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We have created a new transgenic model in which the human Epstein-Barr virus receptor, CR2 (CD21), has been expressed in pancreatic beta-cells. Mice derived from three transgenic founders bred into H-2b (C57BL/6) or H-2k (CBA) genetic backgrounds did not become spontaneously diabetic. After transplantation of CR2-expressing islets under the kidney capsule of genetically matched recipients, a histologic picture of peri-insulitis was found. However, animals did not manifest cellular invasion of the islets, destruction of the islets, or diabetes for at least 230 days. Significant numbers of both T and B lymphocytes were found in the cell population surrounding the islets. A pronounced serologic response to CR2 was also present and appeared to precede the onset of peri-insulitis. Thus, in this model, we have separated the process of diabetes induction into at least two phases. One is associated with peri-islet infiltration and an antibody response. However, at least one second signal is likely necessary for the process of islet destruction to follow.

We stably expressed human complement receptor 2 ([CR2] CD21 C3d/Epstein-Barr virus [EBV] receptor) on the rat insulinoma cell line RINm5F with a recombinant retroviral vector. CR2-expressing RINm5F cells secreted 78–33% less insulin than parental cells or cells transduced with an antisense vector and could be infected with high-titer EBV. We tested whether human CR2 expression on RINm5F cells would affect tumorigenesis after transplantation to syngeneic New England Deaconess Hospital rats. Non-CR2–expressing antisense-transduced RINm5F cells rapidly grew tumors and caused hypoglycemia, hyperinsulinemia, and the death of the animals after 15.7 ± 0.7 days. CR2-expressing RINm5F cells were infiltrated by mononuclear cells at an early stage and eventually caused noninfiltrated tumors and the death of the animals after 33.0 ± 0.4 days. These tumors were CR2− and are believed to have arisen from a minor CR2− population of tumor cells. The pancreatic islets were histologically normal at all time points. We conclude that expression of a xenoantigen on a rat insulinoma cell line induces an immune response in syngeneic rats but does not result in breakage of tolerance to parental or revertant cells.

Purified porcine islets were prepared by collagenase digestion and density gradient purification, and transplanted under the kidney capsule of C57B/B6 mice with streptozotocin-induced diabetes which were receiving varying temporary immunosuppressive therapies. Islets that had been cultured for 1 day at 37 degree C were rejected after : 9+/-0.1 (mean+/-SE) days in control mice: 14+/-3 days in mice receiving mouse antilymphocyte serum (MLS) plus porcine antilymphocyte serum (PLS) on day of transplant (day 0); 43+/-6 days in mice treated for 1 week with anti-CD4 antibody (aCD4); 36+/-4 days in mice given aCD4 for 1 week plus PLS on days 0 and 7; 47+/-3 days in mice treated with aCD4 for 1 week plus MLS and PLS on day 21. Porcine islet survival in these latter three groups was significantly (P

It is still a controversial question whether insulin suppresses its own secretion. We prepared pure human islets from three pancreases by collagenase digestion and density gradient purification. Aliquots of 200 islet equivalents (IE, 150-μm sized-islets) were sequentially perifused at 37°C with 3.3 mmol/l glucose (3.3G, 40 min), 16.7 mmol/l glucose (16.7G, 30 min) and again 3.3G (30 min) after 24 h, 37°C culture in CMRL 1066 medium with or without the addition of either 200 or 400 μU/ml human insulin in the incubation medium (6 replicates each). Insulin secretion was assessed by C-peptide (Cp) measurement in the perufusate. Without added insulin (C) and with 200 (Ins200) or 400 (Ins400) μU/ml added insulin, basal Cp release was 0.12±0.03, 0.14±0.02 and 0.14±0.04 ng/ml, respectively. At 16.7G, the first-phase secretion peak (expressed as Cp value) was significantly lower with Ins200 (0.47±0.13 ng/ml,P

INCE 1985,27 transplants have occurred at Washington University School of Medicine (WUMS) in St. Louis and 4 transplants in conjunction with the University of Western Ontario, London, Ontario. The majority of suc- cessful transplants have occurred since 1989 when we entered into the era of better immunosuppression, early intensive insulin therapy, purer preparations, immunoalter- ation by 24°C culture, and cryopreservation resulting in larger transplanted islet preparations. The two groups of patients most extensively followed at WUMS were patients with established kidney grafts followed by islet transplant (EKI, n = 8) and those receiving simultaneously trans- planted kidney and islet transplant (SKI, n = 9). Several controversies concerning islet transplantation still exist. For example, should islet transplantation occur simultaneous with or following renal transplant? Does cryopreservation and/or islet culture make a difference in islet function? What is the role of human leukocyte antigen (HLA) matching? Does islet mass improve or hamper long-term islet function? Presented here are the results of a retrospec- tive study addressing these questions in regard to our EKI and SKI patients.

A QUESTION that has plagued islet transplantation since its inception is whether islet failure is the result of exhaustion or rejection. Attempts have been made to immunomodulate islet preparations prior to transplantation’ as well as improve immunosuppression’,’ to lessen the risk of rejection. However, clinical islet transplantation has not achieved the desired success rate and the cause of failure still remains unanswered. We have retrospectively analyzed seven of our islet recipients for anti-donor HLA sensitization. Our data suggest that there is a strong correlation between islet allograft failure, documented by a decrease in Sustacal Stimulation Indices (SSI), and a rise in anti-donor HLA sensitization as detected by panel reactive antibody (PRA) testing. Furthermore, the data imply that donor-specific T cells are present in these patients and may be responsible for loss of islet function.

Indefinite survival of concordant xenogeneic Wistar Furth (WF) rat islet survival was obtained by intrahepatic transplants of cultured WF islets and a single injection of antilymphocyte sera in C57BL/6 mice. Adoptive transfer of splenocytes from mice with established WF islet xenografts produced a marked prolongation of survival of WF islets transplanted under the kidney capsule of diabetic irradiated (600 rads), naive C57BL/6 recipients (mean survival time = 48.9 +/- 17.1 days), and three of the recipients were still normoglycemic at 100 days after transplantation. Adoptive transfer of an equal mixture (3 x 10(7) cells each) of these splenocytes with normal splenocytes also prolonged survival of the kidney capsule islet xenografts (mean survival time = 26.5 +/- 7.8 days vs. 15.2 +/- 5.3 days for controls). In vitro studies on lymphocyte proliferation demonstrated a low rate of proliferation of splenocytes from established islet xenografts in the presence of irradiated WF splenocytes (stimulation index = 1.6 vs. 16.2 for naive C57Bl/6 mice), and mixing the cells with control splenocytes also decreased the proliferation of splenocytes as compared with controls (stimulation index = 5.4 vs. 16.2 in controls). The inhibitory effect was not species specific, since splenocytes from mice with established islet xenografts also produced a 42% inhibition of proliferation in the presence of irradiated Lewis splenocytes. These findings demonstrate that concordant, islet xenograft, immune unresponsiveness can be adoptively transferred by splencotyes from mice with established islet xenografts.

It is obvious that islet transplantation as an isograft can produce normoglycemia and can prevent and reverse early complications of diabetes in rodents. It is possible to produce normoglycemia in immunosuppressed diabetic patients with human islet transplants. It is possible to prevent rejection of islets in animals without continuous immunosuppression by immunoalteration of the islets, and this approach will be attempted in human diabetes. Finally, the encapsulation approach for prevention of rejection of islets is promising. Large animals will be used to test prototypes of the hollow fibers for humans, and if these experiments are successful, it will be possible to progress rapidly to human diabetes. The first approach with encapsulation in human diabetes would be to use human islets, then encapsulated pig islets, and finally genetically engineered beta cell lines when they become available. I hope that these approaches in islet transplantation will prevent the devastating diabetic complications that Henry Dolger demonstrated many years ago could occur in diabetic patients even though those patients are taking insulin.

Up-regulation of maternal islet function is essential to accommodate the increased demand for insulin during pregnancy. Previously, we suggested that lactogenic activity regulates islet function during pregnancy. However, this hypothesis was based on the effect of homologous PRLs on islets, since the homologous placental lactogens (or islets) were unavailable. In this study we examine the direct effects of homologous placental lactogens (PL), PRL, and GH on insulin secretion and B-cell division in rat, mouse, and human islets in vitro. Neonatal rat islets were cultured for 8 days in the presence of 0-1000 ng/ml rat PL-I (rPL-I), rPRL, or rGH. Media were changed daily, and the insulin concentration was determined. rPL-I and rPRL (500 ng/ml) treatment resulted in a 2-fold increase in insulin secretion. rGH (1000 ng/ml) elicited a 30% increase in insulin secretion. Similarly, cell replication, as indicated by BrdU incorporation into B-cells, was increased 4-fold in the presence of rPL-I and rPRL. The ED50 for insulin secretion and 5'-bromo-2'-deoxyuridine (BrdU) incorporation was 70 ng/ml for rPL-I and 150 ng/ml for rPRL. Similarly, in adult rat islets, insulin secretion was increased 1.6-fold, and B-cell replication increased 3-fold in the presence of the lactogenic hormones. Neonatal mouse islets were cultured for 8 days in the presence of 500 ng/ml mouse (m) PL-I, mPL-II, mPRL, or mGH. mPL-I, mPL-II, and mPRL treatment resulted in a 2-fold increase in insulin secretion. mGH elicited a 30% increase in insulin secretion. BrdU incorporation into B-cells was increased 3-fold in the presence of mPL-I and mPRL and 2-fold in the presence of mPL-II. Adult human islets were cultured for 8 days in the presence of 1 microgram/ml human (h) PL, hPRL, or hGH. For human islets isolated from six pancreata obtained from females, hPL (138 +/- 10%), hPRL (133 +/- 9%), and hGH (117 +/- 3%) significantly increased insulin secretion compared to that from control islets. This study compares the direct effects among homologous PLs, PRLs, and GHs on insulin secretion and B-cell division in rat, mouse, and human islets. The results indicate that placental lactogen directly regulates islet function in several species and is probably the principal hormone responsible for the increased islet function observed during normal pregnancy.

Hepatic insufficiency, which continues to be a source of morbidity after portacaval shunt (PCS), can be prevented by syngeneic pancreatic islet transplantation into the portal vein before PCS. This study investigated the ability of syngeneic pancreatic islet transplantation after PCS to prevent hepatic atrophy and rescue hepatocellular function.Approximately 1200 to 1400 syngeneic rat pancreatic islets were transplanted through a heparinized catheter into the left lobes of the liver 3, 7, and 21 days after end-to-side PCS. Normal rats received no treatment, and PCS control rats received PCS only, without islet transplantation. Hepatocellular function (caffeine clearance) and hepatic blood flow (indocyanine green clearance) were analyzed at 42 and 49 days after PCS. On day 51 after PCS, the left and right lobes of the liver were divided, weighed, and sectioned for histologic studies.Caffeine clearance in the animals at 3 days (p less than 0.05) and at 7 days (p less than 0.05) after end-to-side PCS was significantly improved versus control PCS animals, indicating that hepatocellular function could be rescued after creation of a PCS. Indocyanine green clearance of all groups with PCS was significantly (p less than 0.001) decreased versus normal animals, showing that hepatic blood flow was uniformly decreased by PCS in all groups. The weight of the transplanted left lobes was significantly greater than the untransplanted right lobes of the groups at 3 days (p less than 0.01) and at 7 days (p less than 0.05) after end-to-side PCS compared with control animals, indicating that liver atrophy was prevented in the islet-transplanted lobes but not in those lobes without a transplant.Islet transplantation early after PCS can prevent liver atrophy and significantly improve hepatocellular function.

We sought to determine if the incidence of renal hydronephrosis in male C57BL/KsJ mice increased with age and if grossly normal kidneys would develop hydronephrosis over time. Spontaneous hydronephrosis was found incidentally in 32% of 234 male C57BL/KsJ mice killed as pancreas donors for islet transplantation experiments. The incidence of hydronephrosis increased with age; the incidence was 15% in 6- to 8-week-old mice, 52% in 8- to 10-week-old mice and 63% in 11- to 15-week-old mice (P less than 0.001). Additional mice received islet isografts beneath the renal capsule. Only mice with grossly normal kidneys received islet grafts. These same kidneys were then re-examined when the graft recipients were killed at the end of the experiment and the incidence of hydronephrosis was determined. The conversion of normal kidneys to hydronephrotic kidneys increased with the time since islet transplantation. Kidneys re-examined less than 4 weeks since transplantation had only 5.8% new hydronephrosis, while those re-examined later than 4 weeks after transplantation had a new hydronephrosis incidence rate of 40% (P less than 0.001). Our findings suggest that hydronephrosis is hereditary but not congenital, that it develops rapidly, and that it can complicate experiments using this strain. This may also represent a useful new animal model of progressive hydronephrosis.

In vitro culture of rat islets at 24 degrees C for 7 days in tissue culture medium CMRL 1066 almost completely eliminated lymphoid cells from the islets. Immunostaining of the islets with monoclonal antibody OX4 for demonstration of class II major histocompatibility complex (MHC)-expressing cells revealed a decrease from 13.1 +/- 0.6 positive cells per islet on day 0 to 0.7 +/- 0.1 cells per islet on day 7. A comparable decrease was found using OX1 for demonstration of all leukocytes. In contrast, culture of rat islets at 24 degrees C for 7 days with tissue culture Roswell Park Memorial Institute (RPMI) 1640 medium was not as effective in eliminating lymphoid cells as in medium CMRL 1066 (3.0 +/- 0.2 class II MHC positive cells per islet at 7 days). Effective elimination of intraislet lymphoid cells apparently is due to the combined effect of low temperature culture and the tissue culture medium CMRL-1066. The second goal of the study was to determine whether the destructive effect of interferon gamma (IFN-gamma) on rat islets in culture was due to intraislet lymphoid cells. In vitro culture of rat islets with IFN-gamma (1000 units/ml) at 37 degrees C caused almost complete destruction of the islets at 7 days. If intraislet lymphoid cells were eliminated from the islets by in vitro culture at 24 degrees C followed by exposure to IFN-gamma (1000 units/ml) for 7 days at 37 degrees C, then IFN-gamma did not cause destruction of the islets and transplants of the treated islets produced normoglycemia in diabetic recipient mice. These findings indicate that intraislet lymphoid cells are responsible for destruction of islet cells when these cells (presumably macrophages) are activated by IFN-gamma. Intraislet lymphoid cells may play a significant role in destroying islet cells in autoimmune diabetes.

Life-long hormone replacement therapy is today the only form of therapy for several endocrine insufficiencies. Transplantation of endocrine tissues could represent a more physiological approach to the treatment of different syndromes such as adrenal insufficiency, diabetes or hypoparathyroidism. Successful experimental islet transplantation across major histocompatibility barriers using cultural pretreatment of the insulin producing tissue in conjunction with temporary immunosuppression of the recipients, encouraged us to attempt similar approaches with parathyroids, adrenal cortex and adrenal medulla. Either bilateral parathyroidectomy or bilateral adrenalectomy were performed on the recipient rats before transplantation, followed by the implantation of parathyroid, adrenal cortical or adrenal medullary tissue beneath the renal capsule. The tissue was transplanted either immediately after its dissection or after pretreatment with 1 week culture. Some of the recipients received a short term cyclosporine treatment. The results indicated that parathyroid and adrenal medulla transplants were often infiltrated by mononuclear cells 30 days after transplantation. In contrast, adrenal cortical tissue was well preserved 4 weeks after transplantation, without showing any sign of rejection when a combination of pre-transplant culture of the adrenal tissue and temporary cyclosporine treatment of the recipients was used.

Inseltransplantation als eine zuverlassige alternative Therapie fur Typ I-Diabetes Mellitus bleibt eine vielversprechende Moglichkeit. Es gibt aber keine gut dokumentierten Falle von erfolgreichen klinischen Versuchen. Trotzdem wurden entscheidende Fortschritte gemacht, um die Isolation der menschlichen Langerhansschen Inseln zu verbessern. Inselautotransplantation nach vollstandiger oder nahezu vollstandiger Pankreatektomie macht eine Langzeitfunktion transplantierter Inseln wahrscheinlich. Anfangliche klinische Versuche mit allogenen Inseltransplantaten haben eine kurzfristige endokrine Funktion aufgezeigt.

Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-Km, low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. We isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus.