Solid organ transplantation is currently standard therapy for those individuals suffering from kidney, liver, pancreas, heart and lung failure. Kidney transplantation affords improved quality of life in the short term and increased survival in the long term. Unfortunately, the compelling benefits afforded to patients by transplantation are tempered by the requirement for indefinite immunosuppression that can be associated with an increased susceptibility to certain infections, malignancy and renal failure secondary to the nephrotoxicity of current mainline agents. This project aims to develop a regimen that exposes the transplant recipient to a transient period of immunosuppression at the time of transplant, but then obviates the need for chronic, nonspecific immunosuppression while still preventing rejection. The state in which the recipient no longer demonstrates a propensity to reject an allograft in spite of the removal of immunosuppression is known as tolerance. The immunological state of tolerance was first described by Medawar in the late 1940's and since then, dozens of methods of establishing a tolerant state have been described in rodents. Tolerance becomes progressively more difficult to induce however, as the animals in the model advance up the phylogenetic scale. Only two approaches have demonstrated significant success at the primate level. These include the depletion/reconstitution model and the costimulation blockade model. Our approach concentrates on the former. In this model, the recipient's immune system is partially ablated and then exposed to donor bone marrow. In our model polyclonal antilymphocyte serum and sirolimus are used to prepare the recipient for donor bone marrow infusion. This approach has been demonstrated in various models to induce the development of recipient regulatory cells that inhibit rejection and induce the deletion of recipient allospecific T cells. This project has 5 parts: (1) identify the cell populations in donor bone marrow responsible for the efficacy of this approach, (2) identify and isolate the specific actions of polyclonal anti-lymphocyte serum that are responsible for the efficacy of this approach, (3) develop a nonhuman primate model to address specific pre-clinical questions, (4) initiate human protocols utilizing this approach, and (5) develop methods of culturing, storing, amplifying and enriching bone marrow to permit application of this protocol to all potential organ recipients from a single cadaveric donor. Since the inception of this project one year ago; the following actions have been taken. A rodent protocol was written and approved by the Armed Forces Radiobiology Research Institute IACUC. This protocol outlines experiments designed to identify those components of donor bone marrow and polyclonal ALS that are responsible for the efficacy of this approach towards tolerance induction. A MCRADA with Wyeth Ayerst was executed to support this protocol. A primate protocol was written and approved by the same IACUC. This protocol outlines experiments designed to test translational issues such as dosage and timing of administration of the components of the regimen in order to facilitate the application of this protocol in humans. A three way MCRADA with Wyeth Ayerst and Sangstat was executed to support these studies. Two post-doctoral fellows have been hired. The bulk of their work over the past six months has been related to familiarizing the fellows with the model and developing proficiency with the various laboratory methods that will be used to study it. Our early rodent studies have: (1) confirmed the efficacy of the protocol in a completely mismatched skin allograft model, (2) demonstrated the depth, breadth and duration of peripheral white blood cell depletion associated with tolerance induction, (3) clarified the magnitude and duration of exposure to sirolimus associated with this protocol, (4) quantitated the depletional effects of multiple monoclonal antibodies that will ultimately be used in lieu of the polyclonal preparation, (5) established cell culture techniques designed to identify which hematopoetic stem cell lines are capable of supporting tolerance induction and (6) established a collaborative effort with Dr. John Chute of the National Naval Medical Research Center to explore the potential use of cultured multipotential stem cells have in this approach to tolerance induction. Three kidney transplants have been done in rhesus primates using this approach to tolerance induction over the past year. Important observations include: (1) the markedly reduced efficacy of rabbit anti-human thymocyte globulin (Thymoglobulina) in non-human primates, (2) the high incidence of gastrointestinal complications (i.e. colitis, diarrhea) associated with the use of sirolimus in primates and (3) the relative ease of obtaining sufficient quantities of unfractionated bone marrow from the primate humerous as a survival procedure. In order to facilitate the collection of data, we have submitted and the IACUC has approved an amendment that permits us to convert to a more widely available cynomolgous model. The laboratory work is designed to support the conduct of tolerance induction protocols in the clinical center. Toward that end, we have written a human use protocol. We have enlisted the cooperation of the clinical center's transfusion service for processing and storage of harvested bone marrow. In addition, we have initiated a close collaboration with the bone marrow transplant service in order to maximize the safety of the protocol and to ensure comprehensive followup. This protocol has completed the first phase of outside scientific review and is currently being revised to accommodate the suggestions of the reviewers. It will subsequently be submitted to the IRB for internal review.
Showing the most recent 10 out of 17 publications
