Hierarchical clustering of immunophenotypes reveals subgroups of ALS patients with distinct clinical characteristics.

<p>Immunophenotypes of ALS patients and HVs were analyzed by unsupervised hierarchical clustering and principal component analysis as outlined in the methods. <b>A.</b> Dendrogram of hierarchical clustering of ALS patients (blue) and HVs (yellow) were grouped into Profile 1 (purple lines) and Profile 2 (green lines). 1 HV and 3 ALS patients were not clustered into a defined profile and 1 ALS patient was excluded due to insufficient data. <b>B.</b> Table of clinical characteristics of ALS patients subgrouped by profile. Familial ALS patients included only definite or probable for this analysis. <b>C.</b> Survival curves of patients in the two different profiles (Profile 1 with purple line and Profile 2 in green line). HR = hazard ratio and the 95% CI represents the confidence interval of the ratio.</p

List of peripheral blood leukocyte biomarkers relevant to ALS.

<p>List of peripheral blood leukocyte biomarkers relevant to ALS.</p

Clinical characteristics of patients with ALS.

<p>Clinical characteristics of patients with ALS.</p

Two immune profiles of ALS patients reveal significant phenotypic differences.

<p>Leukocyte phenotypes from HVs and ALS patients sub-grouped into Profiles 1 and 2 were compared. <b>A.</b> Box-and-whisker plots of total white blood cells (WBCs) and mononuclear cells (MNCs) are shown. The boxes indicate the 25-75^th percentile, the horizontal line indicates the median, and the whiskers represent minimum and maximum values. <b>B.</b> Pie graphs depicting the peripheral blood leukocyte compartment of the three groups. The HV pie graph was set to 100% and the other pie graphs were sized in relation to the HV. <b>C.</b> Box-and-whisker plots of immunophenotypes associated with principal components. *** = p value <0.001, and * = p value <0.05.</p

Additional phenotypic characterizations of CD3⁺CD56⁺ T cells.

<p><b>A.</b> CD3⁺CD56⁺ T cell counts plotted against age (years) for sub-grouped ALS patients and healthy volunteers. <b>B.</b> Correlation between CD8⁺ T cell counts and CD3⁺CD56⁺ T cell counts (left) and the CD4:CD8 ratios are lower in CD56⁺ than CD56^- T cells. <b>C.</b> Comparison of the percentage of CD28 negative cells in CD56^- (filled shapes) and CD56⁺ cells (open shapes) for both CD8 (circles) and CD4 (squares) subsets. <b>D.</b> Examples of CD28/CD56 dot plots gated from CD4 and CD8 subsets from two ALS patients.</p

Different immunophenotypic biomarkers associate with clinical parameters in the two ALS immune profiles.

<p>Immune phenotypes were plotted against ALSFRS-R score or slope (ALSFRS-R points/month). XY graphs of correlations show p-value and Spearman r value. Lines represent the best fit resulting from linear regression analysis. Closed circles represent healthy volunteers, open squares represent ALS patients in Profile 1, and open diamonds represent ALS patients in Profile 2. <b>A.</b> Correlations of selected immunophenotypes to ALSFRS-R score in Profile 1 patients (top row) versus Profile 2 (bottom row). <b>B.</b> Correlations of selected immunophenotypes to slope in ALS Profile 1 patients. <b>C.</b> Survival curves of patients in each profile sub-grouped by cut-off values for PD-1⁺ CD4⁺ T cells. 19.7% was the cut-off value representing the median value for Profile 1 patients (Hi PD-1≥ 19.7; Lo PD-1< 19.7) and 19.5% was the cut-off value for Profile 2 patients. For CD3⁺CD56⁺ T cells, 104.62 cells/μl was used as a cut-off value for Profile 2 patients and 28.21 cells/μl was used for Profile 1. <b>D.</b> CD4⁺CD45RA⁺ naïve T cells show dissimilar age related associations between Profile 2 ALS patients, Profile 1 ALS patients, and healthy volunteers.</p

ALS patients exhibit elevated cell counts of granulocytes, NK cells and T cells.

<p>Major leukocyte populations from 80 ALS patients and 50 healthy volunteers were assessed by flow cytometry. <b>A.</b> Comparisons of granulocytes, NK cells, T cells, CD4⁺ T cells, and CD8⁺ T cells between healthy volunteers (dark circles), and ALS patients (open circles). <b>B.</b> CD3⁺CD56⁺ T cells shown as cell counts (cells/μl), percentage of total T cells, and a representative dot plot. <b>C.</b> Correlations of CD4⁺ T cells and CD4:CD8 ratios are altered in ALS patients. <b>D.</b> NK cells and granulocyte counts inversely correlate with ALSFRS-R score.</p

Allele distribution in gastroparetic and control subjects.

<p>Allele distribution of long (L), medium (M) and short (S) alleles a) for subjects with gastroparesis is significantly different from the distribution for control subjects (Chi² P value = 0.019) and b) for subjects with idiopathic gastroparesis compared to non-diabetic controls (Chi² P value = 0.049). Short alleles were defined as shorter than 29 polyGT repeats and long alleles were defined as longer than 32 repeats (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187772#pone.0187772.g001" target="_blank">Fig 1a</a> for definition).</p

Data on subjects that were studied.

<p>Data on subjects that were studied.</p

Additional file 5: of Transcriptomic signatures reveal immune dysregulation in human diabetic and idiopathic gastroparesis

Table S5. Differentially expressed genes common in diabetic gastroparesis and diabetic controls and idiopathic gastroparesis and controls. (XLSX 107 kb