Renal Subcapsular Space Research Articles

Autotransplantation of rat parathyroid glands: a study on morphological changes.

Autotransplantation of parathyroid glands in man is performed to preserve parathyroid function after surgery. In a rat model, we performed autotransplantation into the renal subcapsular space to examine reinnervation and changes in cell activity in the transplanted glands. Parathyroids grafted for 1-20 weeks were examined immunocytochemically for general and specific neuroendocrine markers to visualize nerve fibers and glandular cells and for bromodeoxyuridine to determine cell proliferation. In situ hybridization was used to localize and quantitate chromogranin A and parathyroid hormone (PTH) mRNA expression. Reinnervation was observed as early as 1 week after transplantation in that nerve fibers containing the general neuronal marker protein gene product 9.5 appeared along blood vessels. During the following 20 weeks, the nerve fiber density increased gradually. One week after transplantation, the immunoreaction intensity for PTH, chromogranin A, and pancreastatin was lower than in control glands. Bromodeoxyuridine-labeled cells were fewer than in control glands at 1 week and at 5-10 weeks after transplantation. The density of PTH mRNA labeling was lower than in control glands during the whole time period studied and reached a minimum after 10 weeks. The density of chromogranin A mRNA labeling was unaffected at 1 and 3 weeks after transplantation and then decreased to a minimum at 10 weeks after transplantation; at 20 weeks, the chromogranin A mRNA labeling had again reached the level in control glands. The changes in PTH and chromogranin A immunoreaction intensity and mRNA density indicate reduced hormone production for several weeks after transplantation. Our results using transmitter-specific markers indicate a rapid ingrowth of mostly sympathetic nerve fibers, preferentially around blood vessels. Later on, parasympathetic and sensory nerve fibers reached the grafts. The parathyroid innervation may be of importance for parathyroid hormone regulation, and the finding of an early reinnervation could be of clinical importance.

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 μg/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 μg/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 μg/mL or more failed to restore normoglycemia in the diabetic recipient mice after transplantation. Viability of islets incubated with doses up to 100 μg/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 μg/mL was significantly decreased. Thus, a therapeutic window for MMC efficacy was found at concentrations from 10 μg/mL to 100 μg/mL. This modality is simple and effective and underlying molecular mechanisms need to be determined in the future.

Successful islet auto- and allotransplantation in diabetic pigs.

Because of its anatomical and physiological similarities to humans, the pig appears to be a suitable large animal model for preclinical studies of islet transplantation. The aim of this study was to investigate islet auto- and allotransplantation in a pig model with diabetes induced by total pancreatectomy. Porcine islets were isolated by a continuous digestion-filtration device at 32 degrees C and purified by a discontinuous iso-osmolar Ficoll-sodium-diatrizoate gradient on a Cobe 2991. The purified islets were autografted into the liver or the renal subcapsular space. The liver appears to be a more suitable site for the islet grafts than the renal subcapsular space, and the minimal amount of islets for reversal of diabetes is >5 microl/kg of body weight. Persistent normoglycemia (fasting blood glucose level: 72.4+/-44.38 mg/dl) with a normal insulin secretion response to glucose stimulation was successfully achieved in five of six diabetic pigs by implanting a sufficient islet mass into the liver. Triple-drug immunosuppressive therapy with cyclosporine, azathioprine, and prednisolone did not prevent porcine islet allografts from experiencing early failure. However, the addition of 15-deoxyspergualin to the triple-drug immunosuppressive regimen significantly prolonged the function of the islet allografts. When antithymocyte globulin was added to the above-mentioned immunosuppressive drug regimen, the normoglycemic period was prolonged to more than 1 month (fasting blood glucose level: 75.4+/-17 mg/dl). We conclude that autotransplantation with a sufficient islet mass can induce normoglycemia with a normal insulin secretion response to glucose stimulation in pancreatectomized diabetic pigs and that allotransplantation can be successfully achieved when 15-deoxyspergualin and antithymocyte globulin are combined with the triple-drug immunosuppression described above. However, this immunosuppressive protocol results in a high rate of infectious complications.

Glucose turnover and insulin sensitivity in rats with pancreatic islet transplants.

To study the metabolic effects of insulin derived from islet grafts, oral glucose tolerance (OGT) and glucose turnover were examined in streptozotocin-induced diabetic Lewis rats rendered normoglycemic by syngeneic islet grafts in the renal subcapsular space (REN), in REN with renal vein-to-mesenteric vein anastomosis (REN-RMA), in the liver (intrahepatic [IH]), or in a parahepatic omental pouch (POP) and compared with normal rats. Normal OGT was found at 1 month posttransplant in all animals receiving approximately 3,000 islets, with hyperinsulinemic responses in the REN group compared with the other groups, and with higher C-peptide responses in the IH group than in the other groups (P < 0.05 by one-way analysis of variance). Glucose turnover studies in the insulin-stimulated steady state (INS-SS; infusion of insulin at 10 pmol x kg(-1) x min(-1)) at 2 months posttransplant showed that whole body glucose disappearance rates (Rd) were similar in all groups, but the REN group had higher steady-state insulin levels than the other groups. Glucose infusion rates (GIRs) were lower in the REN and IH groups than in the other groups. Apparent endogenous glucose production (EGP) was not completely inhibited in the REN and IH groups, while complete inhibition was observed in the other groups. When INS-SS insulin levels were matched to the level in REN rats by increasing the insulin infusion rate to 20 pmol x kg(-1) x min(-1) in REN-RMA, IH, and normal rats, GIR and Rd were elevated, exceeding those values in REN rats, but GIR in IH rats was still lower than in REN-RMA and normal rats. Thus, 1) in the REN group, impairment of inhibition of EGP and of stimulation of Rd by exogenous insulin contribute to insulin resistance; 2) in the IH group, incomplete inhibition of EGP is the major determinant of insulin resistance; and 3) with portal delivery of insulin in the REN-RMA and POP groups, normal insulin sensitivity is preserved. The present study confirms that hepatic portal delivery of islet secretions is necessary for physiological regulation of glucose metabolism. The study also suggests the IH grafts do not provide physiological regulation of glucose metabolism, raising the question of whether the liver is an appropriate site for insulin-secreting tissue replacement therapy in diabetes.

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.

PREVENTION OF PRIMARY ISLET ISOGRAFT NONFUNCTION IN MICE WITH PRAVASTATIN1

Nonspecific inflammatory damage in the early stages of transplantation is the major cause of primary islet graft nonfunction. Using murine isografts, we attempted to prevent this islet graft damage by treating recipients with pravastatin (Pravacol), a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor. Nicotinamide was also tested to determine the synergistic effect of both agents. Unpurified newborn BALB/c islets, ranging in number from 1800 to 2500, were transplanted into the left renal subcapsular space of a syngeneic adult mouse made diabetic with streptozotocin. Recipient mice were divided into the following four groups, based on treatment protocols: treatment with 40 mg/kg pravastatin (group 1), 500 mg/kg nicotinamide (group 2), 40 mg/kg pravastatin and 500 mg/kg nicotinamide (group 3), and vehicle alone (group 4). Pravastatin and nicotinamide were administered orally every day for 14 days, starting on the day of transplantation (day 0). Nonfasting blood glucose levels, urine glucose levels, and the intravenous glucose tolerance test were used to monitor the diabetic state. The reversal of diabetes was defined by normoglycemia and negative urine glucose maintained for more than 7 days. After islet transplantation, levels of blood and urine glucose were significantly lower in groups 1 and 3, compared with those in group 4. K-values of an intravenous glucose tolerance test performed on day 14 were significantly higher in groups 1 and 3 than those of group 4. Reversal of diabetes had occurred in 63% of mice in group 1 and 67% in group 3, levels that were higher than those in group 2 (17%) and group 4 (0%) (P<0.02, groups 1 and 3 vs. group 4). Histological examination of grafts, biopsied on day 21, revealed well preserved islets with little sign of inflammation in groups 1 and 3, whereas grafts in groups 2 and 4 contained broken, smaller islets surrounded by severe fibrosis and mononuclear cell infiltration. Our results in mice have shown the effectiveness of pravastatin for protecting islets from nonspecific inflammatory damage. Nicotinamide did not show a synergistic effect with pravastatin at the dosage used in this study. These results indicate that pravastatin may be a useful agent for clinical islet transplantation.

Purified Murine Islet Allografts: Islet Engraftment as Influenced by Implantation Site and Glucotoxicity

A UNIQUE and natural approach to immunoisolation of islet grafts was explored by investigating different immunoprivileged sites of the body: the brain, the testis, and the renal subcapsular space. For the intraabdominally placed testis, immunologic privilege has been, undoubtfully, demonstrated and attributed to locally produced factors that inhibit the immune response. However, the clinical inapplicability and the possible malignant transformation of the germ cells stand as major drawbacks of grafting the islets in such an unconventional organ site. The data about renal subcapsular space are contradictory. Whereas some investigators reported failure of islet allografts to function and survive in the renal subcapsular space, others demonstrated a remarkable success of islet allografting in this place. We investigated the engraftment, survival, and metabolic function of murine islet allografts when placed in two organ sites, the intraabdominally placed testis and the renal subcapsular space, without immunosuppression.

Follow-up study of the revascularization process of purified rat islet beta-cell grafts

The revascularization of islets of Langerhans transplanted in heterotopic sites like the liver by portal vein embolization or the renal subcapsular space is a major process necessary for the viability of grafted cells. This process has been extensively studied by different techniques and the results have shown that islet revascularization is an early phenomenon that takes place soon after transplantation. In this report we have analyzed by a double indirect immunofluorescence technique, the revascularization process of purified endocrine islet beta-cells transplanted in the renal subcapsular space of syngeneic rats. Lewis rats were grafted with islets cultured for 24 h, with a suspension of purified beta-cells cultured for 24 h, and with a suspension of purified beta plus nonbeta-cells cultured for 24 h. Rats were killed at different days after implantation and the kidney bearing the grafts were snap frozen and immunohistochemically stained with a rabbit anti factor VIII antiserum (which labels endothelial cells). Immunocytochemical analysis revealed that cultured islets completed revascularization by days 3–5 after transplantation, as shown by the detection of capillary endothelial cells within and surrounding the islets. Within purified endocrine beta-cell grafts, the presence of numerous endothelial cells was not observed until days 10–14, indicating that revascularization of beta-cells with host vessels is not such an early phenomenon as it takes place in whole isolated islets. Conversely, the addition of a population of endocrine nonbeta-cells to the purified islet cell grafts, partially accelerated the revascularization of pure beta-cell grafts, which showed the presence of abundant capillary endothelial cells already at day 7 after transplantation, indicating that some other unidentified factors besides the absence of endothelial cells may explain the retardation of beta-cell grafts revascularization.

Awareness of donor alloantigens in antiadhesion therapy induces antigen-specific unresponsiveness to islet allografts.

Antiadhesion therapy using monoclonal antibodies (mAbs) to adhesion molecules in vivo has been shown to produce significant prolongation of graft survival in various transplantation models. However, it remains unclear whether antiadhesion therapy operates by merely blocking adhesion between antigen-presenting cells and T cells physically and/or by blocking costimulatory signals while preserving signals mediated through T-cell receptors in vivo. We examined antigen-specific T-cell responses during and after antiadhesion therapy. BALB/c islets were transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 mice given anti-lymphocyte function-associated antigen (LFA)-1 and/or anti-intercellular adhesion molecule-1 mAb treatment. The animals bearing surviving islet allografts were challenged with BALB/c or third-party islets on day 7 or more than 100 days after transplantation. Islet allografts were acutely rejected in untreated animals, with a mean survival time (MST) of 19+/-8 days. Administration of anti-LFA-1 mAb induced significant prolongation of graft survival with a mean survival time of 72+/-33 days, and half of the allografts showed indefinite survival. The animals given anti-LFA-1 mAb alone 7 days before transplantation showed acute rejection of BALB/c islets, whereas a significant number of animals given anti-LFA-1 mAb and the BALB/c islet allograft simultaneously accepted secondary BALB/c islets, but rejected third-party islets. Likewise, most of the animals bearing long-term functioning BALB/c allografts for more than 100 days accepted secondary BALB/c islets, but rejected C3H islets acutely. Interestingly, the spleen cells from these animals transferred unresponsiveness to BALB/c islets into the 2.5-Gy x-irradiated recipients, whereas those from naive animals induced acute rejection. These results indicate that anti-LFA-1 mAb treatment prevents T-cell activation leading to rejection, but results in a T-cell receptor engagement leading to antigen-specific unresponsiveness maintained by transferrable suppressor cells.

Transplantation of CD95 ligand-expressing grafts: influence of transplantation site and difficulty in protecting allo- and xenografts.

CD95 and its ligand (CD95L) have been implicated in the regulation of immune responses. Recently, it was reported that CD95L expression prevented rejection of allogeneic grafts transplanted under the kidney capsule. In contrast, we reported that enforced CD95L expression in subcutaneously grafted cells induced acute rejection even in the syngeneic or immunodeficient hosts. In this study, we investigated whether the CD95L-expressing cells could be protected from rejection when transplanted under the kidney capsule. CD95-negative cells (baby hamster kidney and L5178Y lymphoma cells) were transfected with CD95L cDNA to express functional CD95L. The cells were transplanted into skin or renal subcapsular space of immunocompetent or T cell-deficient nu/nu mice. The parental cells grew well in nu/nu or syngeneic mice but were rejected in allogeneic or xenogeneic immunocompetent mice. The CD95L transfectants were rejected when transplanted subcutaneously in all types of mice studied. However, when transplanted under the kidney capsule, they survived in nu/nu or syngeneic mice but were rejected in allogeneic or xenogeneic immunocompetent mice. These results imply that CD95L expression may not be sufficient to protect the grafts from rejection, and the survival of CD95L-bearing grafts is substantially influenced by the site of transplantation.

Efficacy of immunosuppressive drugs in islet xenotransplantation: leflunomide in combination with cyclosporine and mycophenolate mofetil prevents islet xenograft rejection in the pig-to-rat model.

Morphological characteristics of islet xenograft rejection differ from those of islet allograft rejection. Therefore, prevention of islet xenograft rejection probably requires a different type of immunosuppression from that used in allogeneic transplantation. Fetal porcine islet-like cell clusters (ICC) were transplanted into the renal subcapsular space of rats treated with different immunosuppressive protocols. The existence of a cellular infiltrate or deposits of antibodies and complement in the grafts was evaluated at different times after transplantation using immunohistochemistry. Treatment with leflunomide (LEF), cyclosporine (CsA), mycophenolate mofetil (MMF), 15-deoxyspergualin, and rapamycin alone or in combination had an insufficient inhibitory effect on ICC xenograft rejection. However, in animals treated with LEF+CsA, the rejection process was markedly inhibited. However, some macrophages and T cells were still present, and at 24 days, the xenografts were destroyed. In LEF+CsA-treated animals that were given sera containing an excessive amount of rat anti-porcine xenoreactive antibodies, marked deposits of IgG, and to some extent C3 as well, were detected along the border between intact ICC, and the xenografts were surrounded by macrophages. However, almost no cells infiltrated the grafts, and there were many intact ICC. In animals treated with LEF+CsA+MMF, only occasional infiltrating cells were seen at 12 and 24 days after transplantation, and the endocrine tissue was completely intact. LEF+CsA+MMF prevented rejection of porcine ICC xenografts in the rat for up to 24 days after transplantation.

Cyclosporine does not inhibit the process of revascularization of pancreatic islet transplantation.

The immunosuppressive drug cyclosporin-A (CsA) has been widely used to prevent pancreatic islet allograft rejection. Because it has been suggested that CsA may inhibit the process of revascularization of transplanted islets, the purpose of the study was to analyze by a double indirect immunofluorescence technique the revascularization process of isolated islets grafted in the liver and in the renal subcapsular space of rats treated with immunosuppressive doses of CsA. Lewis rats were grafted with either Lewis (isografts) or Wistar (allografts) pancreatic islets obtained by collagenase digestion. Rats were killed at different days after implantation and the liver and kidney bearing the grafted islets were snap frozen and immunohistochemically stained with a double immunofluorescence technique using a rabbit antifactor-VIII antiserum (which labels endothelial cells) and a guinea pig antiinsulin antibody. Islets implanted into nonimmunosuppressed hosts completed revascularization by days 3-7 after transplantation, as shown by the detection of endothelial cells within and surrounding the islets. The identical staining pattern of revascularization was observed in nonrejecting allografts as well as in isografts treated with CsA. We conclude that CsA did not inhibit the process of revascularization of rat islets after free transplantation. This finding is relevant for human islet transplantation, where CsA is currently employed to prevent kidney and islet allograft rejection.

Cyclosporine does not inhibit the process of revascularization of pancreatic islet transplantation

The immunosuppressive drug cyclosporin-A (CsA) has been widely used to prevent pancreatic islet allograft rejection. Because it has been suggested that CsA may inhibit the process of revascularization of transplanted islets, the purpose of the study was to analyze by a double indirect immunofluorescence technique the revascularization process of isolated islets grafted in the liver and in the renal subcapsular space of rats treated with immunosuppressive doses of CsA. Lewis rats were grafted with either Lewis (isografts) or Wistar (allografts) pancreatic islets obtained by collagenase digestion. Rats were killed at different days after implantation and the liver and kidney bearing the grafted islets were snap frozen and immunohistochemically stained with a double immunofluorescence technique using a rabbit antifactor-VIII antiserum (which labels endothelial cells) and a guinea pig antiinsulin antibody. Islets implanted into nonimmunosuppressed hosts completed revascularization by days 3–7 after transplantation, as shown by the detection of endothelial cells within and surrounding the islets. The identical staining pattern of revascularization was observed in nonrejecting allografts as well as in isografts treated with CsA. We conclude that CsA did not inhibit the process of revascularization of rat islets after free transplantation. This finding is relevant for human islet transplantation, where CsA is currently employed to prevent kidney and islet allograft rejection.

The effect of preexisting long-term diabetes on the outcome after islet transplantation in rats.

Studies of islet transplantation conducted immediately following diabetes induction may not accurately reflect the clinical situation. Long term preexisting diabetes with generalized microvasculature complication might adversely affect the outcome after islet transplantation. The present study testing this hypothesis by evaluating the effect of long-term preexisting diabetes on glucose-induced insulin secretion up to 6 months after transplantation of two different quantities of islets. One thousand two hundred or 2,400 islets were isotransplanted into the left renal subcapsular space at 10 days (acute diabetes), 3 months (chronic diabetes), or 6 months (long-term diabetes) after diabetes induction by streptozotocin in the rat. In addition, one group of diabetic rats in which normoglycemia was maintained with exogenous insulin treatment for 6 months was then transplanted with 1,200 islets. Intravenous glucose tolerance tests were performed at 10, 90, and 180 days after islet transplantation. Islet transplantation normalized the basal blood glucose levels within 24-48 h in all transplanted groups that remained normal for the entire study period of 6 months, with no differences among acute, chronic, or long-term diabetes. Basal plasma insulin levels were also normalized in all transplanted groups. Diabetic (acute, chronic, or long-term) rats transplanted with 2,400 islets achieved normal glucose-induced insulin secretion at 10 and 90 days after transplantation. In contrast, glucose-induced insulin secretion was impaired in rats transplanted with only 1,200 islets, with no differences among acute, chronic, and long term diabetes. However, at 180 days after transplantation, long term diabetic rats transplanted with 2,400 islets had impaired insulin secretion compared to normal controls. Insulin-treated long-term diabetic rats transplanted with 1,200 islets had normal glucose-induced insulin secretion at 10 days after transplantation. However, at 90 and 180 days after transplantation, insulin-treated long-term diabetic rats had impaired glucose-induced insulin secretion which was not different from nontreated transplanted long-term diabetic rats. It is concluded that long-term preexisting diabetes has no impact on the early outcome after islet transplantation. However, it may adversely affect the long-term function of the transplanted islets. Also, transplantation of a sufficient islet mass is the critical factor in achieving complete glucose homeostasis.

Follow-Up Study of the Revascularization Process of Cryopreserved Islets of Langerhans

Cryopreservation of islets of Langerhans is a necessary procedure since human pancreatic islet transplantation has become a reality for the clinical treatment of Type I, insulin-dependent diabetes mellitus. Although successful cryopreservation of rodent and human islets is a well-established technique for islet storage after isolation and purification, little is known about the influence of the freeze–thaw procedure on the islets’ potential to induce angiogenesis and revascularization, a major process necessary for the viability of grafted cells. In this study, the revascularization process of cryopreserved islets transplanted in the liver and in the renal subcapsular space of diabetic and nondiabetic rats is analyzed by a double indirect immunofluorescence technique. Frozen–thawed pancreatic islets were cooled slowly to −40°C, stored at −196°C, and thawed rapidly. Lewis rats were grafted with either Lewis (isografts) or Wistar (allografts) overnight-cultured and frozen–thawed islets obtained by collagenase digestion. Rats were killed different days after implantation, and the livers and kidneys bearing the grafted islets were snap-frozen and immunohistochemically stained with a double immunofluorescence technique using a rabbit anti-factor VIII antiserum (which labels endothelial cells) and a guinea pig anti-insulin antibody. Overnight-cultured islet grafts completed revascularization by Days 4–7 after transplantation, as shown by the detection of endothelial cells within and surrounding the islets. The identical staining pattern of revascularization was observed in islets frozen–thawed before transplantation. It is concluded that islet cryopreservation is a suitable technique for long-term storage prior to transplantation since it does not interfere with the neovascularization process of islet grafts.

IMMUNOMODULATION OF PANCREATIC ISLET ALLOGRAFTS IN MICE WITH CTLA4Ig SECRETING MUSCLE CELLS

In an effort to create a model of in vivo production of immunosuppressants, we have transfected C2C12 muscle cells (H-2k) with the cDNA for CTLA4Ig, a fusion protein that prevents the activation of T cells by blocking the costimulatory signal transduced by the T cell receptors CD28 and CTLA4. CTLA4Ig-secreting clones were cotransplanted with islets as composite grafts in the renal subcapsular space of diabetic mice. When the myoblasts were syngeneic to C3H/HeJ hosts (H-2k), there was a significant prolongation of survival of allogeneic C57Bl/6J (H-2b) islets from a mean 11.0 days to 31.7 days. When the graft was completely allogeneic (H-2k myoblasts and islets into H-2b recipients), there was no benefit in survival. A transient blockade of LFA-1 with the mAb M17 was synergistic in this combination: 8 out of 12 C57Bl/6J recipients achieved long-term acceptance. Systemic CTLA4Ig levels were detected up to 60 days after transplantation. In conclusion, we have shown that C2C12 muscle cells can be genetically engineered to secrete functional CTLA4Ig and that they can be used as a gene reservoir for the continuous in vivo production of CTLA4Ig to modulate the survival of islet cell allografts.

9a Techniques of pancreas and islet transplantation

Techniques for vascularized pancreas transplantation are relatively standardized, whereas those for human islet isolation and transplantation are rapidly changing and evolving. The commonest method for transplanting the vascularized pancreas is to use the entire pancreas together with a segment of donor duodenum, and to anastomose this to the recipient bladder. This technique offers the advantages of technical ease, the maximum β-cell mass is transplanted and graft function can be monitored by measuring urinary amylase levels. Human islet isolation requires that the pancreas is dissociated, the islets purified and then transplanted to a wellvascularized location. The pancreas is dispersed by a combination of the intraductal injection of collagenase and gentle mechanical agitation, and the islets separated from contaminating exocrine tissue by density-gradient centrifugation. Once purified, the islets can be placed into tissue culture or cryopreserved. The commonest site for human islet transplantation is intraportal injection so that the islets are embolized throughout the liver. Alternatives include transplantation to the renal subcapsular space or the spleen.

Initiation of autoimmune type 1 diabetes and molecular cloning of a gene encoding for islet cell-specific 37kd autoantigen.

Insulin-dependent diabetes mellitus (IDDM) is believed to be an autoimmune disease, characterized by lymphocytic infiltration of the islets and the presence of islet cell autoantibodies. Autoimmunity may result from an intrinsically abnormal immune system, primary alterations of the target beta cell or both. However, the initial event that causes the beta cell-specific autoimmunity remains unknown. Our recent experimental results showed that islet grafts from neonatal BB rats remained intact without insulitis when transplanted into the renal subcapsular space of acutely diabetic BB rats. In contrast, islet grafts from adult BB rats (which had been treated with silica for the prevention of insulitis) revealed severe insulitis and were rapidly destroyed. These results suggest that the delayed expression of a beta cell-specific autoantigen may result in the initiation of beta cell-specific autoimmunity. The islet cell-specific 37 kd autoantigen is not expressed early in the life of BB rats, but is expressed at around 30 days of age. This islet-specific autoantigen might be recognized and attacked by the immune effectors. In contrast, nondiabetic Wistar Furth rats express the autoantigen from birth.

Progressive morphogenesis in vivo after transplantation of cultured small bowel epithelium.

An experimental model for the primary culture and transplantation of late foetal rat small intestinal epithelium is described. Multicellular aggregates of mucosal epithelium containing pre-crypt proliferative cells were isolated from 20-day foetal rat intestine by enzymatic disaggregation. Cellular aggregates, which we refer to as "epithelial organoids," attached readily in culture, proliferated, and spread to produce coalescing colonies within 10 days. Enterocytes were maintained in culture for 3 days, removed as cell sheets, and incubated overnight with foetal mesenchyme. Fourteen recombinant preparations were then grafted to the renal subcapsular space of adult nude mice. Four of six grafts retrieved after 1 wk had developed. Histology demonstrated the formation of simple tubular structures lined by a polarized columnar epithelium. At 14 days, two of eight grafts had developed and demonstrated temporal progression of morphogenesis. Histology showed rudimentary crypts and villi lined by different epithelial cell types, including enterocytes and goblet cells. Small bowel proliferative cells within "epithelial organoids" from 20-day foetal intestine, may be maintained in primary culture for up to four days. After short term primary culture, these proliferative cells retain the capacity for progressive organotypic morphogenesis and pluripotent cytodifferentiation, after transplantation to adult recipients.

Fetal rat pancreas transplantation in BB rats: immunohistochemical and functional evaluation.

Spontaneously diabetic BB/Wor rats received either a syngeneic fetal pancreas transplant or adult islets. In the former, 4-8 fetal pancreases were transplanted, and in the latter, 3-5000 islets. Transplantation was performed by transferring a blood clot containing the pancreases or islets to the renal subcapsular space. Insulin therapy was undertaken postoperatively, except in one experiment with adult islets. Of the fetal pancreas transplanted BB rats, 52% became normoglycaemic, and 21% remained so throughout an observation period of 10 months. Nephrectomy caused a prompt return of diabetes. The histological appearance of the grafts transplanted to the diabetic animals closely resembled that of grafts transplanted to normal rats in a parallel series. For comparison a group of BB rats received a syngeneic transplant of isolated adult islets from WF rats or BBW rats. Following adult islet transplantation, 5 out of 6 animals became hyperglycaemic after a median of 20.5 days when no insulin was given post-transplantation. Four out of 5 animals became hyperglycaemic after a median of 23 days when supportive insulin therapy was administered after the transplantation. The results indicate that recurrent diabetes is not inevitable following syngeneic fetal pancreas transplantation to spontaneously diabetic BB rats. Recurrent diabetes was only occasionally associated with mononuclear cell infiltration. Transplanted tissue was well-preserved and vascularized; mega-islets were a constant finding.