J Immunol 198:1263C1273. saline solution. Serum samples obtained 28?days postimmunization were diluted 1:100 in 1% BSACPBS, and 100-l volumes of samples were incubated overnight with antigen-coupled microspheres (Luminex, Austin, TX), washed, detected with 1:100 anti-mouse IgGCPEC1% BSACPBS, washed, and analyzed around the Luminex 200 platform. Data corresponding to (a) percent amino acid identity and (b) median fluorescence intensity (MFI) are shown for the exotoxin antigens tested. (c) C57BL/6 mice were infected intravenously with the Newman wild-type WT strain, a protein A-null (SSTI patients showed changes in neutrophil counts and serum cytokines in the acute phase of contamination that resolved in convalescence, suggesting a systemic innate immune response. (a) Acutely infected SSTI patients (= 53) were compared to other patients from the same cohort, including patients with Streptococcus sp. SSTI (= 12), coagulase-negative staphylococcus SSTI (= 12), and no-culture-growth SSTI (= 19) and emergency room (ER) uninfected controls (= 12). Grouped analysis was SMYD3-IN-1 performed with analysis of variance (ANOVA) (Kruskal-Wallis test with Dunns multiple-comparison test) (*, < 0.05; **, < 0.01; ***, < 0.001; ****, < 0.0001). To generate SMYD3-IN-1 the data shown in panels b and c, SSTI patient absolute Mouse monoclonal to EGF neutrophil counts were assessed at each visit (acute phase, 6-week-convalescent phase [= 38], and 6-month-convalescent phase [= 17]) and analyzed for longitudinal fluctuations by the use of Graphpad Prism by the Wilcoxon matched-pair signed-rank test. Lower and upper normal ranges for absolute neutrophil counts are marked by red lines corresponding to 1 1.8 and 9.0?k/l, respectively. (d) Assessment of longitudinal modulation of serum cytokines in SSTI patients. Sera from SSTI patients (= 39) at the acute-phase and 6-week-convalescent-phase time points were assayed using a LEGENDplex SMYD3-IN-1 human T helper cytokine panel 13-plex kit (BioLegend), followed by four-parameter logistic curve fitting performed using BioLegend LEGENDplex Data Analysis software and extrapolation of values (in picograms per milliliter). These values were assessed for changes using the following equation: acute-phase values in picograms per milliliter ? convalescent-phase values in picograms per milliliter. Data are presented as delta values in picograms per milliliter, with a positive value reflecting a higher cytokine concentration at the acute phase and a negative value reflecting a higher cytokine concentration at 6?weeks of convalescence. Calculations performed on the basis of the Pearson clustering method in R showed groups that had IL-22 and IL-13 values that were higher in the acute phase; IL-6 and IL-2 values that were higher in the acute phase; IL-2 values that were higher and IL-6 and IL-22 values that were lower in the acute phase; IL-6 values that were higher and IL-2, IL-22, and IL-13 values that were lower in the acute phase; interferon gamma (IFN-gamma) and IL-17A values that were lower in the acute phase; and IL-2 values that were lower in the acute phase; and some groups of patients with broad responses (99624, 79414, 10732, and 44570). Download FIG?S2, PDF file, 0.4 MB. Copyright SMYD3-IN-1 ? 2018 Pelzek et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. TABLE?S3? Antigens in multiplex panel for assessment of immunoglobulin binding. Download TABLE?S3, PDF file, 0.2 MB. Copyright ? 2018 Pelzek et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S3? Analysis of genomes for the presence of toxin genes in infecting and colonizing isolates from human SSTI patients. Whole-genome sequencing was performed for infecting strains from 38 patients (= 40 strains) and colonizing strains from 19 patients (= 20 strains), and the results were analyzed by a custom BLAST-style alignment strategy (tBLASTn, translated nucleotides using a protein query) against a query amino acid sequence for each gene of interest, with NCBI protein accession numbers indicated in parentheses. The best match by percent amino acid identity within each genome is usually shown, with the exception of phenol-soluble modulins (PSM) alpha 2, 3, and 4, which were not detected in the tBLASTn strategy but instead SMYD3-IN-1 with DNA-DNA BLAST. Download FIG?S3, PDF file, 0.3 MB. Copyright ? 2018 Pelzek et al. This.