The goal of this project is to determine the feasability of xenogeneic pig thymus to engraft in an SIV infected macaque, and to determine if this has any impact on T cell reconstitution. The initial aim was to establish baseline assays to determine the degree of immunosupression as a result of SIV. We have conducted comprehensive analysis to determine the optimal in vitro conditions to determine both alloantigen and xenoantigen responses in macaques. Our data demonstrate poor to absent xeno and allo- responses in macaques with advanced SIV disease, as well as 1-2 log reduction in the proliferation response to lectin based on the poor in vitro proliferative responses we wished to examine whether pig thymic tissue might engraft in SIV-infected macaques without any conditioning. We therefore performed transplants of pig thymic tissue in 2 SIV-infected macaques with advanced disease (CD4<200 mm3). The animals were transplanted with pig fetal thymic tissue (approximately 20 slices) in the quadriceps muscle. Prohpylactic treatment for opportunistic infections (PCP and mycobacterial infection) was initiated 2 weeks prior to transplant, and antiretroviral treatment (AZT and 3TC) was initiated 2 weeks post-transplant. No detectable changes in CD4 count were noted. In addition, no change was observed in the low level proliferative responses to xenoantigens or ConA up to 3 months post-transplant. Biopsy of the transplant site at 30 days revealed lymphoid aggregates in both animals, with only minimal inflammatory changes outside the lymphoid aggregates. The origin of the lymphoid aggregates was investigated by immunocytochemistry using a relatively large panel of antibodies specific for either pig or monkey tissue. No evidence for engraftment of pig thymic tissue was observed (negative results for pig cytokeratin, MHC class I, MHC class II, and CD2). Lymphoid aggregates werre made up of mature rhesus T lymphocytes (positive for CD2 and CD3, negative for CD1). Similarly, no evidence for circulating pig lymphocytes was observed in peripheral blood. These results suggest that even SIV-infected animals with advanced disease are able to mount sufficient immune responses to reject swine thymus. Six weeks after transplant, the first animal we studied developed acute bilateral hind limb paralysis. The animal was euthanized and a complete autopsy performed. Examination of the spinal cord revealed inflammation (transverse mylelitis) with granuloma formation. Samples of spinal cord and muscle adjacent to the biopsy site were negative for evidence of pig endogenous retrovirus by PCR (performed by BioTransplant, Inc.). Immunocytochemistry revealed the presence of SIV-infected cells at the site of spinal cord inflammation. At present, the cause of the transverse myelitis cannot be determined with certainty, but appears to be consistent with SIV-induced transverse myelitis. There is currently no evidence that this event occurred as a result of the transplant. Results of these studies should yield valuable information on the potential of xenogeneic thymic transplantation as a strategy to reconstitute immune function in HIV-infected people.
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