Volume 62, Issue 5, Pages (November 2002)

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Volume 62, Issue 5, Pages 1638-1649 (November 2002) Expression profile of leukocyte genes activated by anti-neutrophil cytoplasmic autoantibodies (ANCA)  Jia Jin Yang, Gloria A. Preston, David A. Alcorta, Iwao Waga, William E. Munger, Susan L. Hogan, Stephen B. Sekura, Brian D. Phillips, Robin P. Thomas, J. Charles Jennette, Ronald J. Falk  Kidney International  Volume 62, Issue 5, Pages 1638-1649 (November 2002) DOI: 10.1046/j.1523-1755.2002.00619.x Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 1 In vitro analysis of the time course of changes in the relative mRNA level of (A andB)DIF-2, (C andD)COX-2 and (E andF)IL-8 by TaqMan®-PCR. TNF-α-primed leukocytes from a healthy volunteer were stimulated by normal or ANCA IgG (A-C) or their respective F(ab′)2 fragments (D-F). The values represent the fold-change compared to non-incubated, untreated controls. Each point represents the average value of two treatments with different IgG or F(ab′)2 preparations and all experiments were performed in duplicate. Symbols are: (•) untreated; (♦) normal IgG; (□) MPO-ANCA F(ab′)2; (▵) PR3-ANCA F(ab′)2. Kidney International 2002 62, 1638-1649DOI: (10.1046/j.1523-1755.2002.00619.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 2 In vitro analysis of ANCA stimulated effects. (A) The variability in individual IgG and F(ab′)2 preparations to stimulate superoxide O2- release by SOD-inhibitable reduction of ferricytochrome C. Each symbol represents the average value from six separate experiments with 6 different leukocyte donors. (B) The variability in individual IgG and F(ab′)2 preparations to induce an increase in DIF-2 mRNA levels. Symbols are: (⋄) Normal 1, MPO-ANCA 1, PR3-ANCA 1; (▪) Normal 2, MPO-ANCA 2, PR3-ANCA 3; (▵) Normal 3, MPO-ANCA 3, PR3-ANCA 3; (♦) Normal 4, MPO-ANCA 4, PR3-ANCA 4; (□) MPO-ANCA 5, PR3-ANCA 5; (▴) MPO-ANCA 6, PR3-ANCA 6. Each symbol represents the average value from eight experiments with eight individual donors. All experiments were performed in duplicate. Kidney International 2002 62, 1638-1649DOI: (10.1046/j.1523-1755.2002.00619.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 3 Analysis of DIF-2 (A and D), COX-2 (B) and IL-8 (C) expression in leukocytes from healthy volunteers (normal), from patients with active ANCA disease (ANCA-A), from patients with ANCA disease but in remission (ANCA-R), and from patients with SLE or IgA nephropathy. Overall ANOVA on ranked values was used to compare patient groups: DIF-2 (A and D, P < 0.05), COX-2 (B, P < 0.05) and IL-8 (C, P > 0.05); *P < 0.05 for specific patient groups versus normal group. The bold line represents the median value. Kidney International 2002 62, 1638-1649DOI: (10.1046/j.1523-1755.2002.00619.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 4 DIF-2 protein levels. (A) In vitro exposure of cells to ANCA- or normal-IgG: lane 1,TNF-α-primed control; lane 2, normal IgG (N1); lane 3, MPO-ANCA IgG(P1); lane 4, MPO-ANCA (P3); lane 5, PR3-ANCA IgG(C1). (B) Analysis of leukocytes from ANCA patients at different stages of disease: lanes 1, severe disease activity (A+++); lane 2, moderate disease activity (A++); lanes 3-5, mild disease activity (A+); lane 6, remission (R); lanes 7-9, healthy volunteers (N). Proteinase 3 (PR3) Western blots are given as a reference for protein loading comparisons. Kidney International 2002 62, 1638-1649DOI: (10.1046/j.1523-1755.2002.00619.x) Copyright © 2002 International Society of Nephrology Terms and Conditions