Robert A. Cina

Identification of Exogenous Forms of Human-Tropic Porcine Endogenous Retrovirus in Miniature Swine

ABSTRACT The replication of porcine endogenous retrovirus subgroup A (PERV-A) and PERV-B in certain human cell lines indicates that PERV may pose an infectious risk in clinical xenotransplantation. We have previously reported that human-tropic PERVs isolated from infected human cells following cocultivation with miniature swine peripheral blood mononuclear cells (PBMC) are recombinants of PERV-A with PERV-C. Here, we report that these recombinants are exogenous viruses in miniature swine; i.e., they are not present in the germ line DNA. These viruses were invariably present in miniature swine that transmitted PERV to human cells and were also identified in some miniature swine that lacked this ability. These data, together with the demonstration of the absence of both replication-competent PERV-A and recombinant PERV-A/C loci in the genome of miniature swine (L. Scobie, S. Taylor, J. C. Wood, K. M. Suling, G. Quinn, C. Patience, H.-J. Schuurman, and D. E. Onions, J. Virol. 78:2502-2509, 2004), indicate that exogenous PERV is the principal source of human-tropic virus in these animals. Interestingly, strong expression of PERV-C in PBMC correlated with an ability of the PBMC to transmit PERV-A/C recombinants in vitro, indicating that PERV-C may be an important factor affecting the production of human-tropic PERV. In light of these observations, the safety of clinical xenotransplantation from miniature swine will be most enhanced by the utilization of source animals that do not transmit PERV to either human or porcine cells. Such animals were identified within the miniature swine herd and may further enhance the safety of clinical xenotransplantation.

In utero bone marrow transplantation induces kidney allograft tolerance across a full major histocompatibility complex barrier in Swine.

Background. In utero hematopoietic stem-cell transplantation has been shown to induce donor-specific tolerance in small-animal models. However, tolerance has been difficult to achieve in large-animal studies. Methods. Outbred swine underwent in utero transplantation of fully major histocompatibility complex (MHC)-mismatched CD3-depleted bone marrow mixed with fresh bone marrow to achieve a final CD3 content of 1.5%. Transplantation was performed at 50 to 55 days’ gestation and two animals survived long term and demonstrated multilineage peripheral blood hematopoietic chimerism. These two long-term survivors were analyzed for in vitro evidence of donor-specific tolerance by mixed leukocyte reaction (MLR), cell-mediated lysis (CML), and antibody testing and in vivo by kidney transplantation. Results. Both animals demonstrated in vitro donor-specific unresponsiveness by MLR and CML and did not demonstrate anti-donor antibody production. Donor matched kidney transplants were performed without immunosuppression and functioned for more than 100 days, with no evidence for rejection. Conclusions. The authors demonstrate conclusively that in utero transplantation of fully MHC-mismatched bone marrow in swine can lead to engraftment and stable multilineage hematopoietic chimerism and tolerance to postnatal donor MHC-matched kidney transplantation without the need for immunosuppression.

Predictors of organ allograft tolerance following hematopoietic cell transplantation.

Using the miniature swine large animal model we have attempted to determine the relationship between tolerance and the presence of donor cells in the bone marrow, thymus and lineages of peripheral blood in a series of hematopoietic cell transplant recipients receiving delayed donor allografts without immunosuppression. Twenty‐two animals receiving hematopoietic cell transplantation and a delayed organ allograft were analyzed. Assays for presence of donor CFUs in bone marrow (by PCR), thymic chimerism (by FACS and PCR/Southern Blot), peripheral blood chimerism (by FACS), and in vitro responsiveness to donor MHC were performed. Presence of donor BM CFUs, thymic chimerism and multilineage peripheral blood chimerism at the time of organ transplantation all correlated precisely with subsequent allograft tolerance (p < 0.001, p < 0.001, p < 0.005 respectively). These parameters were therefore accurate predictors (Positive Predictive Value (PPV) = 100% in all) of tolerance. In vitro assays of responsiveness were also highly associated (p < 0.002, p < 0.002 respectively), but were not as accurate predictors of subsequent organ tolerance (CML PPV = 80%). Engraftment, as indicated by the presence of donor derived CFU in the bone marrow, detectable thymic chimerism and multilineage peripheral blood chimerism are reliable predictors of subsequent donor allograft acceptance in hematopoietic cell transplant recipients.

Stable multilineage chimerism without graft versus host disease following nonmyeloablative haploidentical hematopoietic cell transplantation.

Background. Hematopoietic cell transplantation may offer the only cure for patients with hematological diseases. The clinical application of this therapy has been limited by toxic conditioning and lack of matched donors. Haploidentical transplantation would serve to extend the potential donor pool; however, transplantation across major histocompatibility complex barriers is often associated with severe graft-versus-host disease. Here we evaluate a novel protocol to achieve engraftment across mismatch barriers without toxic conditioning or significant posttransplant complications. Methods. Nine major histocompatibility complex (MHC)-defined miniature swine received haploidentical hematopoietic cell transplantation following standard myeloablative conditioning. Nine additional animals received haploidentical hematopoietic cell transplantation following a minimally myelosuppressive regimen, consisting of 100 cGy total body irradiation, immunotoxin mediated T-cell depletion, and a short course of cyclosporine. Donor cell engraftment and peripheral chimerism was assessed by polymerase chain reaction and flow cytometry. Graft-versus-host disease was monitored by clinical grading and histology of skin biopsy specimens. Results. All animals conditioned for haploidentical hematopoietic cell transplantation using myeloablative conditioning were euthanized within 2 weeks due to engraftment failure or graft-versus-host disease. All animals conditioned with the nonmyeloablative regimen developed multilineage peripheral blood chimerism during the first 2 months following transplantation. Six animals evaluated beyond 100 days maintained multilineage chimerism in the peripheral blood and lymphoid tissues, showed evidence of progenitor cell engraftment in the bone marrow, and had minimal treatment-related complications. Conclusions. Here we report that stable multilineage chimerism and engraftment can be established across haploidentical major histocompatibility complex barriers with minimal treatment-related toxicity and without significant risk of graft-versus-host disease.

Risk factors for the development of post-transplant lymphoproliferative disorder in a large animal model.

A high incidence of a post‐transplant lymphoproliferative disorder (PTLD) is observed in miniature swine conditioned for allogeneic hematopoietic cell transplantation using a protocol involving T‐cell depletion and cyclosporine therapy. This study was designed to assess contributing factors to disease development. Forty‐six animals were studied including 12 (26%) that developed PTLD. A number of risk factors for PTLD were examined, including degree of immunosuppression, degree of MHC mismatch and infection by a porcine lymphotrophic herpesvirus (PLHV‐1). Flow cytometry was used to measure host and donor T‐ and B‐cell levels in the peripheral blood. Porcine lymphotrophic herpesvirus viral load was determined by quantitative PCR. Animals developing PTLD had significantly lower levels of T cells on the day of transplant. Cyclosporine levels did not differ significantly between animals with and without PTLD. Animals receiving transplants across a two‐haplotype mismatch barrier showed an increased incidence of PTLD. All animals with PTLD had significant increases in PLHV‐1 viral loads. Porcine lymphotrophic herpesvirus viral copy numbers remained at low levels in the absence of disease. The availability of a preclinical large‐animal model with similarities to PTLD of humans may allow studies of the pathogenesis and treatment of that disorder.

Immunological unresponsiveness in chimeric miniature swine following MHC-mismatched spleen transplantation

Background. In rodents, spleen allotransplantation (SpTx) induces tolerance. We investigated the induction of chimerism and donor-specific unresponsiveness following pig SpTx. Methods. Thirteen pigs underwent splenectomy (day 0); all received a blood transfusion. In 11/13 pigs, SpTx was performed across a MHC class I (n=1) or full (n=10) barrier; two control pigs received no SpTx. All pigs were monitored for chimerism, and anti-donor immune responses, including suppressor assays. Four pigs (two asplenic controls and two with SpTx) underwent delayed donor-matched kidney transplantation without immunosuppression. Results. Six of the 11 spleen grafts were lost from rejection (n=5) or splenic vein thrombosis (n=1), and five remained viable. All 11 SpTx recipients developed multilineage chimerism, but chimerism was rapidly lost if the graft failed. Two control pigs showed <6% blood chimerism for 4 and 11 days only. Pigs with functioning spleen grafts had multilineage chimerism in blood, thymus and bone marrow for at least 2–6 months, without graft-versus-host disease. These pigs developed in vitro donor-specific hyporesponsiveness and suppression. In 2 pigs tolerant to the spleen graft, donor MHC-matched kidney grafts survived for >4 and >7 months in the absence of exogenous immunosuppression; in two asplenic pigs, kidney grafts were rejected on days 4 and 15. Conclusions. Successful SpTx can result in hematopoietic cell engraftment and in vitro donor-specific unresponsiveness, enabling prolonged survival of subsequent donor-matched kidney grafts without immunosuppression.

Perioperative β-Blockade to Reduce Cardiac Morbidity in Noncardiac Surgery

Although numerous studies have addressed the identification of patients at high risk for perioperative myocardial ischemia, far fewer data exist on methods to decrease risk in these patients. To this end, a major area of interest has centered on the perioperative use of -blockers as a cardioprotectant. The perioperative use of -blockers was based in part on the survival benefit of long-term -blockers noted in nonsurgical patients previously diagnosed with myocardial infarction (MI). This information has been well documented by several large trials. Recent investigation has shown this survival advantage to be durable and to exist for lowand high-risk patients. Given this potential to easily institute a therapy that could vastly decrease morbidity and mortality, there has been much interest in proving a similar survival benefit in the noncardiac surgical population. Several studies in the past half-decade have made great strides in producing a clearer understanding of the causes and modifiable risk factors for decreasing morbidity and mortality in noncardiac surgery of the cardiac patient. Through an in-depth review evaluation of these studies, we will attempt to develop guidelines for use of -blockade to decrease perioperative risk.

Evidence for a gene controlling the induction of transplantation tolerance.

Class I mismatched kidney transplantation in Massachusetts General Hospital MHC‐defined miniature swine has been studied extensively as a model for induction of systemic allograft tolerance. In a large series of juvenile swine, long‐term graft acceptance has been observed consistently following a 12‐day course of cyclosporine. It was therefore surprising when three of five recipients in one of our studies rejected their grafts. Examination of the origins of the rejecting animals revealed that they were derived from a subline of the SLAdd miniature swine herd that was intentionally being inbred toward full homozygosity and had been inbred for eight generations prior to these experiments. A blinded study of additional class I mismatched renal transplants into animals from this subline confirmed the genetic basis of this rejection. We present here preliminary evidence suggesting that a likely explanation for this phenomenon is that the rejectors in this subline are homozygous for a recessive mutant allele of a gene normally involved in the induction of tolerance. Subsequent studies will be directed toward identification and characterization of the gene(s) involved, since existence of a similar genetic locus in humans might have implications for assessing an individuals likelihood of graft rejection versus tolerance induction prior to organ transplantation.