MOLECULAR ANALYSIS OF HUMAN ANTIGBM ANTIBODIES

  • Madaio, Michael P., (PI)

Project: Research project

Description

The overall aim of this project is to develop a better understanding of the cellular events within the interstitium in murine lupus nephritis. The hypothesis that a subset of self-reactive CD4 T cells escapes normal tolerance and reacts directly, in an Ag-specific manner, with resident cells within the kidney, will be tested. The experimental approach will emphasize analysis of pathologically relevant cells. For this purpose a preexisting panel of autoreactive T cell clones (ARTC), tubular cell lines and APC, derived from lupus-prone MRL-lpr/lpr mice, will be utilized to pursue the following Specific Aims: 1. To determine whether interstitial T cells represent either a restricted population of autoAg-- specific infiltrating T cells or result from polyclonal (random) T cell infiltration. Two experimental strategies will be utilized: analysis of TcR receptor use of nephritogenic T cells, and examination of the Ag requirements for T cells cultured from the kidneys of mice with active nephritis. In the former approach, TcR V gene analysis of a subset of T cell clones derived from the kidneys of nephritic mice will be determined, and probes specific for relevant TcR sequences (oligonucleotides and/or mAb) will be used to compare the TcR gene repertoire of T cells within the interstitium to those in the spleen and thymus of diseased mice. The latter approach will employ tubular cell lines and professional APC derived from lupus-prone mice to examine the Ag/cellular requirements for activation of nephritogenic T cells. Ultimately the capacity of CD44+ ARTC to transfer disease to naive or pre-disease mice will be evaluated 2. To examine the hypothesis that impaired signal transduction through CD4 results in a defect in tolerance and the emergence of autoreactive and tissue specific T cells in murine lupus. The rationale for this approach is derived from the observation that CD4-mediated tyrosine phosphorylation is abnormal in a MRL-lpr/lpr-derived CD4+ ARTC clone, and this is associated with reduced constitutive expression of lck protein. To further evaluate this abnormality, the nature of this defect will be further defined using the ARTC clone, and the results of these studies will be applied to a more general examination of CD44+ cells in MRL-lpr/lpr mice. Analyses will involve: evaluation signal transduction through CD4-lck, including determination of functional lck activity and the level lck transcription, and evaluation of signaling through other T cell receptors, including lL-2R and CD3. This approach represents a natural evolution of the specific aims of the previous proposal. The focused is shifted from analysis of the role of ARTC in B cell activation to a more direct evaluation of the L cellular and molecular interactions involved in T cell-mediated interstitial nephritis. The shift in direction has been facilitated by isolation of the disease-relevant cell lines and the movement of my laboratory to an environment more conducive to the analysis of autoimmune interstitial nephritis.
StatusFinished
Effective start/end date9/1/976/30/06

Funding

  • National Institutes of Health: $245,677.00
  • National Institutes of Health
  • National Institutes of Health: $245,677.00
  • National Institutes of Health: $257,949.00
  • National Institutes of Health: $202,986.00
  • National Institutes of Health: $39,625.00
  • National Institutes of Health: $302,735.00
  • National Institutes of Health: $245,677.00
  • National Institutes of Health: $250,436.00

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Antibodies
Glomerular Basement Membrane
T-Lymphocytes
Clone Cells
Kidney
Anti-Glomerular Basement Membrane Disease
T-Cell Receptor Genes
Interstitial Nephritis
Nephritis
Glomerulonephritis
Cell Line
Signal Transduction
Lupus Nephritis
Collagen Type IV
T-Lymphocyte Subsets
T-Cell Antigen Receptor
Basement Membrane
Oligonucleotides
Thymus Gland
Cell Movement

ASJC

  • Medicine(all)