William A. Rudert

Baboon-to-human liver transplantation

Our ability to control both the cellular and humoral components of xenograft rejection in laboratory experiments, together with an organ shortage that has placed limits on clinical transplantation services, prompted us to undertake a liver transplantation from a baboon to a 35-year-old man with B virus-associated chronic active hepatitis and human immunodeficiency virus infection. Liver replacement was performed according to conventional surgical techniques. Immunosuppression was with the FK 506-prednisone-prostaglandin regimen used routinely for hepatic allotransplantation, to which a daily non-myelotoxic dose of cyclophosphamide was added. During 70 days of survival, there was little evidence of hepatic rejection by biochemical monitoring or histopathological examination. Products of hepatic synthesis, including clotting factors, became those of the baboon liver with no obvious adverse effects. Death followed a cerebral and subarachnoid haemorrhage that was caused by an angioinvasive aspergillus infection. However, the underlying cause of death was widespread biliary sludge that formed in the biliary tree despite a seemingly satisfactory choledochojejunostomy. During life and in necropsy samples, there was evidence of the chimerism that we believe is integral to the acceptance of both xenografts and allografts. Our experience has shown the feasibility of controlling the rejection of the baboon liver xenograft in a human recipient. The biliary stasis that was the beginning of lethal infectious complications may be correctable by modifications of surgical technique. In further trials, the error of over-immunosuppression should be avoidable.

Systemic chimerism in human female recipients of male livers

We have previously reported data from clinical and laboratory animal observations which suggest that organ tolerance after transplantation depends on a state of balanced lymphodendritic cell chimerism between the host and donor graft. We have sought further evidence to support this hypothesis by investigating HLA-mismatched liver allograft recipients. 9 of 9 female recipients of livers from male donors had chimerism in their allografts and extrahepatic tissues, according to in-situ hybridisation and molecular techniques 10 to 19 years posttransplantation. In 8 women with good graft function, evidence of the Y chromosome was found in the blood (6/8), skin (8/8), and lymph nodes (7/8). A ninth patient whose transplant failed after 12 years from recurrent chronic viral hepatitis had chimerism in her lymph nodes, skin, jejunum, and aorta at the time of retransplantation. Although cell migration is thought to take place after all types of transplantation, the large population of migratory cells in, and the extent of their seeding from, hepatic grafts may explain the privileged tolerogenicity of the liver compared with other organs.

Chimerism and donor-specific nonreactivity 27 to 29 years after kidney allotransplantation

Chimerism was demonstrated with immunocytochemical and/or polymerase chain reaction techniques in kidney allografts and in the native skin, lymph nodes, or blood of 5 of 5 patients who received continuously functioning renal transplants from 1 or 2 haplotype HLA mismatched consanguineous donors (4 parents, 1 aunt) 27–29 years ago. In the 4 cases where the kidney donor still was alive to provide stimulator lymphocytes for testing, these provoked no (n=2) or modest (n=2) MLR in contrast to vigorous MLR to third party lymphocytes. In all 4 cases, the donor cells failed to generate in vitro cytotoxic effector cells (cell-mediated lymphocytotoxicity). These findings are in accord with the hypothesis that cell migration, repopulation, and chimerism are seminal events that define graft acceptance and ultimately can lead to acquired donor-specific nonresponsiveness (tolerance).

Bone marrow augmentation of donor-cell chimerism in kidney, liver, heart, and pancreas islet transplantation

We have previously postulated that donor cell chimerism in organ transplantation is needed to attain a tolerant state. Here we show that donor cell chimerism can be augmented in organ recipients if they are infused perioperatively with 3 x 10(8) per kg of unmodified donor bone marrow cells and are kept on a conventional immunosuppressive regimen of tacrolimus (FK506) and prednisolone. 36 patients took part, of whom the first 18 patients have good transplanted kidney (n = 10), liver (n = 7), and heart (n = 7) function when followed up between 4 and 16 months. All patients are well. We found persistent multilineage leucocyte chimerism in blood of 17 recipients by flow cytometry and PCR techniques to detect donor alleles or Y chromosomes in female recipients of male organs. The use of the 5-antigben HLA matched same sex donor precluded detection of chimerism in one patient.

Chimerism after liver transplantation for type IV glycogen storage disease and type 1 Gaucher's disease.

BACKGROUND Liver transplantation for type IV glycogen storage disease (branching-enzyme deficiency) results in the resorption of extrahepatic deposits of amylopectin, but the mechanism of resorption is not known. METHODS We studied two patients with type IV glycogen storage disease 37 and 91 months after liver transplantation and a third patient with lysosomal glucocerebrosidase deficiency (type 1 Gauchers disease), in whom tissue glucocerebroside deposition had decreased 26 months after liver replacement, to determine whether the migration of cells from the allograft (microchimerism) could explain the improved metabolism of enzyme-deficient tissues in the recipient. Samples of blood and biopsy specimens of the skin, lymph nodes, heart, bone marrow, or intestine were examined immunocytochemically with the use of donor-specific monoclonal anti-HLA antibodies and the polymerase chain reaction, with preliminary amplification specific to donor alleles of the gene for the beta chain of HLA-DR molecules, followed by hybridization with allele-specific oligonucleotide probes. RESULTS Histopathological examination revealed that the cardiac deposits of amylopectin in the patients with glycogen storage disease and the lymph-node deposits of glucocerebroside in the patient with Gauchers disease were dramatically reduced after transplantation. Immunocytochemical analysis showed cells containing the HLA phenotypes of the donor in the heart and skin of the patients with glycogen storage disease and in the lymph nodes, but not the skin, of the patient with Gauchers disease. Polymerase-chain-reaction analysis demonstrated donor HLA-DR DNA in the heart of both patients with glycogen storage disease, in the skin of one of them, and in the skin, intestine, blood, and bone marrow of the patient with Gauchers disease. CONCLUSIONS Systemic microchimerism occurs after liver allotransplantation and can ameliorate pancellular enzyme deficiencies.

Protection of Human Islets from the Effects of Interleukin-1β by Adenoviral Gene Transfer of an IκB Repressor

Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that inhibits β cell function and promotes Fas-triggered apoptosis. IL-1β is thought to act early in the initiation of the autoimmune destruction of pancreatic β cells in type I diabetes. IL-1β promotes β cell impairment, in part, by activating NF-κB transcription factor-dependent signaling pathways. We have examined whether β cells could be protected from the effects of IL-1β by overexpressing an inhibitor of NF-κB activity, IκB, by adenoviral gene transfer to intact human islets in culture. Infection of islets with an adenoviral vector encoding a non-phosphorylatable, non-degradable variant of IκBα resulted in normal insulin responses to glucose in the presence of IL-1β. Furthermore, nitric oxide production was prevented and, more importantly, Fas-triggered apoptosis was inhibited following IκBα gene transfer. These results suggest that blocking the NF-κB pathway might prevent cytokine-induced β cell impairment as a means of facilitating islet transplantation.

Thymus‐specific deletion of insulin induces autoimmune diabetes

Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance towards pancreatic β‐cells. To further explore the function of this ectopic insulin expression, we knocked out the mouse Ins2 gene specifically in the Aire‐expressing medullary thymic epithelial cells (mTECs), without affecting its expression in the β‐cells. When further crossed to the Ins1 knockout background, both male and female pups (designated as ID‐TEC mice for insulin‐deleted mTEC) developed diabetes spontaneously around 3 weeks after birth. β‐cell‐specific autoimmune destruction was observed, as well as islet‐specific T cell infiltration. The presence of insulin‐specific effector T cells was shown using ELISPOT assays and adoptive T cell transfer experiments. Results from thymus transplantation experiments proved further that depletion of Ins2 expression in mTECs was sufficient to break central tolerance and induce anti‐insulin autoimmunity. Our observations may explain the rare cases of type 1 diabetes onset in very young children carrying diabetes‐resistant HLA class II alleles. ID‐TEC mice could serve as a new model for studying this pathology.

Prevention of beta cell dysfunction and apoptosis activation in human islets by adenoviral gene transfer of the insulin-like growth factor I.

Interleukin-1β is a potent pro-inflammatory cytokine that has been shown to inhibit islet β cell function as well as to activate Fas-mediated apoptosis in a nitric oxide-dependent manner. Furthermore, this cytokine is effective in recruiting lymphocytes that mediate β cell destruction in IDDM onset. The insulin-like growth factor I (IGF-I) has been shown to block IL-1β actions in vitro. We hypothesized that gene transfer of the insulin-like growth factor I to intact human islets could prevent IL-1β-induced β cell dysfunction and sensitization to Fas-triggered apoptosis activation. Intact human islets were infected with adenoviral vectors encoding IGF-I as well as β-galactosidase and enhanced green fluorescent protein as controls. Adenoviral gene transfer of human IGF-I prevented IL-1β-mediated nitric oxide production from human islets in vitro as well as the suppression of β cell function as determined by glucose-stimulated insulin production. Moreover, IGF-I gene transfer prevented IL-1β-induced, Fas-mediated apoptosis. These results suggest that locally produced IGF-I from cultured islets may be beneficial in maintaining β cell function and promoting islet survival before and following islet transplantation as a potential therapy for type I diabetes.

Immunosuppressive effects of glucosamine.

Glucosamine is a naturally occurring derivative of glucose and is an essential component of glycoproteins and proteoglycans, important constituents of many eukaryotic proteins. In cells, glucosamine is produced enzymatically by the amidation of glucose 6-phosphate and can then be further modified by acetylation to result in N-acetylglucosamine. Commercially, glucosamine is sold over-the-counter to relieve arthritis. Although there is evidence in favor of the beneficial effects of glucosamine, the mechanism is unknown. Our data demonstrate that glucosamine suppresses the activation of T-lymphoblasts and dendritic cells in vitroas well as allogeneic mixed leukocyte reactivity in a dose-dependent manner. There was no inherent cellular toxicity involved in the inhibition, and the activity was not reproducible with other amine sugars. More importantly, glucosamine administration prolonged allogeneic cardiac allograft survival in vivo. We conclude that, despite its documented effects on insulin sensitivity, glucosamine possesses immunosuppressive activity and could be beneficial as an immunosuppressive agent.

Molecular Basis of Evolutionary Loss of the α1,3-Galactosyltransferase Gene in Higher Primates

Galactose-α1,3-galactose (αGal) epitopes, the synthesis of which requires the enzyme product of α1,3-galactosyltransferase (α1,3GT), are sugar chains on the cell surface of most mammalian species. Notable exceptions are higher primates including Old World monkeys, apes, and humans. The αGal-negative species as well as mice with deletion of the α1,3GT gene produce abundant anti-αGal antibodies. The evolutionary loss of αGal epitopes has been attributed to point mutations in the coding region of the gene. Because no transcripts could be found in the higher primate species with Northern blot analysis, a potential alternative explanation has been loss of upstream regulation of the gene. Here, we have demonstrated that the rhesus promoter is functional. More importantly, a variety of full-length transcripts were detected with sensitive PCR-based methods in the tissues of rhesus monkeys, orangutans, and humans. Five crucial mutations were delineated in the coding region of the human and rhesus and three in the orangutan, any one of which could be responsible for inactivation of the α1,3GT gene. Two of the mutations were shared by all three higher primates. These findings, which elucidate the molecular basis for the evolutionary loss of αGal expression, may have implications in medical research.