Early-Life Gut Bacteria Associate with IL-4?, IL-10? and IFN-γ Production at Two Years of Age

Microbial exposure early in life influences immune maturation and potentially also the development of immune-mediated disease. Here we studied early-life gut colonization in relation to cytokine responses at two years of age. Fecal samples were collected from infants during the first two months of life. DNA was extracted from the fecal samples and Bifidobacterium (B.) adolescentis, B. breve, B. bifidum, a group of lactobacilli (L. casei, L. paracasei and L. rhamnosus) as well as Staphylococcus (S.) aureus were detected with real time PCR. Peripheral mononuclear cells were stimulated with phytohaemagglutinin (PHA) and numbers of IL-4−, IL-10− and IFN-γ secreting cells were evaluated using ELISpot. We further stimulated peripheral blood mononuclear cells with bacterial supernatants in vitro and assessed the IL-4−, IL-10− and IFN-γ inducing capacity by flow cytometry and ELISA. Early S. aureus colonization associated with higher numbers of IL-4− (p = 0.022) and IL-10 (p = 0.016) producing cells at two years of age. In contrast to colonization with S. aureus alone, co-colonization with lactobacilli associated with suppression of IL-4− (p = 0.004), IL-10− (p = 0.004) and IFN-γ (p = 0.034) secreting cells. In vitro stimulations of mononuclear cells with bacterial supernatants supported a suppressive role of L. rhamnosus GG on S. aureus-induced cytokine responses. We demonstrate that the early gut colonization pattern associates with the PHA-induced cytokine profile at two years of age and our in vitro findings support that specific bacterial species influence the T helper cell subsets. This suggests that dysbiosis in the early microbiota may modulate the risk of developing inflammatory conditions like allergy.     

Fecal Calprotectin Excretion in Preterm Infants during the Neonatal Period

Background

Fecal calprotectin has been proposed as a non-invasive marker of intestinal inflammation in inflammatory bowel disease in adults and children. Fecal calprotectin levels have been reported to be much higher in both healthy full-term and preterm infants than in children and adults.

Objective

To determine the time course of fecal calprotectin (f-calprotectin) excretion in preterm infants from birth until hospital discharge and to identify factors influencing f-calprotectin levels in the first weeks of life, including bacterial establishment in the gut.

Methodology

F-calprotectin was determined using an ELISA assay in 147 samples obtained prospectively from 47 preterm infants (gestational age, and birth-weight interquartiles 27–29 weeks, and 880–1320 g, respectively) at birth, and at 2-week intervals until hospital discharge.

Principal Findings

Although median f-calprotectin excretion was 138 µg/g, a wide range of inter- and intra-individual variation in f-calprotectin values (from day 3 to day 78) was observed (86% and 67%, respectively). In multivariate regression analysis, f-calprotectin correlated negatively with ante and per natal antibiotic treatment (p = 0.001), and correlated positively with the volume of enteral feeding (mL/kg/d) (p = 0.009), the need to interrupt enteral feeding (p = 0.001), and prominent gastrointestinal colonization by Clostridium sp (p = 0.019) and Staphylococcus sp (p = 0.047).

Conclusion

During the first weeks of life, the high f-calprotectin values observed in preterm infants could be linked to the gut bacterial establishment.     

Breast Milk and Gut Microbiota in African Mothers and Infants from an Area of High HIV Prevalence

Background

Human milk and infant gut microbiota are essential for the immune system maturation and protection against infections. There is scarce information on the microbiological composition of breast milk in general, and none from developing countries. The objective of the study was to characterize the breast milk and gut microbiota from mothers and infants from southern Mozambique, where infections and breastfeeding are prevalent.

Methods

A community-based study was undertaken among 121 pairs of women and infants. Breast milk and infant''s faeces were analyzed by bacterial culture and molecular methods. Breast milk samples were screened for HIV RNA by RT-PCR.

Results

The most frequent bacterial groups isolated by culture media in breast milk were Staphylococci (96.4%), Streptococci (92.7%) and Lactobacilli (56.4%). HIV RNA was detected in 24% of the samples. Staphylococcus hominis, S. aureus, and S.parasanguis were more frequently isolated in infants ≤14 days of life. Women on exclusive breastfeeding presented higher proportion of S. parasanguis in breast milk than those on mixed infant feeding (36.4% versus 11.1%, p = 0.035). Bacterial diversity (mean number of bacterial species isolated by sample: 10.4 versus 8.5; p = 0.004) and the frequency of Lactobacillus spp (75.9% versus 36%, p = 0.003) were higher in the specimens with HIV RNA than in those without it. The main bacterial groups found in infant''s faeces were Bifidobacterium, Streptococci and Enterococci.

Conclusions

Women with HIV RNA in breast milk had a different pattern of microbiological composition, suggesting specific immunopathological phenomena in HIV-infected women. Both breast milk and faecal microbiota composition varied with lactation period, which might be related to changes in the type of feeding over time and/or in the milk''s biochemical characteristics. These findings provide insights into interactions between commensal bacteria and HIV infection in human milk and the role of these bacteria in mucosal protection against infections in breastfed infants.     

Short-Term Effect of Antibiotics on Human Gut Microbiota

From birth onwards, the human gut microbiota rapidly increases in diversity and reaches an adult-like stage at three years of age. After this age, the composition may fluctuate in response to external factors such as antibiotics. Previous studies have shown that resilience is not complete months after cessation of the antibiotic intake. However, little is known about the short-term effects of antibiotic intake on the gut microbial community. Here we examined the load and composition of the fecal microbiota immediately after treatment in 21 patients, who received broad-spectrum antibiotics such as fluoroquinolones and β-lactams. A fecal sample was collected from all participants before treatment and one week after for microbial load and community composition analyses by quantitative PCR and pyrosequencing of the 16S rRNA gene, respectively. Fluoroquinolones and β-lactams significantly decreased microbial diversity by 25% and reduced the core phylogenetic microbiota from 29 to 12 taxa. However, at the phylum level, these antibiotics increased the Bacteroidetes/Firmicutes ratio (p = 0.0007, FDR = 0.002). At the species level, our findings unexpectedly revealed that both antibiotic types increased the proportion of several unknown taxa belonging to the Bacteroides genus, a Gram-negative group of bacteria (p = 0.0003, FDR<0.016). Furthermore, the average microbial load was affected by the treatment. Indeed, the β-lactams increased it significantly by two-fold (p = 0.04). The maintenance of or possible increase detected in microbial load and the selection of Gram-negative over Gram-positive bacteria breaks the idea generally held about the effect of broad-spectrum antibiotics on gut microbiota.     

Disruption of the Human Gut Microbiota following Norovirus Infection

The gut microbiota, the collection of all bacterial members in the intestinal tract, plays a key role in health. Disruption of the indigenous microbiota by a variety of stressors, including antibiotic therapy and intestinal infections, is associated with multiple health problems. We sought to determine if infection with Norovirus disrupts the gut microbiota. Barcoded pyrosequencing of the 16S rRNA-encoding gene was used to characterize the stool microbiota in Norovirus-infected human patients (n = 38). While the microbiota in most infected patients (n = 31) resembled that seen in uninfected healthy controls, a minority of patients (n = 7) possessed a significantly altered microbiota characterized by reduced relative numbers of Bacteriodetes and a corresponding increase in Proteobacteria. In these patients, the increase in Proteobacteria was due to a single operational taxonomic unit (OTU) of Escherichia coli. We cultured E. coli from Norovirus-infected patients and characterized them using PCR-ribotyping and virulence factor analysis. Multiple ribotypes were encountered, but none possessed typical virulence factors commonly carried by enteropathogenic E. coli strains. Microbiota disruption and elevated Proteobacteria were not significantly correlated to patient age, gender, sampling time following illness onset, or overall gut inflammation. These results demonstrate that some patients have a disrupted microbiota following Norovirus infection, and therefore may be at elevated risk for long-term health complications.     

A Possible Link between Food and Mood: Dietary Impact on Gut Microbiota and Behavior in BALB/c Mice

Major depressive disorder is a debilitating disease in the Western World. A western diet high in saturated fat and refined sugar seems to play an important part in disease development. Therefore, this study is aimed at investigating whether saturated fat or sucrose predisposes mice to develop behavioral symptoms which can be interpreted as depression-like, and the possible influence of the gut microbiota (GM) in this. Fourty-two mice were randomly assigned to one of three experimental diets, a high-fat, a high-sucrose or a control diet for thirteen weeks. Mice on high-fat diet gained more weight (p = 0.00009), displayed significantly less burrowing behavior than the control mice (p = 0.034), and showed decreased memory in the Morris water maze test compared to mice on high-sucrose diet (p = 0.031). Mice on high-sucrose diet burrowed less goal-oriented, showed greater latency to first bout of immobility in the forced swim test when compared to control mice (p = 0.039) and high-fat fed mice (p = 0.013), and displayed less anxiety than mice on high-fat diet in the triple test (p = 0.009). Behavioral changes were accompanied by a significant change in GM composition of mice fed a high-fat diet, while no difference between diet groups was observed for sucrose preferences, LPS, cholesterol, HbA1c, BDNF and the cytokines IL-1α, IL-1β, IL-6, IL-10, IL-12(p70), IL-17 and TNF-α. A series of correlations was found between GM, behavior, BDNF and inflammatory mediators. In conclusion, the study shows that dietary fat and sucrose affect behavior, sometimes in opposite directions, and suggests a possible association between GM and behavior.     

Comparison of Lower Genital Tract Microbiota in HIV-Infected and Uninfected Women from Rwanda and the US

Introduction

Previous studies have shown that alterations of the bacterial microbiota in the lower female genital tract influence susceptibility to HIV infection and shedding. We assessed geographic differences in types of genital microbiota between HIV-infected and uninfected women from Rwanda and the United States.

Methods

Genera of lower genital tract bacterial microbiota were identified by high-throughput pyrosequencing of the 16S rRNA gene from 46 US women (36 HIV-infected, 10 HIV-uninfected) and 40 Rwandan women (18 HIV-infected, 22 HIV-uninfected) with similar proportions of low (0–3) Nugent scores. Species of Lactobacillus were identified by assembling sequences along with reference sequences into phylogenetic trees. Prevalence of genera and Lactobacillus species were compared using Fisher''s exact tests.

Results

Overall the seven most prevalent genera were Lactobacillus (74%), Prevotella (56%), Gardnerella (55%), Atopobium (42%), Sneathia (37%), Megasphaera (30%), and Parvimonas (26%), observed at similar prevalences comparing Rwandan to US women, except for Megasphaera (20% vs. 39%, p = 0.06). Additionally, Rwandan women had higher frequencies of Mycoplasma (23% vs. 7%, p = 0.06) and Eggerthella (13% vs. 0%, p = 0.02), and lower frequencies of Lachnobacterium (8% vs. 35%, p<0.01) and Allisonella (5% vs. 30%, p<0.01), compared with US women. The prevalence of Mycoplasma was highest (p<0.05) in HIV-infected Rwandan women (39%), compared to HIV-infected US women (6%), HIV-uninfected Rwandan (9%) and US (10%) women. The most prevalent lactobacillus species in both Rwandan and US women was L. iners (58% vs. 76%, p = 0.11), followed by L. crispatus (28% vs. 30%, p = 0.82), L. jensenii (20% vs. 24%, p = 0.80), L. gasseri (20% vs. 11%, p = 0.37) and L. vaginalis (20% vs. 7%, p = 0.10).

Discussion

We found similar prevalence of most major bacterial genera and Lactobacillus species in Rwandan and US women. Further work will be needed to establish whether observed differences differentially impact lower genital tract health or susceptibility to genital infections.     

A Complex Interplay between Personality Domains,Marital Status and a Variant in CHRNA5 on the Risks of Cocaine,Nicotine Dependences and Cocaine-Induced Paranoia

Background

Personality correlates highly with both cocaine and nicotine dependencies (CD, ND), and their co-morbid psychopathologies. However, little is known about the nature of these relationships. This study examined if environment (marriage) or genetics (a single SNP, CHRNA5*rs16969968) would moderate the correlation of personality with CD, ND and cocaine-induced paranoia (CIP) in African and European Americans (AAs, EAs).

Methods

1432 EAs and 1513 AAs were examined using logistic regression. Personality was assessed by NEO-PI-R, while CD, ND and CIP were diagnosed according to DSM-IV. ND and CD were examined as binary traits and for the analysis of CIP, subjects were divided into 3 groups: (A) Controls with no CIP; (B) CD cases without CIP; and (C) CD cases with CIP. Multiple testing was Bonferroni-corrected.

Results

For CD and ND in the EA population, marital status proved to be a significant moderator in their relationship with openness only (OR = 1.90, 95%CI = 1.36–2.64, p = 1.54e-04 and OR = 2.12, 95%CI = 1.52–2.90, p = 4.65e-06 respectively). For CIP, marriage was observed to moderate its correlation with openness and neuroticism (OR = 1.39, 95%CI = 1.18–1.63, p = 7.64e-04 and OR = 1.26, 95%CI = 1.12–1.42, p = 1.27e-03 respectively). The correlations moderated by rs16969968 were those of conscientiousness and CD (OR = 1.62, 95%CI: 1.23–2.12, p = 8.94e-04) as well as CIP (OR = 1.21, 95%CI: 1.11–1.32, p = 4.93e-04 when comparing group A versus group C). No significant interactions were observed in AA population. The Bonferroni-corrected significance threshold was set to be 1.67e-03.

Conclusion

The role of personality in CD and CIP may be interceded by both environment and genetics, while in ND by environment only.     

Culture-Independent Evaluation of the Appendix and Rectum Microbiomes in Children with and without Appendicitis

Purpose

The function of the appendix is largely unknown, but its microbiota likely contributes to function. Alterations in microbiota may contribute to appendicitis, but conventional culture studies have not yielded conclusive information. We conducted a pilot, culture-independent 16S rRNA-based microbiota study of paired appendix and rectal samples.

Methods

We collected appendix and rectal swabs from 21 children undergoing appendectomy, six with normal appendices and fifteen with appendicitis (nine perforated). After DNA extraction, we amplified and sequenced 16S rRNA genes and analyzed sequences using CLoVR. We identified organisms differing in relative abundance using ANOVA (p<0.05) by location (appendix vs. rectum), disease (appendicitis vs. normal), and disease severity (perforated vs. non-perforated).

Results

We identified 290 taxa in the study''s samples. Three taxa were significantly increased in normal appendices vs. normal rectal samples: Fusibacter (p = 0.009), Selenomonas (p = 0.026), and Peptostreptococcus (p = 0.049). Five taxa were increased in abundance in normal vs. diseased appendices: Paenibacillaceae (p = 0.005), Acidobacteriaceae GP4 (p = 0.019), Pseudonocardinae (p = 0.019), Bergeyella (p = 0.019) and Rhizobium (p = 0.045). Twelve taxa were increased in the appendices of appendicitis patients vs. normal appendix: Peptostreptococcus (p = 0.0003), Bilophila (p = 0.0004), Bulleidia (p = 0.012), Fusobacterium (p = 0.018), Parvimonas (p = 0.003), Mogibacterium (p = 0.012), Aminobacterium (p = 0.019), Proteus (p = 0.028), Actinomycineae (p = 0.028), Anaerovorax (p = 0.041), Anaerofilum (p = 0.045), Porphyromonas (p = 0.010). Five taxa were increased in appendices in patients with perforated vs. nonperforated appendicitis: Bulleidia (p = 0.004), Fusibacter (p = 0.005), Prevotella (p = 0.021), Porphyromonas (p = 0.030), Dialister (p = 0.035). Three taxa were increased in rectum samples of patients with appendicitis compared to the normal patients: Bulleidia (p = 0.034), Dialister (p = 0.003), and Porphyromonas (p = 0.026).

Conclusion

Specific taxa are more abundant in normal appendices compared to the rectum, suggesting that a distinctive appendix microbiota exists. Taxa with altered abundance in diseased and severely diseased (perforated) samples may contribute to appendicitis pathogenesis, and may provide microbial signatures in the rectum useful for guiding both treatment and diagnosis of appendicitis.     

Human Gut Microbiota Changes Reveal the Progression of Glucose Intolerance

To explore the relationship of gut microbiota with the development of type 2 diabetes (T2DM), we analyzed 121 subjects who were divided into 3 groups based on their glucose intolerance status: normal glucose tolerance (NGT; n = 44), prediabetes (Pre-DM; n = 64), or newly diagnosed T2DM (n = 13). Gut microbiota characterizations were determined with 16S rDNA-based high-throughput sequencing. T2DM-related dysbiosis was observed, including the separation of microbial communities and a change of alpha diversity between the different glucose intolerance statuses. To assess the correlation between metabolic parameters and microbiota diversity, clinical characteristics were also measured and a significant association between metabolic parameters (FPG, CRP) and gut microbiota was found. In addition, a total of 28 operational taxonomic units (OTUs) were found to be related to T2DM status by the Kruskal-Wallis H test, most of which were enriched in the T2DM group. Butyrate-producing bacteria (e.g. Akkermansia muciniphila ATCCBAA-835, and Faecalibacterium prausnitzii L2-6) had a higher abundance in the NGT group than in the pre-DM group. At genus level, the abundance of Bacteroides in the T2DM group was only half that of the NGT and Pre-DM groups. Previously reported T2DM-related markers were also compared with the data in this study, and some inconsistencies were noted. We found that Verrucomicrobiae may be a potential marker of T2DM as it had a significantly lower abundance in both the pre-DM and T2DM groups. In conclusion, this research provides further evidence of the structural modulation of gut microbiota in the pathogenesis of diabetes.     

Dietary Patterns Differently Associate with Inflammation and Gut Microbiota in Overweight and Obese Subjects

Background

Associations between dietary patterns, metabolic and inflammatory markers and gut microbiota are yet to be elucidated.

Objectives

We aimed to characterize dietary patterns in overweight and obese subjects and evaluate the different dietary patterns in relation to metabolic and inflammatory variables as well as gut microbiota.

Design

Dietary patterns, plasma and adipose tissue markers, and gut microbiota were evaluated in a group of 45 overweight and obese subjects (6 men and 39 women). A group of 14 lean subjects were also evaluated as a reference group.

Results

Three clusters of dietary patterns were identified in overweight/obese subjects. Cluster 1 had the least healthy eating behavior (highest consumption of potatoes, confectionary and sugary drinks, and the lowest consumption of fruits that was associated also with low consumption of yogurt, and water). This dietary pattern was associated with the highest LDL cholesterol, plasma soluble CD14 (p = 0.01) a marker of systemic inflammation but the lowest accumulation of CD163+ macrophages with anti-inflammatory profile in adipose tissue (p = 0.05). Cluster 3 had the healthiest eating behavior (lower consumption of confectionary and sugary drinks, and highest consumption of fruits but also yogurts and soups). Subjects in this Cluster had the lowest inflammatory markers (sCD14) and the highest anti-inflammatory adipose tissue CD163+ macrophages. Dietary intakes, insulin sensitivity and some inflammatory markers (plasma IL6) in Cluster 3 were close to those of lean subjects. Cluster 2 was in-between clusters 1 and 3 in terms of healthfulness. The 7 gut microbiota groups measured by qPCR were similar across the clusters. However, the healthiest dietary cluster had the highest microbial gene richness, as evaluated by quantitative metagenomics.

Conclusion

A healthier dietary pattern was associated with lower inflammatory markers as well as greater gut microbiota richness in overweight and obese subjects.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01314690","term_id":"NCT01314690"}}NCT01314690     

Functional Characterization of TLR4 +3725 G/C Polymorphism and Association with Protection against Overweight

Subclinical low-grade systemic inflammation has been associated with obesity, insulin resistance and metabolic syndrome (MS). Recent studies have highlighted the role of gut microbiota in these disorders. The toll-like receptor 4 (TLR4) plays a key role in the innate immune response activation. We studied two polymorphisms (+3725G/C and 11350G/C) in the 3′ untranslated region (3′UTR) of the TLR4 gene that may alter its expression and their association with metabolic disorders related to systemic inflammation. We cloned the 3′UTR into a luciferase reporter system and compared wild-type 3′UTR (WT) and +3725C variant (MUT) constructs luciferase activities. MUT construct reduced the reporter gene activity by 30% compared to WT (P = 0.0001). To evaluate the association between these polymorphisms with biochemical and clinical overweight related variables, we conducted a population cross-sectional study in 966 men of Argentine general population. Considering smoking as a confounding variable that causes systemic inflammation, we studied these possible effects in both, smokers and nonsmokers. The 11350G/C polymorphism was not detected in our sample whereas the CC genotype of +3725 polymorphism was associated with lean subjects (p = 0.011) and higher Adiponectin levels (p = 0.021). Subjects without any NCEP/ATP III MS component were associated with this genotype as well (p = 0.001). These results were strengthened in nonsmokers, in which CC genotype was associated with lean subjects (p = 0.003) and compared with G carriers showed significantly lower BMI (25.53 vs. 28.60 kg/m2; p = 0.023) and waist circumference (89.27 vs. 97.51 cm; p = 0.025). None of these associations were found in smokers. These results showed that +3725C variant has a functional effect down-regulating gene expression and it could be considered as a predictive factor against overweight, particularly in nonsmokers. Considering the role of TLR4 in inflammation, these findings would suggest that the presence of +3725C variant could predict a lower prevalence of chronic metabolic disorders.     

The Proneural Molecular Signature Is Enriched in Oligodendrogliomas and Predicts Improved Survival among Diffuse Gliomas

The Cancer Genome Atlas Project (TCGA) has produced an extensive collection of ‘-omic’ data on glioblastoma (GBM), resulting in several key insights on expression signatures. Despite the richness of TCGA GBM data, the absence of lower grade gliomas in this data set prevents analysis genes related to progression and the uncovering of predictive signatures. A complementary dataset exists in the form of the NCI Repository for Molecular Brain Neoplasia Data (Rembrandt), which contains molecular and clinical data for diffuse gliomas across the full spectrum of histologic class and grade. Here we present an investigation of the significance of the TCGA consortium''s expression classification when applied to Rembrandt gliomas. We demonstrate that the proneural signature predicts improved clinical outcome among 176 Rembrandt gliomas that includes all histologies and grades, including GBMs (log rank test p = 1.16e-6), but also among 75 grade II and grade III samples (p = 2.65e-4). This gene expression signature was enriched in tumors with oligodendroglioma histology and also predicted improved survival in this tumor type (n = 43, p = 1.25e-4). Thus, expression signatures identified in the TCGA analysis of GBMs also have intrinsic prognostic value for lower grade oligodendrogliomas, and likely represent important differences in tumor biology with implications for treatment and therapy. Integrated DNA and RNA analysis of low-grade and high-grade proneural gliomas identified increased expression and gene amplification of several genes including GLIS3, TGFB2, TNC, AURKA, and VEGFA in proneural GBMs, with corresponding loss of DLL3 and HEY2. Pathway analysis highlights the importance of the Notch and Hedgehog pathways in the proneural subtype. This demonstrates that the expression signatures identified in the TCGA analysis of GBMs also have intrinsic prognostic value for low-grade oligodendrogliomas, and likely represent important differences in tumor biology with implications for treatment and therapy.     

Body Size Is a Significant Predictor of Congruency in Species Richness Patterns: A Meta-Analysis of Aquatic Studies

Biodiversity losses over the next century are predicted to result in alterations of ecosystem functions that are on par with other major drivers of global change. Given the seriousness of this issue, there is a need to effectively monitor global biodiversity. Because performing biodiversity censuses of all taxonomic groups is prohibitively costly, indicator groups have been studied to estimate the biodiversity of different taxonomic groups. Quantifying cross-taxon congruence is a method of evaluating the assumption that the diversity of one taxonomic group can be used to predict the diversity of another. To improve the predictive ability of cross-taxon congruence in aquatic ecosystems, we evaluated whether body size, measured as the ratio of average body length between organismal groups, is a significant predictor of their cross-taxon biodiversity congruence. To test this hypothesis, we searched the published literature and screened for studies that used species richness correlations as their metric of cross-taxon congruence. We extracted 96 correlation coefficients from 16 studies, which encompassed 784 inland water bodies. With these correlation coefficients, we conducted a categorical meta-analysis, grouping data based on the body size ratio of organisms. Our results showed that cross-taxon congruence is variable among sites and between different groups (r values ranging between −0.53 to 0.88). In addition, our quantitative meta-analysis demonstrated that organisms most similar in body size showed stronger species richness correlations than organisms which differed increasingly in size (r_adj ² = 0.94, p = 0.02). We propose that future studies applying biodiversity indicators in aquatic ecosystems consider functional traits such as body size, so as to increase their success at predicting the biodiversity of taxonomic groups where cost-effective conservation tools are needed.     

The Microbiota Mediates Pathogen Clearance from the Gut Lumen after Non-Typhoidal Salmonella Diarrhea

Many enteropathogenic bacteria target the mammalian gut. The mechanisms protecting the host from infection are poorly understood. We have studied the protective functions of secretory antibodies (sIgA) and the microbiota, using a mouse model for S. typhimurium diarrhea. This pathogen is a common cause of diarrhea in humans world-wide. S. typhimurium (S. tm ^att, sseD) causes a self-limiting gut infection in streptomycin-treated mice. After 40 days, all animals had overcome the disease, developed a sIgA response, and most had cleared the pathogen from the gut lumen. sIgA limited pathogen access to the mucosal surface and protected from gut inflammation in challenge infections. This protection was O-antigen specific, as demonstrated with pathogens lacking the S. typhimurium O-antigen (wbaP, S. enteritidis) and sIgA-deficient mice (TCRβ^−/−δ^−/−, J_H ^−/−, IgA^−/−, pIgR^−/−). Surprisingly, sIgA-deficiency did not affect the kinetics of pathogen clearance from the gut lumen. Instead, this was mediated by the microbiota. This was confirmed using ‘L-mice’ which harbor a low complexity gut flora, lack colonization resistance and develop a normal sIgA response, but fail to clear S. tm ^att from the gut lumen. In these mice, pathogen clearance was achieved by transferring a normal complex microbiota. Thus, besides colonization resistance ( = pathogen blockage by an intact microbiota), the microbiota mediates a second, novel protective function, i.e. pathogen clearance. Here, the normal microbiota re-grows from a state of depletion and disturbed composition and gradually clears even very high pathogen loads from the gut lumen, a site inaccessible to most “classical” immune effector mechanisms. In conclusion, sIgA and microbiota serve complementary protective functions. The microbiota confers colonization resistance and mediates pathogen clearance in primary infections, while sIgA protects from disease if the host re-encounters the same pathogen. This has implications for curing S. typhimurium diarrhea and for preventing transmission.     

Gut Microbiota in Human Adults with Type 2 Diabetes Differs from Non-Diabetic Adults

Background

Recent evidence suggests that there is a link between metabolic diseases and bacterial populations in the gut. The aim of this study was to assess the differences between the composition of the intestinal microbiota in humans with type 2 diabetes and non-diabetic persons as control.

Methods and Findings

The study included 36 male adults with a broad range of age and body-mass indices (BMIs), among which 18 subjects were diagnosed with diabetes type 2. The fecal bacterial composition was investigated by real-time quantitative PCR (qPCR) and in a subgroup of subjects (N = 20) by tag-encoded amplicon pyrosequencing of the V4 region of the 16S rRNA gene. The proportions of phylum Firmicutes and class Clostridia were significantly reduced in the diabetic group compared to the control group (P = 0.03). Furthermore, the ratios of Bacteroidetes to Firmicutes as well as the ratios of Bacteroides-Prevotella group to C. coccoides-E. rectale group correlated positively and significantly with plasma glucose concentration (P = 0.04) but not with BMIs. Similarly, class Betaproteobacteria was highly enriched in diabetic compared to non-diabetic persons (P = 0.02) and positively correlated with plasma glucose (P = 0.04).

Conclusions

The results of this study indicate that type 2 diabetes in humans is associated with compositional changes in intestinal microbiota. The level of glucose tolerance should be considered when linking microbiota with metabolic diseases such as obesity and developing strategies to control metabolic diseases by modifying the gut microbiota.     

Inflammation and Airway Microbiota during Cystic Fibrosis Pulmonary Exacerbations

Background

Pulmonary exacerbations (PEx), frequently associated with airway infection and inflammation, are the leading cause of morbidity in cystic fibrosis (CF). Molecular microbiologic approaches detect complex microbiota from CF airway samples taken during PEx. The relationship between airway microbiota, inflammation, and lung function during CF PEx is not well understood.

Objective

To determine the relationships between airway microbiota, inflammation, and lung function in CF subjects treated for PEx.

Methods

Expectorated sputum and blood were collected and lung function testing performed in CF subjects during early (0–3d.) and late treatment (>7d.) for PEx. Sputum was analyzed by culture, pyrosequencing of 16S rRNA amplicons, and quantitative PCR for total and specific bacteria. Sputum IL-8 and neutrophil elastase (NE); and circulating C-reactive protein (CRP) were measured.

Results

Thirty-seven sputum samples were collected from 21 CF subjects. At early treatment, lower diversity was associated with high relative abundance (RA) of Pseudomonas (r = −0.67, p<0.001), decreased FEV_1% predicted (r = 0.49, p = 0.03) and increased CRP (r = −0.58, p = 0.01). In contrast to Pseudomonas, obligate and facultative anaerobic genera were associated with less inflammation and higher FEV₁. With treatment, Pseudomonas RA and P. aeruginosa by qPCR decreased while anaerobic genera showed marked variability in response. Change in RA of Prevotella was associated with more variability in FEV₁ response to treatment than Pseudomonas or Staphylococcus.

Conclusions

Anaerobes identified from sputum by sequencing are associated with less inflammation and higher lung function compared to Pseudomonas at early exacerbation. CF PEx treatment results in variable changes of anaerobic genera suggesting the need for larger studies particularly of patients without traditional CF pathogens.     

Partitioning the Heritability of Tourette Syndrome and Obsessive Compulsive Disorder Reveals Differences in Genetic Architecture

The direct estimation of heritability from genome-wide common variant data as implemented in the program Genome-wide Complex Trait Analysis (GCTA) has provided a means to quantify heritability attributable to all interrogated variants. We have quantified the variance in liability to disease explained by all SNPs for two phenotypically-related neurobehavioral disorders, obsessive-compulsive disorder (OCD) and Tourette Syndrome (TS), using GCTA. Our analysis yielded a heritability point estimate of 0.58 (se = 0.09, p = 5.64e-12) for TS, and 0.37 (se = 0.07, p = 1.5e-07) for OCD. In addition, we conducted multiple genomic partitioning analyses to identify genomic elements that concentrate this heritability. We examined genomic architectures of TS and OCD by chromosome, MAF bin, and functional annotations. In addition, we assessed heritability for early onset and adult onset OCD. Among other notable results, we found that SNPs with a minor allele frequency of less than 5% accounted for 21% of the TS heritability and 0% of the OCD heritability. Additionally, we identified a significant contribution to TS and OCD heritability by variants significantly associated with gene expression in two regions of the brain (parietal cortex and cerebellum) for which we had available expression quantitative trait loci (eQTLs). Finally we analyzed the genetic correlation between TS and OCD, revealing a genetic correlation of 0.41 (se = 0.15, p = 0.002). These results are very close to previous heritability estimates for TS and OCD based on twin and family studies, suggesting that very little, if any, heritability is truly missing (i.e., unassayed) from TS and OCD GWAS studies of common variation. The results also indicate that there is some genetic overlap between these two phenotypically-related neuropsychiatric disorders, but suggest that the two disorders have distinct genetic architectures.     

Direct Evidence for the Presence of Human Milk Oligosaccharides in the Circulation of Breastfed Infants

Background

It has been hypothesized that human milk oligosaccharides (HMOs) confer systemic health benefits to breastfed infants; however, plausible mechanisms for some effects, such as systemic immunomodulation, require HMOs to access the bloodstream of the developing infant. While small concentrations of HMOs have been detected in the urine of breastfed infants there are no published studies of these oligosaccharides accessing the plasma compartment of breastfed infants. Here we determined the relative fractions of several ingested HMOs in infant urine and plasma. Plasma from formula-fed infants was used as a control.

Methods

Using gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry/tandem mass spectrometry (LC/MS/MS), and high performance liquid chromatography (HPLC), we analyzed the urine and plasma from 17 healthy formula-fed infants and 16 healthy breast-fed infants (and the milk from their mothers).

Results

Multiple HMOs were detected in the urine and plasma of breastfed infants, but not in formula-fed infants. Levels of 2′-fucosyllactose (2′FL), 3FL and lacto-N-neotetraose (LNnT) in both plasma (r = 0.98, p<0.001; r = 0.75, p = 0.002; r = 0.71, p = 0.004) and urine (r = 0.81, p<0.001; r = 0.56, p = 0.026; NS) correlated significantly with concentrations in the corresponding breast milk. The relative fractions of HMOs were low, 0.1% of milk levels for plasma and 4% of milk levels for urine. Within the breastfed cohort, there were significant differences between secretor and nonsecretor groups in levels of several fucosylated HMOs.

Conclusion

At least some ingested HMOs are absorbed intact into the circulation and excreted in the urine and their concentrations in these fluids correlate with levels of the corresponding mother''s milk. While relative fractions of absorbed HMOs were low, these levels have been shown to have biological effects in vitro, and could explain some of the postulated benefits of human milk.