Principal Coordinate Analysis (PCoA) of unweighted UniFrac distances between samples.

<p>A. Samples are colored by time points, red, 7 d and 30 d, green, 6 m and 8 m, light blue, 10 m and 12 m, dark blue, 18 m and 24 m. B. Samples are colored by intervention groups. Group A: red, Group B: green. Blue: 7 d and 30 d time points prior to diet intervention. A and B: Gold, adult controls: subject 34 gluten-free diet for over 24 weeks and HLA DQ2/8⁺; subject 67 HLA DQ2/8−; subject 103 HLA DQ2/8⁺; subject 86; diagnosed with CD and HLA DQ2/8⁺.</p

¹H-NMR metabolomics analysis.

<p>A. PCA analysis of metabolomic profiles (A). Relative concentration of major metabolites measured from stool NMR profiles in selected subjects over the 24-month study period (B–F). B. Succinate; C. Acetate; D. Lactate; E. Butyrate and F. Propionate. The chemical shift (ppm) value of each metabolite is indicated above each panel.</p

Heatmaps of relative abundance of bacterial phylum in the GI microbiota of samples collected longitudinally from 7 d to 24 months of age in DQ2⁺/DQ8⁺ infants (color key is indicated on the right).

<p>A. Samples are grouped by subjects ID and intervention groups. B. Samples are grouped by timepoints. Red bars indicate samples from subjects in intervention group B. Missing data point are indicated with a white vertical line. Stool samples collected from adult subjects that were processed in parallel are included for comparison (from left to right): subject 69: gluten-free diet for more than 24 weeks and HLA DQ2/8⁺; subject 103: HLA DQ2/8⁺, subject 67 HLA DQ2/8−; subject 86: diagnosed with CD and HLA DQ2/8⁺; subject 34: gluten-free diet for more than 24 months and HLA DQ2/8⁺; subject 36: HLA DQ2/8−; subject 6: HLA DQ2/8⁺; subject 49: HLA DQ2/8−.</p

Cumulative incidence of AGA antibodies.

<p>Percent of AGA positive subjects enrolled in the study in each intervention group. * denotes time of gluten introduction, red, group B and blue, group A.</p

Heatmap of relative abundance of bacterial phylum of longitudinal samples from DQ2⁺/DQ8⁺ infants analyzed in this study and those of Palmer <i>et al.</i>[<b>28</b>] (D), Color keys are indicated on the upper right corner.

<p>A. Complete linkage clustering based on the phylum composition and abundance of GI microbiota. B. Color depicts the study and intervention group of the samples. C. Colors depict the time point at which the samples were collected. Time points D and E were omitted as no corresponding samples were collected in the Palmer <i>et al.</i> study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033387#pone.0033387-Palmer1" target="_blank">[28]</a>.</p

Quantitative real-time PCR of total 16S rRNA gene copies (red) and Bacteroidetes 16S gene copies (blue) for all samples analyzed at each time point in triplicate.

<p>Quantitative real-time PCR of total 16S rRNA gene copies (red) and Bacteroidetes 16S gene copies (blue) for all samples analyzed at each time point in triplicate.</p

Heat map showing all significant partial Pearson correlations of barrier and systemic biomarkers with HAZ or WAZ at enrollment (ie. study start, ss) or with changes in (Δ) HAZ or WAZ.

<p>Significant correlations (at p<0.05; * = p<0.01) between biomarkers and growth, controlling for child age and gender. HAZ = height for age Z score; WAZ = weight for age z score; ss = study start; Δ = change in HAZ or WAZ at 2-6m followup; numbers range from 230 to 292 except for zonulin at age >12m where n = 172. Full r, p and df values are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158772#pone.0158772.s002" target="_blank">S1 Table</a>.</p

Fecal MPO, Fecal alpha-1-antitrypsin (A1AT), and plasma LPS, FABP and SAA each predicts subsequent growth impairment.

<p>a: For MPO, <i>p</i> = 0.028; n = 266 when correcting for age and gender, and independent of breastfeeding status (that showed no correlation in these 6-26m old children) and of age. b: For A1AT, n = 237; <i>p</i> = 0.042; and A1AT also correlates with “catchup WAZ” as well, <i>p</i> = 0.035 after correcting for age and gender. c: For urine L/M, higher values correlated (controlling for age and gender) with impaired growth (delta HAZ) (<i>r</i> = -0.173; <i>p</i> = 0.009; n = 230). d: For plasma LPS (ie lower LUM), higher values correlated with impaired growth (delta HAZ) (<i>r</i> = 0.151; <i>p</i> = 0.017; n = 251). e: For plasma FABP, higher values correlated with impaired growth (delta HAZ) (r = -0.134; <i>p</i> = 0.042; n = 231). f: For plasma SAA, higher values correlated with impaired growth (delta HAZ) (r = -0.132; p = 0.046; n = 231).</p

Path model, using Principle Components Analyses (Equamax rotation solution maximizing independence of groups) showing associations among 1) Barrier (green), 2) Local Gut (orange) and 3) Systemic (pink) sets of biomarkers, as well as their predictive utility regarding linear growth.

<p>Path model, using Principle Components Analyses (Equamax rotation solution maximizing independence of groups) showing associations among 1) Barrier (green), 2) Local Gut (orange) and 3) Systemic (pink) sets of biomarkers, as well as their predictive utility regarding linear growth.</p

Cluster dendrogram with Pearson correlations among those biomarkers with ≥274 values showing three main groups: 1) translocation markers, LPS and IgA and IgG anti-LPS or FliC as well as zonulin and the 2 potential predictors of subsequent growth, tryptophan and citrulline; 2) predominantly systemic responses to disrupted barrier function and translocation; and 3) markers of specific intestinal barrier disruption or local intestinal inflammation.

<p>As discussed, groups 1 and 3 may predispose to group 2 systemic responses in associating with each other as shown in the heat map as would fit our concept of the pathogenesis of enteropathy.</p