Orthotopic liver transplantation is a common therapy for many acquired and inherited disorders. The extent of initial liver damage mediated by ischemia and reoxygenation has been proposed to substantially contribute to acute rejection and graft failure. The mechanisms of cellular damage following ischemia/reperfusion (I/R) in the liver have been extensively studied and are likely multifactorial involving both acute ischemic mediated cellular damage and subacute inflammatory responses. Therapeutic intervention has traditionally centered around the amelioration of free radicals generated by reperfusion and inhibition of neutrophil recruitment to the target organ. Despite numerous studies, effective therapies have been hindered by a lack of understanding regarding the primary mechanisms which initiate organ damage following I/R. To this end, reactive oxygen species (ROS) have been hypothesized to be a major component associated with cellular toxicity. In addition, neutrophil recruitment via cellular responses in the liver, i.e., TNF and IL-1 production by Kupffer cells, has also been hypothesized to play an important role in initiating and/or perpetuating liver damage. This proposal attempts to more closely define the primary mechanism of ischemic induced liver damage associated with the generation of ROS in a mouse model of lobar I/R injury. Utilizing genetically defined knockout strains of mice including Ragl-, B2m-, Class II-, and nu/nu mice, the applicant plans to analyze the involvement of various hematopoietic lineages in the recruitment of neutrophils following I/R injury in the liver. Preliminary studies have demonstrated significantly reduced I/R induced liver damage in nu/nu balb c, as compared to immune competent balb c mice. These findings have provided new insights into the potential involvement of T-cell lineages in the recruitment and amplification of neutrophils following I/R. By evaluating adoptive transfer of T-cell subsets in athymic mice and the cytokine patterns of expression during the acute phases of I/R injury in immune competent mice, they will attempt to delineate the T-cell lineages (Th1, Th2, or Tc) involved in the initial stages of subacute inflammatory responses. Additionally, they propose to study the potential mechanisms by which liver originating ROS following I/R elicit direct hepatocellular toxicity and lead to recruitment of inflammatory cells. To this end, they will utilize a genetic approach to study the effects of reducing ROS formation in the liver following I/R by ectopically expressing free radical scavengers, MnSOD, Cu/ZnSOD, and/or catalase with recombinant adenoviruses. Such an approach will provide an experimental paradigm for linking ROS formation in the liver with subsequent inflammation. Moreover, these studies may provide clinically relevant gene therapy approaches for minimizing organ damage following transplantation which may ultimately increase the graft survival in orthotopic liver transplantation.
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