Project description:Significant gut microbiota heterogeneity exists amongst UC patients though the clinical implications of this variance are unknown. European and South Asian UC patients exhibit distinct disease risk alleles, many of which regulate immune function and relate to variation in gut microbiota β-diversity. We hypothesized ethnically distinct UC patients exhibit discrete gut microbiotas with unique luminal metabolic programming that influence adaptive immune responses and relate to clinical status. Using parallel bacterial 16S rRNA and fungal ITS2 sequencing of fecal samples (UC n=30; healthy n=13), we corroborated previous observations of UC-associated depletion of bacterial diversity and demonstrated significant gastrointestinal expansion of Saccharomycetales as a novel UC characteristic. We identified four distinct microbial community states (MCS 1-4), confirmed their existence using microbiota data from an independent UC cohort, and show they co-associate with patient ethnicity and degree of disease severity. Each MCS was predicted to be uniquely enriched for specific amino acid, carbohydrate, and lipid metabolism pathways and exhibited significant luminal enrichment of metabolic products from these pathways. Using a novel in vitro human DC/T-cell assay we show that DC exposure to patient fecal water led to MCS -specific changes in T-cell populations, particularly the Th1:Th2 ratio, and that patients with the most severe disease exhibited the greatest Th2 skewing. Thus, based on ethnicity, microbiome composition, and associated metabolic dysfunction, UC patients may be stratified in a clinically and immunologically meaningful manner, providing a platform for the development of FMC-focused therapy. Fecal microbiome was assessed with Affymetrix PhyloChip arrays from patients with ulcerative colitis and healthy controls.
Project description:Objective Crohn’s Disease (CD) and Ulcerative Colitis (UC) are chronic inflammatory diseases of the gastrointestinal tract. Reliable diagnosis of these diseases requires a comprehensive examination of the patient, which include invasive endoscopy. This study assesses whether non-invasive LC-MS/MS based analysis of microbial and human proteins from feces may support the diagnosis of the diseases. Design In order to mimic a representative clinical background for this study, we investigated 17 healthy controls, 11 CD patients, 14 UC patients, also 13 Irritable Bowel Disease (IBS) patients, 8 Colon Adenoma (CA) patients, and 8 Gastric Carcinoma (GCA) patients. The proteins were extracted from the fecal samples with liquid phenol in a ball mill. Subsequently, the proteins were digested tryptically to peptides and analyzed by liquid chromatography coupled to an Orbitrap MS/MS. For protein identification and interpretation of taxonomic and functional results, the MetaProteomeAnalyzer software and the UniProtKB/SwissProt database and several metagenomes from human fecal samples were used. Results Cluster analysis and ANOSIM show a separation of healthy controls from patients with CD and UC as well as from patients with GCA. Among others, UC and CD correlated with an increase of neutrophil extracellular traps and immunoglobulins G (IgG) as well as a decrease of IgA. A specific marker metaprotein for CD was an increase of the human enzyme sucrose-isomaltase. IBS and CA patient’s fecal metaproteome showed only minor alterations. Conclusion Metaproteome analysis distinguished between patients with UC, CD and healthy controls and is therefore useful as a non-invasive tool for routine diagnostics in hospitals.
Project description:Circulating antibody secreting cells are present in peripheral blood of healthy individuals reflecting continued activity of the humoral immune system. Antibody secreting cells typically express CD27. We describe and characterize a small population of antibody secreting class switched CD19+CD43+ B cells that lack expression of CD27 in peripheral blood of healthy subjects. Class switched CD27-CD43+ B cells possess characteristics of conventional plasmablasts as they spontaneously secrete antibodies, are morphological similar to antibody secreting cells, show downregulation of B cell differentiation markers. In order to further characterize the cells in comparison to other B cell subtypes we performed gene expression studies. RNA was isolated from sort-purified IgA-expressing and IgG-expressing B cell subpopulations [CD27+CD43-, CD27+CD43+, CD27-CD43-, CD27-CD43+] obtained from 3 healthy adults, and microarray analysis was performed. Data from IgA-expressing B cell populations and from IgG-expressing B cell populations were normalized (separately).
Project description:Maternal secretor status is one of the determinants of human milk oligosaccharides (HMOs) composition, which in turn changes the gut microbiota composition of infants. To understand if this change in gut microbiota impacts immune cell composition, intestinal morphology and gene expression, day 21-old germ-free mice were transplanted with fecal microbiota from infants whose mothers were either secretors (SMM) or non-secretors (NSM) or from infants consuming dairy-based formula (MFM). For each group, one set of mice was supplemented with HMOs. HMO supplementation did not significantly impact the microbiota diversity however, SMM mice had higher abundance of genus Bacteroides, Bifidobacterium, and Blautia, whereas, in the NSM group, there were higher abundance of Akkermansia, Enterocloster, and Klebsiella. In MFM, gut microbiota was represented mainly by Parabacteroides, Ruminococcaceae_unclassified, and Clostrodium_sensu_stricto. In mesenteric lymph node, Foxp3+ T cells and innate lymphoid cells type 2 (ILC2) were increased in MFM mice supplemented with HMOs while in the spleen, they were increased in SMM+HMOs mice. Similarly, serum immunoglobulin A (IgA) was also elevated in MFM+HMOs group. Distinct global gene expression of the gut was observed in each microbiota group, which was enhanced with HMOs supplementation. Overall, our data shows that distinct infant gut microbiota due to maternal secretor status or consumption of dairy-based formula and HMO supplementation impacts immune cell composition, antibody response and intestinal gene expression in a mouse model.
Project description:Metaproteomic portrait of the healthy human gut microbiota. Re-analysis of existing datasets, selected based on the following inclusion criteria: human cohort including at least 5 healthy (clearly not labeled as diseased) adult (>18 years old) individuals; data derived from LC-MS/MS DDA label-free analysis of fecal samples (with neither subcellular fractionation of microbial cells nor offline fractionation of peptides); availability of raw MS data on public repositories.
Project description:This study aimed to analyze changes in gut microbiota composition in mice after transplantation of fecal microbiota (FMT, N = 6) from the feces of NSCLC patients by analyzing fecal content using 16S rRNA sequencing, 10 days after transplantation. Specific-pathogen-free (SPF) mice were used for each experiments (N=4) as controls.
Project description:Significant gut microbiota heterogeneity exists amongst UC patients though the clinical implications of this variance are unknown. European and South Asian UC patients exhibit distinct disease risk alleles, many of which regulate immune function and relate to variation in gut microbiota β-diversity. We hypothesized ethnically distinct UC patients exhibit discrete gut microbiotas with unique luminal metabolic programming that influence adaptive immune responses and relate to clinical status. Using parallel bacterial 16S rRNA and fungal ITS2 sequencing of fecal samples (UC n=30; healthy n=13), we corroborated previous observations of UC-associated depletion of bacterial diversity and demonstrated significant gastrointestinal expansion of Saccharomycetales as a novel UC characteristic. We identified four distinct microbial community states (MCS 1-4), confirmed their existence using microbiota data from an independent UC cohort, and show they co-associate with patient ethnicity and degree of disease severity. Each MCS was predicted to be uniquely enriched for specific amino acid, carbohydrate, and lipid metabolism pathways and exhibited significant luminal enrichment of metabolic products from these pathways. Using a novel in vitro human DC/T-cell assay we show that DC exposure to patient fecal water led to MCS -specific changes in T-cell populations, particularly the Th1:Th2 ratio, and that patients with the most severe disease exhibited the greatest Th2 skewing. Thus, based on ethnicity, microbiome composition, and associated metabolic dysfunction, UC patients may be stratified in a clinically and immunologically meaningful manner, providing a platform for the development of FMC-focused therapy.