Michael T. Bailin

Removal of anti-porcine natural antibodies from human and nonhuman primate plasma in vitro and in vivo by a Galalpha1-3Galbeta1-4betaGlc-X immunoaffinity column.

BACKGROUND Natural antibodies (NAbs) against a terminal alpha1-3 galactosyl (alphaGal) epitope have been identified as the major human anti-pig NAbs. METHODS AND RESULTS We used two synthetic alphaGal trisaccharides--type 6 (alphaGal6) and type 2(alphaGal2)--linked to an inert matrix to remove NAbs from human plasma in vitro. Flow cytometry indicated that an average of 85% of the NAb binding activity was depleted by adsorption with alphaGal6. By measuring the binding of NAbs to pig peripheral blood mononuclear cells and bone marrow cells, we demonstrated that alphaGal6 was more effective than alphaGal2 in removing NAbs, and the combination of alphaGal6 + alphaGal2 did not further increase removal of NAbs. The specificity of the removal of NAbs (IgM and IgG) reactive with the alphaGal epitope by alphaGal6 matrix was shown by enzyme-linked immunosorbent assay. In vivo studies in nonhuman primates compared plasma perfusion through a alphaGal6 immunoaffinity column with hemoperfusion through a pig liver for changes in blood pressure, hematocrit, platelets, and NAb adsorption. CONCLUSIONS Both methods reduced the level of anti-pig IgM and IgG xenoreactive antibodies to nearly background, but column perfusion caused less hypotension and reduction in platelets than liver perfusion. Four pig kidneys transplanted into monkeys after column perfusion did not undergo hyperacute rejection, remaining functional for 2-10 days, with a mean functional period of 7 days, demonstrating that a pig kidney can support renal function in a primate.

Xenotransplantation of pig kidneys to nonhuman primates: I. Development of the model

Abstract: Long‐term survival of discordant xenografts will require control of both humoral and cellular aspects of the immune response. Because cellular responses to xenografts appear to be more potent than those encountered by allografts, recipients of xenogeneic tissues are likely to require more immunosuppression, with potential for unacceptably high complication rates. We have therefore directed our attention toward the induction of tolerance to some or all of the xenogeneic antigens recognized in the primate anti‐pig cellular immune response, utilizing mixed lymphohematopoietic chimerism as the means for tolerance induction. We report here our initial series of 16 animals in which the conditions for application of this treatment regimen were established. In 14 cynomolgus monkey recipients, induction therapy consisted of low dose whole body irradiation, thymic irradiation, and ATG, followed by infusion of pig bone marrow and a pig kidney transplant. Other aspects of the regimen included splenectomy and removal of monkey anti‐porcine natural antibodies by extracorporeal perfusion of the recipients blood through a pig liver. Two control animals received either no treatment or extracorporeal perfusion but no additional induction therapy. Five of the experimental animals were treated posttransplant with an anti‐IgM monoclonal antibody, five with Cyclosporin A, and two with a combination of both immunosuppressants. Both IgM and IgG natural antibodies were removed effectively by liver perfusion in all but one monkey, as determined by flow cytometry. Antibody liters remained low for 5–7 days, but increased progressively thereafter. The longest kidney survival in this series was 13 days, in an animal which maintained excellent kidney function for the first 11 days posttransplant. Peripheral chimerism was detected only transiently on day 10 in the peripheral blood of this recipient. We conclude that extracorporeal perfusion by this technique removes natural antibodies and prevents hyperacute rejection, permitting maintenance of excellent renal xenograft function for at least 11 days. Additional manipulations appear to be required to achieve mixed chimerism and tolerance of the cellular immune system in this model.

Long-term discordant xenogeneic (porcine-to-primate) bone marrow engraftment in a monkey treated with porcine-specific growth factors.

BACKGROUND Mixed allogeneic hematopoietic chimerism has previously been reliably achieved and shown to induce tolerance to fully MHC-mismatched allografts in mice and monkeys. However, the establishment of hematopoietic chimerism has been difficult to achieve in the discordant pig-to-primate xenogeneic model. METHODS To address this issue, two cynomolgus monkeys were conditioned by whole body irradiation (total dose 300 cGy) 6 and 5 days before the infusion of pig bone marrow (BM). Monkey anti-pig natural antibodies were immunoadsorbed by extracorporeal perfusion of monkey blood through a pig liver, immediately before the intravenous infusion of porcine BM (day 0). Cyclosporine was administered for 4 weeks and 15-deoxyspergualin for 2 weeks. One monkey received recombinant pig cytokines (stem cell factor and interleukin 3) for 2 weeks, whereas the other received only saline as a control. RESULTS Both monkeys recovered from pancytopenia within 4 weeks of whole body irradiation. Anti-pig IgM and IgG antibodies were successfully depleted by the liver perfusion but returned to pretreatment levels within 12-14 days. Methylcellulose colony assays at days 180 and 300 revealed that about 2% of the myeloid progenitors in the BM of the cytokine-treated recipient were of pig origin, whereas no chimerism was detected in the BM of the untreated control monkey at similar times. The chimeric animal was less responsive by mixed lymphocyte reaction to pig-specific stimulators than the control monkey and significantly hyporesponsive when compared with a monkey that had rejected a porcine kidney transplant. CONCLUSION To our knowledge, this is the first report of long-term survival of discordant xenogeneic BM in a primate recipient.

TRANSFER OF SWINE MAJOR HISTOCOMPATIBILITY COMPLEX CLASS II GENES INTO AUTOLOGOUS BONE MARROW CELLS OF BABOONS FOR THE INDUCTION OF TOLERANCE ACROSS XENOGENEIC BARRIERS

BACKGROUND The present study examined the potential role of gene therapy in the induction of tolerance to anti-porcine major histocompatibility complex (SLA) class II-mediated responses after porcine renal or skin xenografts. METHODS Baboons were treated with a non-myeloablative or a myeloablative preparative regimen before bone marrow transplantation with autologous bone marrow cells retrovirally transduced to express both SLA class II DR and neomycin phosphotransferase (NeoR) genes, or the NeoR gene alone. Four months or more after bone marrow transplantation, the immunological response to a porcine kidney or skin xenograft was examined. Both the renal and skin xenografts were SLA DR-matched to the transgene, and recipients were conditioned by combinations of complement inhibitors, adsorption of natural antibodies, immunosuppressive therapy, and splenectomy. RESULTS Although the long-term presence of the SLA transgene was detected in the peripheral blood and/or bone marrow cells of all baboons, the transcription of the transgene was transient. Autopsy tissues were available from one animal and demonstrated expression of the SLA DR transgene in lymphohematopoietic tissues. After kidney and skin transplantation, xenografts were rejected after 8-22 days. Long-term follow-up of control animals demonstrated that high levels of induced IgG antibodies to new non-alphaGal epitopes developed after organ rejection. In contrast, induced non-alphaGal IgG antibody responses were minimal in the SLA DR-transduced baboons. CONCLUSIONS Transfer and expression of xenogeneic class II DR transgenes can be achieved in baboons. This therapy may prevent late T cell-dependent responses to porcine xenografts, which include induced non-alphaGal IgG antibody responses.

Tolerance in a concordant nonhuman primate model.

BACKGROUND We have previously demonstrated that induction of mixed lymphohematopoietic chimerism resulted in donor specific renal allograft tolerance without the need for chronic immunosuppression in nonhuman primates. Here we have tested whether tolerance can be similarly induced for baboon to cynomolgus renal xenografts. METHODS After preconditioning with anti-thymocyte globulin (ATG), nonlethal total body irradiation, and thymic irradiation, cynomolgus monkeys underwent splenectomy, native nephrectomies, and baboon marrow and renal transplants. Postoperative cyclosporine was given for 28 days. RESULTS In Group 1 (n=2, survival= 13, 14 days), both animals developed anti-donor immunoglobulin G, had biopsy findings consistent with humoral rejection, and showed rapidly progressive xenograft failure. In Group 2 (n=5, survival=1, 16, 33, 112, 190 days), 15-deoxyspergualine was added to the regimen (Day 0-13). In one long-term survivor, donor specific hyporesponsiveness was first observed (mixed lymphocyte culture [(MLR]) on Day 48. MLR reactivity returned on Day 64 together with the development of anti-donor antibody and subsequent xenograft failure on Day 112. Donor specific T-cell hyporesponsiveness was detected in the other long-term survivor for the first 133 days, after which a donor-specific skin xenograft was placed, (survival 24 days). Following the skin graft rejection, a rise in the MLR, development of anti-donor antibody and progressive rejection of the renal xenograft were observed. CONCLUSIONS Antibody-mediated rejection seems to constitute the major difference between concordant xenografts and allografts. Addition of 15-deoxyspergualine for 2 weeks posttransplant extended concordant primate xenograft survival to 6 months without chronic immunosuppression. In contrast to the allogeneic model, renal transplant acceptance in this xenogeneic system was interrupted by placement of a donor-specific skin graft.

A study of tolerance in a concordant xenograft model

Antibody-mediated rejection appears to constitute the major difference between concordant xenografts and allografts in nonhuman primates. Consistent with its known effect on antibody responses, 5-7 addition of DSG to the conditioning regimen has extended concordant primate xenograft survival for up to 6 months after discontinuation of conventional immunosuppression. In contrast to our observations in recipients of renal allografts, donor-specific skin graft rejection can occur and even in long-term recipients may induce rejection of a previously accepted renal xenograft.

Photosensitivity and Perioperative Polyneuropathy Complicating Orthotopic Liver Transplantation in a Patient with Erythropoietic Protoporphyria

ERYTHROPOIETIC protoporphyria (EPP) is an inherited disorder of porphyrin metabolism. Common manifestations of EPP include photosensitivity and mild hepatic dysfunction. Occasionally, in patients with EPP, end-stage hepatic failure and, very rarely, neurologic dysfunction develop.2-7 We describe the perioperative treatment of a patient with EPP and end-stage liver disease, necessitating liver transplantation, whose care was complicated by photosensitivity and severe polyneuropathy.

Decision support using anesthesia information management system records and accreditation council for graduate medical education case logs for resident operating room assignments.

Our goal in this study was to develop decision support systems for resident operating room (OR) assignments using anesthesia information management system (AIMS) records and Accreditation Council for Graduate Medical Education (ACGME) case logs and evaluate the implementations. We developed 2 Web-based systems: an ACGME case-log visualization tool, and Residents Helping in Navigating OR Scheduling (Rhinos), an interactive system that solicits OR assignment requests from residents and creates resident profiles. Resident profiles are snapshots of the cases and procedures each resident has done and were derived from AIMS records and ACGME case logs. A Rhinos pilot was performed for 6 weeks on 2 clinical services. One hundred sixty-five requests were entered and used in OR assignment decisions by a single attending anesthesiologist. Each request consisted of a rank ordered list of up to 3 ORs. Residents had access to detailed information about these cases including surgeon and patient name, age, procedure type, and admission status. Success rates at matching resident requests were determined by comparing requests with AIMS records. Of the 165 requests, 87 first-choice matches (52.7%), 27 second-choice matches (16.4%), and 8 third-choice matches (4.8%) were made. Forty-three requests were unmatched (26.1%). Thirty-nine first-choice requests overlapped (23.6%). Full implementation followed on 8 clinical services for 8 weeks. Seven hundred fifty-four requests were reviewed by 15 attending anesthesiologists, with 339 first-choice matches (45.0%), 122 second-choice matches (16.2%), 55 third-choice matches (7.3%), and 238 unmatched (31.5%). There were 279 overlapping first-choice requests (37.0%). The overall combined match success rate was 69.4%. Separately, we developed an ACGME case-log visualization tool that allows individual resident experiences to be compared against case minimums as well as resident peer groups. We conclude that it is feasible to use ACGME case-log data in decision support systems for informing resident OR assignments. Additional analysis will be necessary to assess the educational impact of these systems.

Medical information technology: a vehicle for change

Medicine is under extreme pressure to reinvent itself into a system based on standard metrics that are repeatable and open to outside inspection and criticism. In this milieu, information technology takes a central role in the ongoing evolution of health care, simultaneously empowering clinicians and making them more accountable to the public, the government and their patients. The application of information technology in medicine, such as the development of the electronic medical record, clinical workstations and telemedicine, promises to redefine the practice of medicine.