Intra- & Inter-assay Variance for the flow cytometric analysis of STAT5Ptyr in human peripheral blood monocytes.

<p>Whole blood from T1D (#418, 237,327, and 434) and non-autoimmune healthy controls (75, 365, and 236) was analyzed for STAT5 activation as described in the Methods & Materials. Each sample aliquot was analyzed 4 to 5 times during each run of the analyses. <b>A. Intra-assay Variance</b>: There was an assay variance of <5% in between control samples and intra-assay from 0 to 6.3% for a given control sample’s analysis; whereas, there was an assay variance of <13% between T1D samples with an intra-assay 1 to 7.9% for a given T1D sample’s analysis. There was a significant (>13-fold) difference between the % STAT5Ptyr/CD14+ cells detected in the T1D samples tested (mean 65.85%, SD 12.79%, n = 4) as compared to controls analyzed in the same assays (mean 4.952%, SD 4.547%, n = 3). <b>B. </b><b>Inter-assa. </b><b>Variance:</b> Each sample analyzed 24 hr to 48 hr after shipment and storage at 4°C. An inter-assay variance of 2-6-fold between 24 hr (filled circles) and 48 hr (clear circles) assay runs of 3 control samples and a 1.1-fold variance in 3 T1D samples was seen between 24 and 48 hrs. At 24 hr, there was a significant (>13-fold, *p = 0.0017) difference between the % STAT5Ptyr/CD14+ cells of T1D samples (mean 65.85%, SD 12.79%, n = 4) as compared to controls (mean 4.952%, SD 4.547%, n = 3) in same assays. However, this significant difference decreased to 5.4-fold at 48 hr (*p = 0.013), due to the increased inter-assay variability seen in control samples after storage.</p

Characteristics of patient, control and relative samples collected for <i>CSF2</i> and <i>PTGS2</i> sequencing analysis.

*<p>Individuals with overt hyperglycemia or abnormal GTT/FPIR; mean disease duration = 8.5 years ±7.65 SD.</p

STAT5 Phosphorylation in CD14+ Peripheral Blood Monocytes and Correlation of STAT5Ptyr with Duration of Disease.

<p><b>A.</b> Unactivated monocytes from 53 T1D patients had significantly higher activated STAT5 without <i>ex vivo</i> activation than monocytes from 37 healthy, non-autoimmune controls as analyzed by intercellular flow cytometry using small volume (<100 µl) whole peripheral blood samples (control mean 8.46%±7.83 SD, T1D 30.97%±18.00 SD, p<0.0001, Mann Whitney U test). Gender did not affect these data, though a trend for higher values in female T1D monocytes was seen: control females mean 8.904% ±7.445 SD, n = 17 vs. control males mean 8.904% ±8.242 SD, n = 20, (p = 0.6463, Student t test); female T1D subjects 32.30% ±24.37 SD; n = 28, vs.T1D male subjects 24.62% ±15.07 SD; n = 25, (p = 0.4820, Mann-Whitney U test). Overall, 59.65% of T1D subjects had %STAT5Ptyr/CD14 levels at or above the control mean +2 standard deviations (24.12%; indicated by the dashed line) compared to 2.44% of controls. <b>B.</b> No correlation with age was seen in any of the test groups; however, a trend for increasing %STAT5Ptyr levels with age was noted. This trend revealed a significant correlation of %STAT5Ptyr levels with disease duration within the T1D subject group (p = 0.0005, linear regression, R² value = 0.0784). Due to the very low sample volumes tested, most but not all samples were analyzable in duplicate from a single random blood draw. Data points on graph depict average of replicates for each individual.</p

T1D Patient/Control Sequence Analysis for Upstream Regulatory Regions of the <i>CSF2</i> and <i>PTGS2</i> genes. A.

<p>Schematic representation and representative sequence data of the human promoter region upstream of <i>CSF2,</i> the gene encoding GM-CSF. The location of the amplified region is delineated by forward and reverse primers, FP1 and RP1, respectively. Also defined are the STAT5 and STAT6 binding sites (boxed) within the region. No T1D-specific polymorphisms have been detected in the 24 samples tested. <b>B.</b> Schematic representation and representative sequence data of the enhancer region upstream of the PGS2/COX2 gene, <i>PTGS2.</i> The location of the amplified region is delineated by forward and reverse primers, FP1 and RP1, respectively. Also defined are the STAT5 and STAT6 binding sites within the region (boxed). No T1D-specific polymorphisms have been detected in the 24 samples tested.</p

Human Subject Population Characteristics for Peripheral Blood Samples collected over the 3 year study period (2006–2009).

*<p>individuals with no overt symptoms of disease and with no family history of T1D or other autoimmune diseases.</p>†<p>Individuals with overt hyperglycemia or abnormal GTT/FPIR</p>§<p>Most individuals were analyzed only one time and the assay run in duplicate for each sample</p

Multiplex ChIP analysis of Protein Binding, Epigenetic Modification, and Transcription at the <i>PTGS2</i> enhancer region.

<p>Peripheral blood PBMC monocytes were isolated and incubated at 37°C/5%CO₂ for 2 hr in the continuous presence of (A) media alone (0) or media supplemented with (B) 5.6 nM GM-CSF (GM-CSF), (C) 500 U/ml IL-4 (IL4), (D) 100 µM dexamethasone (DEX), or (E) 1 ng/ml endotoxin (LPS). This timeframe is used to allow for optimization of the negative (DEX) and positive (LPS) control stimulation. The cells were washed and extracted for ChIP analysis as described in the Materials and Methods and used in multiplex ChIP Analysis of STAT5Ptyr, STAT6Ptyr, acetylase (P300), and deacetylase (SMRTe) binding, histone H3 acetylation, and the presence of RNA Polymerase II on or near the STAT5/STAT6 binding sites in the <i>IL10</i> to <i>PTGS2</i> enhancer region shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076919#pone-0076919-g003" target="_blank">Figure 3B</a>. Each ChIP multiplex analysis depicted represents the mean of 3 to 5 runs of the analyses on each of control and T1D samples of peripheral blood monocytes. Data is presented on a log scale as an R value = 2^(ΔCt_{nonspecific IgG}^−ΔCt_{specific Ab}⁾ for each specific antibody used <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076919#pone.0076919-Nelson1" target="_blank">[19]</a>. Individual samples (not pooled) were run in at least duplicate and represent data from 5 T1D and 2 Control group members. Due to the low sample size, the apparent trends for high binding of STAT5Ptyr and the other epigenetic modification components seen, when the data were analyzed by ANOVA and pairwise Mann-Whitney U test, no statistically significant differences were found between T1D and control samples for any of proteins examined binding on the <i>PTGS</i> enhancer.</p