Xenotransplantation of adult porcine islets in diabetic mice. A study of UVB irradiation, cryopreservation and immunosuppression on graft survival time.

The major obstacle for successful xenotransplantation of islets to large animals and human diabetics is the host rejection. To address the rejection problem, we studied the efficacy of UV-B irradiation, cryopreservation and immunosuppression on the in vivo functional time and immunogenicity of adult porcine islets (PI) in outbred CD1 mice. Exposure of PI to UV-B irradiation between 300-1800J/M2 did not affect the cellular viability as assessed by fluorescein diacetate or their daily insulin secretion in vitro. Fresh PI normalized the blood glucose (BG) of diabetic CD1 mice for 3.1+/-0.6 (n = 8, mean+/-SEM) days. Islets treated with 600J/M2 UV-B irradiation or cryopreservation had similar graft functional times to fresh islets upon transplantation in diabetic CD1 mice. Immunosuppression with cyclosporin A (CsA), antilymphocyte serum (ALS) and FK506 prolonged the functional time of fresh pig islets to 7.9+/-0.9 (n = 9), 6.2+/-1.3 (n = 5) and 24.2+/-10.4 (n = 12) days, respectively. However, additional pretransplant treatment with either UV-B irradiation or cryopreservation did not further increase the functional time of pig islets in mice immunosuppressed with CsA. Furthermore, there was no apparent difference in the frequency of appearance of cytotoxic antibodies and antibody titers in the recipients of UV-B irradiated or cryopreserved pig islet compared with non-treated islets. The UV-B irradiation and cryopreservation of PI before transplantation with the present protocols did not appear to have significant effect on the islet immunogenicity when assessed by in vivo survival duration and anti-donor antibody titer production.

Prolongation of pig islet xenograft survival in rats immunosuppressed with FK506.

FK506 a new and potent immunosuppressive agent has been shown to be effective in prolonging pancreatic islet allograft survival. The present study was to determine its efficacy in prolonging pig islet xenotransplantation in two different strains of rat recipients. A total of two dosages of FK506 at 1 or 2 mg/kg per day for 2 weeks and then at weekly intervals were tested as monotherapy for their effect on the survival of renal subcapsular xenografts of purified or impure adult pig islets in inbred ACI and outbred Wistar rats. Histological assessment indicated that FK506 at 2 mg/kg per day significantly prolonged purified pig islet xenograft survival and to 7.5 months in two of three ACI recipients. Monotherapy with a lower dosage of FK506 or transplantation with impure pig islets resulted in increased graft survival time over controls, but less than that with the 2 mg/kg per day FK506. The viable pig islet xenografts showed a normal appearance and were readily identified by immunohistochemical staining for insulin and glucagon and further confirmed by immunohistochemical staining with anti-pig islet specific monoclonal antibody clone P44, developed in our laboratory. Mononuclear cell infiltration, mainly of the CD8-positive T-cell subset, increased with the duration of the graft in the recipient. By 7.5 months the majority of the xenografted islet cells were enclosed by the cellular infiltrate. The in vitro perfusion study of pig islets that had survived for 1 or 2 months in vivo showed that they were responsive to glucose stimulation with increase in insulin secretion into the perfusate. The results demonstrated that FK506 significantly prolonged pig islet survival in two rat strains and suggested that it is an effective immunosuppressant for the xenotransplantation model.

Effect of FK506 on rat Leydig cell function--in vivo and in vitro study.

FK506, a macrolide antibiotic, is a potent immunosuppressant and has a biological effect similar to that of cyclosporin A (CsA). In this study, the in vivo and in vitro effects of FK506 on rat Leydig cell function were investigated. In vivo, basal testosterone levels and secretion in response to human chorionic gonadotropin (hCG) stimulation in ACl rats treated with intramuscular (IM) injections of FK506 at a dosage of 1 or 2 mg/kg/d for 14 days were not different from those of age-matched normal controls. Testicular weights (g) from rats treated with 14 injections of 1 mg/kg/d FK506 (1.08 +/- 0.08, n = 14) were similar to weights from age-matched controls (1.04 +/- 0.08, n = 14). Similarly, Wistar (Wi) rats treated with FK506 at a dosage of 1 mg/kg/d for 2 weeks showed basal testosterone and luteinizing hormone (LH) levels and secretion in response to hCG stimulation similar to those of normal controls. Histologically, the Leydig cells and germ cells in FK506-treated animals appeared normal. In vitro, basal testosterone production and response to hCG stimulation by both ACI and Wi rat Leydig cells exposed to overnight treatment of FK506 (10 to 1,000 ng/mL) were not significantly different from those of control Leydig cells. Furthermore, the viability of the Leydig cells cultured for 3 days in FK506 was not significantly different from that of controls, and they continued to secrete testosterone at a rate similar to that of controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Cryopreservation of rat Leydig cells for in vitro and in vivo studies.

Leydig cells lose their ability to secrete testosterone following short-term in vitro culture. A procedure for effective storage of these cells would be useful. In this study, rat Leydig cells were cryopreserved in the presence of 10, 15 or 20% dimethylsulfoxide (DMSO) at approximately 1 degree C/min to -70 degrees C and then stored in LN2. After thawing, the cells cryopreserved in the presence of 15% DMSO showed the highest viability of over 75%. These cells secreted basal levels of testosterone in vitro as well as responded to hCG stimulation by secreting over 9-fold increase in testosterone. The viability of these cells was further confirmed by the demonstration of 3 beta-HSD positive cells under the kidney capsule of rats isografted with cryopreserved Leydig cells. This study demonstrated that purified rat Leydig cells can be cryopreserved and the cryopreserved cells retained normal function and were responsive to hCG stimulation. Cryopreservation is a simple procedure for long-term storage of functional Leydig cells.

Successful islet allotransplantation in diabetic rats immunosuppressed with FK506: a functional and immunological study.

The effect of a novel immunosuppressive agent, FK506, on fresh islet allografts was evaluated in diabetic rats across major histocompatibility complex (MHC) barriers with respect to the transplantation (TR) site, islet source, treatment regimen, and antidonor antibody (Ab) titers of the recipients after TR. The functional periods of Wistar (Wi) islets transplanted under kidney capsule (KC) or intraportally (IPo) and of a mixture of Wi and Lewis (Le) islets under KC or IPo in nonimmunosuppressed ACI rat recipients were 6.9 +/- 0.4 (n = 7), 6.4 +/- 0.5 (n = 7), 5.6 +/- 0.4 (n = 7), and 6.2 +/- 0.4 (n = 5) days, respectively. FK506 treatment at 1 mg/kg/d intramuscularly (IM) for 2 weeks (protocol I) following islet TR under KC and IPo significantly prolonged the allograft function to more than 71.8 +/- 11.3 (n = 10) and 161.7 +/- 18.6 (n = 11) days, respectively. Additional treatment with FK506 at 1 mg/kg/wk (protocol II) further increased the islet survival under KC to more than 212.6 +/- 22.3 (n = 8) days. With this FK506 treatment protocol, the Wi + Le mixed-islet allograft function was extended to more than 106.1 +/- 10.5 (n = 7) and 167.9 +/- 28.6 (n = 7) days under KC and IPo, respectively. Nephrectomy in 8/8 ACI rats with long-term-functioning Wi (n = 6) and Wi + Le (n = 2) islet allografts resulted in their return to hyperglycemia. Immunohistochemical staining showed abundant insulin-positive cells at the graft site, with small numbers of CD4- and CD8-positive cells present in the vicinity of the normal-appearing islets. Macrophages were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

A diabetic rabbit model for pig islet xenotransplantation.

An alloxan-diabetic rabbit model was established for the testing of the function of discordant xenogeneic pig islets isolated and purified from adult pig pancreata. The functional state of the pig islet transplants and immunological state of the rabbit recipients were assessed. Intraportal transplantation of 0.47 +/- 0.01 ml of pig islets with estimated 57418 +/- 5020 in number containing an estimated insulin content of 33.93 +/- 2.97 units (n = 7; mean +/- SEM) resulted in the normalization of blood glucose with a corresponding rise in insulin levels in the diabetic rabbit recipients for 2 days. An intravenous glucose tolerance test performed in 4 recipients during the normoglycemic period resulted in an improved K rate (2.5 +/- 0.4) over the diabetic controls, but this was significantly lower than the normal control animals (K rate = 4.5 +/- 0.4; n = 8). In vitro studies demonstrated that the preformed antibodies detected in the rabbit recipients were cytotoxic to the pig islet cells and lymphocytes. Heat treatment at 56 degrees C and mercaptoethanol treatment markedly reduced the cytotoxic activities of the sera. These findings implicated involvement of complement and IgM class antibodies in the killing of the pig islet cells. Furthermore, pig islet transplants at the kidney capsule site were coated with IgM class antibodies. This study has demonstrated that pig islets can be successfully isolated and purified in sufficient numbers for xenotransplantation studies in alloxan-diabetic rabbit. The porcine islet-to-alloxan diabetic rabbit combination can serve as a highly stringent and useful discordant model for assessing the effectiveness of various immunomodulation and immunosuppressive regimens. The finding of an optimal approach to immunorejection would potentially be applicable to actual clinical islet xenotransplantation in diabetic patients.