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Principal Investigator: Dr. Donna Muscarella
Contact Information: E-mail: dem10@cornell.edu - Phone: 253-4047
Sponsor: Johns Hopkins Center/Alternatives to Animal Testing
Title: An in Vitro Model System to Evaluate Drug Effects on B-Lymphocyte Survival and Antibody Production
Annual Direct Cost: $34,783
Project Period: 02/01/05-01/31/06
The production of the diverse repertoire of antibodies required to recognize foreign antigens is achieved by the positive selection of specific clonal populations of B-lymphocytes (BLs). Equally important is the elimination, by negative selection, of autoreactive BLs which have the potential to cause autoimmune disease. These fundamental processes of negative and positive selection occur within the germinal centers of lymph nodes. There, BLs are either eliminated by apoptosis following activation of the surface IgM/B-cell receptor (IgM/BCR) alone, or are rescued from apoptosis by IgM/BCR-activation in combination with appropriate co-stimulatory survival signals. These survival signals include the activation of the CD40 receptor on the BL surface and by the association/adhesion of BLs with follicular dendritic cells within the germinal center. Animal models have been instrumental in identifying potentially immunotoxic chemicals, but such assays are costly, may lack sensitivity, and generally do not reveal mechanisms of immunotoxicity. Thus, there is a need for a model system to screen the numerous drugs and chemicals produced and in development to provide information on specific mechanisms and for use in risk assessment.
Our experimental system is comprised of a panel of germinal center-derived human BL-cell lines that undergo apoptosis following engagement of the surface IgM/BCR receptor (a model of negative selection), and are rescued from IgM/BCR induced apoptosis following co-stimulation with CD40 ligand and co-culture with the human follicular dendritic cell line, HK (a model of positive selection). Importantly, the BL-cell lines show differences in their sensitivity to apoptosis induction due to the altered expression of survival genes, such as Bcl-2. These genes are differentially expressed at specific stages of normal BL maturation within the germinal center. Thus, this model system will also allow us to identify individual and combinations of drug exposure that may modulate the sensitivity of susceptible and resistant BL populations to apoptotic cell death.
We propose to use this system to examine the effects of potentially immunotoxic chemicals on three essential components of the germinal center reaction: (1) the generation of survival signals following the adhesion of BLs with HK dendritic cells, (2) the induction of apoptosis and/or rescue from apoptosis induction following activation of the surface IgM/BCR and/or CD40 receptors, and (3) the effects on antibody class switching by BLs - a process that must occur in order to generate different classes of antibodies. This model system is expected to provide an alternative means to assess and predict the immunotoxic potential of drugs and chemicals, alone or in combination, and to identify the molecular mechanisms underlying the toxicity of these agents on BL survival, antibody production and the generation of antibody diversity.
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