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Autoantigen microarrays reveal autoantibodies associated with proliferative nephritis and active disease in pediatric systemic lupus erythematosus.

Haddon DJ, Diep VK, Price JV, Limb C, Utz PJ, Balboni I - Arthritis Res. Ther. (2015)

Bottom Line: We also compared pSLE patients with biopsy-confirmed class III or IV proliferative nephritis (n = 23) and without significant renal involvement (n = 18).High levels of anti-BAFF were associated with active disease.Our model, based on ELISA measurements and clinical variables, correctly identified patients with proliferative nephritis with 91 % accuracy.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, 291 Campus Drive, Stanford, CA, 94305, USA. djhaddon@stanford.edu.

ABSTRACT

Introduction: Pediatric systemic lupus erythematosus (pSLE) patients often initially present with more active and severe disease than adults, including a higher frequency of lupus nephritis. Specific autoantibodies, including anti-C1q, anti-DNA and anti-alpha-actinin, have been associated with kidney involvement in SLE, and DNA antibodies are capable of initiating early-stage lupus nephritis in severe combined immunodeficiency (SCID) mice. Over 100 different autoantibodies have been described in SLE patients, highlighting the need for comprehensive autoantibody profiling. Knowledge of the antibodies associated with pSLE and proliferative nephritis will increase the understanding of SLE pathogenesis, and may aid in monitoring patients for renal flare.

Methods: We used autoantigen microarrays composed of 140 recombinant or purified antigens to compare the serum autoantibody profiles of new-onset pSLE patients (n = 45) to healthy controls (n = 17). We also compared pSLE patients with biopsy-confirmed class III or IV proliferative nephritis (n = 23) and without significant renal involvement (n = 18). We performed ELISA with selected autoantigens to validate the microarray findings. We created a multiple logistic regression model, based on the ELISA and clinical information, to predict whether a patient had proliferative nephritis, and used a validation cohort (n = 23) and longitudinal samples (88 patient visits) to test its accuracy.

Results: Fifty autoantibodies were at significantly higher levels in the sera of pSLE patients compared to healthy controls, including anti-B cell-activating factor (BAFF). High levels of anti-BAFF were associated with active disease. Thirteen serum autoantibodies were present at significantly higher levels in pSLE patients with proliferative nephritis than those without, and we confirmed five autoantigens (dsDNA, C1q, collagens IV and X and aggrecan) by ELISA. Our model, based on ELISA measurements and clinical variables, correctly identified patients with proliferative nephritis with 91 % accuracy.

Conclusions: Autoantigen microarrays are an ideal platform for identifying autoantibodies associated with both pSLE and specific clinical manifestations of pSLE. Using multiple regression analysis to integrate autoantibody and clinical data permits accurate prediction of clinical manifestations with complex etiologies in pSLE.

No MeSH data available.


Related in: MedlinePlus

ELISA validation of candidate autoantigens identified by microarray. Significance analysis of microarrays (SAM) identified 13 autoantigens as having significantly different IgG reactivity between pSLE patients with and without proliferative nephritis, of which seven were selected for further validation using indirect serum ELISA. Purified or recombinant forms of the autoantigens shown above were used to coat 96-well ELISA plates. Serum from 42 pSLE patients was used to probe them in duplicate, and europium-labeled anti-human IgG (Fcγ specific) was used as a secondary reagent. Time-resolved fluorescent counts for six of the seven selected autoantigens (minus signal from BSA-coated wells) are shown above. Mann–Whitney tests were used to compare reactivity between groups (bars show mean ± SEM). BSA bovine serum albumin, ELISA enzyme-linked immunosorbent assay, IgG immunoglobulin G, pSLE pediatric systemic lupus erythematosus
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Fig4: ELISA validation of candidate autoantigens identified by microarray. Significance analysis of microarrays (SAM) identified 13 autoantigens as having significantly different IgG reactivity between pSLE patients with and without proliferative nephritis, of which seven were selected for further validation using indirect serum ELISA. Purified or recombinant forms of the autoantigens shown above were used to coat 96-well ELISA plates. Serum from 42 pSLE patients was used to probe them in duplicate, and europium-labeled anti-human IgG (Fcγ specific) was used as a secondary reagent. Time-resolved fluorescent counts for six of the seven selected autoantigens (minus signal from BSA-coated wells) are shown above. Mann–Whitney tests were used to compare reactivity between groups (bars show mean ± SEM). BSA bovine serum albumin, ELISA enzyme-linked immunosorbent assay, IgG immunoglobulin G, pSLE pediatric systemic lupus erythematosus

Mentions: We selected eight candidate proliferative nephritis-associated autoantigens that were identified by microarray, and utilized indirect serum ELISA to validate our findings. The autoantigens were: dsDNA, complement C1q, collagens IV and X, aggrecan, histone H1, histone H2B, and histones H2A and H4. Serum from the 42 new-onset pSLE patients was assayed for reactivity to each of the autoantigens, and we found a high level of agreement between the microarrays and ELISAs. Six of eight of the autoantigens tested, dsDNA, C1q, collagens IV and X, aggrecan, and histone H1, had significantly higher reactivity in patients with biopsy-proven proliferative nephritis than in those without (Fig. 4). Levels of anti-BAFF, as measured by ELISA, did not show an association with proliferative nephritis (data not shown). These findings confirm anti-dsDNA and anti-C1q are associated with LN in pSLE, identify aggrecan, collagen IV, and collagen X autoantibodies as novel markers of LN in pSLE, and validate autoantigen microarray as a platform for discovery of candidate biomarkers of LN.Fig. 4


Autoantigen microarrays reveal autoantibodies associated with proliferative nephritis and active disease in pediatric systemic lupus erythematosus.

Haddon DJ, Diep VK, Price JV, Limb C, Utz PJ, Balboni I - Arthritis Res. Ther. (2015)

ELISA validation of candidate autoantigens identified by microarray. Significance analysis of microarrays (SAM) identified 13 autoantigens as having significantly different IgG reactivity between pSLE patients with and without proliferative nephritis, of which seven were selected for further validation using indirect serum ELISA. Purified or recombinant forms of the autoantigens shown above were used to coat 96-well ELISA plates. Serum from 42 pSLE patients was used to probe them in duplicate, and europium-labeled anti-human IgG (Fcγ specific) was used as a secondary reagent. Time-resolved fluorescent counts for six of the seven selected autoantigens (minus signal from BSA-coated wells) are shown above. Mann–Whitney tests were used to compare reactivity between groups (bars show mean ± SEM). BSA bovine serum albumin, ELISA enzyme-linked immunosorbent assay, IgG immunoglobulin G, pSLE pediatric systemic lupus erythematosus
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4493823&req=5

Fig4: ELISA validation of candidate autoantigens identified by microarray. Significance analysis of microarrays (SAM) identified 13 autoantigens as having significantly different IgG reactivity between pSLE patients with and without proliferative nephritis, of which seven were selected for further validation using indirect serum ELISA. Purified or recombinant forms of the autoantigens shown above were used to coat 96-well ELISA plates. Serum from 42 pSLE patients was used to probe them in duplicate, and europium-labeled anti-human IgG (Fcγ specific) was used as a secondary reagent. Time-resolved fluorescent counts for six of the seven selected autoantigens (minus signal from BSA-coated wells) are shown above. Mann–Whitney tests were used to compare reactivity between groups (bars show mean ± SEM). BSA bovine serum albumin, ELISA enzyme-linked immunosorbent assay, IgG immunoglobulin G, pSLE pediatric systemic lupus erythematosus
Mentions: We selected eight candidate proliferative nephritis-associated autoantigens that were identified by microarray, and utilized indirect serum ELISA to validate our findings. The autoantigens were: dsDNA, complement C1q, collagens IV and X, aggrecan, histone H1, histone H2B, and histones H2A and H4. Serum from the 42 new-onset pSLE patients was assayed for reactivity to each of the autoantigens, and we found a high level of agreement between the microarrays and ELISAs. Six of eight of the autoantigens tested, dsDNA, C1q, collagens IV and X, aggrecan, and histone H1, had significantly higher reactivity in patients with biopsy-proven proliferative nephritis than in those without (Fig. 4). Levels of anti-BAFF, as measured by ELISA, did not show an association with proliferative nephritis (data not shown). These findings confirm anti-dsDNA and anti-C1q are associated with LN in pSLE, identify aggrecan, collagen IV, and collagen X autoantibodies as novel markers of LN in pSLE, and validate autoantigen microarray as a platform for discovery of candidate biomarkers of LN.Fig. 4

Bottom Line: We also compared pSLE patients with biopsy-confirmed class III or IV proliferative nephritis (n = 23) and without significant renal involvement (n = 18).High levels of anti-BAFF were associated with active disease.Our model, based on ELISA measurements and clinical variables, correctly identified patients with proliferative nephritis with 91 % accuracy.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, 291 Campus Drive, Stanford, CA, 94305, USA. djhaddon@stanford.edu.

ABSTRACT

Introduction: Pediatric systemic lupus erythematosus (pSLE) patients often initially present with more active and severe disease than adults, including a higher frequency of lupus nephritis. Specific autoantibodies, including anti-C1q, anti-DNA and anti-alpha-actinin, have been associated with kidney involvement in SLE, and DNA antibodies are capable of initiating early-stage lupus nephritis in severe combined immunodeficiency (SCID) mice. Over 100 different autoantibodies have been described in SLE patients, highlighting the need for comprehensive autoantibody profiling. Knowledge of the antibodies associated with pSLE and proliferative nephritis will increase the understanding of SLE pathogenesis, and may aid in monitoring patients for renal flare.

Methods: We used autoantigen microarrays composed of 140 recombinant or purified antigens to compare the serum autoantibody profiles of new-onset pSLE patients (n = 45) to healthy controls (n = 17). We also compared pSLE patients with biopsy-confirmed class III or IV proliferative nephritis (n = 23) and without significant renal involvement (n = 18). We performed ELISA with selected autoantigens to validate the microarray findings. We created a multiple logistic regression model, based on the ELISA and clinical information, to predict whether a patient had proliferative nephritis, and used a validation cohort (n = 23) and longitudinal samples (88 patient visits) to test its accuracy.

Results: Fifty autoantibodies were at significantly higher levels in the sera of pSLE patients compared to healthy controls, including anti-B cell-activating factor (BAFF). High levels of anti-BAFF were associated with active disease. Thirteen serum autoantibodies were present at significantly higher levels in pSLE patients with proliferative nephritis than those without, and we confirmed five autoantigens (dsDNA, C1q, collagens IV and X and aggrecan) by ELISA. Our model, based on ELISA measurements and clinical variables, correctly identified patients with proliferative nephritis with 91 % accuracy.

Conclusions: Autoantigen microarrays are an ideal platform for identifying autoantibodies associated with both pSLE and specific clinical manifestations of pSLE. Using multiple regression analysis to integrate autoantibody and clinical data permits accurate prediction of clinical manifestations with complex etiologies in pSLE.

No MeSH data available.


Related in: MedlinePlus