Ashley's Paper

Glomerular expression and elevated serum Bcl-2 and Fas proteins in lupus nephritis: preliminary findings

Introduction:
• Autoimmune disease
• May effect many body parts
• Several different types of lupus
• Symptom
o Most common- fatigue, arthritits, fever, skin rahes, and kidney prolems
o Other- chest pain, hair loss, and renaud
• Objective of study:
o To research if proteins play a role in lupus nephritis: Bcl-2 and Fas
Materials and methods:
• Obtained kidney biopsies of 36 patients with SLE
• Renal function tests
Immunohistochemical evaluation Most important technique of paper
o Put tissue in wax to solidify - helps tissue keep shape, able to see distinct cells
o Slice into thin pieces put on glass slides and remove wax
o Remove peroxidase within cells by soaking in H202
o Expose tissue to goat serum - antibodies within serum bind to everything not Bcl-2 and Fas
o Incubate with mouse monoclonal antibodies - binds to specific epitope on Bcl-2
o Rabbit polyclonal antibodies - bind to many epitopes specific to Fas
o Secondary antibodies specific to Bcl-2 and Fas primary antibodies tagged with peroxidase enzyme
o specimen then soaked in H202 to break down peroxidase enzyme tag - turns tagged cells brown
Bcl-2 = brown cytoplasmic cells, Fas = brown cell membrane cells

Results
• Clinicopathological features (see table 1)
o Control groups:
 Serum Bcl-2: 148.6
 Serum Fas: 552
• Glomerular expression of Bcl-2 and Fas proteins in lupus nephritis (see table 2)
o Control group had no Bcl-2 or Fas in renal cells
• Figure 1
o Test subjects have Bcl-2 and Fas
 With lupus has higher concentration
o Control has none of proteins
• Table 3:
o WHO classification deciphers lupus into 5 categories
 1-none
 5-excessive protein loss, edema
 Chronicity depends on length of time having disease
 R value
• coefficient correlation; measure of how closely the points fit to the linear line
 P value
• probability; chance that your allege difference in numerical data can be explained by chance (if 5% or less chance that difference explained by randomness, it’s OKAY)
Discussion:
• Hypothesized that alteration of Bcl-2 and Fas protein expression are associated with lupus nephritis
• Levels of Bcl-2 and Fas may be used as diagnostic markers for those with SLE
• Co-expression of Bcl-2 and Fas suggest that apoptosis and proliferation both contribute to nephritis

  • Reason Bcl-2 and Fas play role in lupus nephritis - Bcl-2= anti-apoptotic, Fas= apoptosis promoter
    • Fas induces apoptosis when kidney cells are damaged by autoantibodies
    • with lupus, patients are unable to "clean up" cells that have been apoptosed which leads to increased levels of Bcl-2 in order to try and stop apoptosis of cells damaged by autoantibodies

Questions:
o polyclonal expansion:
o Inject antigen into animal and take a blood sample after week(s) and spin for serum
o Purify serum to get ABS against your antigen (primary ABS)
o Polyclonal ABS:
 Antibody production come from a mixed B-cell populations and likely against more than one epitope
o Monoclonal ABS
 Wait only a couple of days and get B-cells then solidify B-cells to get ONLY one with particular immunoglobulin
 Take single B-cell and grow it in culture
 Purify ABS from cell culture fluid from cultured B-cells
 Monoclonal: against single epitope

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