Nts from Mabtech, Sweden except recombinant IFN-c which was obtained from National Biological Standards Board), according to the manufacturer’s instructions. The limit of detection was 8 pg/ml for IL-4, 3 pg/ml for IL-10 and 20 pg/ml for IFN-c.Kruskal-Wallis ANOVA was used to investigate differences cytokine numbers with regards to co-colonization with the different species investigated and also for the bacterial supernatant stimulated PBMCs. If significant, Mann Whitney U-test was used to investigate which groups differed. Only Salmon calcitonin web results where trends and differences were consistent over time are reported.Results StatisticsTo assess differences in cytokine secreting cells, among colonized and non-colonized infants, Mann Whitney U-test was used. Further, to investigate correlations between cytokine secreting cells and the relative amounts of the bacterial species, Spearman Rank Correlation test was employed. Additionally,Lactobacilli-, bifidobacteria- and S. aureus colonization during the first months of lifeInfant colonization during the first two months of life, was determined using RT-PCR with specific primer pairs and is shown in Table 1. Out of the species investigated, the Lactobacillus groupFigure 2. S. aureus colonization at 2 weeks of age in relation to cytokine secreting cells, after in vitro PHA stimulation at age two. Infants with (n = 19) or without (n = 8) S. aureus at 2 weeks of age in relation to (A) IL-42 and (B) IL-10 producing cells after PHA stimulation. Boxes cover 25th to 75th percentile and the central square being the median value. Whiskers extend to non-outlier maximum and minimum, squares and stars represents outliers and extremes, respectively. doi:10.1371/journal.pone.0049315.gEarly Gut Bacteria and Cytokine Responses at TwoFigure 3. Co-colonization of lactobacilli and S. aureus at 2 weeks and cytokine secreting cells, after in vitro PHA stimulation at age two. Infants colonized with both S. aureus and lactobacilli (n = 7), none (n = 6), only S. aureus (n = 12) or only lactobacilli (n = 2) at 2 weeks of age in relation to (A) IL-42, (B) IL-102, and (C) IFN-c producing cells. Triangles represent each individual and lines represent median value. doi:10.1371/journal.pone.0049315.gconsisting of L. casei, L. paracasei, and L. rhamnosus, was the least detected one, found only in 21,4 of the fecal samples and S. aureus was the most frequently detected species found in 53,6 of the samples the first week of life. The lactobacilli frequency increased during the first two months of age and was found in 50 of the fecal samples at two-month olds, as did S.aureus frequency, which increased, to approximately 70 . Of the bifidobacteria investigated, B. adolescentis was the most common at 15900046 one week of age, detected in 39,3 of the infants, whereas B. bifidum was the most frequently detected bacterium at two months of age. The bifidobacteria frequencies remained DprE1-IN-2 web stable throughout the two months, except B. bifidum frequency, which increased to 53,4 at the age of two months. B. breve was detected in approximately 25?0 of the infants throughout the first two months after birth.C). Similarly, lactobacilli colonization at one month tended to associate with lower numbers of IL-4 (p = 0.065) and IL-10 (p = 0.075), whereas lactobacilli colonization at the other time points investigated did not associate with lower numbers of cytokine-producing cells. Moreover, the relative amounts of lactobacilli at two weeks (p = 0.042), one month (.Nts from Mabtech, Sweden except recombinant IFN-c which was obtained from National Biological Standards Board), according to the manufacturer’s instructions. The limit of detection was 8 pg/ml for IL-4, 3 pg/ml for IL-10 and 20 pg/ml for IFN-c.Kruskal-Wallis ANOVA was used to investigate differences cytokine numbers with regards to co-colonization with the different species investigated and also for the bacterial supernatant stimulated PBMCs. If significant, Mann Whitney U-test was used to investigate which groups differed. Only results where trends and differences were consistent over time are reported.Results StatisticsTo assess differences in cytokine secreting cells, among colonized and non-colonized infants, Mann Whitney U-test was used. Further, to investigate correlations between cytokine secreting cells and the relative amounts of the bacterial species, Spearman Rank Correlation test was employed. Additionally,Lactobacilli-, bifidobacteria- and S. aureus colonization during the first months of lifeInfant colonization during the first two months of life, was determined using RT-PCR with specific primer pairs and is shown in Table 1. Out of the species investigated, the Lactobacillus groupFigure 2. S. aureus colonization at 2 weeks of age in relation to cytokine secreting cells, after in vitro PHA stimulation at age two. Infants with (n = 19) or without (n = 8) S. aureus at 2 weeks of age in relation to (A) IL-42 and (B) IL-10 producing cells after PHA stimulation. Boxes cover 25th to 75th percentile and the central square being the median value. Whiskers extend to non-outlier maximum and minimum, squares and stars represents outliers and extremes, respectively. doi:10.1371/journal.pone.0049315.gEarly Gut Bacteria and Cytokine Responses at TwoFigure 3. Co-colonization of lactobacilli and S. aureus at 2 weeks and cytokine secreting cells, after in vitro PHA stimulation at age two. Infants colonized with both S. aureus and lactobacilli (n = 7), none (n = 6), only S. aureus (n = 12) or only lactobacilli (n = 2) at 2 weeks of age in relation to (A) IL-42, (B) IL-102, and (C) IFN-c producing cells. Triangles represent each individual and lines represent median value. doi:10.1371/journal.pone.0049315.gconsisting of L. casei, L. paracasei, and L. rhamnosus, was the least detected one, found only in 21,4 of the fecal samples and S. aureus was the most frequently detected species found in 53,6 of the samples the first week of life. The lactobacilli frequency increased during the first two months of age and was found in 50 of the fecal samples at two-month olds, as did S.aureus frequency, which increased, to approximately 70 . Of the bifidobacteria investigated, B. adolescentis was the most common at 15900046 one week of age, detected in 39,3 of the infants, whereas B. bifidum was the most frequently detected bacterium at two months of age. The bifidobacteria frequencies remained stable throughout the two months, except B. bifidum frequency, which increased to 53,4 at the age of two months. B. breve was detected in approximately 25?0 of the infants throughout the first two months after birth.C). Similarly, lactobacilli colonization at one month tended to associate with lower numbers of IL-4 (p = 0.065) and IL-10 (p = 0.075), whereas lactobacilli colonization at the other time points investigated did not associate with lower numbers of cytokine-producing cells. Moreover, the relative amounts of lactobacilli at two weeks (p = 0.042), one month (.
