Pseudomonas exotoxin A (ETA), one of several potential virulence products produced by Pseudomonas aeruginosa is a three domain bacterial toxin that kills mammalian cells by gaining entry to the cytosol and inhibiting protein synthesis. The toxin binds to a surface receptor on susceptible cells, is internalized via coated pits and endosomes, and enters the cytosol. During this journey, the toxin is cleaved to generate an enzymatically active fragment which ADP-ribosylates elongation factor 2. The pathway ETA takes to reach its target is only partially elucidated. Recently the ETA receptor has been isolated and shown to be similar to the alpha2macroglobulin receptor/LDL related protein (alpha2MR/LRP) The long-term goals of this research are to fully understand the cellular action of ETA, including how it is processed, and to determine its interaction with its receptor on toxin-sensitive cells.
The specific aims of this proposal are: 10 Confirm the relationship between the ETA receptor and the alpha2MR/LRP. The investigator will identify sequences in human alpha2MR/LRP. The investigator will identify sequences in human alpha2MR/LRP. The investigator will identify sequences in human alpha2MR/LRP receptor that correspond to ETA binding sites; 2) Determine the basis for cellular susceptibility to toxins. This includes a) determining ETA receptor level in toxin sensitive (LM and Chang) and resistant cells (OVCAR and Hela); b) establishing the level of alpha2MR/LRP receptor associated protein (RAP) in various mammalian cells; c) identifying and localizing the compartments where ETA is proteolytically processed, is activated, and enters the cytosol. Where feasible, these studies will include diphtheria toxin (DT), to determine if a second bacterial toxin is processed in a manner similar to ETA. 3) Define the intracellular pathway taken by the ETA receptor in sensitive cells. This work is significant because it will increase understanding of the pathophysiology of pseudomonas infections, help in the construction of efficient chimeric toxins, and help to answer the question of how toxins have adapted to take advantage of well conserved cellular components such as the alpha2MR/LRP. GRANTS=R29AI35994 Primary biliary cirrhosis (PBC) and autoimmune hepatitus (AI-CAH) ar two chronic diseases of the liver, believed to be autoimmune in nature, with etiologies and pathogenesis that remain unknown. These disorders affect predominantly women, mostly in th prime of their life. For example, over 90% of patients with PBC are women between the ages of 20-70. Liver transplantation ha s proven to be an effective treatment for both AI-CAH and PBC. However, disease recurrence remains an important concern. The goal of this project is to elucidate the role of cytokines and cytotoxic mediators in autoimmune liver disease. Specifically, we propose to: 1) characterize the cytokine profiles within the liver of patients with PBC and AI-CAH 20 identify the cytotoxic mediators 30 characterize the functional properties of lymphoid cells that have infiltrated the liver and 40 apply this information to establish immunological parameters that distinguish disease recurrence from allograft rejection. Molecular methodologies will be utilized to determine and compare the cytokine arrays in normal liver and liver explant tissue from patients with PBC and AI-CAH. RNA obtained from liver tissue will be reverse transcribed, and analyzed for cytokine gene expression using semi- quantitative PCR. In situ hybridization and immunohistochemical techniques will identify the cellular source of cytokine message within the liver. Evidence for specific cellular pathways of liver tissue injury will be obtained by immunohistochemical techniques and Western blot hybridization analysis for serine esterases, perforin, eosinophil cytotoxic proteins and autoantibodies. Direct in vitro examination of infiltrating cells recovered from the PBC and AI-CAH liver will establish the diversity of T cells within the infiltrate, the cytotoxic activity and specificity of T lymphocytes, the frequency of specific cytokine producing cells, the arrays of intrahepatic autoantibodies, and the presence of non-specific effector cells including NK cells and eosinophils. The effect of recombinant cytokines on NHC antigen expression by target tissue will be tested on epithelial and hepatocyte- derived cell lines. Finally, patterns of cytokine production in PBC and AI-CAH will be compared with those of patients who have received liver transplants because of autoimmune liver disease in an effort to distinguish disease recurrence from allograft rejection. Taken together, these studies will provide important mechanistic insights into these two enigmatic autoimmune diseases as well as clarify th issue of disease recurrence post-orthotopic transplantation.
