The phosphotranferase activity of EGFR plays a key role in SSc IgG-induced early signaling events in VSMCs.

<p><b>A</b>, Quiescent vascular smooth muscle cells (VSMCs) were pre-treated for 30 minutes with vehicle (0.01% DMSO) or PDGFR-inhibitor AG1296 (5 µM) before stimulation with 50 ng/mL PDGF (P) or 200 µg/mL control (Ct) or systemic sclerosis (SSc) patient IgG. <b>B</b>, Quiescent VSMCs were pre-treated for 60 minutes with 10 µM imatinib mesylate (Imat; +) or with 0.01% DMSO (−), followed by 5-minute stimulation with 50 ng/mL PDGF or 200 µg/mL IgG from control or SSc patients. <b>C</b>, Quiescent VSMCs were pre-treated with inhibitors (Inh; 10 µM imatinib mesylate (im), 1 µM irbesartan (ir), 250 nM AG1478 (A)), or 0.01% DMSO (−) for 30 minutes prior to stimulation for 5 minutes with 50 ng/mL PDGF (P), 100 nM angiotensin II (AngII), 100 ng/mL epidermal growth factor (EGF), or 200 µg/mL SSc or control IgG. All results shown are representative of at least two experiments with similar results.</p

Characteristics of our SSc cohort (N = 23).

<p>Characteristics of our SSc cohort (N = 23).</p

Systemic sclerosis (SSc) IgG causes growth and pro-fibrotic responses in vascular smooth muscle cells (VSMCs).

<p><b>A</b>, Protein synthesis was measured by [³H]-leucine incorporation in quiescent VSMCs stimulated with IgG buffer, angiotensin II (AngII), or with 200 µg/mL SSc or control (Ct) IgG (left), and mean protein synthesis was compared for Ct-IgG and SSc-IgG treated cells (right). Protein synthesis is shown as a fold-change with respect to basal levels. ** <i>p</i> = 0.0008. <b>B</b>, Expression of transforming growth factor-β1 (TGF-β1) mRNA (<i>Tgfb1</i>) in VSMCs upon treatment with IgG buffer (buff), 50 ng/mL platelet-derived growth factor (PDGF), 10 ng/mL TGF-β1, or 200 µg/mL IgG for 2 hours (left). Results are normalized to <i>β-actin</i> expression and expressed as a fold-change with respect to untreated VSMCs. Mean TGF-β1 expression in Ct-IgG-treated VSMCs compared with that in SSc-IgG treated cells (right). ** <i>p</i><0.0001. <b>C</b>. Expression of TGF-β2 mRNA (<i>Tgfb2</i>) in VSMCs upon treatment with IgG buffer, 50 ng/mL PDGF, 10 ng/mL TGF-β1, or IgG for 2 hours (left). Results are normalized to <i>β-actin</i> and expressed as a fold-change with respect to untreated VSMCs. Mean TGF-β2 expression in Ct-IgG-treated VSMCs compared with that in SSc-IgG treated cells (right). ** <i>p</i> = 0.0009. All results shown are representative of at least two experiments. Error bars represent standard deviation.</p

PLA₂R schematic plot of the seven potential antigen determinants identified by epitope mapping.

<p>All of the determinants identified by epitope mapping were located in the extracellular domain of PLA₂R and are ∼10 to 25 aa long. Only one epitope is not in the C-type lectin like domains of the receptor. <i>[C-R,cysteine-rich region; FNII, fibronectin type II domain; CTLDs, C-type lectin like domains; N, N-terminal end; C, C-terminal end].</i></p

An Anti-Phospholipase A₂ Receptor Quantitative Immunoassay and Epitope Analysis in Membranous Nephropathy Reveals Different Antigenic Domains of the Receptor

<div><p>The phospholipase A₂ receptor (PLA₂R) was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN). Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA) utilizing indirect immunofluorescence (IIF) on tissue culture cells transfected with the PLA₂R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA₂R autoantibodies on an addressable laser bead immunoassay (ALBIA) platform. Since reactive domains of PLA₂R (i.e. epitopes) could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA₂R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA₂R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA₂R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA₂R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA₂R.</p></div

Amino Acid Sequences of Consensus PLA₂R Epitopes Identified by SPOT.

<p>Amino Acid Sequences of Consensus PLA₂R Epitopes Identified by SPOT.</p

ELISA of synthesized PLA₂R peptides.

<p>To verify potential epitopes, synthetic peptides (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061669#pone-0061669-t001" target="_blank">Table 1</a>) were tested by ELISA. Absorbance of patient samples tested positive on the CB-IIF assay was higher than of patient samples tested negative and normal healthy control samples but the difference was not statistically significant (p>0.05).</p

Illustrative Inhibition studies of anti-PLA₂R antibodies using synthetic peptides with an anti-PLA₂R positive serum sample.

<p><i>Panel A:</i> Different concentrations of a mixture of PLA₂R derived peptides were used to inhibit the reactivity to the PLA₂R whole molecule in an addressable laser bead assay. The reactivity showed a significant, dose dependent inhibition. The inhibition with a control peptide (GE-1) was significantly lower. All values are expressed as residual reactivity after inhibition (in %) compared to the sample without inhibitor or control. <i>Panel B:</i> The peptide mixture together with seven individual PLA₂R peptides and a control peptide were used at a concentration of 126 µg/mL. Besides the peptide mixture, peptide 3 and peptide 4 showed inhibition of anti-PLA₂R antibodies. All values are expressed as residual reactivity after inhibition (in %) compared to the sample without inhibitor or control. <i>Panel C:</i> The peptide mixture together with seven individual PLA₂R peptides and a control peptide were used at a concentration of 126 µg/mL. Besides the peptide mixture, peptide 3 and peptide 4 showed inhibition of anti-PLA₂R antibodies. All values are expressed as ALBIA median fluorescence intensities (MFI) or titer by indirect immunofluorescence on cell-based assay (IIF-CBA).</p

Clinical and Serological Features of Patients Referred through a Rheumatology Triage System because of Positive Antinuclear Antibodies

<div><p>Background</p><p>The referral of patients with positive anti-nuclear antibody (ANA) tests has been criticized as an inappropriate use of medical resources. The utility of a positive ANA test in a central triage (CT) system was studied by determining the autoantibody profiles and clinical diagnoses of patients referred to rheumatologists through a CT system because of a positive ANA test.</p><p>Methods</p><p>Patients that met three criteria were included: (1) referred to Rheumatology CT over a three year interval; (2) reason for referral was a “positive ANA”; (3) were evaluated by a certified rheumatologist. The CT clinical database was used to obtain demographic and clinical information and a serological database was used to retrieve specific ANA and/or extractable nuclear antigen (ENA) test results. Clinical information was extracted from the consulting rheumatologist's report.</p><p>Results</p><p>15,357 patients were referred through the CT system; 643 (4.1%) of these because of a positive ANA and of these 263 (40.9%) were evaluated by a certified rheumatologist. In 63/263 (24%) of ANA positive patients, the specialist provided a diagnosis of an ANA associated rheumatic disease (AARD) while 69 (26.2%) had no evidence of any disease; 102 (38.8%) had other rheumatologic diagnoses and 29 (11%) had conditions that did not meet AARD classification criteria. Of ANA positive archived sera, 15.1% were anti-DFS70 positive and 91.2% of these did not have an AARD.</p><p>Conclusions</p><p>This is the first study to evaluate the serological and clinical features of patients referred through a CT system because of a positive ANA. The spectrum of autoantibody specificities was wide with anti-Ro52/TRIM21 being the most common autoantibody detected. Approximately 15% of referrals had only antibodies to DFS70, the vast majority of which did not have clinical evidence for an AARD. These findings provide insight into the utility of autoantibody testing in a CT system.</p></div

Epitope Mapping of PLA₂R Peptides with anti-human IgG₄.

<p>Grey scale heat map representation of results from SPOT used to detect PLA₂R epitopes. Peptide membranes were probed with 10 randomly selected IMN samples that had previously been tested by IIF-CBA for anti-PLA₂R antibodies (7 positive (IMN+) and 3 negative (IMN-)), as well as 5 normal healthy controls (NHC). The positive control rabbit antibody to PLA₂R reacted with the expected peptide used as the immunogen. Consensus epitopes and their respective PLA₂R domains derived from this analysis are illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061669#pone-0061669-g003" target="_blank">Figure 3</a> and summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061669#pone-0061669-t001" target="_blank">Table 1</a>.</p