Additional file 1 of N-acetylglucosamine inhibits inflammation and neurodegeneration markers in multiple sclerosis: a mechanistic trial

Additional file 1: Figure S1. Baseline HexNAc and cytokine levels. A–E) Average HexNAc (A) and cytokine (B–E) levels from the 4 weekly visits prior to GlcNAc treatment separated by MS subtype. HexNAc measured by LC–MS/MS and IFNg, IL-6, IL-17 and IL-10 levels measured by sandwich ELISA. Only cytokine levels above the lower limit of quantification (LLOQ) were included in analysis. Each dot represents an individual subject. Error bars reflect SEM. P values were measured by two-tailed t test with Welch’s correction. Figure S2. Lymphocyte flow cytometry gating strategy. Activated blasting T cells were identified among the large cells. All CD4 and CD8 cells identified in the large gate were confirmed to be T cells by staining for CD3. L-PHA staining of large CD4+CD25+ blasting T cells with pretreatment in black and during GlcNAc treatment in red. Figure S3. N-glycan branching on resting lymphocytes with oral GlcNAc treatment. A–L) Averaged change in L-PHA binding to resting CD4+CD25–T cells, resting CD8+ T cells and resting CD19+ B cells by flow cytometry assessed before (visits 1–4), during (visits 5–8), and after (visits 9–11) GlcNAc treatment individually (A–C, G–I) or combined (D–F, J–L). Change in L-PHA MFI is relative to baseline (V1–4). P value by linear mixed models (two-tailed) using all subjects who completed the study. Figure S4. Changes in serum neurofilament light chain (sNfl) and clinical disability (EDSS) with oral GlcNAc . A–C) Average sNfL levels measured by SIMOA from the 4 visits prior to GlcNAc treatment separated by HexNAc (A) MS subtype (B) or baseline HexNAc (C). Error bars are SEM. P values by two-tailed t test with Welch’s correction. D–G) Averaged sNfL before (V1–4), during (V5–8), and after (V9–11) oral GlcNAc individually (D,E) or combined (F,G) in subjects with median baseline sNfL < 11.07pg/ml. n=8 and n=8 in the 6g (D,F) and 12g (E,G) cohorts, respectively. sNfL measured by SIMOA. P value by linear mixed modeling (one-tailed) in subjects who completed the study. H–I) Shown is EDSS scores at baseline (average of V1 and V4), after 4 weeks of GlcNAc (visit 8) or 4 weeks after stopping GlcNAc (visit 11). P value by one-tailed Wilcoxon paired test in the intention to treat cohort ITT. Table S1. Modified CONSORT checklist for pilot randomized clinical trials (Sections 8–12 on randomization removed).

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