and these hurdles must be overcome before it can be considered a practical treatment for the millions of individuals with diabetes, be it type 1 or type 2. Most obvious is the gross disparity between the number of islets available for clinical transplantation and the number of patients with diabetes who might benefit. Other important limitations, too often lost in the discussion, include complications associated with the technique itself, the toxicity of currently available immunosuppressive drugs, and the imperfect glycemia control achieved in most patients. In fact, our ongoing analysis as to whether transplantation-based therapy improves survival for patients with type 1 diabetes suggests that, for many at least, the opposite may be true. Two variables, as yet undefined, also need to be considered: (1) can the procedure, when done well, prevent or reverse diabetes-associated complications and (2) what are the long-term consequences of intrahepatic islets?

Acceptance of islet allografts in the liver of mice by blockade of an inducible costimulator.

An inducible costimulator (ICOS) has been found to be a novel costimulator for T-cell activation, although its precise role in transplant immunobiology remains unclear. This study determined whether ICOS plays an essential role in rejection of intrahepatic islet allografts in streptozotocin-induced diabetic mice. Mononuclear cells in the liver of mice were isolated and examined by flow cytometry with respect to expression of ICOS in association with rejection, and the effects of in vivo treatment with an anti-ICOS antibody on survival of intrahepatic islet allografts were determined. RESULTSFlow cytometric analysis of mononuclear cells in the liver of normal untreated mice revealed that ICOS is expressed on CD4+CD3int natural killer T cells. The expression of ICOS was up-regulated on CD4+CD3bright T cells and expanded CD8 T cells in the liver in association with rejection. Posttransplant short-term administration of anti-ICOS antibody alone produced a significant prolongation of islet allograft survival. Administration of the antibody in conjunction with a subtherapeutic regimen of FK506 prevented rejection, leading to the acceptance of islet allografts. ICOS has an essential role in rejection of intrahepatic islet allografts and the blockade of ICOS interaction might be a novel approach for preventing islet allograft rejection.

Intrahepatic islet transplantation in type 1 diabetic patients does not restore hypoglycemic hormonal counterregulation or symptom recognition after insulin independence.

Islet allotransplantation can provide prolonged insulin independence in selected individuals with type 1 diabetes. Whether islet transplantation also restores hypoglycemic counterregulation is unclear. To determine if hypoglycemic counterregulation is restored by islet transplantation, we studied hormone responses and hypoglycemic symptom recognition in seven insulin-independent islet transplant recipients using a 3-h stepped hypoglycemic clamp, and compared their responses to those of nontransplanted type 1 diabetic subjects and nondiabetic control subjects. Glucagon responses of islet transplant recipients to hypoglycemia were significantly less than that observed in control subjects (incremental glucagon [mean +/- SE]: -12 +/- 12 vs. 64 +/- 22 pg/ml, respectively; P < 0.05), and not significantly different from that of nontransplanted type 1 diabetic subjects (-17 +/- 10 pg/ml). Epinephrine responses and symptom recognition were also not restored by islet transplantation (incremental epinephrine [mean +/- SE]: 195 +/- 128 [islet transplant recipients] vs. 238 +/- 73 [type 1 diabetic subjects] vs. 633 +/- 139 pg/ml [nondiabetic control subjects], P < 0.05 vs. control; peak symptom scores: 3.3 +/- 0.9 [islet transplant recipients] vs. 3.1 +/- 1.1 [type 1 diabetic subjects] vs. 6.7 +/- 0.8 [nondiabetic control subjects]). Thus the results indicate that despite providing prolonged insulin independence and near-normal glycemic control in these patients with long-standing type 1 diabetes, hypoglycemic hormonal counterregulation and symptom recognition were not restored by intrahepatic islet transplantation.

Histopathological study of intrahepatic islets transplanted in the nonhuman primate model using edmonton protocol immunosuppression.

While islet cell transplantation is a promising way to restore insulin independence to patients with type I diabetes mellitus, a detailed histological analysis of the transplanted, intraportal islets has not yet been reported. Rhesus macaques underwent total pancreatectomy, then had allogeneic isolated islets infused into their portal vein, followed by daclizumab, tacrolimus, and sirolimus to prevent islet rejection. Islets were evenly distributed among the liver lobes. Liver sections from a primate given allogeneic islets 5 d earlier did not display any islet capillary formation, whereas intrahepatic islets transplanted 30 and 90 d before euthanasia showed an abundant capillary supply. Localized hepatocellular glycogenosis was observed surrounding the islets in a primate with functioning islets 7 months post transplant. Liver sections from a primate that rejected islets transplanted 2 months prior displayed only islet remnants with prominent local lymphohistiocytic inflammation and an occasional capillary. We conclude that islets develop an abundant vascular supply within 30 d following transplant and because capillaries persist even following rejection, that the vascular cells are likely from the recipient. While transplanted islets were not vascularized early post transplant, the primates remained insulin independent. The long-term consequence of islets in the liver, marked by the glycogenosis, remains unknown and warrants further study.

Decreased survival of islet allografts in rats with advanced chronic complications of diabetes.

Successful islet transplantation has been possible in experimental animals in contrast to humans. One difference between animal models of diabetes and human islet transplantation is the presence of advanced chronic complications in humans. Even longer-term follow-up of islet transplantation in humans according to the Edmonton protocol suggests that advanced chronic complications may adversely affect allograft survival with the glucocorticoid-free immunosuppressive regimen as well. We developed a rat model of chronic complications of diabetes and compared islet allograft survival in rats with advanced chronic complications to age-matched control rats with acute onset diabetes. Islets were transplanted at either the renal supcapsular, intrahepatic, or intramuscular location. The survival of islet allografts in rats with chronic complications was decreased at all sites compared with the age-matched controls. The best survival in the rats with advanced chronic complications occurred at the renal subcapsular site. Blood sugar measurements indicated impaired glucose tolerance in most of the rats with chronic complications and surviving renal subcapsular islet allograft. Histological and gross examination of the surviving renal subcapsular islet allografts indicated disordered angiogenesis in the rats with chronic complications. Rats with successful intrahepatic islet allografts and the respective age-matched controls had comparable blood sugars. Survival of islet allografts at the intramuscular site was poor in rats with chronic complications or acute onset diabetes. We conclude that the structural or metabolic abnormalities associated with chronic poor control of diabetes impair islet allograft survival and function. This should be considered as a possible explanation for failure of islet allograft survival in human islet transplantation.

Islet transplantation: travels up the learning curve.

Great excitement was generated in 2000 by a report from the University of Alberta in Edmonton, Canada, that seven of seven type I diabetic patients transplanted with intrahepatic cadaveric islets were normal glycemic, 1-year post-transplantation without the use of exogenous insulin treatment. The follow-up information from the same researchers with a larger group of patients indicated that in a group of 12 alloislet recipients, five had impaired glucose tolerance and three had post-transplantation diabetes. Great attention is now being directed toward understanding why alloislet recipients who are initially successful may later develop partial failure. At the same time, the Immune Tolerance Network is sponsoring a multicenter trial using the Edmonton protocol to ascertain whether these results can be replicated by other transplant groups in the United States, Canada, and Europe. Detailed studies of islet beta-cell function have revealed intact insulin secretion in autoislet and alloislet transplant recipients. In contrast, glucagon responses to insulin-induced hypoglycemia are absent from islets transplanted intrahepatically; however, alpha cells within intrahepatic islets are capable of releasing glucagon in response to intravenous arginine. Although many technical refinements are underway to make this procedure even more efficacious, supply and demand issues are a major concern and must be dealt with before the procedure of islet transplantation can be considered generally available for patients with diabetes.

Prevention of diabetes for up to 13 years by autoislet transplantation after pancreatectomy for chronic pancreatitis.

Patients with chronic pancreatitis who undergo total pancreas resection inevitably become diabetic unless their islets are autotransplanted to prevent diabetes. We studied patients who underwent this procedure to assess its long-term efficacy in providing stable glucose regulation. Six patients were followed for up to 13 (6.2 +/- 1.7) years after intrahepatic islet autotransplantation. From 290,000 to 678,000 islets were transplanted and no patients received drugs to control glucose levels postoperatively. Islet function was assessed by measurements of fasting plasma glucose (FPG), intravenous glucose disappearance rate (KG), HbA1c, insulin responses to intravenous glucose and to arginine, and insulin secretory reserve. Patients were studied two to four times each to obtain longitudinal data. Five of six patients remained free of insulin treatment and maintained FPG <126 mg/dl and HbA1c levels <6.5%. As a group, they maintained stable insulin secretory reserve, but insulin responses to glucose tended to decrease over time in three patients. KG values correlated significantly with the number of islets originally transplanted. These data indicate that intrahepatic autoislet transplantation can successfully maintain stable beta-cell function and normal levels of blood glucose and HbA1c for up to 13 years after total pancreatectomy as treatment for chronic painful pancreatitis. This usually overlooked procedure of intrahepatic islet transplantation designed to prevent diabetes in patients undergoing pancreatectomy for chronic pancreatitis should be considered more often.

A Coculture Model of Intrahepatic Islet Transplantation: Activation of Kupffer Cells by Islets and Acinar Tissue.

Clinical and experimental studies of intrahepatic islet transplantation have allowed histological and systemic observations to be made, but the location of the transplanted islets makes it difficult to assess direct effects on the cells of the liver. An in vitro coculture model of Kupffer cells with islets or pancreatic acinar tissue is described, using porcine tissue and measuring the secretion of thromboxane B2, prostaglandin E2, 6-keto-prostaglandin F1α, and prostaglandin F2α as an indicator of Kupffer cell stimulation. The results have demonstrated activation of Kupffer cells in the presence of acinar or islet tissue, both when the cells were in direct contact and when separated by a membrane. This indicated that the stimulation was due to a soluble factor or factors, and was confirmed by the culture of Kupffer cells with acinar conditioned medium. The degree of stimulation was much greater with acinar tissue than with islets. In subsequent experiments, aprotinin, an enzyme activation inhibitor, was added to the cocultures in an attempt to reduce Kupffer cell activation. This had no effect, possibly due to the fact that the endogenous pancreatic enzymes may already be activated during digestion of the pancreas. Aprotinin alone caused an increase in secretion of eicosanoids from Kupffer cells. The high response to acinar tissue is of particular relevance to islet autotransplantation in which unpurified pancreatic digest is often transplanted. The clinical effectiveness of aprotinin in the light of these results is discussed. In conclusion, although unable to mimic the complex situation following intrahepatic islet transplantation, the coculture model described here allows the opportunity to assess the events relating to specific cell types, and will provide the scope to undertake more detailed studies on the mechanisms involved. The same model could be applied to the coculture of pancreatic tissue with hepatocytes to determine any effects on the normal function of hepatocytes.

Insights from a Successful Case of Intrahepatic Islet Transplantation into a Type 1 Diabetic Patient

We report a case of long-term (>4 yr) successful intrahepatic islet transplantation into a type 1 diabetic patient chronically immunosuppressed for a prior kidney graft. The exogenous insulin requirement decreased progressively after transplantation, and insulin treatment was withdrawn at 6 months. Glycosylated hemoglobin levels were in the normal range at 1 and 2 yr (5.3%) and increased slightly above the upper normal limit at 3 and 4 yr (6.3% and 6.4%). Fasting C peptide levels remained stable during the entire follow-up, but the proinsulin to insulin ratios increased dramatically at yr 3. Glycemic levels after an oral glucose tolerance test showed a diabetic profile at 1 yr, a normal profile at 2 yr, and an impaired glucose tolerance profile at 3 yr. Intravenous glucose tolerance test-induced first phase insulin release, present at 1 and 2 yr, disappeared at 3 yr. Diabetes-related autoantibodies (islet cell antibodies, glutamic acid decarboxylase antibodies, and tyrosine phosphatase-like protein antibodies) were undetectable before transplantation and remained so during the entire follow-up. The patient died of myocardial infarction 50 months after transplantation while she was still in good metabolic control (glycosylated hemoglobin, <6.8%) in the absence of exogenous insulin administration. The autoptic liver showed well granulated islets, richly vascularized and without evidence of lympho-mononuclear cell infiltration. The morphometrically extrapolated intrahepaticβ -cell mass was 99.9 mg. In conclusion, this successful islet graft showed a bell-shaped clinical effect, maximal at 2 yr after transplantation, followed by a slow progressive decline. The absence of allo- and autoreactivities against the transplanted islets points to a nonimmune-mediated β-cell loss as the cause of graft functional deterioration.

Prolongation of rat islet xenograft survival in the liver of IFN-gamma-deficient mice.

The cellular mechanisms involved in islet xenograft rejection remain undetermined. In the present study, the role of interferon-gamma (IFN-gamma) in rat islet xenograft rejection was examined with the use of IFN-gamma-deficient mice as recipients and the results were compared with allografts. There was no significant difference in the survival of intrahepatic islet allografts in IFN-gamma-deficient mice compared with that in wild-type mice. In contrast, a marked prolongation of rat islet xenograft survival was obtained in IFN-gamma-deficient mice without immunosuppression when compared with the survival in wild-type mice. In order to dissect the difference, infiltrating cells in the liver in association with rejection were examined with flow cytometry. An expansion of CD8 T cells was seen in the liver of wild-type mice rejecting xenografts compared with isografts. There was no significant change in other cell populations. In IFN-gamma-deficient mice, the expansion of CD8 T cells was seen in the liver rejecting xenografts; however, the time of development was markedly delayed by the time of rejection. These findings suggest that the acute rejection of rat islet xenografts in mice is IFN-gamma-dependent although the exact mechanisms remain unknown.

CD4(+) Valpha14 natural killer T cells are essential for acceptance of rat islet xenografts in mice.

Pancreatic islet transplantation represents a potential treatment for insulin-dependent diabetes mellitus. However, the precise cellular and molecular mechanisms of the immune reactions against allogeneic and xenogeneic transplanted islets remain unclear. Here, we demonstrate that CD4(+) Valpha14 natural killer T (NKT) cells, a recently identified lymphoid cell lineage, are required for the acceptance of intrahepatic rat islet xenografts. An anti-CD4 mAb, administrated after transplantation, allowed islet xenografts to be accepted by C57BL/6 mice, with no need for immunosuppressive drugs. The dose of anti-CD4 mAb was critical, and the beneficial effect appeared to be associated with the reappearance of CD4(+) NKT cells at around 14 days after transplantation. Interestingly, rat islet xenografts were rejected, despite the anti-CD4 mAb treatment, in Valpha14 NKT cell-deficient mice, which exhibit the normal complement of conventional lymphoid cells; adoptive transfer of Valpha14 NKT cells into Valpha14 NKT cell-deficient mice restored the acceptance of rat islet xenografts. In addition, rat islet xenografts were accepted by Valpha14 NKT mice having only Valpha14 NKT cells and no other lymphoid cells. These results indicate that Valpha14 NKT cells play a crucial role in the acceptance of rat islet xenografts in mice treated with anti-CD4 antibody, probably by serving as immunosuppressive regulatory cells.

Hepatocyte growth factor is essential for amelioration of hyperglycemia in streptozotocin-induced diabetic mice receiving a marginal mass of intrahepatic islet grafts.

It is crucial for clinical islet transplantation to find a procedure to improve the success rate of insulin independence after islet transplantation. In the present study, we determined whether hepatocyte growth factor (HGF) has a favorable effect on amelioration of hyperglycemia in streptozotocin (STZ, 200 mg/kg)-induced diabetic mice (C57BL/6) receiving a marginal mass of intrahepatic islet isografts. Isolated syngeneic islets were transplanted into the liver of recipients. HGF with dextran sulfate (DS) was administered intraperitoneally once a day at day 0, 2, 4, 6, and 8 relative to islet transplantation. DS has been known to enhance the effect of HGF. It was found that the number of 250 islets was a marginal mass as donor islets in this model, in which 2 out of 14 diabetic mice receiving 250 islets became normoglycemic by 90 days after transplantation. The treatment with HGF (100 microg) in conjunction with DS (200 microg) produced normoglycemia in all mice (n = 5). Morphological study as well as intraperitoneal glucose tolerance test revealed the beneficial effects of HGF. To our surprise, six out of nine mice receiving 250 islets and treated with DS alone became normoglycemic. Additional anti-HGF antibody treatment (100 microg, day -1, 0, 2, 4, 6, and 8) abolished the effects of DS, indicating that the effect by DS is mediated via the endogenous HGF. The effects of DS were not observed when the renal subcapsular space was the site of islet transplantation. There was a significant increase in plasma HGF levels in mice after the intrahepatic grafts but not the renal subcapsular one. These findings demonstrate that HGF is essential for amelioration of hyperglycemia in STZ-induced diabetic mice when a marginal mass of islets was grafted into the liver. As the liver is the site of clinical islet transplantation and the inability to achieve insulin independence after transplantation is a major obstacle for successful transplantation, HGF may facilitate to overcome such an important issue for clinical islet transplantation.

Effects of graft-versus-host reaction on intrahepatic islet transplants.

The use of donor-specific bone marrow transplantation might represent a valuable approach for the induction of hyporesponsiveness to concurrent allogeneic organ and tissue grafts. In this setting, the occurrence of subclinical forms of graft-versus-host reaction (GVHR) can seldom be formally ruled out. When systemic administration of donor-specific bone marrow is performed together with intraportal transplantation of islets of Langerhans, GVHR might damage the implanted islets through an innocent bystander mechanism. The liver is, in fact, a prominent target of GVHR, and islets are sensitive to the noxious effects of proinflammatory mediators, including selected cytokines produced during GVHR. A parent (Lewis) to F1 (Lewis x Brown Norway) splenocyte transfer model was used to induce GVHR in rats. Islets were also obtained from Lewis donors and implanted into F1 recipients to examine the effects of GVHR on islet function in the absence of rejection. Selected doses of splenocytes were infused in F1 recipients to induce GVHR. When 150 x 10(6) cells were administered, all recipients developed clinical lethal GVHR. When 100 x 10(6) or 80 x 10(6) cells were given, lethal GVHR was observed in 40 and 20% of the F1 animals, respectively. A transient clinical syndrome occurred in the remaining animals and spontaneously subsided; histological findings consistent with persistent low-grade GVHR were observed in these animals at the time of death several months after splenocyte inoculation. When simultaneous splenocyte infusions and islet grafts were performed in chemically diabetic animals, no adverse effect of either clinical or subclinical GVHR was observed on islet function. Blood glucose profiles were normal, as were intravenous glucose tolerance test profiles. In conclusion, GVHR does not seem to adversely influence function of islets of Langerhans implanted intrahepatically in this parent to F1 experimental model.

Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154.

Reported effects of anti-CD154 treatment on autoimmunity, alloreactivity, and inflammatory events mediated by macrophages and endothelial cells indicated that it might be an ideal agent for the prevention of intrahepatic islet allograft failure. This hypothesis was tested in MHC-mismatched rhesus monkeys. Transplantation of an adequate number of viable islets resulted in engraftment and insulin independence in six of six recipients treated with anti-CD154 (hu5c8) induction plus monthly maintenance therapy (post-operative day >125, >246, >266, >405, >419, >476). Anti-CD154 (hu5c8) displayed no inhibitory effect on islet cell function. For monkeys followed for >100 days, continued improvement in graft function, as determined by first phase insulin release in response to intravenous glucose, was observed after the first 100 days post-transplant. No evidence of toxicity or infectious complications has been observed. All recipients treated with anti-CD154 became specifically nonresponsive to donor cells in mixed lymphocyte reactions. Furthermore, three monkeys are now off therapy (>113, >67, and >54 days off anti-CD154), with continued insulin independence and donor-specific mixed lymphocyte reaction hyporeactivity. In striking contrast to all previously tested strategies, transplantation of an adequate number of functional islets under the cover of anti-CD154 (hu5c8) monotherapy consistently allows for allogeneic islet engraftment and long-term insulin independence in this highly relevant preclinical model.

Long-term survival and function of intrahepatic islet allografts in baboons treated with humanized anti-CD154.

Clinical islet cell transplantation has resulted in insulin independence in a limited number of cases. Rejection, recurrence of autoimmunity, and impairment of normal islet function by conventional immunosuppressive drugs, e.g., steroids, tacrolimus, and cyclosporin A, may all contribute to islet allograft loss. Furthermore, intraportal infusion of allogeneic islets results in the activation of intrahepatic macrophages and endothelial cells, followed by production of proinflammatory mediators that can contribute to islet primary nonfunction. We reasoned that the beneficial effects of anti-CD154 treatment on autoimmunity, alloreactivity, and proinflammatory events mediated by macrophages and endothelial cells made it an ideal agent for the prevention of islet allograft failure. In this study, a nonhuman primate model (Papio hamadryas) was used to assess the effect of humanized anti-CD154 (hu5c8) on allogeneic islet engraftment and function. Nonimmunosuppressed and tacrolimus-treated recipients were insulin independent posttransplant, but rejected their islet allografts in 8 days. Engraftment and insulin independence were achieved in seven of seven baboon recipients of anti-CD154 induction therapy administered on days -1, 3, and 10 relative to the islet transplant. Three of three baboons treated with 20 mg/kg anti-CD154 induction therapy experienced delayed rejection episodes, first detected by elevations in postprandial blood glucose levels, on postoperative day (POD) 31 for one and on POD 58 for the other two. Re-treatment with three doses of anti-CD154 resulted in reversal of rejection in all three animals and in a return to normoglycemia and insulin independence in two of three baboons. It was possible to reverse multiple episodes of rejection with this approach. A loss of functional islet mass, as detected by reduced first-phase insulin release in response to intravenous glucose tolerance testing, was observed after each episode of rejection. One of two baboons treated with 10 mg/kg induction therapy became insulin independent post-transplant but rejected the islet graft on POD 10; the other animal experienced a reversible rejection episode on POD 58 and remained insulin independent and normoglycemic until POD 264. Two additional baboon recipients of allogeneic islets and donor bone marrow (infused on PODs 5 and 11) were treated with induction therapy (PODs -1, 3, 10), followed by initiation of monthly maintenance therapy (for a period of 6 months) on POD 28. Rejection-free graft survival and insulin independence was maintained for 114 and 238 days, with preservation of functional islet mass observed in the absence of rejection. Prevention and reversal of rejection, in the absence of the deleterious effects associated with the use of conventional immunosuppressive drugs, make anti-CD154 a unique agent for further study in islet cell transplantation.

HEPATOCYTE GROWTH FACTOR IS ESSENTIAL FOR AMELIORATION OF HYPERGLYCEMIA IN STREPTOZOTOCIN-INDUCED DIABETIC MICE RECEIVING A MARGINAL MASS OF INTRAHEPATIC ISLET GRAFTS

35 Background: The success rate of insulin independence after islet transplantation (Tx) in patients with IDDM has been reported extremely low. One of the major reasons is that the number of grafted islets is not enough to produce normoglycemia after Tx without exogenous insulin. Thus, it is crucial to find a procedure to ameliorate hyperglycemic state when a marginal mass of islets is used as donors for successful clinical islet Tx. Hepatocyte growth factor(HGF) is originally discovered as a factor which promotes regeneration of the liver. Recently, HGF has been reported to have a beneficial effect on other organs. In the present study, we determined whether HGF has a favorable effect on amelioration of hyperglycemia in streptozotocin (STZ)-induced diabetic(DM) mice receiving a marginal mass of intrahepatic islet grafts. Methods: Isolated syngeneic islets were transplanted into the liver(pv) of STZ(200mg/kg)-induced DM mice (C57BL/6). HGF with dextran sulfate (DS) was administered ip once a day at day 0, 2, 4, 6 and 8 relative to islet Tx. DS has been known to enhance the effect of HGF. Results: When 500, 250 and 125 islets were used as donors, 100 % (6/6), 14.3% (2/14) and 0% (0/5) of the recipients, respectively, became normoglycemic by 90 days after Tx. Thus, the number of 250 islets was considered as the marginal mass in this model. Administration of HGF (100 μ g/injection) with DS (200 μ g/injection) produced normoglycemia in all the mice receiving 250 islets at 9.4 ± 3.8 days after Tx (n=5). Morphologically, intact islets were seen in the liver of the recipients at 30 days after Tx. Intraperitoneal glucose tolerance test at 90 days disclosed improved glucose tolerance in the treated recipients compared with those receiving 250 islets without the treatment. When the dosage of HGF was reduced from 100 to 20 μ g /injection, the similar effect was seen (n=6). To our surprise, 6 out of the 9 mice receiving 250 islets and treated with DS alone became normoglycemic (P<0.05, vs the saline control). However, the effect of DS alone was inferior to those of DS with HGF (>49.7 ± 35.2 vs 9.4 ± 3.8 days). To determine whether the effect of DS is mediated via the endogenous HGF, anti-HGF antibody was administered to the mice receiving 250 islets and treated with DS alone. Anti-HGF antibody (100 μ g, day −1, 0, 2, 4, 6, 8) abolished the effect by DS treatment, and all the mice (n=4) remained hyperglycemic for more than 90 days after Tx. Summary: These findings demonstrate that HGF is essential for amelioration of hyperglycemia in the STZ-induced diabetic mice when a marginal mass of islets was grafted into the liver, although the exact mechanisms remain unknown.