Biomedical subjects
David H Sachs
Publications and source records attributed to David H Sachs.
Early weaning of piglets fails to exclude porcine lymphotropic herpesvirus.
BACKGROUND: Xenotransplantation using pigs as source species carries a risk for the activation of latent herpesviruses from the porcine donor and potential transmission to the recipient. In pig-to-baboon xenotransplantation, activation of porcine cytomegalovirus (PCMV) has been associated with xenograft injury and an increased incidence of consumptive coagulopathy and graft loss. Activation of porcine lymphotropic herpesvirus (PLHV)-1 was not observed in pig-to-baboon solid organ xenotransplantation, but was associated with a syndrome of post-transplantation lymphoproliferative disorder (PTLD) after allogeneic stem cell transplantation in pigs. MATERIAL AND METHODS: Early weaning of piglets was used to try to reduce the viral burden of xenograft donors. This consisted of separating the piglets of a litter from the sow within the first 2 weeks after birth and raising them in isolation from the remaining herd. RESULTS: We have previously demonstrated that PCMV could be excluded from source animals by early weaning of piglets. However, early weaning failed to exclude PLHV-1 from source pigs. CONCLUSIONS: This disparity between PCMV and PLHV-1 reflects differing pathogenesis of infection of these herpesviruses. New approaches will be needed to exclude PLHV-1 from pig colonies.
Indirect recognition of MHC class I allopeptides accelerates lung allograft rejection in miniature swine.
The role of indirect allorecognition in graft rejection is examined in two experiments using a swine lung transplantation model. First, two swine received class I mismatched grafts without immunosuppression; another two recipients were treated postoperatively with cyclosporine (CsA). These swine exhibited acute and chronic rejection, respectively. All four recipients developed T-cell reactivity to donor-derived class I major histocompatibility complex (MHC) peptides. Second, six swine were immunized with synthetic donor-derived class I allopeptides prior to transplantation. Control groups consisted of nonimmunized recipients (n = 6) and recipients immunized with an irrelevant peptide (n = 3). These recipients all received a 12-day course of post-operative CsA. Swine immunized with allopeptides exhibited accelerated graft rejection, as compared to both control groups (p < 0.01 and p = 0.03, respectively). Within the experimental group, the dominant histologic finding was acute rejection (AR). Obliterative bronchiolitis (OB) was seen in the graft with the longest survival. Both control groups showed a lesser degree of AR, with four out of six nonimmunized swine ultimately developing OB. These studies suggest that indirect allorecognition is operative during lung allograft rejection, and that pre-transplant sensitization to donor-derived MHC allopeptides can accelerate graft rejection.
Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience.
Hearts from alpha1,3-galactosyltransferase knockout pigs (GalT-KO, n = 8) were transplanted heterotopically into baboons using an anti-CD154 monoclonal antibody-based regimen. The elimination of the galactose-alpha1,3-galactose epitope prevented hyperacute rejection and extended survival of pig hearts in baboons for 2-6 months (median, 78 d); the predominant lesion associated with graft failure was a thrombotic microangiopathy, with resulting ischemic injury. There were no infectious complications directly related to the immunosuppressive regimen. The transplantation of hearts from GalT-KO pigs increased graft survival over previous studies.
Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue.
The use of animal organs could potentially alleviate the critical worldwide shortage of donor organs for clinical transplantation. Because of the strong immune response to xenografts, success will probably depend upon new strategies of immune suppression and induction of tolerance. Here we report our initial results using alpha-1,3-galactosyltransferase knockout (GalT-KO) donors and a tolerance induction approach. We have achieved life-supporting pig-to-baboon renal xenograft survivals of up to 83 d with normal creatinine levels.
T cells from presensitized donors fail to cause graft-versus-host disease in a pig-to-mouse xenotransplantation model.
BACKGROUND: The ability of T cells from pigs, the most suitable donors for clinical xenotransplantation, to induce graft-versus-host disease (GVHD) and to facilitate hematopoietic cell engraftment in highly disparate xenogeneic recipients remains unclear. In this article, the authors address these questions in a presensitized pig-to-mouse transplantation model using porcine cytokine-transgenic mice. METHODS: Swine donors were presensitized by mouse skin grafting and boosted by the injection of mouse cells after the skin graft was rejected. Porcine peripheral blood mononuclear cells (PBMC) and splenocytes were collected at various times after mouse skin grafting, and their potential to induce GVHD and to facilitate donor hematopoietic cell engraftment in conditioned murine recipients was evaluated. RESULTS: Presensitization of donor pigs resulted in marked enhancement of anti-mouse responses, as reflected in augmented anti-mouse mixed lymphocyte responses, cell-mediated cytotoxicity, and antibody production. However, injection of high numbers of PBMC and splenocytes (>1 x 10(8)) with bone marrow cells from the presensitized pigs failed to induce detectable GVHD or long-term chimerism in mice that were treated with depleting anti-T-cell and natural killer cell antibodies, cobra venom factor, medronate-liposomes, and 4 Gy of whole-body and 7 Gy of thymic irradiation. Histologic analysis revealed no mononuclear cell infiltration or GVHD-associated lesions in the liver, intestine, lungs, or skin of the mouse recipients. Furthermore, the recipient mice had no detectable T cells or anti-pig immunoglobulin G antibodies in the blood by 6 weeks after injection of porcine cells. CONCLUSION: These results demonstrate that porcine T-cell function is severely impaired in the xenogeneic murine microenvironment.
Reduction of consumptive coagulopathy using porcine cytomegalovirus-free cardiac porcine grafts in pig-to-primate xenotransplantation.
BACKGROUND: Xenotransplantation using pigs as the source species for organs carries a potential risk for transmission and activation of porcine herpesviruses. Activation of porcine cytomegalovirus (PCMV) in pig-to-baboon xenotransplantation is associated with xenograft injury and possibly an increased incidence of consumptive coagulopathy (CC). METHODS: To further investigate the role of PCMV activation in the occurrence of CC, a strategy to exclude PCMV from the donor was developed. To exclude PCMV, piglets were early-weaned and raised separated from other swine. These piglets were used as donors in an experimental protocol of pig-to-baboon heart xenotransplantation. RESULTS: Early weaning of piglets was successful in excluding PCMV. Use of PCMV-free cardiac porcine xenografts in baboons resulted in prolonged graft survival and prevented consumptive coagulopathy in all recipients. CONCLUSIONS: The use of PCMV-free cardiac grafts is beneficial in reducing the direct effects of PCMV activation in the graft (tissue damage) and the indirect effects of PCMV activation in the recipient (consumptive coagulopathy).
alpha1,3-Galactosyltransferase gene-knockout miniature swine produce natural cytotoxic anti-Gal antibodies.
BACKGROUND: The expression of galactose alpha 1,3 galactose (Gal) in pigs has proved a barrier to xenotransplantation. Miniature swine lacking Gal (Gal pigs) have been produced by nuclear transfer/embryo transfer. METHODS: The tissues of five Gal pigs of SLA dd haplotype (SLA) were tested for the presence of Gal epitopes by staining with the Griffonia simplicifolia IB4 lectin. Their sera were tested by flow cytometry for binding of IgM and IgG to peripheral blood mononuclear cells (PBMC) from wild-type (Gal) SLA-matched pigs; serum cytotoxicity was also assessed. The cellular responses of PBMC from Gal swine toward Gal SLA-matched PBMC were tested by mixed leukocyte reaction and cell-mediated lympholysis assays. RESULTS: None of the tissues tested showed Gal expression. Sera from all five Gal pigs manifested IgM binding to Gal pig PBMC, and sera from three showed IgG binding. In all five cases, cytotoxicity to Gal cells could be demonstrated, which was lost after treatment of the sera with dithiothreitol, indicating IgM antibody-mediated cytotoxicity. PBMC from Gal swine had no proliferative or cytolytic T-cell response toward Gal SLA-matched PBMC. CONCLUSIONS: Gal pigs do not express Gal epitopes and develop anti-Gal antibodies that are cytotoxic to Gal pig cells. The absence of an in vitro cellular immune response between Gal and Gal pigs is related to their identical SLA haplotype and indicates the absence of immunogenicity of Gal in T-cell responses. The model of Gal organ transplantation into a Gal SLA-matched recipient would be a valuable large animal model in the study of accommodation or B-cell tolerance.
Production of alpha-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations.
Hyperacute rejection of porcine organs by old world primate recipients is mediated through preformed antibodies against galactosyl-alpha-1,3-galactose (Galalpha-1,3-Gal) epitopes expressed on the pig cell surface. Previously, we generated inbred miniature swine with a null allele of the alpha-1,3-galactosyltransferase locus (GGTA1) by nuclear transfer (NT) with gene-targeted fibroblasts. To expedite the generation of GGTA1 null pigs, we selected spontaneous null mutant cells from fibroblast cultures of heterozygous animals for use in another round of NT. An unexpectedly high rate of spontaneous loss of GGTA1 function was observed, with the vast majority of null cells resulting from loss of the WT allele. Healthy piglets, hemizygous and homozygous for the gene-targeted allele, were produced by NT by using fibroblasts that had undergone deletional and crossover/gene conversion events, respectively. Aside from loss of Galalpha-1,3-Gal epitopes, there were no obvious phenotypic differences between these null piglets and WT piglets from the same inbred lines. In fact, congenital abnormalities observed in the heterozygous NT animals did not reappear in the serially produced null animals.
Activation of porcine cytomegalovirus, but not porcine lymphotropic herpesvirus, in pig-to-baboon xenotransplantation.
Tissue-invasive disease due to porcine cytomegalovirus (PCMV) has been demonstrated after pig-to-baboon solid-organ xenotransplantation. Porcine lymphotropic herpesvirus (PLHV)-1 is associated with B cell proliferation and posttransplant lymphoproliferative disorder after allogeneic bone marrow transplantation in swine but has not been observed in pig-to-primate xenotransplantation. Activation of PCMV and PLHV-1 was investigated in 22 pig-to-baboon xenotransplants by use of quantitative polymerase chain reaction. PCMV was found in all xenografts; increased viral replication occurred in 68% of xenografts during immunosuppression. PLHV-1 was found in 12 xenografts (55%); no increases in viral replication occurred during immunosuppression. Control immunosuppressed swine coinfected with PCMV and PLHV-1 had activation of PCMV but not PLHV-1. PCMV, but not PLHV-1, is activated in solid-organ xenotransplantation.
Role of the thymus in transplantation tolerance in miniature Swine: IV. The thymus is required during the induction phase, but not the maintenance phase, of renal allograft tolerance.
BACKGROUND: The authors' laboratory previously demonstrated that long-term tolerance to class I-disparate renal allografts in miniature swine can be induced by a short course of cyclosporine A (CsA), and that this stable tolerance is dependent on the presence of an intact thymus. In the present study, the authors have examined the requirement for a thymus during the pretransplant, induction, and maintenance phases of tolerance. METHODS: Twenty-two miniature swine underwent class I major histocompatibility complex-mismatched renal transplantation, with a 12-day course of CsA. Thymectomies were performed on days -21, 0, +8, +21, and greater than or equal to +42, in relation to the day of transplantation. Historical controls consisted of euthymic and sham-thymectomized recipients. RESULTS: Euthymic, sham-thymectomized, and day-greater than or equal to +42 thymectomized recipients demonstrated stable renal function and minimal anti-donor cytotoxic T-lymphocyte (CTL) responses. In contrast, day -21 and day 0 thymectomized recipients demonstrated allograft dysfunction, marked cellular infiltrates, with severe vasculitis and glomerular changes, and strong anti-donor CTL responses. Animals thymectomized on days +8 and +21 did not undergo severe rejection, but likewise did not demonstrate a stable clinical course. CONCLUSIONS: These data indicate that the requirement for thymic function in the induction of rapid and stable tolerance is greatest during the first 8 days and then diminishes over the next 2 weeks posttransplant. Failure of thymectomy to affect the course of tolerance after day +21 suggests that thymic function is not required for the maintenance of tolerance. Understanding the role of the thymus in establishing tolerance may permit the development of tolerance induction strategies, especially for pediatric transplant recipients.
Mixed chimerism and transplantation tolerance.
Establishment of mixed hematopoietic chimerism carries with it the induction of transplantation tolerance to any other tissue or organ from the same donor. This strategy has been studied extensively for induction of tolerance in mice. During the past decade, we have extended the same strategy, with modifications, to cynomolgus monkeys and most recently to renal transplant patients. In this report we review the evolution of these studies from preclinical applications to our current clinical experience with two therapeutic protocols sponsored by the Immune Tolerance Network. The first of these studies is for patients with myeloma and end-stage renal disease with an HLA-matched sibling donor; the second for patients with end-stage renal disease and HLA-mismatched donors. Although it is still early in the course of these studies, the results to date are very encouraging.
Vascularized thymic lobe transplantation in miniature swine: thymopoiesis and tolerance induction across fully MHC-mismatched barriers.
As the major site of self-nonself discrimination in the immune system, the thymus, if successfully transplanted, could potentially carry with it the induction of central tolerance to any other organ or tissue from the same donor. We have recently developed a technique for transplantation of an intact, vascularized thymic lobe (VTL) in miniature swine. In the present study, we have examined the ability of such VTL allografts to support thymopoiesis and induce transplantation tolerance across fully MHC-mismatched barriers. Six miniature swine recipients received fully MHC-mismatched VTL grafts with a 12-day course of tacrolimus. Three of these recipients were thymectomized before transplantation and accepted their VTL allografts long-term, with evidence of normal thymopoiesis. In contrast, three euthymic recipients rejected their VTL allografts. Donor renal allografts, matched to the donor VTL grafts, were transplanted without immunosuppression into two of the three thymectomized recipients, and one of the three euthymic recipients. These renal allografts were accepted by thymectomized recipients, but rejected by the euthymic recipient in an accelerated fashion. This study thus demonstrates that successful transplantation of a vascularized thymus across a fully MHC-mismatched barrier induces tolerance in this preclinical, large-animal model. This procedure should enable studies on the role of the thymus in transplantation immunology as well as offer a potential strategy for tolerance induction in clinical transplantation.
Tolerance to composite tissue allografts across a major histocompatibility barrier in miniature swine.
BACKGROUND: Tolerance to composite tissue allografts might allow the widespread clinical use of reconstructive allotransplantation if protocols to achieve this could be rendered sufficiently nontoxic. The authors investigated whether tolerance could be generated in miniature swine to composite tissue allografts across a major histocompatibility (MHC) barrier. A clinically relevant tolerance protocol involving hematopoietic cell transplantation without the need for irradiation or myelosuppressive drugs was tested. METHODS: Seven recipient animals were transiently T-cell depleted and a short course of cyclosporine was initiated. Twenty-four hours later, a donor hematopoietic cell transplant consisting of cytokine-mobilized peripheral blood mononuclear cells or bone marrow cells and a heterotopic limb transplant were performed. In vitro anti-donor responsiveness was assessed by mixed-lymphocyte reaction and cell-mediated lympholysis assays. Acceptance of the limb allografts was determined by gross and histologic appearance. Chimerism in the peripheral blood and lymphohematopoietic organs was assessed by flow cytometry. RESULTS: All seven experimental animals accepted the musculoskeletal elements but rejected the skin of the allografts. All but one of the animals displayed donor-specific unresponsiveness in vitro. The animals that received cytokine mobilized-peripheral blood mononuclear cells showed chimerism but had clinical evidence of graft-versus-host disease (GVHD). None of the animals that received bone marrow cells showed stable chimerism and none developed GVHD. CONCLUSIONS: This protocol can achieve tolerance to the musculoskeletal elements of composite tissue allografts across an MHC barrier in miniature swine. Stable chimerism does not appear to be necessary for tolerance and may not be desirable because of the risk of GVHD.
Thrombophilia associated with anti-CD154 monoclonal antibody treatment and its prophylaxis in nonhuman primates.
BACKGROUND: The authors previously reported thromboembolic complications associated with anti-CD154 monoclonal antibody (mAb) treatment in nonhuman primates. The underlying mechanisms of this complication and its management have not been established. METHODS: Eighty cynomolgus monkey renal allograft recipients treated with anti-CD154 mAb were studied for the incidence of thrombosis and its prophylaxis. RESULTS: Without anticoagulation prophylaxis, thromboembolic complications were seen in 5 of 11 recipients. With addition of perioperative heparin, the incidence was decreased to 2 of 10. No further improvement was observed by adding intraoperative prostaglandin (PG) E1. However, addition of ketorolac tromethamine to PGE1 and heparin decreased the incidence of thrombosis (one of eight). Most recently, the authors have found that ketorolac administration alone resulted in no thrombosis in 25 consecutive recipients. CONCLUSIONS: Ketorolac is remarkably effective in preventing thromboembolism associated with anti-CD154 mAb treatment, suggesting the mechanism underlying this complication may be related to platelet activation leading to enhanced aggregation.
Induction of human T-cell tolerance to porcine xenoantigens through mixed hematopoietic chimerism.
Xenotransplantation from pigs could provide a potential solution to the severe shortage of allogeneic donor organs. Because xenogeneic tissues are subject to vigorous immune rejection, tolerance induction is likely to be essential to the success of clinical xenotransplantation. Here we explore the possibility of inducing human T-cell tolerance to porcine xenografts through mixed chimerism. We previously showed that NOD/SCID-Tg mice expressing porcine cytokine transgenes permit the induction of durable porcine hematopoietic chimerism. In this study we achieved human T-cell development in these mice by engrafting human fetal thymus/liver tissues. In porcine hematopoietic chimeras, human thymus grafts were populated with porcine class II(high) cells in addition to human cells, and human T cells were tolerant of the porcine hematopoietic donor as measured by mixed lymphocyte reaction assay and skin grafting. This study proves the principle that porcine chimerism induces tolerance of xenoreactive human T cells.
Regulatory T cells generated by the kidney may mediate the beneficial immune effects of combining kidney with heart transplantation.
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Histomorphometric comparison of cardiac allograft vasculopathy in miniature swine.
BACKGROUND: Whether the pathologic characteristics of vascular lesions manifested in recipients with cardiac allograft vasculopathy (CAV) differ with the severity of the histocompatibility barrier crossed at transplantation or with the type or amount of immunosuppression used to prolong graft survival is unclear. We used miniature swine to determine whether a wide variance in heart transplantation protocols, both in histoincompatibility and immunosuppression, affects the histomorphometry of CAV. METHODS: We compared explanted hearts from major histocompatibility complex Class I-disparate recipients who were treated for 12 days with cyclosporine (Group 1) with minor-antigen-disparate hearts transplanted into mixed chimeric recipients previously engrafted with donor hematopoietic progenitor cells (Group 2). We analyzed coronary intimal lesions using computerized morphometry, immunohistochemistry, and TUNEL assay. Myocardial cytokine-gene expression was determined using RNAse protection assays and reverse-transcriptase polymerase chain reaction. RESULTS: The prevalence of CAV in Group 2 was significantly less than that observed in Group 1, but the severity of the lesions in both groups was similar. The vascular lesions that developed in both groups demonstrated the presence of alpha-smooth-muscle-actin-positive spindle cells expanding the intima, with few inflammatory cells. We noted an absence of proliferating cell nuclear antigen activity and TUNEL-positive cells in both groups. We observed prominent myocardial interferon-gamma gene expression only in Group 1. CONCLUSION: Despite differences in myocardial interferon-gamma gene expression, the histology and severity of the vascular lesions in CAV did not vary significantly with different histoincompatibilities or treatment protocols. These results suggest that the origin of CAV cannot be determined by histology alone.