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Subspecificities of anticentromeric protein A antibodies identify systemic sclerosis patients at higher risk of pulmonary vascular disease

View Article: PubMed Central - PubMed

ABSTRACT

Patients with systemic sclerosis (SSc) who express autoantibodies to centromeric proteins (CENPs) are at risk of developing pulmonary vascular disease and pulmonary arterial hypertension without fibrosis. Currently no biomarkers are available to predict these complications. We previously characterized the fine specificity of anti-CENP-A antibodies in SSc by screening a phage display library (expressing random 12-mer peptides), and identified phage clones whose peptides were differentially recognized by patients’ autoantibodies. Here, we examined if subgroups of SSc patients with different anti-CENP-A antibody subspecificities also differ clinically, and if serum reactivity to phage-displayed peptides can predict pulmonary vascular disease.

Clinical data and serum samples were collected from 84 anti-CENP-A-positive SSc patients. Indirect ELISAs were used to test serum reactivity. Pulmonary vascular disease was defined as high systolic pulmonary arterial pressure (sPAP) and low diffusing lung capacity for carbon monoxide (DLCO; percent of predicted values).

Sera were screened for reactivity to peptides expressed by phage clones pc4.2 and pc14.1, confirming our earlier observation of differential specificities. Linear regression showed that the levels of antibodies specific for the 2 phage clones were associated with clinical features of pulmonary vascular disease, but in opposite ways: anti-pc4.2 antibodies were positively associated with sPAP and inversely associated with DLCO, whereas anti-pc14.1 antibodies were inversely associated with sPAP and positively associated with DLCO. Anti-pc4.2 and anti-pc14.1 antibody levels predicted sPAP independently of DLCO. These associations were confirmed by logistic regression using antibodies as predictors and dichotomized sPAP (cutoff, 45 mm Hg) as outcome. The ratio of the 2 antibody levels was a useful marker in predicting high sPAP.

This study demonstrates that some SSc clinical features associate with subspecificities of anti-CENP-A antibodies. Moreover, it shows that a simple, inexpensive phage-based assay can predict which SSc patients have high sPAP and low DLCO, hence who are at greater risk of developing pulmonary arterial hypertension. The ability to identify these at-risk patients can contribute to clinical efficiency and effectiveness. Further research into the peptides expressed by the phage clones may reveal the molecular mechanisms that put some anti-CENP-A-positive patients at greater risk than others for pulmonary vascular disease.

No MeSH data available.


Subspecificities of the 84 SSc patients’ sera for 2 phage-expressed centromeric protein (CENP)-A-derived peptides. (A) Venn diagram showing overlapping sets of patients with antibodies recognizing peptides expressed by 2 phages; sera from 17 patients did not react with either phage clone. (B) Binding of anti-pc4.2 and anti-pc14.1 antibodies expressed as a percentage of the binding obtained with positive control sera from pt4 or pt14. The horizontal bar marks the median and the box indicates the interquartile range; outlier values (more than 1.5 times the interquartile range) are marked with a circle, while extreme outliers values (more than 3 times the interquartile range) are marked with an asterix.
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Figure 2: Subspecificities of the 84 SSc patients’ sera for 2 phage-expressed centromeric protein (CENP)-A-derived peptides. (A) Venn diagram showing overlapping sets of patients with antibodies recognizing peptides expressed by 2 phages; sera from 17 patients did not react with either phage clone. (B) Binding of anti-pc4.2 and anti-pc14.1 antibodies expressed as a percentage of the binding obtained with positive control sera from pt4 or pt14. The horizontal bar marks the median and the box indicates the interquartile range; outlier values (more than 1.5 times the interquartile range) are marked with a circle, while extreme outliers values (more than 3 times the interquartile range) are marked with an asterix.

Mentions: Next, we choose 2 phage clones (pc4.2 and pc14.1) to use in screening sera from the entire SSc study group, in an indirect ELISA with antiphage mAb as capture antibody and phage as antigen. This analysis showed that 49 sera samples recognized both peptides, while 17 bound neither (Fig. 2A). The median binding to pc4.2 was 7.3% of that obtained with pt4 Ig, while the median binding to pc14.1 was 3.8% of that obtained with pt14 Ig, although in both cases there was a broad right skew in the distribution (Fig. 2B). These results therefore confirm, in a larger group of SSc patients, that there are wide subspecificities among the anti-CENP-A antibodies expressed by different patients.


Subspecificities of anticentromeric protein A antibodies identify systemic sclerosis patients at higher risk of pulmonary vascular disease
Subspecificities of the 84 SSc patients’ sera for 2 phage-expressed centromeric protein (CENP)-A-derived peptides. (A) Venn diagram showing overlapping sets of patients with antibodies recognizing peptides expressed by 2 phages; sera from 17 patients did not react with either phage clone. (B) Binding of anti-pc4.2 and anti-pc14.1 antibodies expressed as a percentage of the binding obtained with positive control sera from pt4 or pt14. The horizontal bar marks the median and the box indicates the interquartile range; outlier values (more than 1.5 times the interquartile range) are marked with a circle, while extreme outliers values (more than 3 times the interquartile range) are marked with an asterix.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4998321&req=5

Figure 2: Subspecificities of the 84 SSc patients’ sera for 2 phage-expressed centromeric protein (CENP)-A-derived peptides. (A) Venn diagram showing overlapping sets of patients with antibodies recognizing peptides expressed by 2 phages; sera from 17 patients did not react with either phage clone. (B) Binding of anti-pc4.2 and anti-pc14.1 antibodies expressed as a percentage of the binding obtained with positive control sera from pt4 or pt14. The horizontal bar marks the median and the box indicates the interquartile range; outlier values (more than 1.5 times the interquartile range) are marked with a circle, while extreme outliers values (more than 3 times the interquartile range) are marked with an asterix.
Mentions: Next, we choose 2 phage clones (pc4.2 and pc14.1) to use in screening sera from the entire SSc study group, in an indirect ELISA with antiphage mAb as capture antibody and phage as antigen. This analysis showed that 49 sera samples recognized both peptides, while 17 bound neither (Fig. 2A). The median binding to pc4.2 was 7.3% of that obtained with pt4 Ig, while the median binding to pc14.1 was 3.8% of that obtained with pt14 Ig, although in both cases there was a broad right skew in the distribution (Fig. 2B). These results therefore confirm, in a larger group of SSc patients, that there are wide subspecificities among the anti-CENP-A antibodies expressed by different patients.

View Article: PubMed Central - PubMed

ABSTRACT

Patients with systemic sclerosis (SSc) who express autoantibodies to centromeric proteins (CENPs) are at risk of developing pulmonary vascular disease and pulmonary arterial hypertension without fibrosis. Currently no biomarkers are available to predict these complications. We previously characterized the fine specificity of anti-CENP-A antibodies in SSc by screening a phage display library (expressing random 12-mer peptides), and identified phage clones whose peptides were differentially recognized by patients’ autoantibodies. Here, we examined if subgroups of SSc patients with different anti-CENP-A antibody subspecificities also differ clinically, and if serum reactivity to phage-displayed peptides can predict pulmonary vascular disease.

Clinical data and serum samples were collected from 84 anti-CENP-A-positive SSc patients. Indirect ELISAs were used to test serum reactivity. Pulmonary vascular disease was defined as high systolic pulmonary arterial pressure (sPAP) and low diffusing lung capacity for carbon monoxide (DLCO; percent of predicted values).

Sera were screened for reactivity to peptides expressed by phage clones pc4.2 and pc14.1, confirming our earlier observation of differential specificities. Linear regression showed that the levels of antibodies specific for the 2 phage clones were associated with clinical features of pulmonary vascular disease, but in opposite ways: anti-pc4.2 antibodies were positively associated with sPAP and inversely associated with DLCO, whereas anti-pc14.1 antibodies were inversely associated with sPAP and positively associated with DLCO. Anti-pc4.2 and anti-pc14.1 antibody levels predicted sPAP independently of DLCO. These associations were confirmed by logistic regression using antibodies as predictors and dichotomized sPAP (cutoff, 45 mm Hg) as outcome. The ratio of the 2 antibody levels was a useful marker in predicting high sPAP.

This study demonstrates that some SSc clinical features associate with subspecificities of anti-CENP-A antibodies. Moreover, it shows that a simple, inexpensive phage-based assay can predict which SSc patients have high sPAP and low DLCO, hence who are at greater risk of developing pulmonary arterial hypertension. The ability to identify these at-risk patients can contribute to clinical efficiency and effectiveness. Further research into the peptides expressed by the phage clones may reveal the molecular mechanisms that put some anti-CENP-A-positive patients at greater risk than others for pulmonary vascular disease.

No MeSH data available.