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PubMed1993

Establishment of islet-specific T-cell lines and clones from islet isografts placed in spontaneously diabetic NOD mice.

One impediment to detailed characterization of islet-specific T cells from the NOD mouse model of diabetes is the difficulty encountered in isolation of such cells. This report describes a method that allows routine isolation of relatively large numbers of T cells highly enriched for reactivity toward islet antigens. The method involves renal subcapsular transplantation of spontaneously diabetic NOD mice with NOD islets. These grafts are rapidly destroyed in a tissue-specific manner and this destruction is accompanied by lymphocytic infiltration. Here we demonstrate that the islet graft infiltrates are an excellent source of islet-specific T cells and that islet-specific T-cell lines and clones can be established from these cells. Islet-specific T-cell clones isolated from a T-cell line established from the islet graft infiltrates were capable of adoptive transfer of diabetes to NOD/LtSz-scid recipients.

Animals↗

PubMed1992

Detection of autoantibodies against islet amyloid polypeptide in human serum. Lack of association with type 1 (insulin-dependent) diabetes mellitus, or with conditions favouring amyloid deposition in islets. The Belgian Diabetes Registry.

A radiobinding assay for the detection of autoantibodies against islet amyloid polypeptide was developed, analytically validated, and--in parallel with a similar assay for the detection of autoantibodies against insulin--applied to sera from recent-onset Type 1 (insulin-dependent) diabetic patients and from age- and sex-matched control subjects. There was no difference in islet amyloid polypeptide autoantibody titres between patient groups and matched control subjects, nor within subject groups according to age. At onset of Type 1 diabetes, elevated islet amyloid polypeptide-autoantibody levels (> 97th percentile of control subjects) were only detected in 1 of 30 patients aged 0-19 years and in 2 of 35 patients aged 20-39 years. By contrast, insulin autoantibodies were frequently demonstrated, in particular at onset of diabetes under age 20 (0-19 years: 18 of 30 patients; 20-39 years: 10 of 35 patients; p < 0.01 vs matched control subjects). Islet amyloid polypeptide autoantibodies were not detectable in 3 insulinoma patients nor in 37 patients (aged 33-70 years) with Type 2 diabetes (vs 1 of 40 in matched control subjects). In positive serum, adsorption onto protein A-Sepharose removed islet amyloid polypeptide binding activity, hereby confirming its antibody nature. In conclusion, Type 1 diabetes is associated with an age-dependent autoantibody reaction against insulin but not against islet amyloid polypeptide. Conditions associated with amyloid deposition in islets (Type 2 diabetes, insulinoma and ageing) do not favour the formation of autoantibodies against islet amyloid polypeptide.

Adolescent↗

PubMed1996

Islet amyloid polypeptide (IAPP) secretion from pancreatic islets isolated from non-obese diabetic (NOD) mice.

The secretion of islet amyloid polypeptide (IAPP) during the course of insulin-dependent diabetes mellitus (IDDM) is essentially unknown. In the present study we elucidated this issue by examining IAPP and insulin secretion from isolated pancreatic islets obtained from IDDM-prone female NOD mice aged 6-9 weeks and 12-15 weeks and from non-IDDM-prone male NMRI mice. Basal islet hormone secretion at 1.7 mM glucose and after stimulation with 17 mM glucose or with 17 mM glucose + 5 mM theophylline was studied acutely or after 1 week of tissue culture. The levels of glucose-stimulated insulin release from NOD mouse islets increased after tissue culture, whereas it remained unchanged in NMRI mouse islets. Overall changes in islet insulin secretion were accompanied by similar changes in IAPP secretion. Acute after isolation, islets from NMRI mice displayed a reduced IAPP/insulin secretion ratio in response to the stimulation, which could reflect a destabilized hormone release. When the NOD mouse islets at 6-9 weeks of age were exposed to secretory stimuli the molar ratio of IAPP/insulin secretion declined. At a later stage of advanced insulitis (12-15 weeks) also the basal IAPP/insulin secretory ratio at low glucose tended to decline. If extrapolated to the early prediabetic phase of human IDDM, this would mean that a relative hypersecretion of insulin in relation to IAPP might occur, due to an increased secretory demand for insulin or due to an intrinsic change in the biology of the secretory cells.

Amyloid↗

PubMed2003

Inflammatory mediators expressed in human islets of Langerhans: implications for islet transplantation.

Expression of immune modulating mediators in human Islets of Langerhans could have important implications for development of autoimmunity in type 1 diabetes and influence the outcome of clinical islet transplantation. Islets obtained from five donors were analyzed at various times after isolation using cDNA array technology. The Atlas Human Cytokine/Receptor and Hematology/Immunology nylon membranes representing 268 genes and 406, respectively, were used and the relative expression of each gene analyzed. Of the 51 gene products identified, high mRNA expression of MCP-1, MIF, VEGF, and thymosin beta-10 was detected in all islet samples. IL-8, IL-1-beta, IL-5R, and INF-gamma antagonist were expressed in islets cultured for 2 days. IL-2R was expressed in islets cultured for more than 6 days. In conclusion, several inflammatory mediators were expressed in isolated islets, particularly at an early stage after isolation, indicating that a few days of culture could be beneficial for the outcome of islet transplantation.

Cells, Cultured↗

PubMed2001

Islet amyloid and type 2 diabetes: from molecular misfolding to islet pathophysiology.

Islet amyloid polypeptide (IAPP, amylin) is secreted from pancreatic islet beta-cells and converted to amyloid deposits in type 2 diabetes. Conversion from soluble monomer, IAPP 1-37, to beta-sheet fibrils involves changes in the molecular conformation, cellular biochemistry and diabetes-related factors. In addition to the recognised amyloidogenic region, human IAPP (hIAPP) 20-29, the peptides human or rat IAPP 30-37 and 8-20, assume beta-conformation and form fibrils. These three amyloidogenic regions of hIAPP can be modelled as a folding intermediate with an intramolecular beta-sheet. A hypothesis is proposed for co-secretion of proIAPP with proinsulin in diabetes and formation of a 'nidus' adjacent to islet capillaries for subsequent accumulation of secreted IAPP to form the deposit. Although intracellular fibrils have been identified in experimental systems, extracellular deposition predominates in animal models and man. Extensive fibril accumulations replace islet cells. The molecular species of IAPP that is cytotoxic remains controversial. However, since fibrils form invaginations in cell membranes, small non-toxic IAPP fibrillar or amorphous accumulations could affect beta-cell stimulus-secretion coupling. The level of production of hIAPP is important but not a primary factor in islet amyloidosis; there is little evidence for inappropriate IAPP hypersecretion in type 2 diabetes and amyloid formation is generated in transgenic mice overexpressing the gene for human IAPP only against a background of obesity. Animal models of islet amyloidosis suggest that diabetes is induced by the deposits whereas in man, fibril formation appears to result from diabetes-associated islet dysfunction. Islet secretory failure results from progressive amyloidosis which provides a target for new therapeutic interventions.

Amino Acid Sequence↗

PubMed1990

Survival of islet isografts despite cytotoxicity against pancreatic islets measured in vitro.

In this study the in vivo relevance of spleen cell anti-islet cytotoxicity measured in vitro was examined by transplantation of 1,200 syngeneic islets into the spleen of rats receiving 0.5 ml complete Freund's adjuvant (CFA) 24 h before 25 mg/kg body weight streptozotocin (STZ) was given. Control rats receiving CFA or STZ alone remained normoglycaemic whereas 12 out of 21 CFA/STZ-treated rats developed a severe hyperglycaemia after three combined treatments. After the first and second combined treatment splenocytes showed a significant cytotoxicity (p less than 0.01) against syngeneic islets measured by 51Cr-release. This cytotoxicity was not detectable after the third combined treatment. The CFA/STZ-induced diabetes with a residual pancreatic insulin content of only 5% was permanently reversed by intrasplenic islet isografts, but, surprisingly, syngeneic islets survived too, if transplanted at the time when an anti-islet cytotoxicity was measured in vitro. From our results we conclude that the polyclonal activation by complete Freund's adjuvant potentiates the beta cell-toxic effect of a low dose of streptozotocin and induced a transient splenocyte-mediated anti-islet cytotoxicity not recurrent after islet transplantation. Furthermore, our findings reveal a discrepancy between organ-specific immune reactions measured in vitro and those affecting the beta cells in vivo.

Animals↗

PubMed1991

Pretreatment of rat pancreatic islets with MHC I-A monoclonal antibody: in-vitro and in-vivo effects on islet antigenicity.

Pancreatic islet allografts are rejected very rapidly due to their high immunogenicity. Several attempts have been made to reduce the immunogenicity prior to transplantation. We treated islets with MHC Ia (class II) antigen monoclonal antibody and complement in order to lyse Ia antigen bearing cells within the islets. This treatment caused a distinct reduction of Ia antigen positive cells, which was demonstrated by indirect immunofluorescence tests. A total depletion of the Ia antigens, however, could not be achieved. As a second in-vitro test to evaluate the effect of the antibody treatment a mixed lymphocyte islet culture (MLIC) was performed. The partial Ia antigen reduction provoked a significantly reduced but not completely suppressed allogenic T-cell response. Antibody treatment did neither impair the morphological integrity nor the function of the islets as could be demonstrated by syngenic transplantation of islets pretreated by antibodies in the same way. After allotransplantation of antibody-pretreated islets across a major histocompatibility barrier into non immunosuppressed rats, however, there was no significant prolongation of the graft survival. Thus, a partial reduction of the number of the Ia antigen positive cells within the islets is not sufficient for the prevention of allograft rejection.

Animals↗

PubMed1980

Islet transplantation in experimental diabetes of the rat. VII. Cryopreservation of rat and human islets. Preliminary results.

Islet transplantation in human diabetes at present is confronted with two major obstacles: isolation of a sufficient number of islets and islet graft rejection. "Tissue banking" would enable islet pooling from various donors and offers furthermore the advantage of in vitro manipulations in order to reduce islet immunogenicity. Recently we have reported successful cryopreservation and subsequent transplantation of porcine islets (Bretzel, Beule, Schäfer, Schneider, Pfeiffer and Federlin 1979). These preliminary data deal with cryopreservation and transplantation of isolated rat islets and cryopreservation of isolated human islets.

Animals↗

PubMed2006

An in vitro model of early islet amyloid polypeptide (IAPP) fibrillogenesis using human IAPP-transgenic mouse islets.

The mechanisms underlying insufficient insulin secretion and loss of beta-cell mass in feline and human type 2 diabetes mellitus are incompletely understood. However, islet amyloid polypeptide (IAPP)-derived islet amyloidosis (IA) has been linked to increased rates of beta-cell apoptosis and, therefore, our goal was to develop an in vitro model of IAPP fibrillogenesis using isolated pancreatic islets from mice transgenic for human IAPP (hIAPP Tg mice). Islets from hIAPP Tg mice, from mice transgenic for non-amyloidogenic murine IAPP (mIAPP Tg mice), and from the FVB background strain were exposed to normal (5.5 mM) or high (28 mM) glucose conditions in cell culture for 8 days. On days 0 and 8, islets were collected for electron microscopy (EM). EM showed no abnormalities in the mIAPP Tg or FVB islets at either time point. On day 8, hIAPP Tg islets cultured at high glucose concentration formed extracellular IAPP-derived flocculent deposits. No significant differences in rates of apoptosis were found between groups. Our findings, therefore, show that in vitro culture of hIAPP Tg mouse islets under high glucose conditions produces a readily available and rapidly inducible model of IAPP-derived fibrillogenesis and enables the study of early phases of the molecular pathogenesis of IA.

Amyloid↗

PubMed1989

The specificity of rejection and the absence of susceptibility of pancreatic islet beta cells to nonspecific immune destruction in mixed strain islets grafted beneath the renal capsule in the rat.

The specificity of rejection of isolated pancreatic islets was examined in the rat using a quantitative model in which syngeneic (DA) or a mixture of syngeneic and allogeneic (DA and LEW or PVG) islets were implanted beneath the capsule of the kidney of nondiabetic normal rats (DA). 3 wk after transplantation total insulin extraction assays of the kidney with its islet implant together with immunohistological examination of the site of transplantation for evidence of syngeneic or allogeneic tissue demonstrated the total destruction of allogeneic islets without any evidence of damage to syngeneic islets either distant or in immediate proximity to allogeneic islets. Pancreatic islets, and especially beta cells, appear to be particularly vulnerable to the effector arm of both autoimmune and alloimmune responses, a vulnerability that has been attributed to the cytotoxic effects of lymphokines, notably IL-1, released in both autoimmune and alloimmune responses. The experiments reported here demonstrate not only the exquisite specificity of the allograft reaction but are not compatible with a hypothesis that B cells within an intact islet are nonspecifically susceptible to destruction by lymphokines.

Animals↗

PubMed2001

High glucose concentration favors the selective secretion of islet amyloid polypeptide through a constitutive secretory pathway in human pancreatic islets.

We studied the contribution of the constitutive and the regulated pathways to the total secretion of islet amyloid polypeptide (IAPP) in human pancreatic islets after prolonged culture at either 5.5 or 24.4 mM glucose. In islets cultured in low concentrations of glucose, the secretion of IAPP in response to glucose was unaffected by brefeldin A (BFA) and completely blocked by ethyleneglycoltetraacetic acid. In islets cultured in high glucose concentrations, it was strongly inhibited by both agents. BFA had no effect on the glucose-induced insulin secretion. The determination of the islet peptide contents and the mRNA levels revealed a several-fold increase in the IAPP/insulin molar ratio of islets cultured in high glucose concentrations. Thus, prolonged exposure of human islets to high concentrations of glucose results in an increase in the synthesis of IAPP with respect to insulin. As a result, the release of IAPP through a mechanism sensitive to BFA is favored. These data support the hypothesis that IAPP and insulin are regulated in a noncoordinated way in human pancreatic islets.

Amyloid↗

PubMed1997

Expression of CTLA4-Ig by biolistically transfected mouse islets promotes islet allograft survival.

BACKGROUND: Localized delivery of immunosuppressive molecules, limited to the graft site, may allow transplantation of tissue in the absence of systemic immunosuppressive agents. We tested whether purified mouse islets that had been engineered to produce human CTLA4-Ig locally at the graft site could survive in allogeneic recipients receiving no systemic immunosuppression. METHODS: CBA (H2(k)) islets were subjected to biolistic (gene gun) transfection with a cDNA encoding human CTLA4-Ig under control of the human cytomegalovirus immediate early promoter. After 40-48 hr of culture, the transfected islets (500 per recipient) were transplanted beneath the renal capsule of alloxan-induced diabetic BALB/c (H2(d)) recipients. RESULTS: Control grafts (n=10) consisting of islets biolistically transfected with the expression plasmid alone (i.e., no gene inserted) survived for 12.8+/-3.6 (mean +/- SD) days. In contrast, islets transfected with CTLA4-Ig (n=12) survived 66.8+/-61.5 days (P=0.01), with 50% demonstrating functional survival until follow-up was concluded at 50 (n=2), 130 (n=2), or 165 (n=2) days. Immunohistochemistry on grafts that survived long term showed well-granulated, insulin-positive islets lying adjacent to, but not infiltrated by, dense aggregates of mononuclear cells. CONCLUSIONS: Transfection of allogeneic mouse islets with human CTLA4-Ig results in prolonged allograft survival. Although on histology mononuclear cells are present in the area of the transfected graft, they do not appear to infiltrate or destroy the islet graft.

Abatacept↗

PubMed2002

Adeno-associated virus transduction of islets with interleukin-4 results in impaired metabolic function in syngeneic marginal islet mass transplantation.

Previous studies suggest that therapeutic expression of interleukin (IL)-4 by islet cells improves their efficacy in transplantation models directed at reversing type 1 diabetes. We investigated the effects of introducing IL-4 into islets with recombinant adeno-associated virus (rAAV) on the reversal of hyperglycemia in a syngeneic marginal islet mass transplantation model. C57BL/6 islets were mock-transduced or transduced with rAAV expressing murine IL-4 (rAAV-IL-4) or rAAV expressing green fluorescent protein (rAAV-GFP) before transplantation of a marginal mass into diabetic mice. Normoglycemia was achieved in only 1/7 mice receiving rAAV-IL-4 transduced islets in comparison to 6/6 mock-transduced and 4/6 rAAV-GFP transduced animals. The failure of IL-4 expressing islets was not associated with cellular toxicity of rAAV or impairment of glucose-stimulated insulin release in vitro. Islet expression of IL-4 led to impaired metabolic function in mice receiving a marginal mass of syngeneic islets.

Adenoviridae↗

PubMed2003

Rabbit antithymocyte globulin induction and sirolimus monotherapy supports prolonged islet allograft function in a nonhuman primate islet transplantation model.

BACKGROUND: We reported that rabbit anti-thymocyte globulin (RATG) induction followed by maintenance immunosuppression with sirolimus supports human kidney allograft survival and asked if this combination would promote islet allograft survival in our primate model. METHODS: Using intra-arterial streptozotocin infusion, we rendered four cynomolgus primates diabetic with undetectable C-peptide levels. Animals were maintained on insulin therapy for at least 1 month, and then islets from mixed lymphocyte reaction mismatched primates were infused into the portal vein. Immediately before the islet allotransplant and for 6 additional days, primates were infused with RATG (20 mg/kg) and given a sirolimus dose to achieve a 24-hr trough level of 8 to 14 ng/mL. RESULTS: The regimen resulted in profound peripheral and lymph node lymphocyte depletion for up to 1 month. Repopulation was gradual thereafter. One primate remained insulin-independent for 169 days and rejected after a sirolimus-dose reduction. Two primates died on day 23 while insulin independent because of wound dehiscence, and a third died on day 30 with high sirolimus levels. Liver sections revealed well-vascularized islets with no signs of inflammation. CONCLUSION: Using a nonhuman primate islet transplant model, RATG plus sirolimus supports islet survival as long as proper sirolimus levels are maintained, but the therapy is limited by sirolimus toxicity. Our findings suggest that RATG is not toxic for islets and thus may be considered in future clinical trails while recognizing that sirolimus monotherapy, with its difficult-to-achieve therapeutic dosing, may not be sufficient to maintain long-term islet allograft function in an autoimmune environment.

Animals↗

PubMed2003

Nicotinamide inhibits tissue factor expression in isolated human pancreatic islets: implications for clinical islet transplantation.

BACKGROUND: Islet-produced tissue factor (TF) triggers an adverse clotting reaction, the instant blood-mediated inflammatory reaction (IBMIR), providing a likely explanation for the need of tissue from multiple donors in clinical islet transplantation. In this study, the authors investigated whether compounds previously shown to affect TF and macrophage chemoattractant protein (MCP)-1 expression in monocytes and endothelial cells have the same effect in human islet cells. METHODS: Islets were cultured in the presence of l-arginine, cyclosporine A, enalapril, or nicotinamide for 48 hr, after which the TF content and MCP-1 expression were assessed. The effect of nicotinamide on IBMIR was evaluated by exposing the treated islets to fresh human ABO-compatible blood in an in vitro loop model. RESULTS: Nicotinamide was the only compound that significantly reduced both TF and MCP-1. This reduction was dose-dependent. The level of MCP-1 was strongly correlated with TF expression (r2=0.98). In addition, the level of TF was also correlated with the ability of the islets to initiate IBMIR (r2=0.94). CONCLUSIONS: TF and MCP-1 expression in human islets can be decreased by adding nicotinamide to the culture medium. These observations indicate that the adverse effects of IBMIR in clinical islet transplantation could be reduced without endangering the recipient using antithrombotic drugs.

Arginine↗

PubMed2004

Refinement of the automated method for human islet isolation and presentation of a closed system for in vitro islet culture.

BACKGROUND: The procedure of human islet isolation needs further optimization and standardization. Here, we describe techniques to enhance enzymatic digestion and minimize mechanical forces during the digestion process. The isolation protocol has also been modified to meet current GMP (cGMP) standards. Moreover, the impact of donor- and process-related factors was correlated to the use of islets for clinical transplantation. METHODS: One hundred twelve standardized consecutive islet isolations were evaluated. Metyltioninklorid and indermil (topical tissue adhesive) were applied to detect leakage of collagenase injected and to repair the damaged pancreatic glands. The effects of dye and glue were evaluated in terms of islet yield, islet function using the perifusion assay, and success rate of the isolation. To analyze key factors for successful isolations, both univariate and multivariate regression analysis were performed. RESULTS: Both Metyltioninklorid and Indermil were effective to prevent leakage of enzyme solutions from the pancreatic glands. Both islet yield and success rate were higher when these tools were applied (4,516.1+/-543.0 vs. 3,447.7+/-323.5, P=0.02; 50.0% vs. 21.3%, P=0.02, respectively). No adverse effects on islet function or collagenase activity were observed. Multivariate regression analysis identified the maximal recorded amylase >100 U/L (P=0.026), BMI (P=0.03), and the use of catecholamine (P=0.04) as crucial donor-related factors. In addition, cold ischemia time (P=0.005), the dissection procedure using whole glands with duodenum (P=0.02), and the local procurement team (P=0.03) were identified as crucial isolation-related variables. CONCLUSIONS: A standardized technique of islet isolation is presented applying novel means to improve enzymatic digestion and to meet cGMP standards.

Humans↗

PubMed1996

Islet amyloid and islet amyloid polypeptide in cynomolgus macaques (Macaca fascicularis): an animal model of human non-insulin-dependent diabetes mellitus.

To further characterize spontaneous diabetes mellitus in cynomolgus macaques (Macaca fascicularis) as a model for human non-insulin-dependent diabetes mellitus (NIDDM), we evaluated the morphologic characteristics of the endocrine pancreas of 4 diabetic and 12 age-matched nondiabetic cynomolgus macaques. In addition, the cDNA-predicted amino acid sequence for islet amyloid polypeptide (IAPP) of this species was determined. Islet amyloid deposits exhibiting typical congophilia and green birefringence were found in 4/4 diabetic animals and in 8/12 nondiabetics. Islet amyloid deposits were significantly more extensive in the diabetic macaques (P = 0.001), in which they occupied a mean of 60% of the islet area. In contrast, in the nondiabetic group the maximum islet area occupied by amyloid was 24% (group mean = 6.8%), with four animals having no detectable islet amyloid. Amyloid deposits consistently showed immunoreactivity for IAPP but not for insulin. Comparisons between group means for diabetic versus nondiabetic macaques showed significantly greater islet area (P = 0.01, 85,390 versus 36,540 microns 2) and significantly greater islet area fraction (P = 0.02, 0.065 versus 0.032) for the diabetic group. The cDNA-predicted amino acid sequence for cynomolgus IAPP was identical to that previously reported for pig-tail macaques (M. nemestrina) and had 92%, 86%, and 84% amino acid sequence identity with human, domestic cat, and murine IAPPs, respectively. These findings support the use of cynomolgus macaques as an animal model of human NIDDM.

Amino Acid Sequence↗

PubMedDate not supplied

Pretreatment of crude pancreatic islets with mitomycin C (MMC) prolongs islet graft survival in a xenogeneic rat-to-mouse model.

In this study, we examined the effect of mitomycin C (MMC) treatment on graft survival and evaluated its efficacy in immunomodulation of islet graft for transplantation. Male WS rats were used as islet donors and streptozotocin-induced diabetic C57BL/6 mice as recipients. The isolated islets were treated with MMC at concentrations of 0, 0.1, 1, 3.2, 10, 32, 100, 320, and 1000 microg/mL for 30 min, and were cultured for 20 h. Then, 300-400 islets were transplanted into the renal subcapsular space of diabetic mice. Significant prolongation of graft survival was obtained when the islets were treated with MMC at a concentration of 10, 32, or 100 microg/mL (MST 23 +/- 7.4, 17.5 +/- 5.4, 29.6 +/- 9.7 days: p < 0.003, p < 0.012, p < 0.001, respectively, vs. 12.3 +/- 2.7 days for culturing alone). Islets treated with MMC at a concentration of 320 microg/mL or more failed to restore normoglycemia in the diabetic recipient mice after transplantation. Viability of islets incubated with doses up to 100 microg/mL, assessed under the confocal microscope after propidium iodide and Hoechst 33342 staining, was maintained well comparable to that of freshly isolated islets, while those treated at 320 microg/mL was significantly decreased. Thus, a therapeutic window for MMC efficacy was found at concentrations from 10 microg/mL to 100 microg/mL. This modality is simple and effective and underlying molecular mechanisms need to be determined in the future.

Animals↗