Synthetic Nucleic Acid Antigens in Localized Scleroderma

We investigated the impact of synthetic nucleic acid antigens on the autoantibody profiles in patients with localized scleroderma, an autoimmune skin disease. Anti-DNA antibodies, including double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA), are common among autoimmune diseases, such as systemic lupus erythematosus and localized scleroderma. Based on recent studies, we hypothesized that the sequence of nucleic acid antigens has an impact on the autoimmune reactions in localized scleroderma. To test our hypothesis, we synthesized a panel of DNA and RNA antigens and used them for autoantibody profiling of 70 children with localized scleroderma compared with the healthy controls and patients with pediatric systemic lupus erythematosus (as a disease control). Among the tested antigens, dsD4, which contains the sequence of the human oncogene BRAF, showed a particularly strong presence in localized scleroderma but not systemic lupus erythematosus. Disease activity in patients was significantly associated with dsD4 autoantibody levels. We confirmed this result in vivo by using a bleomycin-induced mouse model of localized scleroderma. When administered intraperitoneally, dsD4 promoted an active polyclonal response in the mouse model. Our study highlights sequence specificity for nucleic acid antigens in localized scleroderma that could potentially lead to developing novel early-stage diagnostic tools

Synthetic Nucleic Acid Antigens in Localized Scleroderma

We investigated the impact of synthetic nucleic acid antigens on the autoantibody profiles in patients with localized scleroderma, an autoimmune skin disease. Anti-DNA antibodies, including double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA), are common among autoimmune diseases, such as systemic lupus erythematosus and localized scleroderma. Based on recent studies, we hypothesized that the sequence of nucleic acid antigens has an impact on the autoimmune reactions in localized scleroderma. To test our hypothesis, we synthesized a panel of DNA and RNA antigens and used them for autoantibody profiling of 70 children with localized scleroderma compared with the healthy controls and patients with pediatric systemic lupus erythematosus (as a disease control). Among the tested antigens, dsD4, which contains the sequence of the human oncogene BRAF, showed a particularly strong presence in localized scleroderma but not systemic lupus erythematosus. Disease activity in patients was significantly associated with dsD4 autoantibody levels. We confirmed this result in vivo by using a bleomycin-induced mouse model of localized scleroderma. When administered intraperitoneally, dsD4 promoted an active polyclonal response in the mouse model. Our study highlights sequence specificity for nucleic acid antigens in localized scleroderma that could potentially lead to developing novel early-stage diagnostic tools. </p

Detection of autoimmune antibodies in localized scleroderma by synthetic oligonucleotide antigens

<div><p>In this study, we developed a series of synthetic oligonucleotides that allowed us to investigate the details on the antigen recognition by autoimmune antibodies in localized scleroderma subjects. Besides dramatically improved analytical specificity of the assay, our data suggests a potential linking for antibodies to DNA to the biological status of disease state in localized scleroderma. Moreover, introducing chemical modifications into short synthetic deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules completely changed the binding titers of corresponding antibodies and their clinical relevance. The strongest observed effect was registered for the localized scleroderma skin damage index (LoSDI) on the IgG antibodies to TC dinucleotide-rich double-stranded antigen (p < 0.001). In addition to providing valuable tools for diagnosis of clinically relevant biomarkers, we believe that this work opens up new opportunities for research on antibodies to nucleic acids in localized scleroderma and other autoimmune diseases.</p></div

Analysis of new antigens in human sera.

<p>The data is presented in a modified box plot with observations outside the range [Q1 − 1.5 IQR, Q3 + 1.5 IQR] being considered outliers and identified using asterisks. Data points for each subject are means for three independent measurements with error < 3%. ELISA was carries out for unmatched healthy controls, n = 60 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195381#sec002" target="_blank">Methods</a> for details). For absorbance values, see data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195381#pone.0195381.s002" target="_blank">S2 Appendix</a>.</p

Antigen sequences and specificity to monoclonal (a-dsDNA/a-RNA, a-β2-micro-globulin), and polyclonal (a-CL) antibodies.

<p>Antigen sequences and specificity to monoclonal (a-dsDNA/a-RNA, a-β2-micro-globulin), and polyclonal (a-CL) antibodies.</p

Current methods for the detection of a-DNA and chemical structures of nucleic acid antigens.

<p>(a) Current detection methods for anti-DNA: <i>HEp-2 cell</i> assay (I), ELISA using plasmid DNA antigen (II), and the novel assay suggested in this work (using synthetic DNA/LNA/RNA; III). (b) Chemical structures of DNA, LNA and RNA nucleotides.</p

Results of statistical analyses of IgG ELISA data.

<p>Effects at a significance level of 0.05 (p<0.05) are shown in red.</p