A Novel Biomarker for Beryllium Sensitization in Humans - Final Report

This research project will determine the T-cell receptor (TCR) gene usages of beryllium reactive T-lymphocytes isolated directly from the peripheral blood of individuals exposed at a U.S. Department of Energy site. The objective is to develop a sensitive and novel biomarker for identifying early human sensitization to environmental beryllium. This is a collaborative project involving the Genetics Laboratory of the University of Vermont and both the Center for Epidemiological Research and the scientific staff of the Cytogenetics Program at the Oak Ridge Institute for Science and Education (ORISE). The > 2000 beryllium exposed workers who have been contacted for participation in the ORISE study ''Follow-up of Beryllium Workers at the Y-12 Plant/Efficacy of the Peripheral Blood Lymphocyte Proliferation (LPT) and other Non-Invasive Procedures for Diagnosis of Chronic Beryllium Disease'' will provide the pool of potential participants for the proposed study. Beryllium reactive T-lymphocytes will be directly isolated from peripheral blood using a novel antigen-independent method of surrogate selection for in vivo arising hprt mutants as representatives of clones that are undergoing chronic proliferation. The T-cells undergoing chronic proliferation in beryllium sensitized individuals will be enriched for beryllium reactive cells. The TCR gene usage of these T-cell isolates will be determined and their junctional (CDR3) regions sequenced. Beryllium reactive T-cell clones will also be recovered following in vitro beryllium stimulation of peripheral blood lymphocytes from these same individuals and the TCR gene CDR3 region sequences similarly determined. The TCR genes used by the beryllium reactive isolates and their CRD3 region sequences will be compared within (in vivo vs. in vitro derived) and among individuals with attention to kinds and durations of beryllium exposure and HPA DPB Glu 69 status. A method for quantitating total body loads of these antigen reactive T-cells in individuals will be developed using quantitative polymerase chain reaction (Q-PCR) amplification of specific TCR gene sequences. Successful achievement of this overall objective will permit future studies aimed at the elucidation of the immunological mechanisms underlying sensitization, the comparison of cells involved in pulmonary and systemic sensitization and the definition of potential targets for immunotherapy