Search

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.

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.

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.

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.

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.

The distribution of insulin injected into normal rats and guinea pigs as the free hormone or as insulin-antibody complexes was studied by extraction of immunoreactive insulin and by following the fate of125I- or131I-labeled insulin in the plasma, liver, kidneys and spleens of the injected animals. Injected free hormone rapidly disappears from the plasma to accumulate transiently in the liver, and, to a greater extent, in the proximal convoluted tubules of the kidneys. When insulin-antibody complexes formedin vitro are injected, the hormone disappears more slowly from the plasma and concentrates in the liver and spleen where it can be demonstrated by autoradiography in reticulo-endothelial cells; little or none is found in the kidneys. After injection into insulin-immunized guinea pigs, the hormone disappears very slowly from the plasma and accumulates almost exclusively in the liver with no evidence of concentration by any particular cell type; little or none is found in the kidneys or spleen.

In the short span of 6 years, remarkable advances have been made in islet transplantation. Techniques have been developed which permit the successful reversal of the diabetic state in rats by transplanting isografts of either adult islets, dispersed neonatal pancreases or fetal pancreatic tissue. Islet transplantation prevents or reverses early microvascular complications of the diabetic state in animals. The immune barrier has been bent by simple in vitro procedures which apparently alter the passenger leucocytes of the islets and thus markedly prolong allograft survival. The safety of islet transplant procedures has been established in man. Hopefully, future investigations will continue the bending and lead to the breaking of the immune barrier so that it can be determined whether islet transplantation in man will prevent, arrest or reverse the microvascular, macrovascular and neurological complications that occur in the diabetic patient.

Monolayer cultures of adult rat islets were used for cinemicrographic studies on the movement of beta granules in the cytoplasm of beta cells. Stimulation of insulin release by glucose produced a significant increase in the amount of beta granule movement in the cultured cells. Disruption of the microtubular system with vinblastine or stabilization of microtubules with deuterium oxide inhibited the induction of granule movement by glucose. Incubation of the cultures in the absence of calcium inhibited glucose-induced movement of beta granules, whereas the subsequent addition of calcium permitted movement to occur. The addition of epinephrine also inhibited glucose-induced beta granule movement. In the presence of nonstimulating levels of glucose, movement of beta granules was present. The amount of movement varied from cell to cell suggesting a difference in base line activity of individual beta cells. The movement of individual beta granules was saltatory in type, and the average rate of movement was 1.5 mum. per second. The study demonstrated that directed movement of beta granules occurs following stimulation with glucose and adds further support to the hypothesis that the microtubular-microfilamentous system is involved in the intracellular translocation of beta granules to the cell surface where they are released by emiocytosis.

The initiation of clinical trials on islet transplantation as a possible therapeutic approach for human diabetes had been blocked by 2 major obstacles. The first was to develop methods that would prevent the rejection of islet transplants in animals without requiring continuous immunosuppression of the recipients. The second was to develop procedures which would permit the isolation of a sufficient number of intact, viable islets from a single human pancreas for transplantation into a diabetic patient. Significant advances have been made in overcoming both of these barriers. A brief review of these advances is presented.

Spontaneous hydronephrosis was observed in 15 of 33 necropsied C57BL/KsJ mice. The gross and histological appearances of these lesions are described. Spontaneous hydronephrosis is rare in mice and has not been previously described in this strain. At least four different mutations are reported to cause hydronephrosis in the mouse; each is associated with a high incidence of severe skeletal defects. Because no skeletal malformations were observed in the present study, it is likely that either a multigenic trait which has been present for a long time but is expressed variably, or a mutation other than those previously described may be responsible for hydronephrosis in C57BL/KsJ mice.

Islets of Langerhans from unrelated donor rhesus monkeys were transplanted into the portal vein of five rhesus monkeys which had been made diabetic by partial pancreatectomy and Streptozotocin. There was definite improvement of the diabetic state with an increase in the mean KT1/2 from 0.71 to 1.52 percent per minute. The islets were isolated by a more efficient digestion-filtration process than previously had been described. This process has allowed, for the first time, successful isolation of large masses of islets permitting islet transplants in subhuman primates.

Three weekly intraperitoneal injections of complete Freund's adjuvant (CFA) and, one day later, 25 mg/kg of STZ have been reported to cause a gradual onset of autoimmune diabetes. We have previously reported that the onset of diabetes in this model occurs too rapidly to be due to a specific immune reaction and that other compounds that activate macrophages also potentiate low-dose STZ in rats. We have proposed that mediators released from activated macrophages may enhance the cytotoxic effect of STZ. In the present study, we have observed that intraperitoneal injections of silica, a macrophage toxin, markedly decrease the incidence and severity of diabetes induced with CFA and low-dose STZ.

The findings in this series of investigations indicate that the passenger leukocyte concept applies to islet transplants. Six methods have been developed which prevent rejection of islet allografts in rats and mice without requiring the continuous use of immunosuppressive drugs in the recipient. Initial studies indicate that the passenger leukocyte concept also applies to the prevention of rejection of islet allografts in the dog. Thus the problems remaining with respect to human application are two. One is to determine which of the six methods will completely prevent rejection of islet allografts in dogs and could serve as a model for human application. The second is to modify and adapt the Velcro technique and the automated procedure for mass isolation of islets to the human pancreas. When we have been successful in modifying these isolation procedures so that we can obtain at least 100-150,000 islets, then human islets will be transplanted into the omentum of diabetic subjects who have received a kidney transplant and are already being treated with immunosuppressive agents. The purpose of these initial studies in humans will be to determine whether a sufficient mass of endocrine islet tissue has been transplanted to maintain normoglycemia and normal carbohydrate metabolism in these individuals. We hope that by the time these studies are completed we will have finished the studies in dogs on the selection of the optimal method for preventing rejection and will then be able to initiate human islet transplants without the use of immunosuppressive drugs. Dr. Davie and I also believe that these methods may be applicable to the prevention of rejection of other tissues and organs. It should be quite easy to apply these procedures to transplants of the parathyroid. Dr. Lafferty already has evidence that parathyroid tissue is present in established allografts of thyroid in mice following in vitro culture of the thyroid in 95% O2. It is possible that the procedures will also apply to other endocrine tissue such as the adrenal, the ovary and neuroendocrine tissue. In addition, some of the procedures that have been developed recently could be used for perfusion of organs such as the heart, kidney and liver before transplantation to determine whether alteration or removal of passenger leukocytes in these large organs would affect the survival of the allografts. It is apparent that these past few years have been extremely exciting and we only hope that the next few years will be even more exciting.

Alloxan was found to inhibit a Ca2+- and calmodulin-dependent protein kinase recently identified in pancreatic islets. This effect of alloxan may be specifically related to the inhibitory action of alloxan on insulin secretion from islets since: 1) in islet-cell subcellular fractions, alloxan at micromolar concentrations irreversibly inhibits the Ca2+- and calmodulin-dependent protein kinase activity; 2) pretreatment of intact islets with alloxan at concentrations that inhibit insulin secretion similarly inhibits the protein kinase activity; and 3) alloxan inhibition of both insulin secretion and protein kinase activity in intact islets can be prevented by D-glucose. This inhibition by alloxan appears to be a direct effect on the enzyme since alloxan treatment of either the islet homogenate or the microsomal fraction enriched in protein kinase activity inhibited the kinase activity with similar concentration dependence. These results suggest that alloxan-induced inhibition of a Ca2+- and calmodulin-dependent protein kinase may represent a critical inhibitory site which mediates alloxan-induced inhibition of insulin secretion.

This report describes a method for monolayer cell culture of adult rat pancreatic islets. In this procedure, the isolated islets of Langerhans were enzymatically dispersed and subsequently cultured for 2 to 3 weeks. Enhanced secretion of insulin was present over the basal secretion rate when the cultures were exposed to a high glucose concentration alone or to a combination of glucose, theophylline and glucagon. The ultrastructural studies of the beta cells in culture demonstrated preservation of their morphological characteristics throughout the culture periods.

Active calcium uptake was demonstrated in a subcellular fraction of islets which was enriched in endoplasmic reticulum. Calcium uptake was stimulated by ATP in a magnesium-dependent manner. The rate of calcium accumulation was sustained by oxalate (10 mM) and uptake was prevented or reversed by addition of the calcium ionophore A23187. This calcium uptake process was not affected by azide or ruthenium red. Direct comparison of calcium uptake by endoplasmic reticulum-enriched and plasma membrane-enriched fractions indicated that the uptake was not due to contamination of the fraction with plasma membrane vesicles. These factors as well as the purity of the fraction indicate that the calcium uptake system resides in the endoplasmic reticulum. The properties of the islet endoplasmic reticulum calcium uptake system are similar to properties reported for endoplasmic reticulum derived from other cell types. These properties include stimulation by potassium and a Km for ionized calcium of 1.5 +/- 0.3 microM. The islet-cell endoplasmic reticulum may play a critical role in cellular calcium homeostasis and contribute to the regulation of insulin secretion.

The effect of cyproheptadine on the structure and function of rat pancreatic beta cells was studied in vitro by culturing isolated islets in media containing the drug (0.5 mM). After 8 days in culture the ultrastructure of islet cells maintained in control media appeared well preserved, being similar to the previously reported for islets kept in long term organ culture. In contrast beta cells from islets incubated in media containing cyproheptadine appeared degranulated and the rough endoplasmic reticulum showed cisternal dilation and vacuole formation. These vacuoles were filled with an electron-dense granular material and their surface was usually studded with ribosomes. These lesions are identical with those produced by the administration of cyproheptadine to rats in vivo. In addition to these findings, increased numbers of lysosomes and myeloid bodies were observed in both alpha and beta cells. Compared with that of the controls, the 24-hour basal insulin secretion of islets cultured in the presence of cyproheptadine was significantly reduced from the 4th day of the study onwards. It is thus concluded that cyproheptadine has direct effects on the morphology and function of the rat pancreatic beta cell.

Aberrant Ia antigen expression has been implicated in the pathogenesis of type 1 diabetes. Ia antigen expression was induced on isolated B10.BR murine islet parenchymal cells by culturing them for 5 days with lymphokine supernatants containing interferon-gamma (IFN-gamma) or with recombinant murine IFN-gamma + recombinant tumor necrosis factor. Ia positivity was confirmed by indirect immunofluorescence. Islets cultured for 5 days without cytokines were Ia-negative. Purified B10.BR islets allografted into the portal veins of C57BL/6J mice do not reject, which allowed the authors to determine whether aberrant expression of Ia on parenchymal cells has a deleterious effect on allograft survival. Ia-positive or Ia-negative islets were transplanted via the portal vein into diabetic C57BL/6J mice. All mice remained normoglycemic until they were killed at 30 to 60 days. Well-granulated islet allografts were identified histologically in all mice. The experiment was repeated using Balb/cJ mice as donors. Purified Balb/cJ islets are rapidly rejected by C57BL/6J mice. Induction of Ia expression on Balb/cJ islets significantly improved allograft survival. These findings indicate that Ia-positive islet cells do not induce rejection in these allograft models but may actually have a protective effect.

Fluorophotometry was used to evaluate the integrity of the blood-ocular barriers to fluorescein in experimental diabetes mellitus in rats. This technique allowed quantitation of ocular fluorescein concentrations following intravenous injection. Streptozotacin-induced diabetes resulted in an increased fluorescein accumulation in the anterior chamber (1.52 +/- 0.17 microgram/ml, mean +/- S.E.M.) and vitreous (0.82 +/- 0.11) over baseline nondiabetic levels (0.68 +/- 0.80 and 0.40 +/- 0.03, respectively). Fluorophotometry was repeated at 5, 13, and 20 days following portal vein pancreatic islet transplantation. At 5 days anterior chamber (1.40 +/- 0.17) and vitreous (0.61 +/- 0.08) fluorescein concentrations remained elevated. However, at 13 and 20 days following islet transplantation, ocular fluorescein concentrations were identical to levels observed prior to the induction of diabetes. Intravenous glucose (0.5 gm/kg) tolerance testing was performed 5 and 13 days following transplantation. The glucose responses to the tolerance test were normal and similar at both times. However, at 5 days the insulin response was abnormal with a decreased initial peak and an absent second peak. At 13 days there was a normal biphasic insulin response. In experimental diabetes mellitus ocular vascular permeability was more closely correlated with insulin than blood glucose abnormalities.

The purpose of this study was to determine the effect of pancreatic islets on the survival of clusters of rat hepatocytes transplanted across a major histocompatibility barrier into an ectopic site. Aggregates of hepatocytes and pancreatic islets were obtained from Wistar-Furth rats by an automated method of collagenase digestion. Approximately 8000 Wistar-Furth hepatocyte clusters (100 to 300 micron diameter) were transplanted beneath the renal capsule of Lewis rats with or without the addition of 800 pancreatic islets. Recipients received subcutaneous injections of cyclosporine A (CsA): 30 mg/kg on days 0, 1, and 2, 10 mg/kg on days 3 to 7, and 10 mg/kg every other day for days 8 to 28. In the absence of CsA recipient treatment, all the allografts were rejected within 7 days. The combined hepatocyte-islet allografts in recipients treated with CsA showed maintenance of morphologic integrity of both hepatocytes and islet cells 1, 2, and 4 weeks after transplantation, whereas in the absence of pancreatic islets, the hepatocellular aggregates degenerated despite CsA treatment, leaving only a thin rim of hepatocytes immediately adjacent to the renal parenchyma. It can be concluded that in immunosuppressed recipients hepatocyte-islet renal subcapsular allografts survived despite the presence of an intact liver, which indicates a possible role of trophic factors released locally from the islets.

Individual rat islets could be aggregated into single megaislets in vitro and the megaislets remained morphologically and functionally intact after a 7-day period of culture in the presence of 95% O2 and 5% CO2. Cultured rat megaislets transplanted beneath the renal capsule of diabetic mice produced normoglycemia in the recipients and the survival of the xenografts was markedly prolonged by the 7-day exposure of a high oxygen tension. A single injection of antilymphocyte sera to mouse and rat lymphocytes into the recipients receiving cultured megaislets did not produce a further increase in the percentage of survival of the grafts at 70 days after transplantation. Lymphoid aggregates were present around xenografts of cultured negaislets at 60 and 90 days after transplantation. This lymphoid reaction did not interfere with the function of the xenografts since the recipients were normoglycemic and removal of the grafts resulted in a rapid return to a diabetic state. Intraportal and intrasplenic transplants of cultured rat megaislets did not survive as long as the xenografts of megaislets transplanted beneath the renal capsule. The renal subcapsule site apparently provided some immunological advantage for delaying acute rejection since transplants of individual, fresh rat islets survived for twice as long under the renal capsule as compared wtih intraportal transplants of fresh rat islets.

The normal beta-cell is in essence a closed-loop system for insulin delivery. The successful transplantation of islets into diabetics should provide a means of maintaining normoglycemia in the recipients and determining whether this would prevent, arrest, or reverse the complications of diabetes. The accomplishments leading toward this idealized objective have shown that in the experimental animal, diabetes can be reversed by islet transplantation. Early microvascular complications of diabetes are reversed. Three pretreatment procedures have been developed that prevent rejection of islets transplanted between strains of animals without immunosuppression of the recipients, and two pretreatment procedures have been shown to prevent immune rejection of rat islets transplanted into diabetic mice without the use of immunosuppressive drugs. The critical problems remaining are the development of procedures for the mass isolation of islets that would be required for human transplantation, and if this is successful, determining whether the pretreatment regimens that prevent rejection in mice and rats will be applicable to man.

The uptake of theophylline and caffeine was determined in isolated pancreatic islets employing a dual isotope procedure with sucrose as an extracellular marker. Islets rapidly accumulated caffeine and theophylline with apparent dissociation constants of approximately 23 and 6 mM, respectively. Theophylline inhibited the uptake of caffeine and caused displacement of caffein from islets. These results indicated a competition by theophylline and caffeine for a common site (binding and/or transport carrier). In addition, theophylline and caffeine inhibited D-glucose transport in a dose-dependent manner and within the limits of the experimental system, this inhibition appeared to be non-competitive. (Bu)2cAMP under similar experimental conditions exerted no effect on D-glucose transport. These results present evidence for a rapid uptake of theophylline and caffeine in pancreatic islets, which is compatible with their immediate cellular effects. In addition, these results demonstrate a direct effect by theophylline and caffeine on D-glucose transport which appears independent of their ability to alter intracellular cAMP levels.

The spleen has been examined as a possible site for transplantation of rat islets into diabetic mice. Marked prolongation of islet xenograft survival in the spleen can be achieved with in vitro culture (24 C) and a single injection of either rabbit antiserum to mouse lymphocytes (MALS) alone or MALS and rabbit antiserum to rat lymphocytes (RALS) into the recipients. The percentage of survival of intrasplenic xenografts at 100 days was 16%, as compared with 70% when rat islet xenografts were transplanted via the portal vein. Further improvement in pretreatment regimens will be needed before the spleen can be used as an effective site for possible future islet allograft or xenograft transplants in man.

Rabbits and guinea-pigs were immunized with various pancreatic antigens in Freund's adjuvant. Rabbits received unfractionated bovine insulin and the "A" component and "single peak" insulin separated from it by gel-filtration. All produced antibodies capable of reacting with porcine insulin but none were found to have pancreatic lesions when killed up to 6 weeks after initial injection. Guinea-pigs immunized with bovine "A" component developed pancreatic peri-ductulitis which appeared most frequently (10/20) in animals killed 30 days after a single injection and less frequently in animals killed after 60 (4/10) and 90(1/10) days. Similar lesions were found in only a small proportion of control animals (2/23) or of guinea-pigs immunized with single peak bovine insulin (3/22). Guinea-pigs immunized with homogenates of homologous and heterologous islets of Langerhans developed signs of peri-ductulitis in a high proportion of animals killed up to about 60 days after first injection (18/26). None of these animals exhibited clearly defined signs of diabetes mellitus and the incidence of induced lesions could not be correlated with levels of circulating insulin-binding antibodies.