Posttransplant oxygen inhalation improves the outcome of subcutaneous islet transplantation: A promising clinical alternative to the conventional intrahepatic site.

Subcutaneous tissue is a promising site for islet transplantation, due to its large area and accessibility, which allows minimally invasive procedures for transplantation, graft monitoring, and removal of malignancies as needed. However, relative to the conventional intrahepatic transplantation site, the subcutaneous site requires a large number of islets to achieve engraftment success and diabetes reversal, due to hypoxia and low vascularity. We report that the efficiency of subcutaneous islet transplantation in a Lewis rat model is significantly improved by treating recipients with inhaled 50% oxygen, in conjunction with prevascularization of the graft bed by agarose-basic fibroblast growth factor. Administration of 50% oxygen increased oxygen tension in the subcutaneous site to 140 mm Hg, compared to 45 mm Hg under ambient air. In vitro, islets cultured under 140 mm Hg oxygen showed reduced central necrosis and increased insulin release, compared to those maintained in 45 mm Hg oxygen. Six hundred syngeneic islets subcutaneously transplanted into the prevascularized graft bed reversed diabetes when combined with postoperative 50% oxygen inhalation for 3 days, a number comparable to that required for intrahepatic transplantation; in the absence of oxygen treatment, diabetes was not reversed. Thus, we show oxygen inhalation to be a simple and promising approach to successfully establishing subcutaneous islet transplantation.

Pharmacological strategies for protection of extrahepatic islet transplantation.

The safety and effectiveness of islet transplantation has been proven through world-wide trials. However, acute and chronic islet loss has hindered the ultimate objective of becoming a widely used treatment option for type 1 diabetes. A large islet loss is attributed, in part, to the liver being a less-than-optimal site for transplantation. Over half of the transplanted islets are destroyed shortly after transplantation due to direct exposure to blood and non-specific inflammation. Successfully engrafted islets are continuously exposed to the liver micro-environment, a unique immune system, low oxygen tension, toxins and high glucose, which is toxic to islets, leading to premature islet dysfunction/death. Investigations have continued to search for alternate sites to transplant islets that provide a better environment for prolonged function and survival. This article gathers courses and conditions that lead to islet loss, from organ procurement through islet transplantation, with special emphasis on hypoxia, oxidative stress, and antigen non-specific inflammation, and reviews strategies using pharmacological agents that have shown effectiveness in protecting islets, including a new treatment approach utilizing siRNA. Pharmacological agents that support islet survival and promote ß-cell proliferation are also included. Treatment of donor pancreata and/or islets with these agents should increase the effectiveness of islets transplanted into extrahepatic sites. Furthermore, the development of methods designed to release these agents over an extended period, will further increase their efficacy. This requires the combined efforts of both islet transplant biologists and bioengineers.

mRNA of the pro-apoptotic gene BBC3 serves as a molecular marker for TNF-α-induced islet damage in humans.

AIMS/HYPOTHESIS: TNF-α plays important roles in the pathogenesis of type 1 and type 2 diabetes mellitus. In light of this, we examined the involvement of a pro-apoptotic gene, BBC3 (also known as PUMA), in TNF-α-mediated beta cell dysfunction and destruction in human islets. METHODS: Human islets were exposed in vitro to TNF-α alone or in combination with IFN-γ. Gene expression was assessed by RT-PCR using a set of single islets. Protein abundance and cellular localisation of BBC3 were assessed by immunoblot and immunohistochemistry. A marginal number of islets were transplanted into diabetic NODscid mice to correlate in vivo islet function with BBC3 expression. RESULTS: BBC3 and IL8 mRNA were upregulated in TNF-α-stimulated islets in a dose-dependent manner and enhanced through addition of IFN-γ, but not upregulated by IFN-γ alone. Immunohistochemistry revealed that TNF-α in combination with IFN-γ upregulated basal BBC3 abundance in the cytoplasm of beta cells along with the perinuclear clustering of mitochondria partially co-localised with BBC3. TNF-α alone did not induce beta cell death, but did abrogate preproinsulin precursor mRNA synthesis in response to high glucose stimulation, which was inversely associated with upregulation of BBC3 mRNA expression by TNF-α. Higher BBC3 mRNA expression in islets correlated with decreased graft function in vivo. CONCLUSIONS/INTERPRETATION: These results suggest that BBC3 mRNA can serve as a molecular marker to detect early TNF-α-induced beta cell stress and may help identify islet-protective compounds for the treatment of diabetes.

Modulating autoimmune responses to GAD inhibits disease progression and prolongs islet graft survival in diabetes-prone mice.

In nonobese diabetic (NOD) mice, beta-cell reactive T-helper type 1 (Th1) responses develop spontaneously and gradually spread, creating a cascade of responses that ultimately destroys the beta-cells. The diversity of the autoreactive T-cell repertoire creates a major obstacle to the development of therapeutics. We show that even in the presence of established Th1 responses, it is possible to induce autoantigen-specific anti-inflammatory Th2 responses. Immune deviation of T-cell responses to the beta-cell autoantigen glutamate decarboxylase (GAD65), induced an active form of self-tolerance that was associated with an inhibition of disease progression in prediabetic mice and prolonged survival of syngeneic islet grafts in diabetic NOD mice. Thus, modulation of autoantigen-specific Th1/Th2 balances may provide a minimally invasive means of downregulating established pathogenic autoimmune responses.

Expression of genetically determined diabetes and insulitis in the nonobese diabetic (NOD) mouse at the level of bone marrow-derived cells. Transfer of diabetes and insulitis to nondiabetic (NOD X B10) F1 mice with bone marrow cells from NOD mice.

The development of autoimmune diabetes in the nonobese diabetic (NOD) mouse is controlled by at least three recessive loci, including one linked to the MHC. To determine whether any of these genetic loci exert their effects via the immune system, radiation bone marrow chimeras were constructed in which (NOD X B10)F1-irradiated recipients were reconstituted with NOD bone marrow cells. Unmanipulated (NOD X B10)F1 mice, or irradiated F1 mice reconstituted with F1 or B10 bone marrow, did not display insulitis or diabetes. In contrast, insulitis was observed in a majority of the NOD----F1 chimeras and diabetes developed in 21% of the mice. These data demonstrate that expression of the diabetic phenotype in the NOD mouse is dependent on NOD-derived hematopoietic stem cells. Diabetogenic genes in the NOD mouse do not appear to function at the level of the insulin-producing beta cells since NOD----F1 chimeras not only developed insulitis and diabetes but also rejected beta cells within pancreas transplants from newborn B10 mice. These data suggest that the beta cells of the NOD mouse do not express a unique antigenic determinant that is the target of the autoimmune response.

Genetic control of diabetes and insulitis in the nonobese diabetic (NOD) mouse.

Genetic analysis of the development of diabetes and insulitis has been performed in the nonobese diabetic (NOD) mouse strain, a model of insulin-dependent (type I) diabetes mellitus. (NOD X C57BL/10)F1, F2, and (F1 X NOD) first-, second-, and third-backcross generations were studied. The data obtained were consistent with the hypothesis that diabetes is controlled by at least three functionally recessive diabetogenic genes, or gene complexes, one of which is linked to the MHC of the NOD. In contrast, pancreatic inflammation leading to insulitis was found to be controlled by a single incompletely dominant gene. One of the two diabetogenic loci that is not linked to the MHC appears to be essential for the development of severe insulitis. This diabetogenic gene may be identical to the gene that controls the initiation of the autoimmune response that progresses to insulitis. Although this gene appears to be functionally recessive in its control of diabetes, it is incompletely dominant in its control of insulitis. The MHC-linked diabetogenic gene, although not required for the development of insulitis, apparently influences the progression of the autoimmune response since NOD MHC homozygotes in the backcross generations displayed the highest incidence and most severe cases of insulitis. Interestingly, we have found two MHC heterozygous backcross females that have become diabetic, suggesting that either the MHC-linked diabetogenic gene is not strictly recessive or that a recombination event has occurred between the diabetogenic gene and the K or I-A regions of the MHC. The third diabetogenic locus appears to influence the progression of severe insulitis to overt diabetes. In animals homozygous at this locus, diabetes may result from a decreased ability to develop a protective suppressor response to the autoimmune process.

Control of experimental diabetes mellitus in rats by transplantation of fetal pancreases.

Experimental diabetes mellitus in young adult Lewis rats was successfully treated by transplantation of fetal pancreases from syngeneic fetuses. Complete or partial control lasting up to 165 days was achieved in 64 percent of recipients by using two to three pancreases of fetal age (15 to 18(1/2) days) placed under each kidney capsule. Islets of Langerhans without exocrine elements were present in the transplants.

Complete reversal of experimental diabetes mellitus in rats by a single fetal pancreas.

Complete reversal of streptozotocin-induced diabetes mellitus in adult rats will follow transplantation of a single fetal pancreas if the organ is first grown in a normal syngeneic carrier before transfer to the diabetic recipient. Careful control of the blood sugar of diabetic recipients may enhance the function of a single donor organ and thus improve histocompatibility matching.

Importance of hepatic portal circulation for insulin action in streptozotocin-diabetic rats transplanted with fetal pancreases.

The importance of the hepatic portal circulation in the response to insulin was assessed in streptozotocin-diabetic rats transplanted with syngeneic fetal pancreases. Partial reversal of diabetes was accomplished by transplantation of two or three fetal pancreases beneath the capsule of the kidney; complete reversal followed shunting of the venous drainage from the transplants to the liver. Plasma glucose after streptozotocin of 509+/-31 mg/dl (mean+/-SEM) fell after transplantation to 395+/-23 and after the shunt to 143+/-5 mg/dl. Urine volume fell from 84+/-4 to 50+/-5 ml/d and then to normal (17+/-1 ml/d) after the shunt. Glucose excretion which was 8.1+/-0.3 g/d after streptozotocin fell after transplantation to 4.8+/-0.3 g/d and after the shunt completely disappeared from the urine. The disappearance rate of glucose injected into the circulation, which was 0.50+/-0.07%/min in untreated diabetes, increased to 1.39+/-0.38%/min after transplantation and to 2.52+/-0.31%/min after the shunt, not different from normal controls (2.79+/-0.25). Plasma immunoreactive insulin (IRI) was below normal (25-35 muU/ml) and unresponsive to glucose in untreated diabetic rats. After transplantation IRI levels ranged from 73-223 muU/ml and there was no rise after glucose injection. After the shunt both the basal IRI (36+/-5 muU/ml) and the peak response to glucose at 10 min (58+/-7 muU/ml) were the same as in normal controls (42+/-4 and 62+/-7 muU/ml, respectively). The fall in IRI after the shunt is explained by increased extraction of insulin passing into the liver and also diminished secretion. After removal of the transplants plasma glucose and urine values returned almost to pretransplant levels. Secretion of insulin by transplanted pancreases into the liver enhances the effectiveness probably by increased extraction and action and reveals the importance of the normal route for insulin delivery.

Transfer of autoimmune diabetes mellitus with splenocytes from nonobese diabetic (NOD) mice.

The nonobese diabetic (NOD) mouse, a model of human type I diabetes, develops insulitis beginning at 4-6 wk of age. By 30 wk of age, 72% of females and 39% of males develop spontaneous diabetes, apparently because of an overwhelming autoimmune response to the insulin-producing beta-cells within the islets. To identify the immune mechanism responsible for destruction of beta-cells in the NOD mouse, we developed an adoptive transfer protocol that induces diabetes in NOD mice at an age when spontaneous diabetes is rarely observed. Splenocytes from overtly diabetic NOD mice were unable to transfer diabetes to very young (less than or equal to 6 wk) irradiated NOD mice but effectively transferred diabetes to irradiated NOD mice greater than 6 wk of age. In such transfers, overt diabetes was induced within 12-22 days in greater than 95% (79/82) of the recipients. Thus, transfer of splenocytes to young mice induces them to become diabetic at a higher frequency and at a younger age than their untreated littermates. Equally successful transfers with as few as 5 X 10(6) spleen cells have been performed in male and female NOD mice, even though males display a lower spontaneous incidence of diabetes than females. Splenocytes obtained from diabetic mice maintained on insulin for up to 2 mo also transferred diabetes. Because NOD mice display increasing levels of insulitis with age, spleen cells obtained from nondiabetic NOD mice of different ages were tested for their ability to transfer diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Normalization of six key hepatic enzymes after fetal pancreas transplantation in diabetic rats.

In diabetic rats transplanted with fetal pancreata we measured the activities of six important enzymes to assess the return of liver metabolism to normal. Comparison was made among the responses of transplanted rats with and without renal-portal vein shunts and of those not transplanted and injected with insulin in varying doses. Insulin supply was not limited since three or four fetal pancreata were first grown in normal rats before transfer into the diabetic animals. Transplantation normalized blood and urine glucose and the rate of disappearance of intravenous glucose. Glucokinase and pyruvate kinase activities in liver rose toward normal at 7 days after transplantation and reached normal levels at 30 and 90 days. The response of the other four enzymes, glucose-6-phosphate dehydrogenase, citric lyase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase, was more rapidly restored to normal at 7 days and remained normal at 30 and 90 days. No difference was observed in the enzyme activities of transplanted-shunted rats to nonshunted animals. Glucokinase activity was restored to normal after 1 wk of daily injections of 1 U of PZI; pyruvate kinase restoration required 3 U/day. Glucose-6-phosphate dehydrogenase and citric lyase required 2 U/day to be restored to normal; 3 U daily resulted in temporary supernormal activities. The gluconeogenic enzymes, fructose-1,6-bisphosphatase and glucose-6-phosphatase, were only partially suppressed toward normal by insulin even with 3 U daily for 3 wk. These findings indicate that pancreas transplantation is a more effective regulator of liver metabolism in diabetes than insulin injections.

Fetal pancreas transplantation for reversal of streptozotocin-induced diabetes in rats.

Streptozotocin-induced diabetes in rats was completely reversed by transplantation of syngeneic fetal pancreases placed beneath the kidney capsule. To accomplish complete reversal of diabetes, four or more pancreases were necessary; three resulted in partial reversal, and two produced a slight but significant effect in some recipients. Removal of the transplants resulted in the prompt return of diabetes. The islets of Langerhans in the transplants functioned homeostatically; this was indicated by regular normal blood glucose values, in addition to normal findings in blood IRI response and glucose disappearance rate after glucose injection. Disappearance of exocrine elements, with only ducts and fibrous tissue remaining, resulted in a pure endocrine organ. The advantages of this technie, such as ease of accessibility for placement, observation, and removal, are of great importance for possible application to humans.

Fetal pancreas transplantation in miniature swine. II. Survival of fetal pig pancreas allografts cultured at room temperature.

Islet-allograft survival has been shown to be markedly prolonged in rodents when donor tissue has been precultured at 24 degrees C. In this study, the feasibility of this approach was tested in NIH minipigs transplanted with fetal pancreases. Collagenase-digested fetal pig pancreatic tissues survived in culture at 24 degrees C for 6-7 days and continued to grow in vitro at 37 degrees C after being transferred. These tissues no longer stimulated allogeneic lymphocytes in vitro, although some tissues cultured at 37 degrees C did. This allogeneic stimulation did not correlate to the number of major histocompatibility complex (MHC) class II-positive cells in stimulator pancreatic cultures. When transplanted into an omentum pouch of normal, nonimmunosuppressed minipigs, fresh fetal pancreatic tissues were rejected within 14 days. Tissues cultured at 24 degrees C grew, and beta-cells proliferated in minipigs treated daily with cyclosporin A (CsA) and azathioprine. Twelve normal minipigs were transplanted with 24 degrees C-cultured fetal pancreases: 8 pigs received no treatment, 2 received 14 mg.kg-1.day-1 CsA for 14 days, and 2 received 6-7 intravenous injections of platelets prepared from pooled farm-pig blood before grafting. Strong lymphocytic infiltration was detected in all grafts removed between 30 and 90 days posttransplantation. However, beta-cells were found on day 45 in one of five minipig pancreas grafts incompatible at the MHC loci and on days 60-90 in all three grafts compatible at MHC but incompatible at minor histocompatibility loci. Short-term CsA treatment did not prolong survival of allografts from farm pigs into minipigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Multipotent progenitor cells isolated from adult human pancreatic tissue.

The supply of islet cells is a limiting factor for the widespread application of islet transplantation of type-1 diabetes. Islets constitute 1% to 2% of pancreatic tissue, leaving approximately 98% as discard after islet isolation and purification. In this report we present our data on the isolation of multipotent progenitor cells from discarded adult human pancreatic tissue. The collected cells from discarded nonislet fractions, after enzymatic digestion and gradient purification of islets, were dissociated for suspension culture in a serum-free medium. The cell clusters grown to a size of 100 to 150 mum contained cells staining for stage-specific embryonic antigens, but not insulin or C-peptide. To direct cell differentiation toward islets, clusters were recultured in a pancreatic differentiation medium. Insulin and C-peptide-positive cells by immunocytochemistry appeared within a week, reaching over 10% of the cell population. Glucagon and somatostatin-positive cells were also detected. The cell clusters were found to secrete insulin in response to glucose stimulation. Cells from the same clusters also had the capacity for differentiation into neural cells, as documented by staining for neural and glial cell markers when cultured as monolayers in media containing neurotrophic factors. These data suggest that multipotent pancreatic progenitor cells exist within the human pancreatic tissue that is typically discarded during islet isolation procedures. These adult progenitor cells can be successfully differentiated into insulin-producing cells, and thus they have the potential for treatment of type-1 diabetes mellitus.

Inhibition of p38 mitogen-activated protein kinase protects human islets from cryoinjury and improves the yield, viability, and quality of frozen-thawed islets.

The development of an optimal islet cryopreservation method will permit transplantation of islets from multiple donors in a single procedure and contribute to alleviation of the islet shortage. In this study, we have improved human islet cryopreservation methods under serum-free conditions using an intracellular-based islet cryopreservation solution (ICS), especially supplemented with a p38 pathway inhibitor (p38IH) to suppress p38 mitogen-activated protein kinase (MAPK) activation. Three different solutions were compared for freezing and thawing of human islets (1) conventional RPMI1640 medium, (2) ICS, and (3) ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Islet cryopreservation with ICS-p38IH significantly improved islet recovery, viability, and quality after thawing of cryopreserved islets. This improvement may allow the use of cryopreserved islets in clinical islet transplantation.

Micro mixed hemadsorption assay--a sensitive method for detecting antibodies against tumor-associated antigens.

Antibodies against methylcholanthrene (MCA)-induced sarcomas in Fischer (F344) rats were detected by a modified micro mixed hemadsorption (MHA)-assay. The assays detected anti-tumor antibodies as titers up to 1 : 320 in sera from hyperimmunized rats and at titers up to 1 : 160 in sera from rats bearing a 5-8 cm3 progressively growing tumor. MHA-titers decreased when sera were absorbed with sarcoma cells prior to MHA-assays. IgG antibodies in sera from tumor bearing rats showed titers of 1:20 and 1:5. These anti-tumor sera formed rosettes on the corresponding sarcoma cells as well as other sarcomes induced by MCA in F344 and Lewis strain rats tested. The assay was modified for a micro technique using a microtest plate (No. 3034, Falcon, CA). This modification yielded as assay requiring only 10 microliter of test sera. The test is quantitative and highly sensitive and results are reproducible. Several critical factors which influence test results in this assay were examined.

Characteristics of specific unresponsiveness toward kidney and skin allografts in adult rats inoculated at birth with allogenic bone marrow or kidney cells across a strong H-1 barrier.

Specific unresponsiveness inducible in newborn Lewis (Le) rats by injection of bone marrow or kidney cells from (Bu x Le)F1 hybrids was tested in adults by allografting of skin or kidneys from strongly incompatible Buffalo (Bu) strain donors. High doses of bone marrow cells (10(7) or more) proved quite effective in procuring indefinite survival of subsequent Bu kidney allografts, but not Bu skin allografts. Kidney allografts remained fully functional without modulation of their inherent immunogenicity regardless whether skin allografts were acutely rejected before or after kidney grafting on bone marrow-tolerant recipients. Lower doses of bone marrow cells were either near threshold for induction of specific Bu kidney allograft tolerance (5 x 10(6)) or, conversely, led to increased immunity(1-5 x 106). A strikingly different pattern of reactivity was found in adult Le recipients of Bu skin allografts as shown by only a moderate, but stepwise increase in survival times as a function of increasing allogeneic cell dosage at birth. Allogeneic kidney cells at all doses were surprisingly ineffective in inducing tolerance of either kidney or skin allografts, so the existence of kidney-specific transplantation antigens remains problematical. Lymphocytes from kidney allograft-tolerant recipients showed much reduced but still significant antidonor activity in several tests of cell-mediated immunity in vitro and in vivo. Blocking antibody activity was also demonstrable in these animals. Acquired tolerance (i.e., essential nonreactivity) may not only exist in degrees in either T or B cell pathways but may coexist with specific immunoblocking reactions in a dynamic equilibrium. Instead of tolerance versus enhancement, a new concept of selective specific immunoregulation emerges.

Prevention of overt diabetes and insulitis by intrathymic injection of syngeneic islets in newborn nonobese diabetic (NOD) mice.

Based on previous studies showing that allogeneic islets transplanted into the thymus can induce donor-specific unresponsiveness, we investigated in the nonobese diabetic (NOD) mouse the effect of intrathymic islet isografts on preventing autoimmunity directed against pancreatic islet antigens. Islets prepared from newborn NOD pancreata were injected into one lobe of the thymus of 10- to 11-day-old female NOD mice (experimental group) with no immunosuppression. PBS alone was used for injection into age- and sex-matched litter mates (control group). Thirty of 32 (94%) experimental mice remained normoglycemic for over 30 weeks. Well-formed islets with no indication of insulitis were found in the thymus of these 30 mice, whereas no grafted islets were found in the 2 mice that became diabetic at 17 and 19 weeks, respectively (technical failures). In the control group, 10 of 32 (31%) mice became diabetic between 20 and 29 weeks. This diabetic incidence was, however, lower than that in our colony female mice. In the pancreas of experimental mice, 90.9% of islets were free of infiltrates, whereas only 13.1% of islets were intact in control mice. The spleens of 30-week-old experimental mice contained a slightly higher percentage of CD8+ T cells (P < 0.05) than those of control mice. Cyclophosphamide injections at 30 weeks induced diabetes in 4 of 9 experimental mice. The 2 lines of evidence, (1) marked reduction in insulitis of intrathymic islet-grafted mice and (2) induction of diabetes after treatment with cyclophosphamide, suggest that both thymic clonal deletion and peripheral tolerance may play a role in preventing diabetes.

Fetal pancreas allografts for reversal of diabetes in rats. I. Allograft survival in nonimmunosuppressed recipients.

Survival times of allogeneic fetal pancreases were determined across various immunogenetic barriers using several inbred rat strains. Pancreases from 17-day-old embryos transplanted beneath the kidney capsule of normal, nonimmunosuppressed recipients were richly vascularized within a short period regardless of histoincompatibilities. The transplants grew progressively, increased beta cell number, and synthesized insulin at a rate similar to that in isografts until rejection intervened. Survival end points were scored by (1) complete disappearance of intrinsic vascularization and necrotic change of tissue by gross observation of grafts, (2) a sharp decrease in insulin content in a graft as compared to that in a control isograft, and (3) destruction and disappearance of beta cells in histological examination. Although fetal pancreases were rejected within 10 days in all strain combinations, there were clear differences in host immune-reactions in terms of immunogenetic barriers between donor and recipient.

Cytotoxic versus immunoblocking effects of specific alloantibodies: effects of IgM, IgG, and IgG2 on rat kidney allograft survival.

Passively introduced IgM alloantibodies of antidonor specificity regularly led to decreased kidney allograft survival times, whereas IgG alloantibodies from the same hyperimmune sera had a specific immunoblocking effect that promoted prolonged allograft survival. These strikingly opposite effects as a function of antibody isotype occurred across both strong H-1 (Ag-B) and moderate non-H-1 histocompatibility barriers. However, IgM and IgG directed against non-H-1 specificities were far more effective, respectively, in either curtailing or prolonging renal allograft survival. In the Fischer to Lewis strain combination, this was reflected in a 4-fold diminution in median survival time of IgM-treated recipients from 115 to 31 days, in contrast to indefinitely prolonged survival of IgG-treated recipients (greater than 350 days). Purified IgG2 alloantibodies proved as effective as the whole IgG fraction in passively promoting long-term renal allograft survival across a strong H-1 barrier. Conflicting data from other sources is evaluated. Possible mechanisms of specific immunoregulation are briefly discussed in relation to antibody properties of specificity, idiotype, class-subclass, and avidity-affinity.