Alpha B crystallin (Cryab), a member of the small heat shock protein family, recently has been shown to be a broad based anti-inflammatory agent effective in animal models of multiple sclerosis, rheumatoid arthritis, ischemia-reperfusion injury, and stroke. The basis of this proposal is to establish whether Cryab is effective in models of LPS toxemia and bacterial sepsis.
The aims of the grant are first to demonstrate that Cryab, and not Cryab with a point mutation at residue 120, can (i.) reduce in a dose dependent manner serological levels of IL-6, serum amyloid A, and C reactive protein in mice injected with LPS and (ii.) extend the lifetime of animals administered lethal doses of LPS compared with untreated animals.
The second aim i s to establish the relevance of the potency of Cryab by comparing the modulation of inflammatory cytokines with that observed for treatment with murine activated protein C. Activated protein C is the only approved drug for sepsis and any prospective therapeutic must be equally or more potent. In addition, we propose to determine whether Cryab and activated protein C could act synergistically by the co-administrating of the two proteins. The last aim is to assay the levels of Cryab in murine plasma at various times after injection with LPS to determine whether the protein is a marker of the severity of the disease and also support the hypothesis that Cryab has a natural role in modulating inflammation. In addition, Cryab levels in plasma from human trauma patients will be monitored to determine whether this protein can be used as a marker for progression of SIRS into septic shock. The mode of action of Cryab appears to be linked with the protein's role as a chaperone. The ability of the small heat shock proteins to bind partially unfolded proteins and lower their concentration at sites of inflammation and consequently limit aggregation and the inflammatory stimulus. Using mass spectrometry we have demonstrated that Cryab binds between 50 and 75 different plasma proteins in a temperature dependent fashion, with the vast majority being acute phase proteins or members of the complement or coagulation cascades. Analyzing the supernatants of these pull down experiments demonstrate that the concentration of many of the plasma proteins was reduced significantly. This data provides scientific foundation that Cryab can be distinguished from the large number of potential therapeutics that have not achieved their clinical milestones in this complex disease. We believe that the broad specificity of alpha B crystallin could enable the protein to be an effective therapeutic for sepsis and SIRS.
Sepsis, a complex dysregulation of inflammation resulting from the host response to a bacterial infection or severe trauma, is responsible for over half of the deaths in intensive care units accounting for more than 200,000 deaths per year. Over 30 different clinical trials of anti-inflammatory agents have failed during the past twenty years. This proposal seeks to provide preclinical support that a novel biotherapeutic, alpha B crystallin, can be an effective drug for sepsis. The intellectual framework for this optimism is the protein's capacity to modulate a far wider range of inflammatory mediators in plasma than the drug candidates used in previous clinical trials resulting in significant reductions of the serological markers of this complex disease.
