The candidate is a molecular biologist who wishes to establish an independent research career in the molecular immunology field. Enhanced training under Dr. Max D. Cooper?s supervision in immunology is proposed for the initial two or three years in order to achieve research independence within the award period. The proposal will focus on the molecular basis and regulatory mechanism of human VH gene replacement and its contribution to normal B cell development, B cell responses, and rheumatoid arthritis in particular. The hypothesis builds on an analysis of a clonal human cell line, EU12, that undergoes continuous in vitro differentiation from proB (CD34+) to preB (CD34 mu SLC+) then to B cells (CD34 mu+LC) and generates intraclonal diversity through serial VH replacements. The ongoing VH replacement in the EU12 cells is verified by the detection of VH replacement excision circles and double stranded DNA breaks at the cryptic RSS sites.
(Aim 1) The EU12 cells will be used as an experimental model to dissect the molecular basis for the VH replacement. Purified RAG 1, RAG 2, and HMG 1 proteins will be used in in vitro studies to define the function of the cryptic RSS site.
(Aim 2) Using the methods and principles derived from the study of the EU 12 model, normal human bone marrow and tonsillar germinal center B lineage cells will be used to investigate the occurrence and stage(s) of VH replacement in humans.
(Aim 3) The regulatory mechanism of VH replacement will be investigated using the EU12 model through evaluating the effects of. (i) modulating surrogate light chain (lamda 5/14. 1) or conventional light chain (kappa and lamdaX) expression, (iii) ligating cell surface receptors (pre BCR, BCR, and CD40), and (iii) stimulating with cytokines (IL 1 beta, TFNaphla, IL 6, and IL 7) on VH replacement.
(Aim 4) Determine if VH replacement occurs in synovial B cells thereby contributing to the generation of autoantibodies and to search for potential VH replacement stimuli in rheumatoid arthritis synovial tissue.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
1K01AR048592-01
Application #
6460285
Study Section
Arthritis and Musculoskeletal and Skin Diseases Special Grants Review Committee (AMS)
Program Officer
Gretz, Elizabeth
Project Start
2002-09-01
Project End
2007-08-31
Budget Start
2002-09-01
Budget End
2003-08-31
Support Year
1
Fiscal Year
2002
Total Cost
$70,200
Indirect Cost
Name
University of Alabama Birmingham
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
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Huang, Lin; Lange, Miles D; Yu, Yangsheng et al. (2013) Contribution of V(H) replacement products in mouse antibody repertoire. PLoS One 8:e57877
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Zhang, Zhixin; Espinoza, Celia R; Yu, Zhihong et al. (2006) Transcription factor Pax5 (BSAP) transactivates the RAG-mediated V(H)-to-DJ(H) rearrangement of immunoglobulin genes. Nat Immunol 7:616-24
Liu, Yanwen; Fan, Run; Zhou, Song et al. (2005) Potential contribution of VH gene replacement in immunity and disease. Ann N Y Acad Sci 1062:175-81
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Zhang, Zhixin; Zemlin, Michael; Wang, Yui-Hsi et al. (2003) Contribution of Vh gene replacement to the primary B cell repertoire. Immunity 19:21-31