Demographic characteristics at enrollment and features of <i>P. falciparum</i> clinical malaria episodes of <i>Schistosoma haematobium</i>-positive and age-matched <i>S. haematobium</i>-negative Malian children contributing PBMC for immunologic analysis.^a

a<p>Urinary egg excretion detected in 10 ml of filtered morning urine.</p>b<p>Results for children who did not develop malaria (n = 14) are not included these calculations. If no statistical difference was noted between children in the 4–8 year old category compared to the 9–14 year old category, the results were combined.</p>c<p>Multivariable Cox regression analysis used controlling for age and schistosoma status.</p>d<p>Geometric mean parasite density per mm³.</p

Memory B cell (MBC) expressed as the mean number of <i>Schistosoma haematobium</i>-specific SFC to soluble egg antigen (SEA) or soluble worm antigen protein (SWAP) compared to total IgG SFC measured in children with (SP) or without (SN) <i>S. haematobium</i> infection and stratified by age group and season (i.e., malaria transmission and dry season).

a<p>Statistical analysis performed between transmission and dry season values using the paired two-tailed ttest. P value deemed statistically significant ≤0.05.</p>b<p>Statistical significance measured between SP and SN values using the Mann Whitney test for values not normally distributed (P value ≤0.05).</p>c<p>Three of fifteen SN children had a positive response (ASC >0.01) to SEA. One child was subsequently determined to have acquired a <i>S. haematobium</i> infection in the interim since screening. All other responders to SWAP or SEA had repeatedly negative urine analyses for egg excretion.</p><p>ASC ratios of >0.01 were defined as positive specific MBC responses. Mean age-stratified group values >0.01 are depicted in bold font.</p

B memory cell responders to <i>S. haematobium</i> antigens.

<p>Shown are the percentages of B memory cell responders to <i>S. haematobium</i> antigens, SEA and SWAP, as determined by ELISpot assay during the malaria transmission season and again during the dry season with significant differences demarcated. Results were stratified by age (A–C) and <i>S. haematobium</i> status [<i>S. haematobium</i> positive (SP) = 54 and <i>S. haematobium</i> negative (SN) = 29] Responders are defined to those individuals with antigen specific cell (ASC) ratios (ratio of spot forming cells (SFC)/10⁶ expanded cells vs. total IgG SFC/10⁶ cells) greater than 0.01.</p

Intracellular cytokine production to malaria and schistosoma antigens after T regulatory cell depletion.

<p>Representative example of intracellular cytokine detection in PBMC, from a child with concomitant <i>S. haematobium</i> and <i>P. falciparum</i> infection, mock or anti-CD25^hi depleted prior to stimulation with malaria or schistosoma antigenic pools. Removal of regulatory T cells appears to reverse suppressed immunologic responses resulting in enhanced intracellular IL-2 and IFN-γ in the anti-CD25hi depleted cell population. Media controls are included for comparison.</p

Geometric mean antibody titers (OD 450 nm) stratified by antigen, season and schistosoma status in children with (SP) and without (SN) <i>S. haematobium</i>.

a<p>Statistical analysis performed between children with and without <i>S. haematobium</i> using the Mann Whitney test. P value deemed statistically significant ≤0.05. Statistical analysis between transmission and dry season values is not shown but no statistical significance was noted.</p>b<p>95% Confidence Interval of Spearman rank correlation test.</p>c<p>Ab refers to geometric mean antibody and age refers to age-in-months. Spearman rank correlation (ρ) is depicted.</p>d<p>Geometric mean antibody titers are reported for negative controls (10 U.S. malaria naïve adult sera) and positive controls (10 pooled Malian adult sera).</p><p>Also depicted are geometric mean negative and positive control values for each antigen. Spearman rank correlation (ρ) testing and 95% confidence interval depicting strength of association between antibody tested and MBC results as well as antibody tested and the child’s age in months.</p

B memory cell responders to <i>P. falciparum</i> antigens.

<p>Shown are the percentages of B memory cell responders to <i>P. falciparum</i> antigens, AMA1 and MSP1, as determined by ELISpot assay during the malaria transmission season and again during the dry season with significant differences demarcated. Results were stratified by age (A–C) and <i>S. haematobium</i> status [<i>S. haematobium</i> positive (SP) = 54 and <i>S. haematobium</i> negative (SN) = 29] Responders are defined as those individuals with antigen specific cell (ASC) ratios (ratio of spot forming cells (SFC)/10⁶ expanded cells vs. total IgG SFC/10⁶ cells) greater than 0.01.</p

Proliferative responses to malaria and schistosoma antigens.

<p>Shown are the geometric mean lymphocyte proliferative responses as determined by [³H]-thymidine incorporation to malaria and schistosoma antigen pool stimulation (4a and b) in PBMC collected during the malaria transmission season and again in the dry season in children with (SP Mal, n = 20) and children without (SN Mal, n = 17) <i>S. haematobium</i>. Responders refer to those individuals with significant proliferative responses to malaria antigen stimulation (n = 8 SP Mal and n = 6 SN Mal, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031647#pone-0031647-g004" target="_blank">Figure 4a</a>) or to schistosoma antigen stimulation during the transmission season (n = 13 SP Mal and n = 1 SN Mal, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031647#pone-0031647-g004" target="_blank">Figure 4b</a>), with their subsequent dry season values depicted. The dotted line depicts the minimally significant SI of 2.0. The asterix depicts significant differences between SP Mal and SN Mal (P<0.001). No differences were noted between mock or anti-CD25^hi depletion experiments (not shown).</p

Memory B cell (MBC) expressed as the mean number of malaria antigen specific SFC cells to apical membrane antigen (AMA1) or merozoite surface protein 1 (MSP1) compared to total IgG SFC (i.e., ASC ratio) measured in children with (SP) or without (SN) <i>S. haematobium</i> infection and stratified by age group and season (i.e., malaria transmission and dry season).

a<p>A total of 53 SP (26 aged 4–8 years, 27 aged 9–14 years) and 29 SN (14 aged 4–8 years, 15 aged 9–14 years) were examined in both the transmission and dry season (Note: Sample for 1 SP and 1 SN were excluded from the wet and from the dry season for a total of 4 samples).</p>b<p>Statistical analysis performed between transmission and dry season values using paired ttest (two-tailed). P value deemed statistically significant ≤0.05.</p>c<p>Statistical significance measured between SP and SN values using the Mann Whitney test for values not normally distributed (P value ≤0.05).</p><p>ASC ratios ≥0.01 were defined as a positive specific MBC responses. Mean age-stratified group values ≥0.01 are depicted in bold font.</p

CD25^hi Depletion Assay Results.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031647#s3" target="_blank">Results</a> of enhanced cytokine expression to antigenic stimulation after CD25^hi depletion of PBMC followed by antigenic stimulation in comparison to mock-depleted populations. The numerator represents the number of children with enhanced Interleukin 2 (IL-2) or Interferon-gamma (IFN-γ) expression after depletion compared to mock-depletion. The denominator is the number of children examined. The net percentage of cytokine expressed in CD3⁺CD4⁺CD8⁻ T cells (i.e., depletion results minus mock-depletion results) after depletion as measured by multiparameter flow cytometry is also depicted in those experiments with significant cytokine expression increase. Cells were acquired from age-matched children aged 4–14 years with or without <i>S. haematobium</i> during the malaria transmission (wet) season and again during a dry season follow-up visit. Also depicted are results from SP children who did not acquire malaria (SP no Mal) during the wet season.</p>a<p>Stratification between younger children aged 4–8 years and older children aged 9–14 years revealed no significant difference so results are reported as a combined age cohort of children aged 4–14 years.</p>b<p>PBMC were stimulated with a <i>P. falciparum</i> antigen pool (Apical Membrane antigen 1 and Merozoite Surface Protein 1) or with a <i>S. haematobium</i> antigen pool (soluble egg antigen and soluble worm antigen protein).</p>c<p>The mean percentage increase represents the average of the net increase in cytokine production (stimulant wells minus media wells) of those experiments with statistically significant results. Significance is defined by 1) net percentage of cytokine producing cells ≥0.05% in CD25hi-depleted compared to mock-depleted PBMC, 2) the number of positive events between depleted and mock-depleted cultures was significant by Chi-square analysis and 3) the number of positive events in the stimulant pool compared to the media control in depleted cultures was significantly increased.</p

Intracellular Cytokine Expression to Antigen Stimulation.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031647#s3" target="_blank">Results</a> of intracellular cytokine expression after antigenic stimulation of PBMC acquired from age-matched children with (SP Mal) or without <i>S. haematobium</i> (SN Mal) during the malaria transmission (wet) season at the time of a malaria episode and again during a dry season follow-up visit. The numerator results represent those individuals with statistically significant increases in the percentage of cytokine expressed compared to media controls after PBMC stimulation and the denomenator represents the total number of individual PBMC samples analyzed.</p>a<p>PBMC were stimulated with a malaria antigen pool (Apical Membrane antigen 1 and Merozoite Surface Protein 1) or with a <i>S. haematobium</i> pool (soluble egg antigen and soluble worm antigen protein). SP no Mal children were not included in this analysis.</p>b<p>χ² analysis, using Mantel-Haenszel or Fisher Exact (two-tailed) as appropriate, was performed between <i>S. haematobium</i> positive (SP Mal) vs. <i>S. haematobium</i> negative (SN Mal) children in each age category. P value significance set at <0.05. Not significant  =  ns.</p>c<p>Three experiments were excluded due to insufficient or poor viability cell quantities to allow proper interpretation of data.</p>d<p>IL-2 and IFN-g production was noted in one SN individual. Urine was negative for eggs both at enrollment and at dry season follow-up suggesting a false positive result.</p