NTS treatment stimulates myeloid colony forming capacity in patient bone marrow cells.

<p>CD34+ cells were cultured in the presence of G-CSF to induce neutrophil differentiation in the absence or presence of 0.5 µM (patient 1) or 1.0 µM (patient 2–3) NTS1 and NTS2. Cells were isolated after 3 days for CFU assays in the presence of G-CSF during 11 days. Data were expressed as the number of CFU-GM (A, upper panel), CFU-G (<b>A, middle panel</b>) and CFU-M (<b>A, lower panel</b>) for each patient and for all patients together (<b>B</b>). Progenitor expansion and terminal differentiation was evaluated after 14 days, Data were expressed as fold induction (<b>C</b>) and the percentage of mature neutrophils and monocytes (<b>D</b>). Error bars represent SEM (between patients). *p = <0.05.</p

NTS1 and NTS2 treatment stimulates myelopoiesis and affects C/EBPα and p38MAPK activity.

<p>BALB/c mice were treated with 150 mg/kg 5FU at day 0, followed by treatment with 1 mg/kg NTS1 or NTS2 once a week. Mice were treated 2, 5, 9, 15, or 21 days (3 per group). Mice only treated with 5FU were used as control (2 per group) (<b>A</b>). Bone marrow mononuclear cells were cultured in the presence of rmGM-CSF and rmG-CSF to induce myeloid colony formation during 7 days. Data were expressed as the cumulative number of colonies (<b>B</b>) and the number of CFU-G and CFU-M per femur at day 2, 5, 9, 15 and 21 (<b>C–D</b>). Error bars represent SEM (between mice). *p = <0.05, **p = <0.01. Data are representative for 2 independent experiments. Ba/F3 cells were starved overnight in the presence of 0.5% FCS. Cells were left untreated or treated with NTS1 or NTS2 for 30 minutes, before stimulation with 10% FCS for 15 minutes. CD34+ cells were cultured in the presence of G-CSF for 6 days in the absence or presence of 0.5 or 5.0 µM NTS1 or NTS2. Protein lysates were prepared and Western Blot analysis was performed with an antibody against phosphorylated ERK1/2, phosphorylated p38MAPK and phosphorylated C/EBPα. Antibodies against ERK1/2, p38MAPK, C/EBPα and tubulin were used as controls (<b>E–F</b>). Data are representative for 3 independent experiments.</p

Treatment with NTS1 and NTS2 does not affect terminal neutrophil differentiation.

<p>CD34+ cells were cultured in the presence of G-CSF to induce neutrophil differentiation during 17 days. Cells were cultured either in the absence or presence of NTS1 (0.5–5 µM) or NTS2 (0.5–5 µM). After 17 days of neutrophil differentiation was determined by cytospin analysis (<b>A</b>) and FACS analysis or intracellular lactoferrin expression. Data were expressed as the percentage of mature neutrophils (banded or segmented nuclei) (N = 3) (<b>B</b>) the mean lactoferrin expression (MFI) (<b>C</b>) (N = 3), and the percentage of mature monocytes (<b>C</b>). Error bars represent SEM (between experiments). *p = <0.05, **p = <0.01.</p

qPCR primers.

<p>qPCR primers.</p

VPA and NAM treatment modulates H3K27 acetylation at the promoters of myeloid specific genes.

<p>UCB-derived CD34+ cells were differentiated towards megakaryocytes. At day 4 of differentiation, cells were treated overnight with 200μM, or 5mM NAM. Next, lysates were prepared, followed by ChIP-sequencing (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196400#sec002" target="_blank">Methods</a>). Data represent a Venn diagram comparing the number of genes identified based on a >2-fold increase or decrease in H3K27 acetylation levels compared to the control (A). Gene ontology analysis of up- and down-regulated genes by VPA (B) and NAM (C) treatment. From the same cells, RNA was isolated, followed by cDNA synthesis and qPCR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196400#sec002" target="_blank">Methods</a>). Data represent the relative mRNA level of GATA1, RUNX1, LMO2 and RUNX2 compared to the control (D). Data are representative for 3 experiments. Error bars represent SEM, * p<0.05, ** p<0.01.</p

VPA and NAM treatment inhibits megakaryocyte progenitor proliferation.

<p>UCB-derived CD34+ cells were differentiated towards megakaryocytes for 11 days in the absence or presence of 100–200μM VPA, or 1-5mM NAM. (A) At all culture time points, trypan blue negative cells were counted. Data represent the fold expansion of megakaryocyte precursors during development. (B) At day 7 and 11, the percentage of apoptotic cells was determined by FACS. Data represent the percentage of Annexin V-positive cells, compared to the control. (C) Absolute numbers of CD61/CD42b positive megakaryocytes were calculated after 11 days of differentiation. Data are representative for 4 independent experiments. Error bars represent SEM, * p<0.05, ** p< 0.01.</p

VPA treatment increases the absolute number of MEP.

<p>UCB-derived CD34+ cells were differentiated towards megakaryocytes for 4 days in the absence or presence of 200μM VPA, or 5mM NAM. Myeloid progenitor staining was performed according to Manz <i>et al</i>. (46), and distinct progenitor populations were analysed by FACS. MEP were gated from the Lin- CD34+, CD38+ CD123- and CD45RA- cell population. Data represent (A) the absolute numbers of CD34+ cells and MEP, compared to the control, and (B) the percentage of CD34+ cells and MEP, compared to the control. Data are representative for 3 independent experiments. Error bars represent SEM, * p<0.05, ** p< 0.01.</p

KDACi treatment differentially modulates megakaryocyte development.

<p>(A) From the peripheral blood the absolute number of platelets was analysed in patients treated with VPA (n = 217) together with plasma VPA concentration, measured at the same day. Data represent linear regression analysis of the absolute number of platelets (dependent variable), and plasma VPA concentration (independent variable). (B) UCB-derived CD34+ cells were differentiated towards megakaryocytes in the absence or presence of 10nM TSA, 250μM SB, 200μM VPA, or 1mM NAM for 11 days. Differentiation was determined based on the surface expression of CD61 and CD42b (percentage of double positive cells compared to the control), and (C) cytospin analysis. Data are representative for 3 independent experiments. Error bars represent SEM, * p<0.05, ** p< 0.01.</p

Additional file 2 of Meningococcal ACWY conjugate vaccine immunogenicity in adolescents with primary or secondary immune deficiencies, a prospective observational cohort study

Supplementary Material

Additional file 1 of Meningococcal ACWY conjugate vaccine immunogenicity in adolescents with primary or secondary immune deficiencies, a prospective observational cohort study

Supplementary Material