Co-incubation of pig islet cells with spleen cells from non-obese diabetic mice causes decreased insulin release by non-T-cell- and T-cell-mediated mechanisms.

In vitro studies were conducted in the non-obese diabetic (NOD) mouse, prone to Type 1 autoimmune diabetes, to investigate the mechanisms involved in cell-mediated rejection of pig islet xenografts. Our previous work concerning the mechanisms of proliferation of xenogeneic lymphocytes to pig islet cells (PIC) was not indicative of PIC impairment. Consequently, a test was developed based on perifusion analysis of the alteration of basal and stimulated insulin release from adult PIC incubated with mouse splenocytes or subsets. Compared with PIC incubation alone or with syngeneic pig splenocytes, co-incubation with mouse whole spleen cells resulted in a decrease of basal and stimulated insulin release (P < 0.001). Two components of this alteration were detected separately: PIC impairment was decreased (P < 0.01) after removal of plastic-adherent cells from spleen cells, but maintained (P < 0.01) when plastic-adherent cells alone were co-incubated with PIC. The increase of murine interleukin-1 beta when mouse plastic-adherent spleen cells were cultured with PIC (P < 0.04) was indicative of macrophage activation. Soluble factors produced during co-incubation of mouse splenocytes or plastic-adherent cells with PIC were involved in the impairment process, since supernatant fluids collected during previous PIC-mouse cell co-incubations directly altered (P < 0.01) insulin release from PIC. Moreover, impairment of PIC by mouse spleen cells was abolished (P < 0.01) by gadolinium chloride (which inhibits macrophages), but not by cyclosporin A. Another mechanism was apparent, since co-incubation of PIC with purified mouse T cells or CD4+ T cells, re-mixed with antigen-presenting cells, led to a decrease (P < 0.01) of insulin release. This model, based on the alteration of dynamic basal and stimulated insulin release, is indicative of in vitro cell-mediated alteration of PIC in the NOD mouse. The effect of whole spleen cells was rapid, and a crucial role was played by plastic-adherent cells. Two mechanisms were responsible for the behaviour of these cells: an early direct effect (at least in part via soluble products); and the indirect presentation of PIC xenoantigens (leading to impairment by CD4+ T lymphocytes).

In vitro xenorecognition of adult pig pancreatic islet cells by splenocytes from nonobese diabetic or non-diabetes-prone mice.

BACKGROUND: In vitro studies of the nonobese diabetic (NOD) mouse prone to type 1 autoimmune diabetes were conducted in order to investigate the mechanisms possibly involved in cell-mediated rejection of adult pig islet xenografts. Mouse cellular proliferation in discordant situations was previously investigated only with stimulator lymphocytes and found to be low in intensity and due to an indirect recognition mechanism involving murine antigen-presenting cells (APC). It was also important to characterize murine anti-pig islet response. METHODS AND RESULTS: In the present study, mouse splenocytes responded to pig islet cells since primary proliferations were detected in non-diabetes-prone Balb/c (P<0.04) or NOD (P<0.001) mice. Moreover, NOD mice displayed a higher (P<0.003) splenocyte response to pig islet cells (stimulation index: 5.8+/-0.7) than did Balb/c mice (stimulation index: 2.3+/-0.3), whereas responses to pig stimulator splenocytes were similar in both strains. The proliferation of NOD splenocytes to pig islet cells was lower (P<0.0001) than the allogeneic response to Balb/c islet cells but similar to syngeneic proliferation to NOD islet cells. In both NOD and Balb/c mice, splenocyte proliferation to pig islet cells was abolished (P<0.01) when CD4+ cells were blocked with antibodies, whereas the blocking of CD8+ cells had a nonsignificant effect. The main T-splenocyte subsets involved were restricted to mouse MHC class II molecules as they did not proliferate in the presence of monoclonal antibodies directed at I-A molecules. NOD and Balb/c splenocyte proliferation to pig islet cells was abolished after removal of plastic-adherent APC, which indicates that the major activation pathway was indirect. Purified CD4+ or CD8- cells alone did not proliferate in response to pig islet cells but recovered a proliferative ability when mixed with APC. CD4- cells, alone or in the presence of APC, were not capable of responding to pig islet cells. Both Th1 and Th2 splenocytes were involved in response to pig islet cells since interferon-gamma (IFN-gamma) and interleukin (IL-)-4 production increased significantly (300-fold and 11-fold, respectively, P<0.02 for both), whereas the increase in IL-10 production was much lower (only 1.5-fold). The IFN-gamma/IL-4 and IFN-gamma/IL-10 ratios stimulated by pig islet cells were not different with NOD and Balb/c splenocytes. CONCLUSION: In conclusion, mouse cell-mediated reaction against xenogeneic adult pig islet cells mainly involves class II-restricted CD4+ T lymphocytes of Th1 and Th2 subtypes, with an indirect pathway for the recognition. Although of low intensity, this cell-mediated reaction constitutes an obstacle to pig islet engraftment in the mouse, although one not necessarily more insurmountable than alloreactivity. The peculiarity of NOD mouse splenocytes, in terms of proliferation against pig islets, suggests that the study of islet xenograft rejection should take the immunogenetic context of diabetes into account, in which case the use of non-diabetes-prone mice has its limitations.

Perifusion analysis of insulin secretion from specific pathogen-free large-white pig islets shows satisfactory functional characteristics for xenografts in humans.

Though the pig appears to be the islet donor of choice for grafts in diabetic patients, there may be a risk of transmission of infectious agents. In this context, we adopted a strategy of islet isolation from pigs raised and killed in specific pathogen-free (SPF) conditions as a minimum with regard to the concept of quality assurance. Accordingly, the present study investigated the function of SPF pig islets to determine whether they react qualitatively and quantitatively to nutriments, hormones and neuromediators with which they would be confronted in man and could therefore provide effective regulation during physiologic or physiopathologic situations. beta cells from 18 Large-White SPF pigs were functionally intact after 7 days in culture. Insulin stimulation indexes (SI) of 3.1 +/- 0.2, 2.2 +/- 0.1, and 4.4 +/- 0.3 were found respectively for 30 mmol/l K+, 100 mumol/l tolbutamide and 10 mmol/l theophylline. Basal insulin secretion (72.2 +/- 7.6 muU/min) had already increased significantly (p < 0.001) with 5.5 mmol/l glucose (184.2 +/- 25.5 muU/min, SI: 2.5 +/- 0.6), indicating that the threshold stimulatory concentration was comparable to that of human islets. Insulin secretion increased in a glucose dose-dependent manner (p < 0.001): SI: 3.1 +/- 0.3 and 3.6 +/- 0.2 with 11.0 mmol/l and 22.0 mmol/l glucose, which showed a satisfactory magnitude with reference to human islets. Even the subtle phenomenon of "glucose memory" was apparent in these pig islets. Arginine stimulated (p < 0.001) insulin secretion dose-dependently (SI: 2.2 +/- 0.3 with 5 mmol/l and 2.9 +/- 0.2 with 10 mmol/l). The ketone body beta-hydroxybutyrate (10 mmol/l) also induced insulin secretion (SI: 4.3 +/- 0.3). Insulin release was stimulated by 4 mumol/l gastric inhibitory peptide, revealing sensitivity to the hormonal enteroinsular axis, and by 2 mumol/l glucagon. Parasympathetic cholinergic influence was studied using 500 mumol/l carbamylcholine, which increased insulin secretion. The influence of orthosympathetic control and of stress situations was also studied. As in human islet response, epinephrine and the alpha 2-agonist clonidine (50 mumol/l) inhibited insulin secretion. Finally pre-culture of islets may be beneficial for graft outcome, provided that no deterioration in islet function occurs. A prolonged 21-day culture of SPF pig islets showed no decrease in insulin response to glucose, arginine and potassium, even with an unaltered threshold stimulatory glucose concentration. Thus, Large-White SPF pigs and the application of our isolation procedure provided islets with functional characteristics reproducibly compatible with potential utilisation for effective regulation of glycaemia under physiologic and physiopathologic situations in humans.

Reversal of diabetes in non-obese diabetic mice by xenografts of porcine islets entrapped in hollow fibres composed of polyacrylonitrile-sodium methallylsulphonate copolymer.

Intraperitoneal xenografting of islets immunoprotected by semipermeable membranes is a potential method of avoiding rejection by reversal of diabetes without immunosuppression. In this preliminary study, a xenograft of porcine islets, immunoprotected in semipermeable hollow fibres composed of a hydrogel of a polyacrylonitrile-sodium methallylsulphonate copolymer (AN 69), was used to reverse autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse. A diabetic state was maintained in all 46 NOD mice which received transplants of empty fibres. Transplantation of encapsulated islets reversed the diabetic state in 37% (18/54) of the recipients. In these mice, nonfasting blood glucose concentration decreased within 24 h. Glycaemia was kept below the diabetic control range and the initial pretransplant value for 6 weeks. Recipient NOD mice suffered from the severe insulitis characteristic of clinical diabetes, confirming that reversal of the hyperglycaemic state was due solely to the xenografts. Pretransplant glycaemia was slightly (p < 0.05) higher in mice which remained diabetic after grafts of fibre-containing islets than in animals which experienced reversal of hyperglycaemia after transplantation) for the peritoneal cavity of recipients which had returned to normoglycaemia after grafting with islet-containing fibres. In all 4 cases, the islets responded to glucose during a perifusion assay. In 2 out of 4 grafts removed from mice which remained hyperglycaemic after grafting with islet-containing fibres (11, 13, 15 and 27 days after transplantation), no basal or stimulated insulin secretion was detectable. Histological sections of a total of 75 fibres retrieved from the peritoneal cavities of recipient NOD mice showed surrounding inflammation, with adherent cells, neovascularisation and fibrotic reaction. These preliminary results are promising for the continued development of this bioartificial pancreas for xenogeneic islet transplantation since they demonstrate that xenogeneic islets can survive in the autoimmune environment of the NOD mouse with spontaneous diabetes mimicking human IDDM).

Minimisation of microbial contamination for potential islet xenografts using specific pathogen-free pigs and a protected environment during tissue preparation.

One major risk of islet xenotransplantation is transmission of infections. We thus compared microbial contamination during preparation of islets from 4 pigs conventionally breeded and slaughtered or 8 specific pathogen free (SPF) pigs, and different environmental conditions during pancreas excision. Pancreas harvested in a slaughterhouse (for conventional pigs) or in a protected autopsy room (for SPF pigs) were soaked in betadine solution and submitted to enzymatic digestion with collagenase. Islets were purified on histopaque gradient with a COBE 2991 processor. For each step of the process, a 10 ml aliquot was harvested and microbial contamination was analysed. For all animals, contamination of livers, which were not soaked in betadine solution, was also examined. Analysis of livers from the 4 conventional pigs showed polymicrobial contaminations (1,122 +/- 841 CFU/mg) with several species of Staphylococcus, Streptococcus, Bacillus and Enterobacteriaceae. For these conventional pigs, soaking of pancreas in betadine solution and presence of antibiotics in all media decreased the pancreatic contamination compared to hepatic contamination, but were unable to suppress it, as transport solution and crude suspension obtained after the digestion step with collagenase showed persistent contamination (9.7 +/- 2.4 and 10.5 +/- 4 CFU/ml, respectively). After islet purification by histopaque gradient, no medium remained contaminated. During analysis of the 8 SPF pigs, no liver exhibited contamination. Analysis of medium from each preparation step showed complete absence of contamination for 7 pancreases. Only one contamination with Staphylococcus simulans was observed for one pancreas in transport solution (6 CFU/ml), and persisted in digestion medium (16 CFU/ml). Finally, all purified suspensions were completely sterile. In conclusion, breeding conditions of pig islet donors, and controlled environment for pancreas excision, considerably influence the risk of microbial contamination. In order to limit the risk, SPF pigs are a suitable and compulsory source of islets.

Prophylactic oral administration of metabolically active insulin entrapped in isobutylcyanoacrylate nanocapsules reduces the incidence of diabetes in nonobese diabetic mice.

Nonobese diabetic (NOD) mice develop an autoimmune disease with a long prodromal period and constitute a model for investigating the prevention of human type 1 diabetes. Since prophylactic insulin injections reduced the incidence of diabetes in NOD mice, we tested a new prophylactic strategy to prevent diabetes in NOD mice consisting of oral administration of insulin, protected in polyalkylcyanoacrylate nanocapsules from degradation in the gastrointestinal tract. In humans, this form of prophylactic insulin administration would be less constraining than insulin injections. Ninety female NOD mice were randomized at weaning and fed once a week (from 60 to 300 days of age) with insulin nanocapsules (100 U/kg) or empty nanocapsules. Within the group fed with insulin nanocapsules, the incidence of diabetes was reduced (38% vs 75%; P < 0.02), the onset of disease was delayed (P < 0.02), and the severity of lymphocytic inflammation of endogenous islets was reduced (P < 0.03). Although the oral treatment was stopped at 300 days of age, the incidence of diabetes at 360 days remained lower in mice previously fed insulin nanocapsules (P < 0.02). Previous feedings with insulin nanocapsules did not protect against cyclophosphamide-induced diabetes, since final incidence of diabetes (sum of the incidence during the initial 360 days and the further CY-induced incidence) reached the final incidence obtained in mice previously fed empty nanocapsules and treated with cyclophosphamide. Intestinal absorption of insulin nanocapsules was evidenced by HPLC separation of human insulin in NOD sera. During cotransfer, T splenocytes from mice fed insulin nanocapsules were able to reduce the capacity of T cells from diabetic donors to adoptively transfer the disease (P < 0.01). Antigens for islet-cell autoantibodies (ICA) in pancreata from both NOD groups were compared by immunofluorescence with the same ICA-positive human sera to ensure that differences were due to quantitative changes in antigen. These antigens, which could serve as an index of a possibly more extended antigen beta-cell rest, were decreased (P < 0.02) and pancreatic insulin content was reduced (P < 0.05) in mice fed with insulin nanocapsules, suggesting a mechanism of 'beta cell rest'. To summarize, early feeding with insulin nanocapsules reduces diabetes and insulitis in the NOD mouse model that mimics human type 1 diabetes. This may be due both to generation of cellular mechanisms that actively suppress disease and a decrease in antigens which makes beta cells less vulnerable to autoimmune aggression.

Prevention of diabetes in the nonobese diabetic mouse by oral immunological treatments. Comparative efficiency of human insulin and two bacterial antigens, lipopolysacharide from Escherichia coli and glycoprotein extract from Klebsiella pneumoniae.

As oral administration of insulin reduces the incidence of diabetes in NOD mice, and to achieve a better approximation of oral insulin trials being developed for human studies which will use human insulin, we attempted to determine the preventive efficacy of oral administration of human insulin rather than resorting to the animal insulins used in previous studies. As the strength of prevention obtained by oral insulin has not been adequately demonstrated, we determined whether the protection persisted after the oral treatment was discontinued and whether it was resistant to a diabetogenic injection of cyclophosphamide (CY). We also determined whether the effect of insulin could be increased by oral administration of lipopolysaccharide from Escherichia coli (LPS) or another immunostimulant (glycoprotein extracts from Klebsiella pneumoniae, GEKP) which may be more feasible for human application. Female NOD mice were fed once a week (from 35 to 300 days of age) with insulin, LPS, GEKP, insulin plus LPS, insulin plus GEKP, or PBS. A decreased incidence of diabetes were observed in animals fed human insulin (p < 0.01 incidence of diabetes at 300 days of age: 31% in mice fed with insulin and 65% in those fed PBS). Prevention by insulin was not enhanced by oral LPS or GEKP. Yet unexpectedly, mice fed with LPS alone or GEKP alone displayed decreases in diabetes incidence (p < 0.01). The severity of insulitis was reduced in animals fed insulin, LPS, GEKP or combinations of insulin and either immunostimulant (p < 0.02). Although the oral treatments were stopped at 300 days of age, the incidence of diabetes at 360 days remained lower in mice previously fed insulin, LPS, GEKP or combinations of insulin and either immunostimulant (p < 0.01). In mice previously fed PBS, CY injection (60 days after withdrawal of the oral treatment) led to a final incidence of diabetes of 90% (sum of the incidence during the initial 360 days and the further CY-induced incidence). Previous feedings with insulin, LPS, GEKP or combinations of insulin and either immunostimulant did not protect against CY-induced diabetes since incidences reached the final control incidence. T splenocytes from animals fed insulin, LPS, or GEKP, similarly reduced the capacity of T cells from diabetic mice to transfer the disease (p < 0.01). It is concluded that oral treatment with human insulin to be used in human trials reduces the incidence of diabetes in NOD mice. Equivalent preventive efficacy was obtained through feedings with LPS or GEKP (even though no cumulative efficiency was observed with insulin). The latter results suggest that it would be advisable to evaluate the efficiency of oral bacterial antigens for the prevention of human Type 1 diabetes. The protection afforded by oral treatments with insulin or bacterial antigens may be attributed to cellular suppression, persists for some time after treatments are stopped, but is not resistant to major immune stimulation such as injection of CY.

Immunization of non-obese diabetic (NOD) mice with glutamic acid decarboxylase-derived peptide 524-543 reduces cyclophosphamide-accelerated diabetes.

NOD mice constitute a model for studying the prevention of human autoimmune type 1 diabetes. Glutamic acid decarboxylase (GAD) could be a key antigen involved in this disease, and GAD65 peptide 524-543 has been implicated in early T cell response in young NOD mice. We performed two i.p. injections of GAD peptide 524-543 (100 micrograms at each injection), together with Freund's incomplete adjuvant (FIA), into female NOD mice at 30 and 45 days old. Diabetes was accelerated 2 weeks later by a single injection of cyclophosphamide (CY), which acts against suppressive mechanisms. Treatment with GAD 524-543 peptide delayed the onset of diabetes and reduced its incidence (28% versus 60%; P < 0.001) compared with control mice injected with FIA alone, or GAD peptide 534-553, or an irrelevant peptide. In the same group, the severity of lymphocytic inflammation of pancreatic islets was reduced (P < 0.03). Up to 3 months after peptide injections, a strong splenocytic proliferative response occurred in immunized NOD mice against the immunizing peptide alone (but not against a panel of seven other GAD65-derived peptides). After peptide challenge of splenocytes in vitro, protection against CY-accelerated diabetes was associated with higher peptide-specific production of T helper type 2 (Th2)-associated interleukins 4 and 10, whereas Th1-associated interferon-gamma and IL-2 were proportionally less represented. During contransfer, T splenocytes from GAD 524-543-immunized mice were able to reduce the capacity of T cells from diabetic donors to transfer the disease adoptively (P < 0.01), demonstrating the generation of cellular mechanisms that actively suppress the disease. It is concluded that immunization of NOD mice with GAD65 peptide 524-543 can counteract CY-accelerated diabetes, possibly through active cellular suppression linked to a shift of Th1/Th2 balance toward the production of Th2 cytokines such as IL-4 and IL-10. This study provides additional support for the notion that GAD, and more precisely its epitope 524-543, could be one of the key targets for the pathogenesis of type 1 diabetes in NOD mice, as well as for the efficacy of disease-specific peptide therapy in type 1 diabetes.

Xenografts of porcine islets immunoprotected in hollow fibres reduce the incidence of diabetes in non-obese diabetic mice.

Non-obese diabetic (NOD) mice develop an autoimmune disease with a long prodromal period and constitute a model for investigating the prevention of human insulin-dependent diabetes mellitus. Since insulin injected prophylactically has been shown to reduce incidence of diabetes in NOD mice, we tested a new strategy consisting of prophylactic xenografts of porcine pancreatic islets immunoprotected in semipermeable hollow fibres. Female NOD mice were transplanted twice (at 60 and 180 days of age) with islet-containing or empty fibres. Within the group grafted with protected islets, the incidence of diabetes was reduced (37 vs 75%; p < 0.01), the onset of disease was delayed (p < 0.02), and the severity of lymphocytic inflammation of endogenous islets was reduced (p < 0.02). When already diabetic mice were not taken into account for analysis, blood glucose level was slightly lower in those grafted with islet-containing fibres (p < 0.04). Graft function was also evidenced by HPLC separation of porcine insulin in NOD sera. Histological and perifusion studies of fibres retrieved from recipients confirmed immunoprotection. During co-transfer, T splenocytes from mice grafted with islet-containing fibres were able to reduce the capacity of T cells from diabetic donors to adoptively transfer the disease (p < 0.01). Antigens for islet-cell autoantibodies (ICA) in pancreata from both groups were compared by immunofluorescence with the same ICA-positive human sera to ensure that differences were due to antigen quantitative changes. These antigens, which could serve as an index of a possibly more extensive antigen beta-cell rest, were decreased (p < 0.01) in mice grafted with protected islets. Reduction of diabetes and insulitis following early islet transplantation may thus be due to generation of cellular mechanisms that actively suppress disease, and possibly in part to a decrease in antigens which make beta cells less vulnerable to autoimmune aggression. These effects can be obtained with xenogeneic islets protected in hollow fibres, thereby eliminating the need for immunosuppression. Based on the concept of prophylactic insulin therapy, this form of insulin administration offers a controlled means of delivering insulin to meet the physiological needs of recipients.