Akkermansia muciniphila Reduces Peritonitis and Improves Intestinal Tissue Wound Healing after a Colonic Transmural Defect by a MyD88-Dependent Mechanism.

Anastomotic leakage is a major complication following colorectal surgery leading to peritonitis, complications, and mortality. Akkermansia muciniphila has shown beneficial effects on the gut barrier function. Whether A. muciniphila reduces peritonitis and mortality during colonic leakage is unknown. Whether A. muciniphila can directly modulate the expression of genes in the colonic mucosa in humans has never been studied. We investigated the effects of a pretreatment (14 days) with live A. muciniphila prior to surgical colonic perforation on peritonitis, mortality, and wound healing. We used mice with an inducible intestinal-epithelial-cell-specific deletion of MyD88 (IEC-MyD88 KO) to investigate the role of the innate immune system in this context. In a proof-of-concept pilot study, healthy humans were exposed to A. muciniphila for 2 h and colonic biopsies taken before and after colonic instillation for transcriptomic analysis. Seven days after colonic perforation, A.-muciniphila-treated mice had significantly lower mortality and severity of peritonitis. This effect was associated with significant improvements of wound histological healing scores, higher production of IL22, but no changes in the mucus layer thickness or genes involved in cell renewal, proliferation, or differentiation. All these effects were abolished in IEC-MyD88 KO mice. Finally, human subjects exposed to A. muciniphila exhibited an increased level of the bacterium at the mucus level 2 h after instillation and significant changes in the expression of different genes involved in the regulation of cell cycling, gene transcription, immunity, and inflammation in their colonic mucosa. A. muciniphila improves wound healing during transmural colonic wall defect through mechanisms possibly involving IL22 signaling and requiring MyD88 in the intestinal cells. In healthy humans, colonic administration of A. muciniphila is well tolerated and changes the expression of genes involved in the immune pathways.

Faecalibacterium prausnitzii increases following fecal microbiota transplantation in recurrent Clostridioides difficile infection.

OBJECTIVE: Fecal microbiota transplantation (FMT) is a highly effective treatment for Clostridioides difficile infection (CDI). However, the fecal transplant's causal components translating into clearance of the CDI are yet to be identified. The commensal bacteria Faecalibacterium prausnitzii may be of great interest in this context, since it is one of the most common species of the healthy gut microbiota and produces metabolites with anti-inflammatory properties. Although there is mounting evidence that F. prausnitzii is an important regulator of intestinal homeostasis, data about its role in CDI and FMT are relatively scarce. METHODS: Stool samples from patients with recurrent CDI were collected to investigate the relative abundance of F. prausnitzii before and after FMT. Twenty-one patients provided fecal samples before the FMT procedure, at 2 weeks post-FMT, and at 2-4 months post-FMT. The relative abundance of F. prausnitzii was determined using quantitative polymerase chain reaction. RESULTS: The abundance of F. prausnitzii was elevated in samples (N = 9) from donors compared to pre-FMT samples (N = 15) from patients (adjusted P<0.001). No significant difference in the abundance of F. prausnitzii between responders (N = 11) and non-responders (N = 4) was found before FMT (P = 0.85). In patients with CDI, the abundance of F. prausnitzii significantly increased in the 2 weeks post-FMT samples (N = 14) compared to the pre-FMT samples (N = 15, adjusted P<0.001). The increase persisted 2-4 months post-FMT (N = 15) compared to pre-FMT samples (N = 15) (adjusted P<0.001). CONCLUSIONS: FMT increases the relative abundance of F. prausnitzii in patients with recurrent CDI, and this microbial shift remains several months later. The baseline abundance of F. prausnitzii in donors or recipients was not associated with future treatment response, although a true predictive capacity cannot be excluded because of the limited sample size. Further studies are needed to discern whether F. prausnitzii plays an active role in the resolution of CDI.

Butyrate Rescues Oxidative Stress-Induced Transport Deficits of Tryptophan: Potential Implication in Affective or Gut-Brain Axis Disorders.

INTRODUCTION: Butyrate is a short-chain fatty acid metabolite produced by microbiota in the colon. With its antioxidant properties, butyrate has also been shown to alter the neurological functions in affective disorder models, suggesting it as a key mediator in gut-brain interactions. OBJECTIVE: Here, we evaluated the negative effect of oxidative stress on the transport of the serotonin precursor tryptophan as present in affective disorders. Butyrate was hypothesized to be able to rescue these deficits due to its antioxidative capacities and its effect on transmembrane transport of tryptophan. Human skin-derived fibroblasts were used as cellular models to address these objectives. METHODS: Human fibroblasts were treated with hydrogen peroxide to induce oxidative stress. Stressed as well as control cells were treated with different concentrations of butyrate. Tryptophan (3H) was used as a tracer to measure the transport of tryptophan across the cell membranes (n = 6). Furthermore, gene expression profiles of different amino acid transporters were analyzed (n = 2). RESULTS: As hypothesized,oxidative stress significantly decreased the uptake of tryptophan in fibroblast cells, while butyrate counteracted this effect. Oxidative stress did not alter the gene expression profile of amino acid transporters. However, treatment of stressed and control cells with different concentrations of butyrate differentially regulated the gene expression of large amino acid transporters 1 and 2, which are the major transporters of tryptophan. CONCLUSIONS: Gut microbiota-derived butyrate may have therapeutic potential in affective disorders characterized by either aberrant serotonergic activity or neuroinflammation due to its role in rescuing the oxidative stress-induced perturbations of tryptophan transport.

Differences in Gut Microbiome Composition between Senior Orienteering Athletes and Community-Dwelling Older Adults.

BACKGROUND: Gastrointestinal (GI) health is an important aspect of general health. Gastrointestinal symptoms are of specific importance for the elderly, an increasing group globally. Hence, promoting the elderly's health and especially gastrointestinal health is important. Gut microbiota can influence gastrointestinal health by modulation of the immune system and the gut-brain axis. Diverse gut microbiota have been shown to be beneficial; however, for the elderly, the gut microbiota is often less diverse. Nutrition and physical activity, in particular, are two components that have been suggested to influence composition or diversity. MATERIALS AND METHODS: In this study, we compared gut microbiota between two groups of elderly individuals: community-dwelling older adults and physically active senior orienteering athletes, where the latter group has less gastrointestinal symptoms and a reported better well-being. With this approach, we explored if certain gut microbiota were related to healthy ageing. The participant data and faecal samples were collected from these two groups and the microbiota was whole-genome sequenced and taxonomically classified with MetaPhlAn. RESULTS: The physically active senior orienteers had a more homogeneous microbiota within the group and a higher abundance of Faecalibacterium prausnitzii compared to the community-dwelling older adults. Faecalibacterium prausnitzii has previously shown to have beneficial properties. Senior orienteers also had a lower abundance of Parasutterella excrementihominis and Bilophila unclassified, which have been associated with impaired GI health. We could not observe any difference between the groups in terms of Shannon diversity index. Interestingly, a subgroup of community-dwelling older adults showed an atypical microbiota profile as well as the parameters for gastrointestinal symptoms and well-being closer to senior orienteers. CONCLUSIONS: Our results suggest specific composition characteristics of healthy microbiota in the elderly, and show that certain components of nutrition as well as psychological distress are not as tightly connected with composition or diversity variation in faecal microbiota samples.

Alterations in the relative abundance of Faecalibacterium prausnitzii correlate with changes in fecal calprotectin in patients with ileal Crohn's disease: a longitudinal study.

Objectives: Crohn's disease is characterized by a gut dysbiosis with decreased abundance of butyrate producers such as Faecalibacterium prausnitzii. Although F. prausnitzii secretes anti-inflammatory molecules, few studies have addressed the importance of F. prausnitzii in a longitudinal setting. We aimed to examine the relationship between temporal profiles of F. prausnitzii, the C. leptum group, overall butyrate production, and inflammatory activity. Material and methods: Fecal samples (n = 59) were collected every third month from nine patients with ileal Crohn's disease. The abundance of F. prausnitzii and C. leptum was quantified relative to the total amount of bacteria using quantitative-PCR. To assess butyrate production of gut microbiota, gene copy numbers of the butyryl-CoA:acetate-CoA transferase (BCoAT) gene were quantified by qPCR. The inflammatory activity was defined by fecal (f)-calprotectin. Results: No correlation between the relative abundance of F. prausnitzii, the C. leptum group, or copy numbers of the BCoAT gene, and f-calprotectin was observed in the total sample set. By analyzing alterations between consecutive samples, a negative correlation between changes in the relative abundance of F. prausnitzii and f-calprotectin was observed (R = -0.39; p = .009). Changes in C. leptum (R = -0.18, p = .23) and number of copies of the BCoAT gene (R = -0.12; p = .42) did not correlate with f-calprotectin. Conclusions: There was an inverse correlation between temporal changes in the relative abundance of F. prausnitzii, but not overall butyrate producing capacity, and changes in inflammatory activity in ileal Crohn's disease. These findings indicate that F. prausnitzii may play a role in gut homeostasis, even though causality is still to be demonstrated.

Impact of Proinflammatory Cytokines on the Virulence of Uropathogenic Escherichia coli.

The effect of a urinary tract infection on the host is a well-studied research field. However, how the host immune response affects uropathogenic Escherichia coli (CFT073) virulence is less studied. The aim of the present study was to investigate the impact of proinflammatory cytokine exposure on the virulence of uropathogenic Escherichia coli. We found that all tested proinflammatory cytokines (TNF-α, IL-1ß, IL-6, IL-8 and IFN-γ) induced an increased CFT073 growth. We also found that biofilm formation and hemolytic activity was reduced in the presence of all proinflammatory cytokines. However, a reduction in siderophore release was only observed in the presence of IL-1ß, IL-6 and IL-8. Real time-qPCR showed that all proinflammatory cytokines except TNF-α significantly increased genes associated with the iron acquisition system in CFT073. We also found that the proinflammatory cytokines induced significant changes in type-1 fimbriae, P-fimbriae and gluconeogenetic genes. Furthermore, we also showed, using a Caenorhabditis elegans (C. elegans) killing assay that all cytokines decreased the survival of C. elegans worms significantly. Taken together, our findings show that proinflammatory cytokines have the ability to alter the virulence traits of UPEC.

Assessing the Committed Effective Dose From 226Ra in Thermal Spring Water From San Diego De Alcala, Chihuahua, Mexico.

The oral administration of mineral-rich spring water is known as hydropinic treatment and is used to treat certain ailments. Health benefits are attributed to thermal spring water containing radioactive elements such as radium; this has popularized use of such radioactive water in various parts of the world, causing those who ingest it to increase their internal radiation dose. The goal of this study was to assess the activity concentrations of Ra present in the thermal spring waters of San Diego de Alcala, in the state of Chihuahua, Mexico, and to estimate the health risk posed to patients by the effective dose received from ingesting this water during hydropinic treatments. Water samples were taken from different areas of the San Diego de Alcala thermal springs, and pH, temperature, electrical conductivity, and total dissolved solids were measured. The Ra activity concentrations were measured with a liquid scintillation counter. The activity concentrations of Ra in sampled water varied from 125 to 452 mBq L with an average of 276 ± 40 mBq L. The committed effective dose from each of the Ra activity concentrations found in samples ranged from 9.80 × 10 to 4.0 × 10 mSv for hydropinic treatments being carried out in San Diego de Alcala thermal spring spas. Different treatments had different intake rates (200, 600, 1,000, and 1,500 mL d) and occurred over periods of 2 or 3 wk. According to the guidelines of the US Environmental Protection Agency, the maximum permissible amount of radium in drinking water is 185 mBq L; the Ra content in most of the collected samples exceeded this limit. The committed effective doses varied with Ra concentration and intake rate; none exceeded the World Health Organization's reference dose for drinking water of 0.1 mSv y, which is the maximum amount to which the population should be exposed.

Qualitative modelling of the interplay of inflammatory status and butyrate in the human gut: a hypotheses about robust bi-stability.

BACKGROUND: Gut microbiota interacts with the human gut in multiple ways. Microbiota composition is altered in inflamed gut conditions. Likewise, certain microbial fermentation products as well as the lipopolysaccharides of the outer membrane are examples of microbial products with opposing influences on gut epithelium inflammation status. This system of intricate interactions is known to play a core role in human gut inflammatory diseases. Here, we present and analyse a simplified model of bidirectional interaction between the microbiota and the host: in focus is butyrate as an example for a bacterial fermentation product with anti-inflammatory properties. RESULTS: We build a dynamical model based on an existing model of inflammatory regulation in gut epithelial cells. Our model introduces both butyrate as a bacterial product which counteracts inflammation, as well as bacterial LPS as a pro-inflammatory bacterial product. Moreover, we propose an extension of this model that also includes a feedback interaction towards bacterial composition. The analysis of these dynamical models shows robust bi-stability driven by butyrate concentrations in the gut. The extended model hints towards a further possible enforcement of the observed bi-stability via alteration of gut bacterial composition. A theoretical perspective on the stability of the described switch-like character is discussed. CONCLUSIONS: Interpreting the results of this qualitative model allows formulating hypotheses about the switch-like character of inflammatory regulation in the gut epithelium, involving bacterial products as constitutive parts of the system. We also speculate about possible explanations for observed bimodal distributions in bacterial compositions in the human gut. The switch-like behaviour of the system proved to be mostly independent of parameter choices. Further implications of the qualitative character of our modeling approach for the robustness of the proposed hypotheses are discussed, as well as the pronounced role of butyrate compared to other inflammatory regulators, especially LPS, NF- κB and cytokines.

Transcriptional Alterations of Virulence-Associated Genes in Extended Spectrum Beta-Lactamase (ESBL)-Producing Uropathogenic Escherichia coli during Morphologic Transitions Induced by Ineffective Antibiotics.

It is known that an ineffective antibiotic treatment can induce morphological shifts in uropathogenic Escherichia coli (UPEC) but the virulence properties during these shifts remain to be studied. The present study examines changes in global gene expression patterns and in virulence factor-associated genes in an extended spectrum beta-lactamase (ESBL)-producing UPEC (ESBL019) during the morphologic transitions induced by an ineffective antibiotic and in the presence of human primary bladder epithelial cells. Microarray results showed that the different morphological states of ESBL019 had significant transcriptional alterations of a large number of genes (Transition; 7%, Filamentation; 32%, and Reverted 19% of the entities on the array). All three morphological states of ESBL019 were associated with a decreased energy metabolism, altered iron acquisition systems and altered adhesion expression. In addition, genes associated with LPS synthesis and bacterial motility was also altered in all the morphological states. Furthermore, the transition state induced a significantly higher release of TNF-α from bladder epithelial cells compared to all other morphologies, while the reverted state was unable to induce TNF-α release. Our findings show that the morphological shifts induced by ineffective antibiotics are associated with significant transcriptional virulence alterations in ESBL-producing UPEC, which may affect survival and persistence in the urinary tract.

Identification and management of prediabetes: results of the Latin America Strategic Prediabetes Meeting.

To understand the status of prediabetes diagnosis and treatment in Latin America and to evaluate the use of metformin for diabetes prevention in this context. A panel of 15 diabetes experts from seven countries in Latin America met on 14 - 15 August 2014 in Lima, Peru, to review the available literature, discuss the role of prediabetes in type 2 diabetes mellitus and cardiovascular disease, analyze collected information, and make conclusions for prediabetes diagnosis and treatment in Latin America. Prediabetes diagnosis, screening, and treatment, including lifestyle changes, pharmacological treatment, and cost-effectiveness were discussed. Five resulting statements were issued for Latin America: prediabetes is a clinical and public health problem; health care systems do not currently diagnose/treat prediabetes; use of prediabetes risk detection tools are needed region-wide; treatment includes lifestyle changes, multidisciplinary education, and metformin; and registries of patient records and further studies should be supported. The expert panel concluded that in Latin America, preventive treatment through lifestyle changes and metformin are cost-effective interventions. It is important to improve prediabetes identification and management at the primary care level.

Comprender el estado del diagnóstico y el tratamiento de la prediabetes en América Latina y evaluar el uso de la metformina para la prevención de la diabetes en este contexto.Un panel de 15 expertos en diabetes de siete países de América Latina se reunió del 14 al 15 de agosto de 2014 en Lima, Perú, para revisar la literatura disponible, discutir el papel de la prediabetes en la diabetes mellitus tipo 2 y la enfermedad cardiovascular, analizar la información recolectada y formular conclusiones para el diagnóstico y el tratamiento de la prediabetes en América Latina.Se analizaron el diagnóstico, el tamizaje y el tratamiento de la prediabetes, inclusive los cambios en el estilo de vida, el tratamiento farmacológico y la relación costo-eficacia. Se emitieron cinco conclusiones para América Latina: la prediabetes es un problema clínico y de salud pública; los sistemas de atención de la salud actualmente no diagnostican o no tratan la prediabetes; el uso de herramientas de detección del riesgo de prediabetes es necesario en toda la región; el tratamiento incluye cambios en el estilo de vida, educación multidisciplinaria y metformina; y se debe brindar apoyo para llevar registros de historias clínicas y realizar estudios adicionales.El panel de expertos concluyó que en América Latina el tratamiento preventivo basado en cambios en el estilo de vida y administración de metformina son intervenciones eficaces en relación al costo. Es importante mejorar la identificación y el manejo de la prediabetes en el nivel de atención primaria.

Entender o estado do diagnóstico e tratamento do prediabetes na América Latina e avaliar o uso de metformina para prevenção de diabetes neste contexto.Um painel de 15 especialistas em diabetes de sete países da América Latina reuniu-se de 14 a 15 de agosto de 2014 em Lima, Peru, para analisar a literatura disponível, discutir o papel do prediabetes em diabetes mellitus tipo 2 e doenças cardiovasculares, analisar informações coletadas e fazer conclusões para o diagnóstico e tratamento do prediabetes na América Latina.O diagnóstico, rastreio e tratamento pré-diabetes, incluindo mudanças de estilo de vida, tratamento farmacológico e custo-efetividade foram discutidos. Foram emitidas cinco conclusões resultantes para a América Latina: o prediabetes é um problema clínico e de saúde pública; os sistemas de saúde atualmente não diagnosticam/tratam prediabetes; o uso de ferramentas de detecção de risco de prediabetes é necessário em toda a região; o tratamento inclui mudanças de estilo de vida, educação multidisciplinar e metformina; e devem ser suportados registros de pacientes e outros estudos.O painel de especialistas concluiu que na América Latina, o tratamento preventivo através de mudanças de estilo de vida e metformina são intervenções efetivas em relação ao custo. É importante melhorar a identificação e gestão do prediabetes no nível de atenção primária.

Identification and management of prediabetes: results of the Latin America Strategic Prediabetes Meeting / Identificación y manejo de la prediabetes: resultados del Encuentro Estratégico de Prediabetes de América Latina / Identificação e gerenciamento de prediabetes: resultados da Reunião Estratégica de Prediabetes da América Latina

ABSTRACT To understand the status of prediabetes diagnosis and treatment in Latin America and to evaluate the use of metformin for diabetes prevention in this context. A panel of 15 diabetes experts from seven countries in Latin America met on 14 - 15 August 2014 in Lima, Peru, to review the available literature, discuss the role of prediabetes in type 2 diabetes mellitus and cardiovascular disease, analyze collected information, and make conclusions for prediabetes diagnosis and treatment in Latin America. Prediabetes diagnosis, screening, and treatment, including lifestyle changes, pharmacological treatment, and cost-effectiveness were discussed. Five resulting statements were issued for Latin America: prediabetes is a clinical and public health problem; health care systems do not currently diagnose/treat prediabetes; use of prediabetes risk detection tools are needed region-wide; treatment includes lifestyle changes, multidisciplinary education, and metformin; and registries of patient records and further studies should be supported. The expert panel concluded that in Latin America, preventive treatment through lifestyle changes and metformin are cost-effective interventions. It is important to improve prediabetes identification and management at the primary care level.(AU)

RESUMEN Comprender el estado del diagnóstico y el tratamiento de la prediabetes en América Latina y evaluar el uso de la metformina para la prevención de la diabetes en este contexto. Un panel de 15 expertos en diabetes de siete países de América Latina se reunió del 14 al 15 de agosto de 2014 en Lima, Perú, para revisar la literatura disponible, discutir el papel de la prediabetes en la diabetes mellitus tipo 2 y la enfermedad cardiovascular, analizar la información recolectada y formular conclusiones para el diagnóstico y el tratamiento de la prediabetes en América Latina. Se analizaron el diagnóstico, el tamizaje y el tratamiento de la prediabetes, inclusive los cambios en el estilo de vida, el tratamiento farmacológico y la relación costo-eficacia. Se emitieron cinco conclusiones para América Latina: la prediabetes es un problema clínico y de salud pública; los sistemas de atención de la salud actualmente no diagnostican o no tratan la prediabetes; el uso de herramientas de detección del riesgo de prediabetes es necesario en toda la región; el tratamiento incluye cambios en el estilo de vida, educación multidisciplinaria y metformina; y se debe brindar apoyo para llevar registros de historias clínicas y realizar estudios adicionales. El panel de expertos concluyó que en América Latina el tratamiento preventivo basado en cambios en el estilo de vida y administración de metformina son intervenciones eficaces en relación al costo. Es importante mejorar la identificación y el manejo de la prediabetes en el nivel de atención primaria.(AU)

RESUMO Entender o estado do diagnóstico e tratamento do prediabetes na América Latina e avaliar o uso de metformina para prevenção de diabetes neste contexto. Um painel de 15 especialistas em diabetes de sete países da América Latina reuniu-se de 14 a 15 de agosto de 2014 em Lima, Peru, para analisar a literatura disponível, discutir o papel do prediabetes em diabetes mellitus tipo 2 e doenças cardiovasculares, analisar informações coletadas e fazer conclusões para o diagnóstico e tratamento do prediabetes na América Latina. O diagnóstico, rastreio e tratamento pré-diabetes, incluindo mudanças de estilo de vida, tratamento farmacológico e custo-efetividade foram discutidos. Foram emitidas cinco conclusões resultantes para a América Latina: o prediabetes é um problema clínico e de saúde pública; os sistemas de saúde atualmente não diagnosticam/tratam prediabetes; o uso de ferramentas de detecção de risco de prediabetes é necessário em toda a região; o tratamento inclui mudanças de estilo de vida, educação multidisciplinar e metformina; e devem ser suportados registros de pacientes e outros estudos. O painel de especialistas concluiu que na América Latina, o tratamento preventivo através de mudanças de estilo de vida e metformina são intervenções efetivas em relação ao custo. É importante melhorar a identificação e gestão do prediabetes no nível de atenção primária.(AU)

Degree of colitis correlates with microbial composition and cytokine responses in colon and caecum of Gαi2-deficient mice.

An altered immune response and gut microbiota have been associated with the pathology of inflammatory bowel diseases (IBDs). However, there is limited knowledge of how inflammation is associated with changes in the microbiota. We studied the microbiota in the intestine and faeces as well as the cytokine gene expressions in caecum and colon of a mouse model (Gαi2(-/-)) of colitis, and analysed them in relation to the degrees of inflammation in the colon. The degree of colitis was associated with general changes in the complexity of the microbiota and was corroborated by quantitative analyses of the Bacteroides and Lactobacillus High gene expression levels of IL-17 and IFN-γ in colon and caecum were detected in Gαi2(-/-) mice with moderate and severe colitis. High IL-27 gene expression in the colon of mice with moderate and severe colitis and in the caecum of mice with moderate colitis was also detected. Negative correlations between IL-27 and Bacteroides and Lactobacillus and between IFN-γ and Lactobacillus were detected in caecum. This research indicates that the degree of colitis in IBD correlates with the gene expression of cytokines and with disturbances in the gut microbiota. Furthermore, the caecum could have an important role in the pathology of IBD.

Cytokine Response after Stimulation with Key Commensal Bacteria Differ in Post-Infectious Irritable Bowel Syndrome (PI-IBS) Patients Compared to Healthy Controls.

BACKGROUND: Microbial dysbiosis and prolonged immune activation resulting in low-grade inflammation and intestinal barrier dysfunction have been suggested to be underlying causes of post-infectious irritable bowel syndrome (PI-IBS). The aim of this study was to evaluate the difference in cytokine response between mucosal specimens of PI-IBS patients and healthy controls (HC) after ex vivo stimulation with key anaerobic bacteria. METHODS: Colonic biopsies from 11 PI-IBS patients and 10 HC were stimulated ex vivo with the commensal bacteria Bacteroides ovatus, Ruminococcus gnavus, Akkermansia muciniphila, Subdoligranulum variabile and Eubacterium limosum, respectively. The cytokine release (IL-1ß, IL-2, IL-8, IL-10, IL-13, IL-17, TNF-α and IFN-γ) in stimulation supernatants was analyzed using the LUMINEX assay. Comparison of cytokine release between PI-IBS patients and healthy controls was performed taking both unstimulated and bacterially stimulated mucosal specimens into account. KEY RESULTS: IL-13 release from mucosal specimens without bacterial stimulation was significantly lower in PI-IBS patients compared to HC (p < 0.05). After stimulation with Subdoligranulum variabile, IL-1ß release from PI-IBS patients was significantly increased compared to HC (p < 0.05). Stimulation with Eubacterium limosum resulted in a significantly decreased IL-10 release in HC compared to PI-IBS patients (p < 0.05) and a tendency to decreased IL-13 release in HC compared to PI-IBS patients (p = 0.07). CONCLUSIONS & INFERENCES: PI-IBS patients differ from HC with regard to cytokine release ex vivo after stimulation with selected commensal bacteria. Hence, our results support that the pathogenesis of PI-IBS comprises an altered immune response against commensal gut microbes.

Aberrant mucosal lymphocyte number and subsets in the colon of post-infectious irritable bowel syndrome patients.

BACKGROUND: Irritable bowel syndrome (IBS) is characterized by chronic abdominal symptoms such as pain, discomfort, and altered bowel habits. A subset of IBS patients, denoted as post-infectious IBS (PI-IBS) patients, develop symptoms after an enteric infection. Distinct abnormalities in the gut mucosa, including mucosal inflammation, have been proposed to contribute to or be the cause of PI-IBS. This study investigated lymphocyte subsets in PI-IBS patients compared to healthy controls. MATERIALS AND METHODS: Ten PI-IBS patients and nine healthy controls participated. All PI-IBS patients met the Rome III diagnostic criteria for IBS and reported sustained symptoms at least 1 year after an episode of acute gastroenteritis. Intraepithelial lymphocytes and lamina propria lymphocytes (LPLs), isolated from mucosal tissue samples, were stained and analyzed for a comprehensive set of cell markers using flow cytometry. RESULTS: The number of LPLs in PI-IBS was significantly increased compared to those in healthy controls (p < 0.05). PI-IBS patients showed significantly increased proportions of CD45RO(+) CD4(+) activated/memory T cells (p < 0.05) and double-positive CD4(+) CD8(+) cells (p < 0.05), respectively, in the lamina propria. The number of CD19(+) LPLs was decreased in PI-IBS patients compared to healthy controls (p < 0.001). CONCLUSION: This study presents new evidence that PI-IBS is associated with a sustained aberrant mucosal immune response and support future studies of anti-inflammatory or immune-modulating treatments in these patients.

Comparison between terminal-restriction fragment length polymorphism (T-RFLP) and quantitative culture for analysis of infants' gut microbiota.

The infantile intestinal microbiota is a major stimulus for immune maturation. Both culture and DNA-based methods can be used for microbiota characterization, but few studies have systematically compared their performance for analysis of the gut microbiota. Here, we examined fecal samples obtained on six occasions between one week and 12 months of age from six vaginally delivered infants. After quantitative aerobic and anaerobic culture of the samples on selective and non-selective media, DNA was extracted from the fecal samples and analyzed regarding 16S rRNA gene polymorphism by terminal-restriction fragment length polymorphism (T-RFLP). A database was constructed for direct identification of T-RFLP peaks by analysis of pure-culture bacteria and analysis of a limited number of samples by 16S rRNA cloning and sequencing. Bacterial genera present at >106 CFU/g feces, as determined by quantitative culture, were generally readily detected by T-RFLP, while culture on selective media was more sensitive in detecting facultative anaerobes with lower population counts. In contrast, T-RFLP more readily than culture detected several anaerobic species, also taxa that could not be identified using the database. T-RFLP readily identified bacteria to the genus level and also provided some sub-genus discrimination. Both T-RFLP and culture identified Bifidobacterium, Clostridium and Bacteroides spp. among the most common colonizers of the infantile microbiota throughout the first year of life. T-RFLP analysis showed that microbiota complexity was high in the first weeks of life, declined to a minimum at 1-2 months of age, and thereafter increased again. Principal component analysis revealed that early samples (1 week-6 months) chiefly differed between individual infants, while 12-month samples were similar between children, but different from the early samples. Our results indicate that T-RFLP has high sensitivity and adequate taxonomic discrimination capacity for analysis of gut microbiota composition, but that both culture and molecular based analysis have limitations and both approaches may be needed to obtain a full picture of the complex gut microbiota.

Interplay between T(h)1 and T(h)17 effector T-cell pathways in the pathogenesis of spontaneous colitis and colon cancer in the Gαi2-deficient mouse.

Gαi2-deficient mice spontaneously develop colitis. Using xMAP technology and RT-PCR, we investigated cytokine/chemokine profiles during histologically defined phases of disease: (i) no/mild, (ii) moderate, (iii) severe colitis without dysplasia/cancer and (iv) severe colitis with dysplasia/cancer, compared with age-matched wild-type (WT) littermates. Colonic dysplasia was observed in 4/11 mice and cancer in 1/11 mice with severe colitis. The histology correlated with progressive increases in colon weight/cm and spleen weight, and decreased thymus weight, all more advanced in mice with dysplasia/cancer. IL-1ß, IL-6, IL-12p40, IL-17, TNF-α, CCL2 and CXCL1 protein levels in colons, but not small intestines increased with colitis progression and were significantly increased in mice with moderate and severe colitis compared with WT mice, irrespective of the absence/presence of dysplasia/cancer. CCL5 did not change during colitis progression. Colonic IL-17 transcription increased 40- to 70-fold in all stages of colitis, whereas IFN-γ mRNA was gradually up-regulated 12- to 55-fold with colitis progression, and further to 62-fold in mice with dysplasia/cancer. IL-27 mRNA increased 4- to 15-fold during the course of colitis, and colonic IL-21 transcription increased 3-fold in mice with severe colitis, both irrespective of the absence/presence of dysplasia/cancer. FoxP3 transcription was significantly enhanced (3.5-fold) in mice with moderate and severe colitis, but not in mice with dysplasia/cancer, compared with WT mice. Constrained correspondence analysis demonstrated an association between increased protein levels of TNF-α, CCL2, IL-1ß, IL-6 and CXCL1 and dysplasia/cancer. In conclusion, colonic responses are dominated by a mixed T(h)1/T(h)17 phenotype, with increasing T(h)1 cytokine transcription with progression of colitis in Gαi2(-/-) mice.