Mandel Te

Pig‐to‐human xenotransplantation: The expression of Galα(l–3)Gal epitopes on pig islet cells

Abstract: Previous histological studies of porcine tissues using the Galα(l–3)Gal specific lectin, IB4‐biotin, and an immunoperoxidase technique demonstrated the widespread distribution of Galα(l–3)Gal in pigs—particularly in the endothelium of all vessels and in the parenchyma of liver and kidney. An exception was the endocrine (islets) and exocrine tissue of the pig pancreas—adult islets did not express Galα(l–3)Gal and apart from ducts, and the exocrine pancreas was also nonreactive. Because of the potential use of islet xenotransplantation for the treatment of diabetes, a more extensive study was undertaken of fetal pancreas and of cultured islet cell clusters. Like the adult pancreas, islets from fetuses at term or earlier, essentially did not express Galα(l–3)Gal; however, during culture, under a variety of conditions, they became strongly reactive with IB4 and expressed large amounts of Galα(l–3)Gal. Staining for pancreatic hormones (insulin, glucagon, and somatostatin) demonstrated that true islets were being examined, although double staining for insulin and Galα(l,3)Gal indicated that cells secreting insulin were not Galα(l–3)Gal positive. The origin of the Galα(l–3)Gal+ cells is not apparent, nor is whether they are precursors of hormone secreting cells. It was of interest that, after transplantation to nude mice and allowing for maturation, the islets resembled the adult phenotype and were Galα(l–3)Gal negative. The implications of the findings are not clear but indicate that some cells in the cultured fetal islet (other than insulin secreting cells) could be the target of antibody mediated destruction.

Organ Culture of Fetal Mouse and Fetal Human Pancreatic Islets for Allografting

In organ culture of fetal human and fetal murine pancreas under “conventional” conditions (10% CO2 in air), the islet cells of both species survive, proliferate and function but the acinar tissue rapidly degenerates. Fetal mouse islet cells also survive in 90% CO2 but nonendocrine cells, including fibroblasts and macrophages, degenerate. Fetal mouse islets grown in 90% O2 show diminished immunogenicity when transplanted into recipients differing across the entire MHC, but a reduced allograft response by the host is frequently still present in the absence of immunosuppresslon. Fetal human islets, grown in 10% C02 in air, produce insulin in vitro for prolonged periods, and as xenografts, differentiate under the kidney capsule of athymlc mice, suggesting that under appropriate conditions both in vitro and in vivo, the fetal human islets can survive. However, fetal human pancreatic cells of all types are highly susceptible to high oxygen concentrations and are rapidly killed. Because of the susceptibility of fetal human pancreas to oxygen, conditions for the culture of fetal human islets for allotransplantation may need to be modified from those tolerated by fetal mouse islets. Fetal human islets may be a useful source of transplant material in human insulin-dependent diabetes, but it is likely that tissue matching and immunosuppression may be required in addition to modification of islet immunogenicity by prior organ culture.

Cellular rejection of fetal pancreas grafts: Differences between alio- and xenograft rejection

Abstract: Hyperacute rejection (HAR) is the major immunologic problem with vascularized xenografts between discordant donor/recipient combinations but does not occur in neovascularized grafts of organ‐cultured fetal pig pancreas in either mice or cynomolgus monkeys. However, a form of cell‐mediated acute rejection with quite different histopathologic features does occur with kinetics that are similar to acute cellular rejection of fetal pancreas allografts in non‐immunosuppressed MHC‐mismatched mice. Xenograft rejection is dominated by non‐lymphoid cells, mostly eosinophils, that appear some days after transplantation. In contrast, in mouse allografts, mononuclear cells are the dominant population throughout the rejection process^. The rejected allograft site rapidly resolves to form a mature non‐irifiltrated scar whereas the infiltrate in the xenograft site remains for weeks and forms a large granuloma before its eventual resolution. There are also differences in the intra‐graft cytokine profile in the graft site between alio‐ and xenografts during acute rejection with an early predominance of IL‐5 and TNF‐α and an absence of TNF‐γ in the xenografts. Immunosuppression with a depleting anti‐CD4 mAb shows that xenograft rejection is more dependent on CD4+ve T cells but xenografts are more difficult to maintain with conventional immunosuppression that is often effective for allografts. Limited studies in primates have shown that the histopathology of fetal pig pancreas rejection is similar to that seen in mice but occurs at a faster tempo. Thus, although HAR may not be a problem in rejection of neovascularised xenografts, a vigorous form of cellular rejection is present that may require different immunosuppression than is usually used for the I control of allograft rejection.

Organ Culture of Fetal Mouse Pancreas: The Effect of Culture Conditions on Insulin and Glucagon Secretion

Seventeen-day-old mouse pancreas was grown in organ culture under various culture conditions and the insulin released into the medium, either chronically or during acute incubations, was measured. Three media, DME, RPMI 1640, and TC199, were tested, each at glucose concentrations of 1, 2, 3, and 4 g/L. Fetal pancreata maintained in RPMI 1640 at 4 g/L glucose secreted the most insulin over a 25-day period. Tissue grown in RPMI 1640 and DME secreted more insulin in higher glucose concentrations, but explants grown in TC199 secreted low levels of insulin regardless of glucose concentration. Fetal pancreata grown in medium supplemented with 5% FCS secreted less insulin in acute incubations than tissues grown in 10%, 15%, 20%, or 30% FCS, which secreted equivalent amounts. There was no difference in the amount of insulin released by islets grown in media supplemented with 15% FCS from four different batches. Although fetal islets grown in high glucose concentrations generally secreted more insulin (and less glucagon) than “normoglycemic” controls, their ability to release insulin in subsequent acute incubations was impaired. Explants, which had been maintained for 2 wk in 2.5 or 4 g/L glucose and a further 3 days in normoglycemic medium, secreted less insulin and glucagon in acute release exeriments than tissue maintained in 1 g/L glucose throughout. Light microscopy of cultured fetal pancreas showed that tissue remained viable at all glucose concentrations tested and differences in response could not be attributed to selective necrosis. Tranplantation of cultured islets indicated that tissue grown under normoglycemic conditions functioned better than tissue grown in hyperglycemic media.

Are any functionally mature cells of medullary phenotype located in the thymus cortex

Experiments were undertaken to test if thymocytes of mature or medullary phenotype were restricted to the medullary area of the thymus. A calculation based on direct cell counts on serial sections indicated that 11.5% of adult male CBA thymic lymphoid cells were within the medullary zone. Since only 3-4% of thymocytes were cortisone resistant, the majority of thymocytes within the medulla were, like cortical thymocytes, cortisone sensitive. A series of cell surface antigenic markers, used alone or in pairs, suggested that 13-15% of thymocytes were of medullary phenotype, somewhat more than the number of thymocytes actually present in the medulla. However, much of this discrepancy could be explained by differential death of cortical cells during isolation and staining, and by the existence in the cortex of a subpopulation of early blast cells which shared some, but not all markers with medullary thymocytes. A direct test for mature or medullary phenotype cells in the cortex involved selective transcapsular labeling of outer-cortical cells with fluorescent dyes, followed by multiparameter immunofluorescent analysis of the 10% labeled population. Outer-cortical thymocytes included some cells (mainly early blasts) sharing some markers with medullary thymocytes, but very few (less than 1%) of these cells expressed all the characteristic mature markers. Limit-dilution precursor frequency studies showed the level of functional cells in the outer cortex was extremely low. The overall conclusion was that the vast majority of cells of complete mature phenotype are confined to the thymic medulla. These findings favor the view that thymus migrants originate from the thymic medulla, but do not exclude a cortical origin. The results also illustrate the need for multiparameter analysis to distinguish medullary thymocytes from early blast cells.

Transplantation of organ cultured fetal pig pancreas in non-obese diabetic (NOD) mice and primates (Macaca fascicularis)

Abstract: Xenografts of organ cultured fetal pig pancreas in prediabetic NOD mice can survive for prolonged periods (>20 weeks) in recipients treated with anti‐T cell monoclonal antibodies (MAb) directed against host CD4 and CD3 cell surface molecules. Anti‐CD4 MAb treatment alone is only partly effective and xenograft rejection occurs over a period of many weeks. In diabetic recipients, by contrast, recurrence of autoimmune disease in isografts is rapid (<28 days) despite similar depletion of CD4+ve T cells. In spontaneously diabetic NOD mice immunosuppressed with anti‐CD3 and anti‐CD4 MAbs xenograft function occurs and the recipients blood glucose levels fall into the pig range. Organ cultured fetal pig pancreas transplanted into cynomolgus monkeys is rapidly but not hyperacutely rejected when azathioprine‐cyclosporin A‐prednisolone immunosuppression is used. Anti‐T‐cell MAb treatment is now being studied in this primate model.

Fetal pig islet xenografts in NOD/Lt mice: The effect of peritransplant anti‐CD4 monoclonal antibody and graft immunomodification on graft survival, and lack of expression of Gal(α1–3)Gal on endocrine cells

Abstract: Transplantation of tissues that are vascularized by ingrowth of host vessels, as opposed to primarily vascularized grafts, may be one way of avoiding hyperacute rejection (HAR). Pretransplant treatment of the graft to eliminate from it highly immunogenic cells may also improve graft survival. We examined organ cultured fetal pig pancreas in prediabetic NOD/Lt female mice treated with peritransplant anti‐CD4 MAb (GK 1.5) and in separate experiments also studied the expression of Gal(α 1–3)Gal, an epitope that may be a major target of a human anti‐pig response. Immunocytochemistry to detect insulin, glucagon, and somatostatin and Gal(α 1–3)Gal showed that differentiated (i.e., hormone containing) endocrine cells were Gal(α 1–3)Gal negative but hormone‐negative ductal cells showed strong Gal(α1–3)Gal staining on their luminal border and interstitial cells were also positive particularly early after transplantation. Rejection occurred in all animals, but its rate was markedly altered by transient immunosuppression with the depleting anti‐CD4 MAb. Cell infiltration was first detected in controls 2 days posttransplant, was pronounced by 4 days, and grafts showed advanced rejection by 7 days whether or not they had been “immunomodified” by 2 days exposure to 90% O2. In contrast, peritransplant immunosuppression with GK 1.5 improved graft survival but rejection was advanced by 28 days. The control graft sites had many eosinophils, macrophages and mast cells by 4 days, but little infiltration was seen until after 14 days in the immunosuppressed mice and was predominantly by mononuclear cells. Use of “immunomodified” grafts was of no benefit. Thus, graft immunomodification per se, that often allows islet allografts to survive in rodents, had no effect on xenograft survival in this model. A major potential target antigen for natural Ab in humans, Gal(α 1–3)Gal, is not present on differentiated graft endocrine cells.

Transplantation treatment for diabetes

Islet transplantation in the treatment of insulin-dependent diabetes mellitus is making rapid progress. Vascularized pancreas allotransplantation is now an accepted treatment but has major limitations. A recent meeting∗ discussed alternative approaches, particularly islet xenotransplantation.

Effect of Culture Conditions on Fetal Mouse Pancreas In Vitro and After Transplantation in Syngeneic and Allogeneic Recipients

Organ culture of fetal mouse pancreas under conditions designed to either maximally preserve islet cell survival or reduce immunogenicity was used to test the efficacy of islet graft function in diabetic syngeneic or fully allogeneic recipients. Organ culture in either 90% O2, or at low temperature (22°C), or in a combination of 90% O2 and 22°C for 14 days not only failed to reduce immunogenicity in fully allogeneic recipients but also diminished endocrine cell survival in grafts in syngeneic recipients. Monitoring of in vitro insulin secretion provided a better guide to future graft function than did the insulin content of the cultured tissue. It is concluded that culture conditions that have been shown to reduce immunogenicity (high O2 concentration and low temperature) are not synergistic when used together and are also potentially damaging to endocrine cells.

Insulin Secretion by Fetal Human Pancreatic Islets of Langerhans in Prolonged Organ Culture

Fetal human pancreata, obtained from legally-induced abortions, were placed in organ culture to study their capacity to produce insulin over periods in vitro of between 18 and 40 days. Specimens obtained by hysterotomy usually showed increasing insulin secretion as measured by the insulin content of the media at each twice-weekly medium change. In most instances, relatively little insulin was produced during the first 7–10 days, but thereafter insulin secretion rapidly increased. In contrast, most specimens from prostaglandin-induced abortions showed high levels of insulin in the medium early in the culture period but little thereafter, indicating rapid release of insulin from damaged tissue. In some instances, after early insulin release by damaged tissue, some recovery of islet function occurred and insulin secretion again increased. The presence of differentiated endocrine cells was confirmed histologically. Tissue from each fetus was placed in a number of petri dishes, and for each individual fetus a qualitatively similar pattern of secretion was noted in each dish. These data suggest that representative and nondestructive monitoring of islet function is possible and may be important before such tissue is considered for use in islet transplantation in insulin-dependent diabetes.