TT-specific CD4+CD154+ CD3+ responses over time following HibMenCY-TT vaccine.

<p>The frequency above background (Δ) of TT-specific CD4+CD154+ memory cells in CD3+ lymphocytes was determined in PBMCs from infants after 2 doses (M6) or 3 doses (M7) of prime, prior to (M12), and 1 month post (M13) a booster dose of HibMenCY-TT. Each point represents the response from 1 individual and the horizontal bars represent median values. Red dots represent paired samples from individuals who had blood taken following 2 doses of HibMenCY-TT prime, whilst black dots represent those samples from individuals who had blood taken after 3 doses of HibMenCY-TT prime. p(*)≤0.05, indicating significance of related samples Wilcoxon signed rank tests.</p

Association of Men C- and Men Y- specific memory B cells with SBA titre pre- and post- boost.

<p>Men C-specific memory B cells at M6 and M7 were plotted against log-transformed M12 Men C SBA (A and B) and M13 SBA (C and D). M12 Men C-specific memory B cells were plotted against log-transformed M13 SBA values (E). Men Y-specific memory B cells at M6 and M7 were plotted against log-transformed M12 Men Y SBA (F and G) and M13 SBA (H and I). M12 Men Y-specific memory B cells were plotted against log-transformed M13 SBA values (J). Linear regression analyses were performed to determine statistically significant associations where r = r² value, n = number of subjects and p(*)≤0.05.</p

Single and double positive TT-specific memory CD4+ T cell cytokine responses following HibMenCY-TT vaccine.

<p>Intracellular TNFα, IFNγ, and IL-2 production by TT-specific memory cells was determined 48 hours after stimulation with TT, in PBMCs from infants after 2 doses (M6) or 3 doses (M7) of prime, and prior to (M12), and 1 month post (M13) a booster dose of HibMenCY-TT. <b>A.</b> single-positive TNFα+, IFNγ+ and IL-2+ cells and <b>B.</b> double-positive TNFα+IFNγ+, IL-2+IFNγ+ and TNFα+IL-2+ cells. Mean + SEM are shown. p(*)≤0.05 indicating significance of related samples Wilcoxon signed rank tests.</p

Association of TT-specific memory T cell responses with SBA titre pre- and post- boost.

<p>TT-specific CD4+CD154+ T cells at M6 and M7 were plotted against log-transformed M12 Men C SBA (A and B) and M13 SBA (C and D). M12 TT-specific CD4+CD154+ T cells were plotted against log-transformed M13 SBA values (E). TT-specific CD4+CD154+ T cells at M6 and M7 were plotted against log-transformed M12 Men Y SBA (F and G) and M13 SBA (H and I). M12 TT-specific CD4+CD154+ T cells were plotted against log-transformed M13 SBA values (J). Linear regression analyses were performed to determine statistically significant associations where r = r² value and n = number of subjects.</p

Men C- and Men Y- specific memory B cell responses following HibMenCY-TT vaccine.

<p><b>A.</b> Men C- and <b>B.</b> Men Y- specific Antibody Forming Cells (AFCs) per 1x10⁶ cultured PBMCs after 2 (M6) or 3 (M7) priming doses, and prior to (M12) and 1 month post (M13) the booster dose of HibMenCY-TT, where each point represents the response from one individual and horizontal bars indicate median values. Individuals with no detectable spots were given an arbitrary value of 1. Red dots represent paired samples from individuals who had blood taken following 2 doses of HibMenCY-TT prime, whilst black dots represent those samples from individuals who had blood taken after 3 doses of HibMenCY-TT prime. The dashed line represents the assay limit of detection. The numbers below the x axis of each graph indicate the percentage of subjects with detectable memory B cells specific for that antigen at that time point. p(**)≤0.01 and p(*)≤0.05 indicating significance of independent samples Mann-Whitney U tests or related samples Wilcoxon signed rank tests.</p

Men C- and Men Y- specific SBA titres following HibMen CY-TT vaccine.

<p><b>A.</b> Men C- and <b>B.</b> Men Y- specific SBA titre after 2 (M6) or 3 (M7) priming doses, and prior to (M12) and 1 month post (M13) the booster dose of HibMenCY-TT vaccine. Red dots represent paired samples from individuals who had blood taken following 2 doses of HibMenCY-TT prime, whilst black dots represent those samples from individuals who had blood taken after 3 doses of HibMenCY-TT prime. Horizontal bars indicate median values. The dashed line represents the assay limit of detection. p(**)≤0.01 and p(*)≤0.05 indicating significance of independent samples Mann-Whitney U tests or related samples Wilcoxon signed rank tests.</p

Otitis-prone children have fewer CD4⁺ T cells but more NK cells, particularly CD56^lo NK cells, than non-otitis-prone controls.

<p>Frequencies of CD4⁺ T cell, CD8⁺ T cell and NK cell (CD56 ^lo, CD56^hi) populations are expressed as: a) proportion of the total lymphocytes or b) cells/μL. Horizontal lines represent the median. **p<0.01 when comparing the proportion of cell subsets between cases and controls.</p

NTHi is a potent stimulator of innate inflammatory mediators regardless of susceptibility to OM.

<p>PBMC from otitis-prone children (cases; black dots) or non-otitis-prone children (controls; grey squares) were challenged with NTHi 86-028NP (NTHi-86), NTHi 289 or SEB and cytokine secretion was measured at 4h (left panel) or 24h (right panel) post-stimulation. Each dot represents a child and the horizontal lines represent the median level of cytokine production for each group. ** = p<0.001, * = p<0.05 when each stimulation was compared with unstimulated PBMCs from the same donors.</p

Bacterial biofilm matrices were demonstrated through staining with lectins.

<p>Concanavalin A (ConA) and Wheat Germ Agglutinin (WGA) (green) and propidium iodide (red) to stain for DNA. Co-localisation of the DNA and lectins are observed (yellow) suggesting that these are part of a biofilm structure. Bacterial biofilm can be observed (arrow) with bacteria being surrounding by a matrix that binds the lectin. Scale 10 µm.</p

Demographics and risk factors for otitis prone and healthy children in this study.

<p>NTHi, nontypeable <i>Haemophilus influenzae</i>; PD, protein D. p<0.05 was considered statistically significant. ^aThe total number of AOM episodes was not recorded for 1 otitis-prone child but they fitted the inclusion criteria of at least 3 doctor-diagnosed episodes of AOM. ^bDay-care attendance was not recorded for 1 child. ^cViral PCR was not conducted on nasopharyngeal (NP) swabs from 3 cases and 1 control. ^dNP swab was not cultured for 1 control. ^eNo serum IgG data for 2 cases and 1 control.</p