Search

A method is described for the isolation of "proislets' from mouse foetal pancreas. Histologically, isolated proislets are composed of undifferentiated tissue which does not stain with aldehyde fuchsin. Following isotransplantation, proislets develop into well differentiated islets with beta cell granulation. In addition, isografts consisting of proislets harvested from 8 foetal mouse pancreases have the functional capacity to reverse Streptozotocin-induced diabetes. Proislets appear to represent foetal precursor islet tissue.

The role of T-lymphocyte subpopulations in the rejection of fetal pig proislet (islet precursor) xenografts in mice was examined with in vivo administration of monoclonal antibodies (MoAbs) to CD4+ (GK 1.5) and CD8+ (49-11.1) T-lymphocyte subsets. The data indicate that the rejection process is T-lymphocyte dependent and mediated by CD4+ T-lymphocytes. The in vivo administration of MoAbs specific for graft-borne pig leukocytes failed to prevent xenograft rejection in fully immunocompetent transplant recipients.

In this paper the theoretical basis of alloreactivity and its relevance to transplantation biology is discussed prior to a review of work showing that culture of adult mouse pancreatic islets for 7 days in 95% O2 and 5% CO2 facilitates successful grafting to nonimmunosuppressed allogeneic recipients. These allografts function by reversing both chemically induced and spontaneous diabetes. The fetal mouse pancreas is more immunogenic than adult islets, and even after a culture period of 10 days in 95% O2 and 5% CO2, BALB/c allografts are consistently rejected by nonimmunosuppressed recipient mice. The immunogenicity of fetal pancreas is thought to be due to the presence of contaminating lymphoreticular cells in the mesentery surrounding the fetal pancreas. Digestion of the fetal pancreas with collagenase allows the isolation of proislets that develop into functional islet tissue on transplantation. Fetal proislets are less immunogeneic than the whole fetal pancreas and may provide a source of tissue for clinical transplantation. Established islet allografts are relatively stable and are not rejected following nonspecific stimulation of the recipient's immune system or following passive transfer of either antibody or antibody and complement. After prolonged residence in the recipient a state of allograft tolerance develops and such grafts resist rejection by specific stimulation of the recipient. The administration of donor antigen in the form of uv-irradiated cells enforces this state of allograft tolerance.

The combination of donor pretreatment with cyclophosphamide, organ culture in 95% O2:5% CO2 for 7-10 days, and short-term immunosuppression of recipients with cyclosporin A (CsA) were necessary to obtain 100% survival of single-cluster BALB/c islet allografts in outbred mice. In vivo and in vitro pretreatment of the donor tissue alone resulted in the acceptance of 45% of the islet allografts in nonimmunosuppressed outbred mice. CsA treatment of recipients alone yielded 40% survival of the untreated allografts. CsA treatment played an important role in maintaining the capacity of islet allografts to function in outbred mice. During CsA treatment, 88% of streptozocin-treated mice showed graft-dependent reversal of diabetes; the remainder showed no evidence of graft function, and CsA treatment failed to prevent acute graft rejection. After withdrawal of CsA immunosuppression, 38% of this total group remained normoglycemic. These findings suggest that modulation of both donor-tissue immunogenicity and recipient responsiveness will be required for successful pancreatic islet transplantation in diabetic humans.

Insulin-dependent diabetes mellitus was induced in the pig by pancreatectomy or by administration of intravenous streptozotocin (150 mg/kg). High post-operative morbidity and mortality in the pancreatectomised animals made this method of inducing diabetes unsuitable for animals to be used in long term studies. By contrast, the good clinical state of animals after streptozotocin and the permanence of their diabetes indicated that these animals were suitable for long term studies such as those involving transplantation of pancreatic islet tissue. Techniques designed to facilitate the assessment and management of these animals included placement of an indwelling jugular venous catheter to enable blood samples to be obtained for metabolic studies, denervation of an area on the flank of the animal to enable insulin administration with minimum discomfort and denervation of an ear to enable blood samples to be obtained from the animal for glucose estimation in long term studies.

Thyroid and thymus tissue autografts were used to examine the suitability of different transplant sites in the pig for endocrine tissue transplantation. The omental pouch, gall bladder fossa and renal subcapsular space showed optimal implantation, preservation and vascularisation of graft tissue. Marked dissemination of graft tissue was observed at intramuscular sites and graft viability at subcutaneous sites in the ear was poor. The omental pouch represents a potentially suitable site for pancreatic islet tissue transplantation studies in the pig.

It has been generally assumed that because streptozotocin is toxic to the adult B cell of most species, it should also damage B cells obtained at earlier stages of development. This paper examines whether this is true for human fetal pancreata obtained from the therapeutic termination of pregnancies during the first half of the second trimester. Experiments were carried out both in vivo and in vitro. For the former experiments diced explants of the human fetal pancreas were grafted beneath the renal capsule of nude mice 3 weeks before streptozotocin was administered to make the animals diabetic. The grafts were removed 1 week, 2-4 weeks or 3 months later, and were found to be of similar weight and insulin content to the control grafts. In 2 of the animals with grafts remaining for 3 months the diabetes had even been reversed by the implant, hyperglycaemia recurring when the graft was removed. In contrast, rat fetal pancreata grafted beneath the renal capsule of nude mice, subsequently rendered diabetic, were adversely affected by streptozotocin, the insulin content of the implants being 12% of levels in control grafts. Adequate uptake of streptozotocin by the implanted human fetal pancreas was established by measuring tissue levels of the drug 30 min after its injection. Histological examination of the grafted human fetal pancreas showed no deleterious effect of streptozotocin on the number of granulated B cells one day after injection, although by this time the host was diabetic.(ABSTRACT TRUNCATED AT 250 WORDS)

Uncultured mouse islet allografts (BALB/c to CBA) are rejected 2 to 4 weeks after transplantation. Allografts, cultured in 95% O2 and 5% CO2 for 7 days before transplantation, show no sign of rejection up to 3 months post-transplantation. However, the cultured allografts are rejected if the CBA recipient is given an i.v. injection of 10(5) peritoneal cells at the time of transplantation. Organ culture of BALB/c fetal pancreas (16 to 17 days gestation) under the same conditions failed to prevent allograft rejection. The immunogenicity of fetal pancreas is reduced if this tissue is cultured in 95% O2 and 5% CO2 for 17 days before transplantation.

Proislets, derived from fetal mouse pancreata by collagenase digestion and subsequent organ culture, can be frozen to -196 degrees C and stored in a viable condition before successful syngeneic transplantation. Cryopreserved proislets are relatively undifferentiated morphologically, but continue to differentiate into mature islets after transplantation.

Tissue-specific differences in immunogenicity were demonstrated following allotransplantation across the same minor histocompatibility barrier (BALB/c----DBA/2). In contrast to the high immunogenicity of fetal pancreas and skin, isolated islets, fetal proislets, and thyroid were weakly immunogenic. These tissue-specific effects were not related to the presence of tissue-specific antigens or the absence of recognizable minor alloantigens from the less immunogenic tissues. There was a strong correlation between tissues that were highly immunogenic and those that contained rich populations of donor leukocytes. The survival of fetal pancreas allografts was significantly improved by pretreating the donor tissue in high-oxygen organ culture and by the preparation of fetal proislets. Using other MHC-compatible strain combinations (B10.D2----BALB/c; BALB/c----B10.D2; C3H.SW----C57/10J) strain-specific effects were observed in the immunogenicity of thyroid allografts. In two of three strain combinations (B10.D2----BALB/c; BALB/c----B10.D2), pretreatment of the donor tissue with cyclophosphamide and organ culture prior to grafting significantly improved graft survival. These findings suggest that donor passenger leukocytes may play an important role in determining the immunogenicity of MHC-compatible allografts.