Faculty Bibliography

BACKGROUND:Transplantation of vascularized donor thymic tissue along with a kidney transplant has markedly improved graft survival across the discordant pig-to-baboon xenogeneic barrier. To quantify the production of baboon T cells by the porcine thymic tissue, we recently developed an assay to measure the excised DNA products of baboon T-cell receptor (TCR) gene rearrangement (signal-joining TCR excision circles, sjTREC). METHODS:Initial polymerase chain reaction (PCR) analysis documented that TCR δREC-ψJα rearrangement occurs in baboons. Primers, specific to baboon sjTREC sequence were designed and used to quantify sjTREC molecules in peripheral blood mononuclear cells and thymic tissue using a quantitative PCR assay. RESULTS:sjTREC levels were higher in phenotypically naïve (CD3CD45RA) T cells (650 copies/100,000 cells) than in phenotypically memory (CD3CD45RA) T cells, with sjTREC below the limit of detection (40 copies/100,000 cells). Surgical removal of the native thymus in two baboons led to a significant decrease of sjTREC in peripheral blood (from 1104 and 920 copies to 184 and 190 copies/100,000 cells, respectively), confirming the role of the thymus in maintaining the peripheral T-cell pool. In two thymectomized baboons that received porcine thymokidney xenografts, sjTREC levels remained low in the peripheral blood (<40 copies/100,000 cells), but increased to 52 and 192 copies/100,000 cells in thymic biopsies, implying that baboon thymopoiesis had begun to occur in the porcine thymic xenografts. CONCLUSIONS:Baboon sjTREC can be quantified by quantitative PCR using primers specific to baboon sequence. Initial results suggest that baboon thymopoiesis occurs in vascularized porcine thymus xenografts.

UNLABELLED:BACKGROUND AND AIMS OF STUDY: We have previously demonstrated a requirement for the presence of a juvenile thymus for the induction of transplantation tolerance to renal allografts by a short-course of calcineurin inhibition in miniature swine. We have also shown that aged, involuted thymi can be rejuvenated when transplanted as vascularized thymic lobes into juvenile swine recipients. The present studies were aimed at elucidating the extrinsic factors facilitating this restoration of function in the aged thymus. In particular, we tested the impact of sex steroid blockade by Luteinizing Hormone-Releasing Hormone (LHRH). MATERIALS AND METHODS/METHODS:30 naive animals (25 males and 5 females) were used for measurement of serum testosterone levels. 3 mature male pigs (aged at 22, 22 and 29 months old) were used to test the effects of Lupron (LHRH analog) injection at 45 mg (per 70-80 kg body weight) as a 3-month depot on testosterone levels and thymic rejuvenation. Thymic rejuvenation was assessed by histology, flow cytometric analysis, morphometric analysis and TREC assays. RESULTS:Hormonal alterations were induced by Lupron and resulted in macroscopic and histologic regeneration of the thymus of aged animals within 2 months, as evidenced by restoration of juvenile thymus architecture and increased cellularity. Two animals that were evaluated for TREC both showed increased levels in the periphery following Lupron treatment. CONCLUSION/CONCLUSIONS:Treatment of aged animals with Lupron leads to thymic rejuventaion in adult miniature swine. This result could expand the applicability of thymus-dependent tolerance-inducing regimens to adult recipients.

The thymus serves as the central organ of immunologic self-nonself discrimination. Thymocytes undergo both positive and negative selection, resulting in T cells with a broad range of reactivity to foreign antigens but with a lack of reactivity to self-antigens. The thymus is also the source of a subset of regulatory T cells that inhibit autoreactivity of T-cell clones that may escape negative selection. As a result of these functions, the thymus has been shown to be essential for the induction of tolerance in many rodent and large animal models. Proper donor antigen presentation in the thymus after bone marrow, dendritic cell, or solid organ transplantation has been shown to induce tolerance to allografts. The molecular mechanisms of positive and negative selection and regulatory T-cell development must be understood if a tolerance-inducing therapeutic intervention is to be designed effectively. In this brief and selective review, we present some of the known information on T-cell development and on the role of the thymus in experimental models of transplant tolerance. We also cite some clinical attempts to induce tolerance to allografts using pharmacologic or biologic interventions.

BACKGROUND:Hematopoietic chimerism induces transplantation tolerance across allogeneic and xenogeneic barriers, but has been difficult to achieve in the pig-to-primate model. We have now utilized swine with knockout of the gene coding for alpha-1,3-galactosyltransferase (GalT-KO pigs) as bone marrow donors in an attempt to achieve chimerism and tolerance by avoiding the effects of natural antibodies to Gal determinants on pig hematopoietic cells. METHODS:Baboons (n = 4; Baboons 1 to 4 = B156, B158, B167, and B175, respectively) were splenectomized and conditioned with TBI (150 cGy), thymic irradiation (700 cGy), T cell depletion with rabbit anti-thymocyte globulin (rATG) and rat anti-primate CD2 (LoCD2b), and received FK506 and supportive therapy for 28 days. All animals received GalT-KO bone marrow (1 to 2 x 10(9) cells/kg) in two fractions on days 0 and 2, and were thereafter monitored for the presence of pig cells by flow cytometry, for porcine progenitor cells by PCR of BM colony-forming units, and for cellular reactivity to pig cells by mixed lymphocyte reaction (MLR). In vitro antibody formation to LoCD2b and rATG was tested by ELISA; antibody reactivity to GalT-KO pig cells was tested by flow cytometry and cytotoxicity assays. Additionally, Baboons 3 and 4 received orthotopic kidney transplants on days 17 and 2, respectively, to test the potential impact of the protocol on renal transplantation. RESULTS:None of the animals showed detectable pig cells by flow cytometry for more than 12 h post-BM infusion. However, porcine progenitor cell engraftment, as evidenced by pig-derived colony forming units in the BM, as well as peripheral microchimerism in the thymus, lymph node, and peripheral blood was detected by PCR in baboons 1 and 2 for at least 28 days post-transplant. ELISA results confirmed humoral immunocompetence at time of transplantation as antibody titers to rat (LoCD2b) and rabbit (ATG) increased within 2 weeks. However, no induced antibodies to GalT-KO pig cells or increased donor specific cytotoxicity was detectable by flow cytometry. In contrast, baboons 3 and 4 developed serum antibodies to pig cells as well as to rat and rabbit immunoglobulin by day 14. Retrospective analysis revealed that although all four baboons possessed low levels of antibody-mediated cytotoxicity to GalT-KO cells prior to transplantation, the two baboons (3 and 4) that became sensitized to pig cells (and rejected pig kidneys) had relatively high pre-transplantation titers of anti-non-Gal IgG detectable by flow cytometry, whereas baboons 1 and 2 had undetectable titers. CONCLUSIONS:Engraftment and specific non-responsiveness to pig cells has been achieved in two of four baboons following GalT-KO pig-to-baboon BMT. Engraftment correlated with absence of preformed anti-non-Gal IgG serum antibodies. These results are encouraging with regard to the possibility of achieving transplantation tolerance across this xenogeneic barrier.

John Jones was a pioneer of American Surgery. Born in Long Island, New York in 1729, he received his medical degree in France from the University of Rheims. He returned to the colonies and helped to establish the medical school that would later become Columbia University's College of Physicians and Surgeons where he was appointed the first Professor of Surgery in the New World. He used his position to assert that surgeons trained in America should be familiar with all facets of medicine and not be mere technicians. Before the outbreak of the American Revolution, he wrote a surgical field manual, which was the first medical text published in America. A believer in the principles of the American Revolution, he would go on to count Benjamin Franklin and George Washington as his patients. Despite achieving many firsts in American medicine, his influence on surgical training is his most enduring legacy.

BACKGROUND AND OBJECTIVE/OBJECTIVE:Clinical intestinal transplantation (Int-Tx) is limited by high rates of rejection, infection, and graft versus host disease. To improve clinical outcomes and eliminate the comorbidities associated with chronic immunosuppression, the induction of donor-specific tolerance to intestinal grafts is desirable, especially in the pediatric population. This study determined the ability of intestinal grafts to facilitate tolerance induction in major histocompatibility complex (MHC)-inbred miniature swine. METHODS:Seven MGH-miniature swine received heterotopic intestinal grafts, two across MHC-matched, minor-antigen disparities, three across a class I MHC disparity with 12 days of cyclosporine A, and two across a class I MHC disparity without an immunosuppressant. Chimerism was assessed by FACS analysis and immunohistochemistry. Cell-mediated lympholysis assays were used to assess antidonor responses. RESULTS:Two animals receiving intestinal grafts without an immunosuppressant developed antidonor IgG in 14 days and rejected these completely. All other grafts were accepted with 12 days of cyclosporine A across both MHC-matched and MHC class I barriers. Cell-mediated lympholysis assays showed donor-specific unresponsiveness by day 30 across MHC class I barriers. Greater than 15% peripheral donor cell chimerism persisted for more than 60 days after MHC-matched Int-Tx. Although less than 1.5% peripheral donor cell chimerism was seen during the maintenance period after class I-mismatched Int-Tx, 5% to 10% myeloid chimerism was found in the peripheral blood 14 to 90 days after Int-Tx. FACS analysis demonstrated that 1% to 2% of lymphocytes in the graft mesenteric lymph nodes were CD4/CD25(HIGH+)/Foxp3(+) cells. CONCLUSION/CONCLUSIONS:To our knowledge, this is the first demonstration of tolerance induction and persistence of chimerism in a large animal intestinal transplant model.

Clinical transplantation for the treatment of end-stage organ disease is limited by a shortage of donor organs. Successful xenotransplantation could immediately overcome this limitation. The development of homozygous alpha1,3-galactosyltransferase knockout (GalT-KO) pigs removed hyperacute rejection as the major immunologic hurdle to xenotransplantation. Nevertheless, GalT-KO organs stimulate robust immunologic responses that are not prevented by immunosuppressive drugs. Murine studies show that recipient thymopoiesis in thymic xenografts induces xenotolerance. We transplanted life-supporting composite thymokidneys (composite thymus and kidneys) prepared in GalT-KO miniature swine to baboons in an attempt to induce tolerance in a preclinical xenotransplant model. Here, we report the results of seven xenogenic thymokidney transplants using a steroid-free immunosuppressive regimen that eliminated whole-body irradiation in all but one recipient. The regimen resulted in average recipient survival of over 50 days. This was associated with donor-specific unresponsiveness in vitro and early baboon thymopoiesis in the porcine thymus tissue of these grafts, suggesting the development of T-cell tolerance. The kidney grafts had no signs of cellular infiltration or deposition of IgG, and no grafts were lost due to rejection. These results show that xenogeneic thymus transplantation can support early primate thymopoiesis, which in turn may induce T-cell tolerance to solid organ xenografts.

C1q nephropathy, first proposed by Jennette and Hipp [Am J Clin Pathol 83:415-420, 1985; Am J Kidney Dis 6:103-110, 1985], was described as a distinct glomerular disease entity characterized by extensive mesangial deposition of C1q, with associated mesangial immune complexes, and the absence of any clinical and laboratory evidence of systemic lupus erythematosus. Now, 20 years since the first report, the disease entity is gradually attaining recognition, particularly in the field of pediatrics. C1q is the subcomponent of C1 in the classical pathway of complement activation. Generally, C1q deposition is caused by the activation of C1 by immunoglobulin G (IgG) and IgM; therefore, C1q nephropathy is considered as an immune complex glomerulonephritis. However, in C1q nephropathy, it remains unclear whether the deposition of C1q in the glomeruli is in response to the deposition of immunoglobulin or immune complex, or whether deposition is non-specific trapping that accompanies increased glomerular protein trafficking associated with proteinuria. Since not only the pathogenesis of C1q deposition in glomeruli but also its significance are still uncertain, it has not yet been established as an independent disease. From recent publications of the clinical and pathological characterizations, C1q nephropathy has been thought to be a subgroup of primary focal segmental glomerular sclerosis. However, many reports describe different symptoms, histopathologies, therapeutic responses and prognoses, suggesting that C1q nephropathy is not a single disease entity, but that it may be a combination of several disease groups. There are many uncertain areas requiring further investigation, though it is hoped that a detailed examination of future cases will clarify the subgroups making up C1q nephropathy and their clinicopathological characteristics, and will lead to the establishment of C1q nephropathy as an independent disease entity.

BACKGROUND:Survival of ABO-mismatched kidneys with stable renal function despite the persistence of anti-ABO antibodies is called accommodation. The mechanism of accommodation is unclear, but may involve complement regulatory proteins such as CD59. The development of alpha-1,3-galactosyltransferase knock-out (GalT-KO) swine that produce anti-Gal antibodies provides a large animal model capable of determining the role of complement regulatory proteins in accommodation. METHODS:ELISA and antibody fluorescence-activated cell sorting were used to examine the rate of anti-Gal antibody expression as a function of age. Major histocompatibility complex-matched kidneys were transplanted from Gal-positive MGH miniature swine to MGH GalT-KO swine with systemic immunosuppression. One recipient underwent adsorbtion of anti-Gal antibodies before transplantation. Graft survival, antibody, and complement deposition patterns and CD59 expression were determined. RESULTS:Three animals rejected Gal-positive kidneys by humoral mechanisms. One animal with low titers of anti-Gal antibody displayed spontaneous accommodation and the animal that was treated with antibody adsorbtion also displayed accommodation. Rejected grafts had deposition of IgM, IgG, C3, and C5b-9 with low expression of CD59, whereas accommodated grafts had low deposition of C5b-9 and high expression of CD59. Retransplantation of one accommodated graft to a naïve GalT-KO animal confirmed that changes in the graft were responsible for the lack of C5b-9 deposition. CONCLUSION/CONCLUSIONS:GalT-KO miniature swine produce anti-Gal antibodies and titers increase with age. These anti-Gal antibodies can cause rejection of major histocompatibility complex-matched kidneys unless accommodation occurs. CD59 up-regulation seems to be involved in the mechanism of accommodation by preventing the formation of the membrane attack complex (MAC) on the accommodated graft.

Xenograft outcomes are dictated by xenoantigen expression, for example, Gal alpha1, 3Gal (Gal), but might also depend on differing vascular responses. We investigated whether differential vascular gene expression in kidney and cardiac xenografts correlate with development of thrombotic microangiopathy (TM) and consumptive coagulation (CC). Immunosuppressed baboons underwent miniswine or hDAF pig kidney (n = 6) or heart (n = 7), or Gal-transferase gene-knockout (GalT-KO) (thymo)kidney transplantation (n = 14). Porcine cDNA miniarrays determined donor proinflammatory, apoptosis-related and vascular coagulant/fibrinolytic gene expression at defined time points; validated by mRNA, protein levels and immunopathology. hDAF-transgenic and GalT-KO xenografts, (particularly thymokidneys) exhibited prolonged survival. CC was seen with Gal-expressing porcine kidneys (3 of 6), only 1 of 7 baboons postcardiac xenotransplantation and was infrequent following GalT-KO grafts (1 of 14). Protective-type genes (heme oxygenase-I, superoxide dismutases and CD39) together with von Willebrand factor and P-selectin were upregulated in all renal grafts. Transcriptional responses in Gal-expressing xenografts were comparable to those seen in the infrequent GalT-KO rejection. In cardiac xenografts, fibrin deposition was associated with increased plasminogen activator inhibitor-1 expression establishing that gene expression profiles in renal and cardiac xenografts differ in a quantitative manner. These findings suggest that therapeutic targets may differ for renal and cardiac xenotransplants.