Genera within the Clostridium cluster XIVa.

<p>The error bar shows the standard error.</p

Intestinal microbiota diversity in healthy young children and adults estimated by Simpson reciprocal index (1/D) and Shannon index.

<p>The whiskers show the highest and lowest value after excluding outliers (dots). The outliers are defined as more than 3/2 times of upper quartile or less than 3/2 times of lower quartile. Boxplot shows 25^th and 75^th percentile, with a line at median. The subjects are divided into two groups Adults (>21Y) and Children (<4Y) (A, C) or four groups‘<2Y’, ‘2-3Y’, ‘3-4Y’ and Adults (>21Y) (B, D). A) Simpson index of diversity shows a significant difference between adults and children (p = 0.007), B) Simpson index of diversity shows significant differences between ‘<2Y’and ‘3-4Y’ age groups and adults (*p = 0.006, **p = 0.002 respectively). C) Shannon index of diversity shows significant difference between adults and children (p = 0.001), D) Shannon index of diversity shows significant difference between ‘<2Y’, ‘3-4Y’ and adults, (*p = 0.003, **p = 0.001 respectively). Significant difference is also observed between ‘2-3Y’and ‘3-4Y’ age groups (***p = 0.043).</p

Principal component analysis (PCA) (A) and redundancy analysis (RDA) (B) of fecal samples from healthy young children and adults at the genus-like level.

<p>Log transformed data were used for analysis. In PCA, the first two principal components capture 33% (PCA1) and 11% (PCA2) of variance respectively. RDA plot shows the result from supervised PCA, where group assignment of subjects (adults or children) was used as a dependent variable. In RDA, first and second ordination axes are plotted, explaing 13% and 64% of the variance. Separation between children and adults is significant (p = 0.003, permutation test).</p

Phylum-level distribution of bacteria in fecal samples of healthy young children and adults.

<p>Phylum-level distribution of bacteria in fecal samples of healthy young children and adults.</p

Comparison of bacterial groups showing significant differences in fecal samples from adults and healthy young children.^*

*<p>Table includes groups at the genus-like level that constitute 0.01% or more of the total signal (total microbiota) in both adults and</p><p>young children, and show significant difference between the study groups.</p>A<p>Relative contribution of genus-like phylogenetic group is the average of relative abundances of the subjects in adults or children groups. </p>B<p>Ratio of the average relative abundance for each genus-like phylogenetic group. A – adults and C – children.</p>C<p>Adjusted p values from two sample t-test followed with the correction by Benjamini-Hochberg false discovery rate correction (p<0.05 significant).</p

Principal component analysis (PCA) (A) and redundancy analysis (RDA) (B) of fecal samples from healthy young children and adults at the phylum-like level.

<p>Log transformed data were used for analysis. In PCA, The first two principal components capture 21% (PCA1) and 16% (PCA2) of variance respectively. RDA plot shows the result from supervised PCA, where group assignment of subjects (adults or children) was used as a dependent variable. In RDA, first and second ordination axes are plotted, explaining 13% and 20% of the variance. Separation between children and adults is significant (p = 0.0002, permutation test).</p

Induction of cytokine production in human moDCs by recombinant SpaCBA-piliated lactococcal cells.

<p>Stimulation of TNF-α, IL-12, IL-10, and IL-6 cytokine production (A, B, C, and D, respectively) in human moDCs by normalized cultures of recombinant SpaCBA-piliated lactococcal cells (GRS1185, GRS1195, GRS1211, and GRS1223) was carried out according to the method described in Materials and Methods. Cells from GRS71 and GRS1052 <i>L. lactis</i> were included as negative controls. RPMI cell culture media and <i>E. coli</i> lipopolysaccharide (LPS; 1 µg/ml) were also included as controls. Measurements were carried out in quadruplicate and each experiment was done three times with moDCs from four different donors, with the data used being the most representative of the experiments. Error bars indicate standard error of mean (SEM).</p

PCR oligonucleotide primers used for lactococcal cloning.

<p>Sequences for <i>Nco</i>I (1984) and <i>Xho</i>I (1987) restriction endonucleases used to facilitate cloning are underlined</p

TLR2-mediated immune responses induced by recombinant SpaCBA-piliated lactococcal cells.

<p>Stimulation of TLR2-dependent NF-κB activation in the HEK-TLR2 cell line by normalized cultures of recombinant SpaCBA-piliated lactococcal cells (GRS1185, GRS1195, GRS1211, and GRS1223) was carried out according to the method described in Materials and Methods. GRS71 and GRS1052 lactococcal cells as well as DMEM cell culture media were included as negative controls. TLR2-agonist lipopeptide Pam3CSK4 (1 ng/ml) was used a positive control. Measurements were done in quadruplicate and the experiment was performed three times. Error bars are standard error of mean (SEM). Differences between data (GRS1185, GRS1195, GRS1211, or GRS1223) from pairwise comparisons against GRS71 data are considered extremely significant (<i>P</i><0.001).</p

Effect of heat-treated WT SpaCBA-piliated lactococcal cells on TLR2-dependent immune activity.

<p>Stimulation of TLR2-dependent NF-κB activation (A) and IL-8 cytokine production (B) in the HEK-TLR2 cell line by either live (-) or heat-treated (100°C for 10 minutes) (+) WT SpaCBA-piliated lactococcal cells (GRS1185) was performed as described in Materials and Methods. Live and heat-treated GRS1052 lactococcal cells were also tested. DMEM cell culture media, Pam3CSK4 (1 ng/ml), and <i>E. coli</i> lipopolysaccharide (LPS; 1 ng/ml) were included as controls. Quadruplicate and duplicate measurements for NF-κB activation and IL-8 production, respectively, were performed. Error bars indicate standard error of mean (SEM). Differences between GRS1185 and GRS1052 data are deemed extremely significant (<i>P</i><0.001).</p