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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.


Differential recognition of affinity-purified anti-Ap1–17 Ig from 12 SSc patients for peptides expressed by 4 phage clones, previously isolated from a phage display library using Ig from 2 of the patients. (A, B) Phage clones isolated using patient pt4 Ig. (C, D) Phage clones isolated using patient pt14 Ig. Each data point is the mean of duplicate wells. The data are representative of 2 experiments.
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Figure 1: Differential recognition of affinity-purified anti-Ap1–17 Ig from 12 SSc patients for peptides expressed by 4 phage clones, previously isolated from a phage display library using Ig from 2 of the patients. (A, B) Phage clones isolated using patient pt4 Ig. (C, D) Phage clones isolated using patient pt14 Ig. Each data point is the mean of duplicate wells. The data are representative of 2 experiments.

Mentions: Affinity-purified anti-Ap1–17 Ig from 12 of the 84 SSc patients was tested for reactivity to peptides expressed by 2 pairs of phage clones previously isolated from a phage display library using similar Ig from patients pt4 and pt14. In indirect ELISAs using the 12 preparations of purified Ig as capture antibodies and 1 of the 2 pt4 phage clones as antigen (Fig. 1A, B), the reactivity of pt4 Ig to both clones was confirmed by strong absorbance; moreover, 6 Ig preparations gave negligible absorbance in both tests, while 5 Ig preparations (pt3, pt5, pt7, pt9, and pt21) gave absorbance values >0.5 for at least 1 phage clone. When the experiment was repeated using the pt14 phage clones (Fig. 1C, D), no reactivity was seen at all, with the exception of pt14 Ig (the exact Ig used to isolate the phage clones). Hence, 12-mer phage-expressed peptides that are specifically bound by pt4 and pt14 Ig are recognized in a differential manner by anti-Ap1–17 Ig purified (on the basis of their affinity to the 17-mer synthetic peptide) from 10 other SSc patients.


Subspecificities of anticentromeric protein A antibodies identify systemic sclerosis patients at higher risk of pulmonary vascular disease
Differential recognition of affinity-purified anti-Ap1–17 Ig from 12 SSc patients for peptides expressed by 4 phage clones, previously isolated from a phage display library using Ig from 2 of the patients. (A, B) Phage clones isolated using patient pt4 Ig. (C, D) Phage clones isolated using patient pt14 Ig. Each data point is the mean of duplicate wells. The data are representative of 2 experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Differential recognition of affinity-purified anti-Ap1–17 Ig from 12 SSc patients for peptides expressed by 4 phage clones, previously isolated from a phage display library using Ig from 2 of the patients. (A, B) Phage clones isolated using patient pt4 Ig. (C, D) Phage clones isolated using patient pt14 Ig. Each data point is the mean of duplicate wells. The data are representative of 2 experiments.
Mentions: Affinity-purified anti-Ap1–17 Ig from 12 of the 84 SSc patients was tested for reactivity to peptides expressed by 2 pairs of phage clones previously isolated from a phage display library using similar Ig from patients pt4 and pt14. In indirect ELISAs using the 12 preparations of purified Ig as capture antibodies and 1 of the 2 pt4 phage clones as antigen (Fig. 1A, B), the reactivity of pt4 Ig to both clones was confirmed by strong absorbance; moreover, 6 Ig preparations gave negligible absorbance in both tests, while 5 Ig preparations (pt3, pt5, pt7, pt9, and pt21) gave absorbance values >0.5 for at least 1 phage clone. When the experiment was repeated using the pt14 phage clones (Fig. 1C, D), no reactivity was seen at all, with the exception of pt14 Ig (the exact Ig used to isolate the phage clones). Hence, 12-mer phage-expressed peptides that are specifically bound by pt4 and pt14 Ig are recognized in a differential manner by anti-Ap1–17 Ig purified (on the basis of their affinity to the 17-mer synthetic peptide) from 10 other SSc 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.