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JY performed the corrected and adjusted analysis of the data and assisted in drafting and editing the manuscript

JY performed the corrected and adjusted analysis of the data and assisted in drafting and editing the manuscript. MCP-1 and IFN-, which has been previously demonstrated in systemic sclerosis, suggesting a shared pathophysiology. Within the LS patients, those with active disease demonstrated IP-10, TNF- and GM-CSF, which may potentially serve as biomarkers of disease activity in the clinical setting. contracture, limb-length discrepancy, facial atrophy etc.) [37]. The LoSDI was created using the sum of individual scores for 18 surface body sites and assessed dermal atrophy (none, shiny skin, Rabbit polyclonal to AKR1A1 visible vessels, cliff drop), subcutaneous atrophy (none, flat, concave, marked atrophy) and maximum hyperpigmentation/hypopigmentation [37]. Other clinical outcome measures that were collected included the following: disease duration, systemic treatment, disease subtype, presence of extracutaneous manifestations (ECMs), patient measured indicators, traditional inflammatory markers (i.e. C sedimentation rate) and antibody status. The patient reported indicators included were the parent and patient Global Assessment of disease impact (Parent-GA, Patient-GA), which are 0 to 100 VAS scales regarding disease severity (no problem = 0, to very severe problem = 100), and the Childhood Dermatology Life Quality Index (CDLQI). The CDLQI is a reliable and valid measure of quality of life impact in pediatric dermatologic diseases [38], including LS [39]. Patients answer 10 questions, which are summed into an overall score, range 0C30. Higher scores indicate that quality of life has been adversely affected. Specific antibodies evaluated for this study included anti-nuclear antibodies (ANA), anti-histone antibodies (AHA) and anti-single stranded DNA antibodies (anti-ssDNA). These have been described in LS as reflecting disease severity [40, 41] and have been associated with other cytokines evaluated in LS [20, 21, 33]. Levels of ANA for all LS subjects MG-101 (healthy controls not tested) were determined using indirect immunofluorescence on HEp-2 cells through the University of Pittsburgh Immunology laboratory. An ANA titer of 1 1:80 or greater was considered positive. Sera samples meeting the cutoff titer of 1 1:80 were serially diluted to 1 1:1280. AHA and anti-ssDNA antibodies were assayed by ELISA through the University of Pittsburgh Immunology laboratory. AHA levels higher than 1. 0 U/mL and levels of anti-ssDNA antibodies higher than MG-101 19 U/mL were considered positive. 2.3. MG-101 Cytokine and chemokine analysis Plasma levels of twenty-nine total TH1, TH2, and TH17-associated cytokines and chemokines were analyzed using Milliplex Luminex xMAP Technology (EMD Millipore, USA) and the Luminex bead immunoassay system (Human Cytokine/chemokine Panel 1 and human TH17 kits; BioRad, Hercules, CA), following the manufacturers instructions. These studies were performed at the University of Pittsburgh Cancer Institute (UPCI) Luminex Core Facility. Plasma samples (both LS and healthy control) were undiluted and compared to a High-PMT-Standard Dilution Series (BioRad, Hercules, CA). Samples were run in duplicate, and inter-panel and intra-assay control plasma samples were included to ensure consistency across panels. The cytokines and chemokines included were macrophage inflammatory protein ?1 (MIP-1), interferon gamma-induced protein 10 (IP-10), interleukin ?5 (IL-5), IL-4, IL-2, IL-17a, IL-13, IL-12p70, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor- (TNF-), IL-8, IL-6, IL-1, IL-10, MG-101 interferon- (IFN-), IFN-2, platelet-derived growth factor-bb (PDGF-bb), IL-1, monocyte chemotactic protein- MG-101 1 (MCP-1), vascular endothelial growth factor (VEGF), IL-17f, IL-22, IL-33, IL-21, IL-23, IL-17e, IL-27, IL-31, and IL-28 (general detection limit 3.2 pg/mL C 10,000 pg/mL). 2.4. Data and statistical analysis All analyses were performed using software (SPSS, Version.20, IBM Corp, Armonk, NY) and R language, version 13.1 (R Core Team, Vienna, Austria). Agreement between duplicate samples was measured using the intra-class correlation coefficient (ICC). Medians and interquartile ranges (IQR) and counts and percentages were used to summarize continuous and categorical variables respectively. Cytokine and chemokine levels in LS patients were compared to levels in healthy controls using Mann-Whitney U tests. Similar analyses were performed on only the LS patients using categorical clinical variables to.