Sarah K. Abey

Elevated circulating miR-150 and miR-342-3p in patients with irritable bowel syndrome.

BACKGROUND AND AIMS MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression and are thus of interest as diagnostic markers, and as clues to etiology and targets of intervention. This pilot study examined whether circulating miRNAs are differentially expressed in patients with IBS. METHODS miRNA microarrays (NanoString) were run on the whole blood of 43 participants. RESULTS hsa-miR-150 and hsa-miR-342-3p were found to be significantly elevated (FDR adjusted p≤0.05, ≥1.6 fold change) in IBS patients compared to healthy controls. Neither of these miRNAs showed any relationship to race or sex. hsa-miR-150 is associated with inflammatory bowel disorders and pain, and interacts with a protein kinase (AKT2) through which it may affect inflammatory pathways. hsa-miR-342-3p is predicted to interact with mRNAs involved in pain signaling, colonic motility, and smooth muscle function. CONCLUSIONS This preliminary study reports the association of two miRNAs, detected in whole blood, with IBS. These miRNAs link to pain and inflammatory pathways both of which are thought to be dysregulated in IBS. Larger sample sizes are needed to confirm their importance and potential as biomarkers.

The microbiome of the oral mucosa in irritable bowel syndrome

abstract Irritable bowel syndrome (IBS) is a poorly understood disorder characterized by persistent symptoms, including visceral pain. Studies have demonstrated oral microbiome differences in inflammatory bowel diseases suggesting the potential of the oral microbiome in the study of non-oral conditions. In this exploratory study we examine whether differences exist in the oral microbiome of IBS participants and healthy controls, and whether the oral microbiome relates to symptom severity. The oral buccal mucosal microbiome of 38 participants was characterized using PhyloChip microarrays. The severity of visceral pain was assessed by orally administering a gastrointestinal test solution. Participants self-reported their induced visceral pain. Pain severity was highest in IBS participants (P = 0.0002), particularly IBS-overweight participants (P = 0.02), and was robustly correlated to the abundance of 60 OTUs, 4 genera, 5 families and 4 orders of bacteria (r2 > 0.4, P < 0.001). IBS-overweight participants showed decreased richness in the phylum Bacteroidetes (P = 0.007) and the genus Bacillus (P = 0.008). Analysis of β-diversity found significant separation of the IBS-overweight group (P < 0.05). Our oral microbial results are concordant with described fecal and colonic microbiome-IBS and -weight associations. Having IBS and being overweight, rather than IBS-subtypes, was the most important factor in describing the severity of visceral pain and variation in the microbiome. Pain severity was strongly correlated to the abundance of many taxa, suggesting the potential of the oral microbiome in diagnosis and patient phenotyping. The oral microbiome has potential as a source of microbial information in IBS.

Letter: gender‐associated cell‐free microRNA profiles in non‐alcoholic fatty liver disease

Sirs; The study by Schwimmer et al. suggested the existence of a gender phenomenon in non-alcoholic fatty liver disease (NAFLD).1 First, the majority of children diagnosed with NAFLD were male. Second, NAFLD boys had significantly higher levels of serum alanine aminotransferases (ALT) than NAFLD girls. This ALT trend, however, was also seen in non-NAFLD children who were obese and overweight. Therefore, ALT alone was likely inadequate, if at all involved, in explaining the biology underlying the gender-skewed prevalence of NAFLD in their study and others.2 We have identified miRNA profiles in fasting serum from NAFLD patients (n = 5) recruited to the National Institutes of Health (NIH) Clinical Research Center between January 2009 and December 2012, compared with age and race matched controls with no NAFLD (n=5) using nCounter® (Nanostring). Male NAFLD patients also had significantly (P < 0.05) upregulated expressions of the hsa-miR-432-5p, hsa-miR-578, and hsa-miR-155-5p compared to female NAFLD patients (See Figure 1). The hsa-miR-432-5p and hsa-miR-301b levels were significantly (P < 0.05) higher in NAFLD male patients compared to male controls. In contrast, female NAFLD patients did not have significantly different miRNA levels compared to female controls. Thus, our pilot study showed empirical evidence for a male NAFLD-associated miRNA profile. Figure 1 MicroRNA Expressions in Male NAFLD versus Female NAFLD Patients. The male NAFLD-associated miRNA species found above are linked to inflammatory cytokines.3 Our findings also add to the literature suggesting the intriguing involvement of oestrogen and endocrine/metabolic signaling in NAFLD pathogenesis. In mice, oestrogen withdrawal leads to alterations in the peroxisome proliferator-activated receptor (PPAR) genes, which are the predicted targets of hsa-miR-142, which in turn is a target of hsa-miR-155. 4,5,6 We suggest that these male NAFLD miRNA profiles provide a starting point not only for the development of biomarkers for male NAFLD screening, but also for understanding the molecular basis of a gender-unique NAFLD pathogenesis.

Structural and functional alterations in the colonic microbiome of the rat in a model of stress induced irritable bowel syndrome

ABSTRACT Stress is known to perturb the microbiome and exacerbate irritable bowel syndrome (IBS) associated symptoms. Characterizing structural and functional changes in the microbiome is necessary to understand how alterations affect the biomolecular environment of the gut in IBS. Repeated water avoidance (WA) stress was used to induce IBS-like symptoms in rats. The colon-mucosa associated microbiome was characterized in 13 stressed and control animals by 16S sequencing. In silico analysis of the functional domains of microbial communities was done by inferring metagenomic profiles from 16S data. Microbial communities and functional profiles were compared between conditions. WA animals exhibited higher α-diversity and moderate divergence in community structure (β-diversity) compared with controls. Specific clades and taxa were consistently and significantly modified in the WA animals. The WA microbiome was particularly enriched in Proteobacteria and depleted in several beneficial taxa. A decreased capacity in metabolic domains, including energy- and lipid-metabolism, and an increased capacity for fatty acid and sulfur metabolism was inferred for the WA microbiome. The stressed condition favored the proliferation of a greater diversity of microbes that appear to be functionally similar, resulting in a functionally poorer microbiome with implications for epithelial health. Taxa, with known beneficial effects, were found to be depleted, which supports their relevance as therapeutic agents to restore microbial health. Microbial sulfur metabolism may form a key component of visceral nerve sensitization pathways and is therefore of interest as a target metabolic domain in microbial ecological restoration.

Gender and Weight Influence Quality of Life in Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by abdominal pain and bowel dysfunction in the absence of structural abnormality. Diagnosis can be challenging and often leads to extensive medical tests, non-effective therapeutic modalities, and reduced quality of life (QOL). Identifying factors associated with dysfunction have the potential to enhance outcomes. Participants with IBS (n = 41) and healthy volunteers (n = 74) were recruited into this cross-sectional, descriptive, natural history protocol at the National Institute of Health, Clinical Center. Demographic characteristics were self-reported. QOL was assessed with the Irritable Bowel Syndrome Quality of Life (IBS-QOL) questionnaire. Statistical analysis included descriptive statistics, factorial ANOVA, and multiple regression. Individuals with IBS reported lower QOL scores across all QOL-subscales compared to healthy controls. Normal-weight women and overweight men with IBS reported the greatest QOL impairment. Body fat percent had confounding effects on the relationship between IBS and QOL. The disparity between QOL scores in participants with IBS by both gender and weight groups may reflect different social pressures perceived by normal and overweight women and men. These findings enhance the recognition of the disparities in patients with chronic symptoms and thereby lead to personalized assessment and interventions to improve their QOL.

The Gastrointestinal Pain Pointer: A Valid and Innovative Method to Assess Gastrointestinal Symptoms.

Abdominal pain is a chronic condition experienced by approximately 20% of individuals in the United States. The purpose of the study was to assess the validity of the Gastrointestinal Pain Pointer as a measure of abdominal pain intensity. A prospective longitudinal time-series study design was utilized. The sample included 93 outpatients (58.1% female). Participants met Rome III criteria for irritable bowel syndrome (n = 32) or were healthy controls (n = 61). The Gastrointestinal Pain Pointer, a new electronic pain assessment tool, was used to assess self-reported abdominal pain intensity among participants before and after ingestion of an intestinal permeability test solution across 11 time points over a 5-hour time period. The results were compared with the Short-Form McGill Pain Questionnaire. The Gastrointestinal Pain Pointer was found to be valid in the assessment of abdominal pain intensity. The tool is a novel and valid measure of abdominal pain intensity that enhances the ability for clinicians to better quantify, in real time, patient-related pain outcomes for both clinical care and research.

Emerging Role of Nutri-Epigenetics in Inflammation and Cancer.

Nutrition is a factor involved in inflammation and a modulator of risk toward some cancers, and the complexity of linkages between dietary components and epigenetics mechanisms (e.g., DNA methylation, histone modification, chromatin remodeling) may affect the inflammation phenotype and the development of cancer. An increasing number of studies support the role of diet in cancer development, prevention, and treatment. Although current knowledge regarding nutri-epigenetics is expanding, more work is needed, and nurse scientists have the potential to significantly contribute to the expansion of this knowledge.

A 14-day Dexamethasone timecourse exposure in Caco-2 monolayers results in differential expression of tight-junction and cytoskeleton regulatory pathway genes

Glucocorticoid (GC) hormones are modulators of endogenous stress responses and are important pharmaceuticals for inflammatory and autoimmune diseases. The gastrointestinal epithelium is a significant tissue target of Glucocorticoids; perturbation of epithelial barrier function during the endogenous stress response plays a major role in the pathophysiology of inflammatory bowel disease. Epithelial permeability and barrier function are mediated by the tight junction protein complex, with a network of molecular/cellular interactions occurring between the actin cytoskeleton, RhoGTPase, Akt/PI3K and growth factor receptor, and inflammatory cytokine signaling, influencing the establishment of a partial-EMT phenotype. To improve our understanding of GC-responsive gene expression in a gastrointestinal epithelial context, we tested polarized Caco-2 monolayer cultures during at 30-day timecourse, with 15-days of continuous Dexamethasone exposure. Trans-epithelial resistance (TEER) was recorded to provide a physiological quantification of barrier function during the timecourse treatments. Presence of intracellular glucocorticoid hormone activates the human glucocorticoid receptor (GCR, human NR3C1 gene) transcription factor, resulting in transcriptional activation and repression of various GCR-responsive genes. We tested for differential gene expression with a multiplexed panel of 250 gene expression panel using the Nanostring nCounter system. Gene panel selection was based on membership of genes in canonical KEGG pathways for tight-junction, adherens junction, focal adhesion, actin cytoskeleton regulation, and colorectal cancer. Our TEER results confirm, as previously reported, that long-term Dexamethasone exposure results in decreased permeability in Caco-2 monolayers ~day 20-25. Culture age and Dexamethasone exposure both contributed to differential gene expression for cell-cell junction, protein kinases, survival- and cancer-associated genes, reported here in the context of their corresponding KEGG pathway representations. The findings give evidence for GC- and time-associated patterns of transcriptional response and provide further insight into long-term glucocorticoid-associated physiological effects on the gastrointestinal epithelia. Future research utilizing more advanced cell culture methods will use this data-set as a reference.The Caco-2 cell line has served a historically important role as in vitro model for molecular and cellular biology of polarized intestinal epithelia, including for effects of glucocorticoid hormone Dexamethasone. Glucocorticoid hormones modulate the endogenous stress response and are important pharmaceuticals for inflammatory diseases including IBD, yet while they significantly affect immune cells, less is known about their specific effects upon epithelial cells and specific effect on epithelial permeability. Previous research showed that DEX exposure does not immediately produce a quantitative effect, but only after a prolonged treatment >10 days. Culture age itself causes marked effects in these non-renewing cell layers which acts as a confounding variable for observed DEX results. To improve resolution of GC-responsive gene expression in this context, we tested polarized Caco-2 monolayer cultures during at 30-day timecourse, with ~15-days of continuous Dexamethasone exposure. We tested differential gene expression using a 250-plex gene expression panel with the Nanostring nCounter® system, with multiple replicates collected periodically over the timecourse. Gene panel was selectively enriched a-priori for KEGG pathway annotations from tight-junction, actin cytoskeleton regulation, colorectal cancer pathways and others, allowing highly focused, gene-set pathway enrichment analyses. Nanostring nSolver™ data modelling algorithm uses an optimization algorithm and mixture negative binomial model to factor for Time and DEX covariate effects during determination of DE. Analysis identifies strong, culture age-associated “EMT-like” signature with upregulation of actomyosin contraction and integrins, while DEX treatment is associated with a subtler, yet significant counter-signal with suppression of actomyosin genes, and selective DE for different RTKs.

Colon Epithelial MicroRNA Network in Fatty Liver

Background & Aims Intestinal barrier alterations are associated with fatty liver (FL) and metabolic syndrome (MetS), but microRNA (miR) signaling pathways in MetS-FL pathogenesis remain unclear. This study investigates an epithelial-focused miR network in colorectal cell models based on the previously reported MetS-FL miR trio of hsa-miR-142-3p, hsa-miR-18b, and hsa-miR-890. Methods Each miR mimic construct of MetS-FL miR trio was transfected into human colorectal cells, CRL-1790 or Caco-2. Global miRNome changes posttransfection were profiled (nCounter® Human v3 miRNA, NanoString Technologies). Changes in barrier (transepithelial electrical resistance, TEER) and epithelial cell junction structure (Occludin and Zona Occludens-1/ZO-1 immunofluorescence staining-confocal microscopy) were examined pre- and posttransfection in Caco-2 cell monolayers. A signaling network was constructed from the MetS-FL miR trio, MetS-FL miR-induced colorectal miRNome changes, ZO-1, and Occludin. Results Transfection of CRL-1790 cells with each MetS-FL miR mimic led to global changes in the cellular miRNome profile, with 288 miRs being altered in expression by more than twofold. Eleven miRs with known cytoskeletal and metabolic roles were commonly altered in expression by all three miR mimics. Transfection of Caco-2 cell monolayers with each MetS-FL miR mimic induced barrier-associated TEER variations and led to structural modifications of ZO-1 and Occludin within epithelial cell junctions. Pathway analysis incorporating the MetS-FL miR trio, eleven common target miRs, ZO-1, and Occludin revealed a signaling network centered on TNF and AKT2, which highlights injury, inflammation, and hyperplasia. Conclusions Colon-specific changes in epithelial barriers, cell junction structure, and a miRNome signaling network are described from functional studies of a MetS-FL miR trio signature.

Eating Behavior, Stress, and Adiposity: Discordance Between Perception and Physiology

The purpose of the study was to examine the interrelationships among stress, eating behavior, and adiposity in a cohort of normal- and overweight individuals. Clinical markers of physiological stress (fasting serum cortisol) and adiposity (body mass index [BMI] and percent body fat) were obtained from participants selected for a natural history protocol (n = 107). Self-reported data on eating behavior (using the Three-Factor Eating Questionnaire subscales such as Cognitive Restraint, Disinhibition, and Hunger) and psychological stress (via the Perceived Stress Scale) were evaluated. Demographic information was incorporated using principal component analysis, which revealed sex- and weight-based differences in stress, adiposity, and eating behavior measures. Following a cross-sectional and descriptive analysis, significant correlations were found between the Disinhibition and Hunger eating behavior subscales and measures of adiposity including BMI (r = .30, p = .002 and r = .20, p = .036, respectively) and percent body fat (r = .43, p = .000 and r = .22, p = .022, respectively). Relationships between stress measures and eating behavior were also evident in the analysis. Disinhibition and Hunger correlated positively with perceived stress (r = .32, p .001 and r = .26, p = .008, respectively). However, Disinhibition varied inversely with serum cortisol levels (r = −.25, p = .009). Future studies are warranted to better understand this paradox underlying the effects of perceived and physiological stress on eating behavior.