Plasma ceramide profile.

<p>Plasma ceramide species were measured by LC/ESI/MS/MS analysis. (A) Ceramide profiles are shown as a heat map similar to above. Ceramide concentrations were log-transformed prior to computing z-scores. Ceramide profile elements (columns) were not clustered. (B) Select plots of individual ceramide species, each symbol represents an individual subject, red lines indicate mean.</p

Increased total peripheral blood B cells in IDUs.

<p>PBMC were analyzed by flow cytometry. (<b>A</b>) Representative plots gated on live, CD14-CD3-CD4- lymphocytes, the gate is colored red to highlight the expanded CD19+CD20+ total B cell population in IDUs. (<b>B</b>) Frequency of CD19+ (live, CD14-CD3-CD4-) populations among lymphocytes defined by FSC and SSC, each symbol is an individual subject, the red lines indicate mean. The singular CD19+ population is the combination of both the CD19+CD20low/neg population and the CD19+CD20+ population. Flow cytometry was conducted once per sample.</p

Substance Use Characteristics.

<p>Substance Use Characteristics.</p

Plasma analyte profile.

<p>Plasma cytokines, chemokines and growth factors were measured by Milliplex Immunoassay and LPS was measured by a limulus assay. (A) Analyte profiles are shown as a heat map similar to above. Analyte concentrations were log-transformed prior to computing z-scores. (B) Select plots of individual analytes, each symbol represents an individual subject, the red lines indicate mean.</p

Principal component analysis of features identified by machine learning pipeline.

<p>Principal component analysis (PCA) of 19 HCs (black circles) and 19 IDUs (red asterisks) based on ten features from the various assays identified by machine learning (CD19+CD20low/negIgD-CD27-, MIP-1β, gp140 IgM, IgG4, CD19+CD20+, sCD40L, TGF-α, MH: d18:1/16:0, TNF-α and MH: d18:1/22:0). PCA was computed on per-feature z-scores (based on all 38 samples) of log transformed data (except for B cell subset frequencies which were not log-transformed).</p

B cell phenotypic profile.

<p>(<b>A</b>) B cell profiles for live, CD14-CD3-CD4- CD19+CD20low/neg (left) and CD19+CD20+ (right). Heat maps show data corresponding to a particular B cell subset (column) and subject (row). Colors correspond to z-scores computed separately for each subset using the mean and standard deviation of the HCs. Both subsets and subjects were clustered hierarchically based on Euclidean distance and complete linkage, although HCs and IDUs were clustered separately. Sample ID, gender (F = magenta, M = blue) and race (W = white, B = black, A = Asian, AI = American Indian,— = unknown) are shown just to the right of each heat map. Cocaine and heroin usage is shown as a bar graph to the right of the injection drug user sample heat maps. Bar plot below each heat map shows the mean z-score for each subset for the IDUs samples only (z-score based on mean, standard deviation of HCs only). Gray traces represent data from individual IDU samples. (<b>B</b>) Representative plots gated on live, CD14-CD3-CD4- CD19+CD20low/neg or CD19+CD20+ B cell populations, the gates are colored red to highlight expanded CD19+CD20low/neg CD27+ subsets in IDUs. (<b>C</b>) Frequency of select CD19+CD20low/neg subsets, each symbol represents an individual subject, the red lines indicates mean.</p

Plasma antibody profile.

<p>Relative concentrations of plasma antibody species was determined by ELISAs. (A) Antibody profiles are shown as a heat maps similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158641#pone.0158641.g001" target="_blank">Fig 1</a>. Antibody concentrations were log-transformed prior to computing z-scores. (B) Plasma concentrations of total antibody isotypes and IgG subclasses and (C) select antigen-specific antibodies. Each symbol represents an individual subject, the red lines indicate mean.</p

Features Identified by Machine Learning Analysis.

<p>Features Identified by Machine Learning Analysis.</p