Donald V. Cramer

Migration of mesenchymal stem cells to heart allografts during chronic rejection

Background. Mesenchymal stem cells (MSC) are pluripotent progenitors for a variety of cell types, including fibroblasts and muscle cells. Their involvement in the tissue repair of allografts during the development of chronic rejection has been hypothesized, but not yet substantiated, by experimental evidence. Methods. Rat MSC were isolated from circulation using an aortic pouch allograft as a trapping device. The plasticity of these cells was examined in differentiation cultures. One of the resulting MSC lines was immortalized and transduced to express a marker lacz gene. The lacz-labeled cells were then transferred to F344 rats bearing Lewis (LEW) cardiac allografts to measure their localization and contribution to graft tissue repair. Results. The MSC isolated from circulation exhibited multipotential for differentiation in culture, developing into various lineages including osteoblasts, lipocytes, chondrocytes, myotubes, and fibroblasts. Intravenous engraftment of the lacz-labeled cells into recipients of heart transplant resulted in migration of the &bgr;-gal+ cells into the lesions of chronic rejection in the cardiac grafts and homing of the cells to the bone marrow. The majority of &bgr;-gal+ cells present in the allografts exhibited fibroblast phenotypes, and a small number of the cells expressed desmin, indicative of myocyte differentiation. Conclusion. MSC vigorously migrated into the site of allograft rejection. This data suggests that they may be attracted to this site to actively participate in tissue repair during chronic rejection. In addition, given the robust migration, the inhibition of MSC differentiation toward fibroblast progeny and induction toward the myocyte lineage may serve as a new strategy for treatment of chronic rejection and allograft tissue repair.

Hamster-to-rat heart and liver xenotransplantation with FK506 plus antiproliferative drugs

Heterotopic hamster hearts transplanted to unmodified LEW rats underwent humoral rejection in 3 days. Survival was prolonged to a median of 4 days with 2 mg/kg/day FK506. As monotherapy, 15 mg/kg/day cyclophosphamide greatly prolonged graft survival--far more than could be accomplished with RS-61443, brequinar (BQR), mizoribine, methotrexate, or deoxyspergualin. However, when FK506 treatment, which was ineffective alone, was combined with a short induction course (14 or 30 days) of subtherapeutic BQR, RS-61443, or cyclophosphamide, routine survival of heart xenografts was possible for as long as the daily FK506 was continued. In addition, a single large dose of 80 mg/kg cyclophosphamide 10 days preoperatively allowed routine cardiac xenograft survival under FK506. The ability of these antimetabolites to unmask the therapeutic potential of FK506 correlated, although imperfectly, with the prevention of rises of preformed heterospecific cytotoxic antibodies immediately postoperatively. As an adjunct to FK506, azathioprine was of marginal value, whereas mizoribine, methotrexate, and deoxyspergualin (DSPG) were of intermediate efficacy. After orthotopic hepatic xenotransplantation, the perioperative survival of the liver with its well-known resistance to antibodies was less dependent than the heart on the antimetabolite component of the combined drug therapy, but the unsatisfactory results with monotherapy of FK506, BQR, RS-61443, or cyclophosphamide were changed to routine success by combining continuous FK506 with a short course of any of the other drugs. Thus, by breaking down the antibody barrier to xenotransplantation with these so-called antiproliferative drugs, it has been possible with FK506 to transplant heart and liver xenografts with consistent long-term survival of healthy recipients.

The major histocompatibility complex of the rat.

Immunogenetic studies in the rat began with the investigation of blood group antigens (1-7) and evolved into the investigation of histocompatibility antigens and transplantation phenomena (8-17). In the course of this work, several nomenclature systems evolved that were eventually reconciled in a series of comparison studies (18-24). The biennial Workshops on Alloantigenic Systems in the Rat held under the aegis of the Transplantation Society (24) have provided the forum for the continued evolution of this field. In addition, several reviews (24-31) and books (32-35) have provided periodic summaries of different aspects of rat immunogenetics. This review will focus on the structure of the major histocompatibility complex (MHC) of the rat from the serological, biochemical, and molecular points of view.

The use of a pig liver xenograft for temporary support of a patient with fulminant hepatic failure

A 26-year-old female patient with fulminant hepatic failure and a history of autoimmune hepatitis was heterotopically transplanted with a pig hepatic xenograft to provide temporary metabolic support prior to transplantation with a human donor organ. Circulating natural antipig antibodies were removed prior to transplantation by plasmapheresis and ex vivo en bloc perfusion of the donor pig kidneys. The liver xenograft functioned after transplantation as measured by active bile production, stabilization of prothrombin levels, and reduction in the circulating levels of lactic acid and the enzymes AST and ALT. Despite the removal of greater than 90% of the recipients natural xenoantibodies prior to transplantation, the levels of antibody rapidly returned and were associated with antibody and complement-mediated rejection of the donor graft. Immunohistochemical evidence of graft rejection could be detected by the deposition of antibody, complement components including properdin, and endothelial swelling as early as 3 hr posttransplantation. These lesions progressed in severity and were accompanied by evidence of thrombosis and ischemic necrosis of the liver xenograft by 34 hrs posttransplantation. The main portal vein, hepatic artery, and vena cava were patent. The placement of the liver graft did not result in any improvement in the neurological status of the patient and she died 34 hr after xenografting due to irreversible brain damage. The information derived from this case has renewed interest in the clinical use of bioartificial devices and whole organ perfusion using xenogeneic tissue for temporary bridging of patients prior to allografting.

Cardiac transplantation in the rat. I: The effect of histocompatibility differences on graft arteriosclerosis

The development of arteriosclerosis is the most serious and common complication in long-term survivors of cardiac transplantation. We have used a variety of inbred rat strains with selected histocompatibility differences to examine the influence of prolonged, mild rejection reactions on the development of pathological changes in long-term cardiac allografts. Heterotopic cardiac allografts were exchanged between rat strains that differed for MHC class I (RT1.A and/or RT1.E) antigens or groups of minor, non-MHC antigens in MHC-compatible congenic combinations. Our results demonstrate that in strain combinations in which the allograft reaction is mild and prolonged, the donor hearts exhibit pathological changes that include a diffuse, interstitial myocardial fibrosis, perivascular fibrosis, and intimal proliferation in arteries of the graft myocardium. The lesions were less prominent in animals with more active rejection and infrequent in strains that differ for class I histocompatibility antigens or syngeneic controls. These results suggest that the comparable pathological changes seen in long-term human cardiac survivors may reflect low-level, persistent allograft reactions rather than factors associated with graft anoxia or effects of immunotherapy to prevent graft rejection.

The use of FK-506 for small intestine allotransplantation : inhibition of acute rejection and prevention of fatal graft-versus-host disease

Small intestine allotransplantation in humans is not yet feasible due to the failure of the current methods of immunosuppression. FK-506, a powerful new immunosuppressive agent that is synergistic with cyclosporine, allows long-term survival of recipients of cardiac, renal, and hepatic allografts. This study compares the effects of FK-506 and cyclosporine on host survival, graft rejection, and graft-versus-host-disease in a rat small intestine transplantation model. Transplants between strongly histoincompatible ACI and Lewis (LEW) strain rats, and their F1 progeny are performed so that graft rejection alone is genetically permitted (F1----LEW) or GVHD alone permitted (LEW----F1) or that both immunologic processes are allowed to occur simultaneously (ACI----LEW). Specific doses of FK-506 result in prolonged graft and host survival in all genetic combinations tested. Furthermore, graft rejection is prevented (ACI----LEW model) or inhibited (rejection only model) and lethal acute GVHD is eliminated. Even at very high doses, cyclosporine did not prevent graft rejection or lethal GVHD, nor did it allow long-term survival of the intestinal graft or the host. Animals receiving low doses of cyclosporine have outcomes similar to the untreated control groups. No toxicity specific to FK-506 is noted, but earlier studies by other investigators suggest otherwise.

Peptides derived from α-helices of allogeneic class I major histocompatibility complex antigens are potent inducers of CD4+ and CD8+ T cell and B cell responses after cardiac allograft rejection

We studied the rejection of cardiac allografts in a rat strain combination (PVG.R8 to PVG.1U) disparate for a single class I MHC antigen (RT1.Aa) to test the extent by which this molecule is recognized as peptides in association with recipient MHC molecules during graft rejection and the contribution of this recognition process to the rejection reaction. Three synthetic peptides that correspond to the portions of α-helices of the al (PI, P2) and α2 (P3) domains of the donor RT1 Aa molecule were used in this study. Splenocytes from heart allograft recipients at rejection responded in a proliferation assay to all 3 peptides and in a cytotoxic assay to peptides PI and P2. The peptide-mediated proliferation and cytolytic reactions were blocked by antibodies against CD4/class II MHC and CD8 molecules. Serum from graft recipients at rejection contained significant titers of antibodies to peptides. Pre-sensitization of graft recipients with the peptides resulted in a marked increase in peptide-mediated T cell and antibody responses. Although all 3 peptides were effective in eliciting active immune responses, the P3-mediated response was minimal when compared with those mediated by PI and P2. Recipients presensitized with the peptides rejected their grafts in 5 days compared with 6 days for unsensitized animals. Recipients presensitized with donor-irradiated splenocytes and aortic endothelial cells, on the other hand, rejected their grafts in 1 and 3 days, respectively, which suggests that immunization with the whole RT1.Aa molecule is required to stimulate accelerated rejection of the graft. This rejection was associated with high titers of donor cell-specific antibodies that exhibited moderate cross-reactivity with the peptides. Our results clearly demonstrate that (1) the donor RT1.Aa molecule is recognized as peptides in the context of recipient class I and class II MHC molecules during the rejection of heart allografts, and (2) peptides derived from this molecule are highly immunogenic in that they contain epitopes recognized by CD4+ and CD8+ T cells and alloantibodies. Immune responses elicited by these peptides, however, did not significantly affect the rate of rejection. These results suggest that acute rejection of allografts may be mediated primarily by the direct recognition of intact MHC molecules.

Suppression of allograft rejection with FK506: I. prolonged cardiac and liver survival in rats following short-course therapy

Heterotopic heart and orthotopic liver grafts from ACI donors were transplanted to Lewis rat recipients that were treated with a 3 (or 4) day course of FK506 IM that was started on postoperative day 0, 2, 3, 4, 5, or 6. Hearts, which rejected after a median of 6 days in untreated controls, always had prolonged survival (median 91 days) when treatment was started on postoperative day 4. The results were inferior when treatment was started earlier or later than this, but even when the first dose of FK506 was on postoperative day 5, one day before rejection was imminent in controls, the median survival was 50 days. The poorest results with a median graft survival of only 36 days were obtained when injections were on days 0–3. Results were similar with liver grafts that rejected after a median time of 10 days in nontreated controls but that usually survived permanently after a 3 (or 4) day FK506 course starting on day 0, 2, 3, or 4. Therapy started on day 6 was too late.

The effect of a new immunosuppressive drug, brequinar sodium, on heart, liver, and kidney allograft rejection in the rat.

Brequinar sodium (BQR) prevents cell proliferation by virtue of its inhibition of de novo pyrimidine biosynthesis. BQR is capable of inhibiting immune responses in vitro and is effective in suppressing the development of contact sensitivity and adjuvant arthritis in rodent models. Based on the antiproliferative and immunosuppressive capacity of BQR, we have evaluated the efficacy of BQR in preventing allograft rejection utilizing experimental models of heterotopic heart and kidney and orthotopic liver transplantation in an MHC and non-MHC mismatched ACI----LEW rat strain combination. The immunosuppressive activity of BQR is illustrated by its ability to inhibit the development of delayed-type hypersensitivity to DNFB in mice. When BQR was administered orally throughout the sensitization and elicitation phases of the DNFB contact sensitivity response, it was found to be a potent immunosuppressant with an ED50 value of 0.5 mg/kg. This immunosuppressive activity is also seen in vitro, where BQR is capable of inhibiting the mixed lymphocyte response between allogeneic ACI and LEW rat strains with an IC50 of 150 ng/ml. The immunosuppressive activity of BQR is highly effective in prolonging heart, liver, and kidney allograft survival in the rat. Cardiac allografts are not rejected during the period of drug treatment at dosage levels of 12 to 24 mg/kg orally three times weekly. The grafts survive until the drug is discontinued (30 days posttransplantation), and the grafts are then rejected approximately 14 days later. Liver and kidney allografts are permanently accepted by approximately 50 to 90% of the recipient rats following 30 days of treatment with BQR at 12 mg/kg. The tolerance that is induced to the liver grafts extends in the majority of animals to greater than 250 days and is specific for the donor ACI strain. Challenge of long-term liver graft survivors with donor cardiac grafts is associated with permanent survival of donor, but not third-party, heart grafts. Combination therapy consisting of suboptimal doses of BQR and CsA demonstrates that the combination of these two immunosuppressive drugs results in an increased efficacy in prolonging graft survival. The results of these allograft experiments demonstrate that this new immunosuppressive agent is highly effective in preventing allograft rejection in the rat. The antiproliferative activity of BQR is effective for inhibiting T-lymphocyte-mediated immune responses, and Brequinar sodium should be an important addition to a polytherapeutic approach in the treatment of organ graft rejection.

Regeneration-induced accelerated rejection in reduced-size liver grafts.

Liver regenerative processes are associated with enhanced expression of alloantigens. Accordingly, we tested the hypothesis that such enhanced surface expression of alloantigens during regeneration of reduced-size liver grafts is associated with accelerated rejection. Our OLT model was LEW (RT1) to BN (RT1n), with donor liver resected by 50%. The study group consisted of reduced-size allografts. Control groups were syngeneic reduced-size isografts and full-size allografts. Reduced-size isograft recipients survived indefinitely. Both isografts and allografts regenerated to their prereduction size within 12 days. Recipients of reduced-size allografts died of accelerated rejection within 12.2 +/- 0.8 days, significantly earlier than recipients receiving full-size allografts (36.2 +/- 4.1 days, P < 0.01). The accelerated rejection in the regenerating allografts was mediated both by cellular and humoral mechanisms, evidenced by earlier lymphocytic invasion of the graft, enhanced donor MHC class II expression, and the emergence of IgM antibodies, directed specifically against donor endothelial antigens. These data suggest that regeneration of reduced-size allografts is accompanied by accelerated cellularly and humorally mediated alloreactivity. Recipients of reduced-size allografts may, therefore, benefit from more potent immunosuppression during the period of active liver regeneration.