Assessment of qPCR Sensitivity for Bacteria Using <i>S</i>. <i>epidermdis</i>.

<p>We assessed the sensitivity of quantitative PCR for detection of bacteria via amplification of the universal bacterial 16s ribosomal RNA gene, using DNA purified from germ free mice skin tissue spiked with serial 10-fold dilutions of quantified <i>Staphylococcus epidermidis</i>. The assay's sensitivity was at or better than 10 colony-forming units per mL, with no evidence of decreasing sensitivity at lower levels.</p

Algorithm for Identification of Confirmed Cellulitis Cases and Controls.

<p>Please see the text for an explanation of the application of this algorithm.</p

Heat Map of a Derivation Set, Comprised of Verified Cases and Inflamed Controls.

<p>This heat map, built on agglomerative clustering with linkage by mean and a Euclidian distance metric, shows a comparison of log(2)-transformed normalized gene expression data from blood. Rows represent individual genes, the symbols of which are shown at right. Columns represent individual participants in our study. Colors represent the z-scores of the counts of mRNA molecules per 100 ng RNA in blood; <i>red</i> represents a high ratio of expression relative to other genes in that participant, and <i>green</i> represents a low ratio of expression relative to other genes in that participant. The left three columns are confirmed bacterial cellulitis cases, and have high expression of genes HLA-DQB1 through sCTLA4, and low expression of genes ABL1 through STAT5A. All of the other columns are inflamed controls, as defined in the text.</p

Comparison of Gene Expression Patterns in Verified Bacterial Cases versus Three Comparison Groups.

<p>This heat map, built on agglomerative clustering with linkage by mean and a Euclidian distance metric, shows a comparison of log(2)-transformed normalized gene expression data from blood. Rows represent individual genes, the symbols of which are shown at right. Each column displays a comparison of gene expression in bacterial cases (B) vs. one of three comparator groups: sterile inflamed controls (ic), indeterminate participants (ind), and normal volunteers (v). Qualitatively, this heat map reveals that the cases differ in a similar way from all three comparison groups. This, in turn, suggests that the cases–which had confirmed bacterial cellulitis based on biopsy–could be detectable by transcription profiling.</p

Photomicrographs from patients diagnosed clinically as having bacterial cellulitis.

<p>Fig 5A: Culture-proven MRSA cellulitis with positive Gram stain. Note intracellular Gram-positive cocci in clusters. Fig 5B: Lymphocytic vasculitis misdiagnosed as cellulitis. Note abundant lymphocytes invading vessel wall.</p

Relationships between co-expression modules, cytokines, and differentially expressed gene signatures.

<p><b>A</b>) Association between gene modules and cytokine production, with the color intensity indicating p-value (–log10, BH-adjusted) from linear regression between the modules’ eigengenes and the level of cytokine, and color indicating the correlation direction (red = positive, blue = negative). <b>B)</b> Enrichment of the co-expression modules in differentially expressed gene signatures, with the color intensity indicating p-value (–log10, BH-adjusted) from Fisher’s exact test for modules’ enrichments in the DEG signatures, and color indicating whether the genes in the signature were up-regulated (red) or down-regulated (blue). Module size (number of genes) is indicated in parenthesis after the label; the color scale (p-value) is truncated at ±10 (max is 163).</p

Immune Annotation analysis of the differentially expressed gene signatures.

<p>The color intensity indicates the Immune Annotation Score and the values are the odds ratios from Fisher’s exact tests with BH adjusted p-value < 0.1 for the given test. A. Level 1 (Cells), B. Level 2 (Cell Subsets), C. Level 3 (Activation Status), D. Level 4 (TLR-activated myeloid cells). The color indicates whether the genes in the signature were up-regulated (red) or down-regulated (blue). The results are shown for twelve of the total 24 DEG signatures used in the analysis, and immune Annotation categories with at least a single category with BH adjusted p-value ≤ 0.1. Colored text highlights significant associations with the matching DEG signature. Inf = Infinite, all observations were within the subgroup.</p

Cytokine secretion by group and stimulation condition.

<p>Cytokine secretion by group and stimulation condition.</p

Enrichment of differentially expressed gene signatures for transcription factor targets.

<p>Enrichment of the DEG signatures (top 200 by FC from top 500 by p-values) for transcriptional factor target gene sets, with Chip-X (ChIP-chip, ChIP-seq, ChIP-PET and DamID) derived ChEA set from human based experimental data in <b>A)</b>, ChEA set from rodent based experimental data in <b>B)</b>, and computationally predicted TRANSFACv.1 set in <b>C)</b>. The color intensity indicates–log10 of the FDR adjusted p-values and the indicated values are odds ratios (Fisher’s exact test). The color indicates whether the genes in the signature were up-regulated (red) or down-regulated (blue). The results are shown for only twelve (out of the total of 24 used in the analysis) DEG signatures, and only the TF target sets with at least a single enrichment with BH adjusted p-value ≤0.0001 for A) and B) and ≤0.05 for C), clustered according to the similarity of the enrichment pattern.</p

Immune Annotation enrichment in five egg allergy-associated co-expression modules.

<p>Enrichment results are shown for A) Level 1 immune cell types; B) Level 2 immune cell sub-types; C) Level 3 activation state of immune cell types; and D) Level 4 myeloid cell stimulation. The color intensity indicates the Immune Annotation Score and the values are the Fisher’s exact tests’ Odds Ratios for tests with BH adjusted p-value ≤ 0.10. Only categories with at least one significant result are included. Inf = Infinite, all observations were within the subgroup.</p