Ziverel for PPI-refractory reflux symptoms: efficacy and mechanisms of action in humans.

OBJECTIVES: It is thought that esophageal hypersensitivity in combination with an impaired mucosal barrier function contributes to PPI-resistant reflux symptoms. Ziverel, a bioadhesive agent that coats the esophageal wall, was shown to have a positive effect on reflux symptoms. However, the mechanisms of action are unclear. We aimed to assess the effect of Ziverel on esophageal sensitivity to acid and mucosal barrier function. METHODS: We performed a double-blind randomized placebo-controlled crossover trial in PPI-refractory patients with reflux symptoms. Patients were assigned (1:1) to 14 days of Ziverel followed by 14 days of placebo or opposite treatment order. The effect was evaluated using acid perfusion tests, an upper endoscopy with electrical tissue impedance spectroscopy (ETIS) and esophageal biopsies. The primary outcome was the esophageal sensitivity based on perfusion sensitivity score. Secondary outcomes included mucosal barrier function and reflux symptoms and correlations between the different outcomes. RESULTS: Perfusion sensitivity score was not significantly different during treatment with Ziverel (106 (73-115)) and placebo (102 (67-110)) (p = 0.508) along with total RDQ score (2.6 (1.9-3.3) vs 2.8 (1.6-3.5) p = 0.456). ETIS showed comparable values during treatment with Ziverel (13514 (8846-19734)Ω·m) and placebo (13217 (9127-24942)Ω·m (p = 0.650)). Comparing Ziverel and placebo no difference was seen in transepithelial electrical resistance (TEER) 203 (163-267) Ω.cm2 vs 205 (176-240) Ω.cm2 (p = 0.445) and fluorescein flux 775 (17-6964) nmol/cm2/h vs 187 (4-12209) nmol/cm2/h (p = 0.638). CONCLUSION: Ziverel did not show a benefit on acid sensitivity, reflux symptoms or esophageal mucosal integrity compared to placebo in PPI-refractory patients with reflux symptoms.Trial registration: Netherlands Trial Register number: NL7670.

Candida spp. in Human Intestinal Health and Disease: More than a Gut Feeling.

Fungi are an essential part of the normal collection of intestinal microorganisms, even though their collective abundance comprises only 0.1-1% of all fecal microbes. The composition and role of the fungal population is often studied in relation to early-life microbial colonization and development of the (mucosal) immune system. The genus Candida is frequently described as one of the most abundant genera, and altered fungal compositions (including elevated abundance of Candida spp.) have been linked with intestinal diseases such as inflammatory bowel disease and irritable bowel syndrome. These studies are performed using both culture-dependent and genomic (metabarcoding) techniques. In this review, we aimed to summarize existing data on intestinal Candida spp. colonization in relation to intestinal disease and provide a brief overview of the biological and technical challenges in this field, including the recently described role of sub-species strain variation of intestinal Candida albicans. Together, the evidence for a contributing role of Candida spp. in pediatric and adult intestinal disease is quickly expanding, even though technical and biological challenges may limit full understanding of host-microbe interactions.

Fecal Filobasidium Is Associated with Clinical Remission and Endoscopic Response following Fecal Microbiota Transplantation in Mild-to-Moderate Ulcerative Colitis.

Fecal microbiota transplantation (FMT) has the potential to restore (bacterial and fungal) microbial imbalance in ulcerative colitis (UC) patients and contribute to disease remission. Here, we aimed to identify fecal fungal species associated with the induction of clinical remission and endoscopic response to FMT for patients with mild-to-moderate ulcerative colitis. We analyzed the internal transcribed spacer 1 (ITS1)-based mycobiota composition in fecal samples from patients (n = 31) and donors (n = 7) that participated previously in a double-blinded randomized control trial evaluating the efficacy of two infusions of donor FMT compared with autologous FMT. The abundance of the yeast genus Filobasidium in fecal material used for transplantation was shown to correlate with clinical remission following FMT, irrespective of its presence in the material of donor or autologous fecal microbiota transfer. The amplified sequence variants within the genus Filobasidium most closely resembled Filobasidium magnum. Monocyte-derived macrophages and HT29 epithelial cells were stimulated with fungal species. Especially Filobasidium floriforme elicited an IL10 response in monocyte-derived macrophages, along with secretion of other cytokines following stimulation with other Filobasidium species. No effect of Filobasidium spp. was seen on epithelial wound healing in scratch assays. In conclusion, the enriched presence of Filobasidium spp. in donor feces is associated with the positive response to FMT for patients with UC and hence it may serve as a predictive fungal biomarker for successful FMT.

Genetic and phenotypic diversity of fecal Candida albicans strains in irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a common disorder characterized by chronic abdominal pain and changes in bowel movements. Visceral hypersensitivity is thought to be responsible for pain complaints in a subset of patients. In an IBS-like animal model, visceral hypersensitivity was triggered by intestinal fungi, and lower mycobiota α-diversity in IBS patients was accompanied by a shift toward increased presence of Candida albicans and Saccharomyces cerevisiae. Yet, this shift was observed in hypersensitive as well as normosensitive patients and diversity did not differ between IBS subgroups. The latter suggests that, when a patient changes from hyper- to normosensitivity, the relevance of intestinal fungi is not necessarily reflected in compositional mycobiota changes. We now confirmed this notion by performing ITS1 sequencing on an existing longitudinal set of fecal samples. Since ITS1 methodology does not recognize variations within species, we next focused on heterogeneity within cultured healthy volunteer and IBS-derived C. albicans strains. We observed inter- and intra-individual genomic variation and partial clustering of strains from hypersensitive patients. Phenotyping showed differences related to growth, yeast-to-hyphae morphogenesis and gene expression, specifically of the gene encoding fungal toxin candidalysin. Our investigations emphasize the need for strain-specific cause-and-effect studies within the realm of IBS research.

Dietary Curdlan Enhances Bifidobacteria and Reduces Intestinal Inflammation in Mice.

ß-glucan consumption is known for its beneficial health effects, but the mode of action is unclear. While humans and mice lack the required enzymes to digest ß-glucans, certain intestinal microbes can digest ß-glucans, triggering gut microbial changes. Curdlan, a particulate ß-glucan isolated from Alcaligenes faecalis, is used as a food additive. In this study we determined the effect of curdlan intake in mice on the intestinal microbiota and dextran sodium sulfate (DSS)-induced intestinal inflammation. The effect of curdlan on the human intestinal microbiota was assessed using i-screen, an assay for studying anaerobic microbial interactions. Mice received oral gavage with vehicle or curdlan for 14 days followed by DSS for 7 days. The curdlan-fed group showed reduced weight loss and colonic inflammation compared to the vehicle-fed group. Curdlan intake did not induce general microbiota community changes, although a specific Bifidobacterium, closely related to Bifidobacterium choerinum, was observed to be 10- to 100-fold more prevalent in the curdlan-fed group under control and colitis conditions, respectively. When tested in i-screen, curdlan induced a global change in the microbial composition of the healthy intestinal microbiota from a human. Overall, these results suggest that dietary curdlan induces microbiota changes that could reduce intestinal inflammation.

Glial A2B Adenosine Receptors Modulate Abnormal Tachykininergic Responses and Prevent Enteric Inflammation Associated with High Fat Diet-Induced Obesity.

The role played by adenosine A2B receptors (A2BRs) in the regulation of enteric glial cell (EGC) functions remains unclear. This study was aimed at investigating the involvement of A2BRs in the control of EGC functions in a model of obesity. C57BL/6 mice were fed with standard diet (SD) or high fat diet (HFD) for eight weeks. Colonic tachykininergic contractions were recorded in the presence of BAY60-6583 (A2BRs agonist), MRS1754 (A2BRs antagonist), and the gliotoxin fluorocitrate. Immunofluorescence distribution of HuC/D, S100ß, and A2BRs was assessed in whole mount preparations of colonic myenteric plexus. To mimic HFD, EGCs were incubated in vitro with palmitate (PA) and lipopolysaccharide (LPS), in the absence or in the presence of A2BR ligands. Toll-like receptor 4 (TLR4) expression was assessed by Western blot analysis. Interleukin-1ß (IL-1ß), substance P (SP), and glial cell derived neurotrophic factor (GDNF) release were determined by enzyme-linked immunosorbent assay (ELISA) assays. MRS1754 enhanced electrically evoked tachykininergic contractions of colonic preparations from HFD mice. BAY60-6583 decreased the evoked tachykininergic contractions, with higher efficacy in HFD mice. Such effects were blunted upon incubation with fluorocitrate. In in vitro experiments on EGCs, PA and LPS increased TLR4 expression as well as IL-1ß, GDNF, and SP release. Incubation with BAY60-6583 reduced TLR4 expression as well as IL-1ß, GDNF, and SP release. Such effects were blunted by MRS1754. The present results suggest that A2BRs, expressed on EGCs, participate in the modulation of enteric inflammation and altered tachykininergic responses associated with obesity, thus representing a potential therapeutic target.

Microbiota-neuroimmune cross talk in stress-induced visceral hypersensitivity of the bowel.

Visceral hypersensitivity of the lower gastrointestinal tract, defined as an increased response to colorectal distension, frequently prompts episodes of debilitating abdominal pain in irritable bowel syndrome (IBS). Although the pathophysiology of IBS is not yet fully elucidated, it is well known that stress is a major risk factor for development and acts as a trigger of pain sensation. Stress modulates both immune responses as well as the gut microbiota and vice versa. Additionally, either microbes themselves or through involvement of the immune system, activate or sensitize afferent nociceptors. In this paper, we review current knowledge on the influence of stress along the gut-brain-microbiota axis and exemplify relevant neuroimmune cross talk mechanisms in visceral hypersensitivity, working toward understanding how gut microbiota-neuroimmune cross talk contributes to visceral pain sensation in IBS patients.

Colonic dysmotility associated with high-fat diet-induced obesity: Role of enteric glia.

The present study was designed to examine the role of enteric glial cells (EGCs) in colonic neuromuscular dysfunctions in a mouse model of high-fat diet (HFD)-induced obesity. C57BL/6J mice were fed with HFD or standard diet (SD) for 1, 2, or 8 weeks. Colonic interleukin (IL)-1ß, IL-6, and malondialdehyde (MDA) levels were measured. Expression of occludin in colonic tissues was examined by western blot. Substance P (SP), S100ß, GFAP, and phosphorylated mitogen-activated protein kinase 1 (pERK) were assessed in whole mount specimens of colonic plexus by immunohistochemistry. Colonic tachykininergic contractions, elicited by electrical stimulation or exogenous SP, were recorded in the presence or absence of fluorocitrate (FC). To mimic exposure to HFD, cultured EGCs were incubated with palmitate (PA) and/or lipopolysaccharide (LPS). SP and IL-1ß levels were assayed in the culture medium by ELISA. HFD mice displayed an increase in colonic IL-1ß and MDA, and a reduction of occludin at week 2. These changes occurred to a greater extent at week 8. In vitro electrically evoked tachykininergic contractions were enhanced in HFD mice after 2 or 8 weeks, and they were blunted by FC. Colonic IL-6 levels as well as substance P and S100ß density in myenteric ganglia of HFD mice were increased at week 8, but not at week 1 or 2. In cultured EGCs, co-incubation with palmitate plus LPS led to a significant increase in both SP and IL-1ß release. HFD-induced obesity is characterized by a hyperactivation of EGCs and is involved in the development of enteric motor disorders through an increase in tachykininergic activity and release of pro-inflammatory mediators.

Miltefosine treatment reduces visceral hypersensitivity in a rat model for irritable bowel syndrome via multiple mechanisms.

Irritable bowel syndrome (IBS) is a heterogenic, functional gastrointestinal disorder of the gut-brain axis characterized by altered bowel habit and abdominal pain. Preclinical and clinical results suggested that, in part of these patients, pain may result from fungal induced release of mast cell derived histamine, subsequent activation of sensory afferent expressed histamine-1 receptors and related sensitization of the nociceptive transient reporter potential channel V1 (TRPV1)-ion channel. TRPV1 gating properties are regulated in lipid rafts. Miltefosine, an approved drug for the treatment of visceral Leishmaniasis, has fungicidal effects and is a known lipid raft modulator. We anticipated that miltefosine may act on different mechanistic levels of fungal-induced abdominal pain and may be repurposed to IBS. In the IBS-like rat model of maternal separation we assessed the visceromotor response to colonic distension as indirect readout for abdominal pain. Miltefosine reversed post-stress hypersensitivity to distension (i.e. visceral hypersensitivity) and this was associated with differences in the fungal microbiome (i.e. mycobiome). In vitro investigations confirmed fungicidal effects of miltefosine. In addition, miltefosine reduced the effect of TRPV1 activation in TRPV1-transfected cells and prevented TRPV1-dependent visceral hypersensitivity induced by intracolonic-capsaicin in rat. Miltefosine may be an attractive drug to treat abdominal pain in IBS.

Rhamnogalacturonan, a chemically-defined polysaccharide, improves intestinal barrier function in DSS-induced colitis in mice and human Caco-2 cells.

Natural polysaccharides have emerged as an important class of bioactive compounds due their beneficial biological effects. Here we investigated the protective and healing effects of rhamnogalacturonan (RGal) isolated from Acmella oleracea (L.) R.K. Jansen leaves in an experimental model of intestinal inflammation in mice and in heterogeneous human epithelial colorectal adenocarcinoma cells (Caco-2). The findings demonstrated that RGal treatment for 7 days reduced the severity of DSS-induced colitis by protecting mice from weight loss, macroscopic damage and reduction of colon length. When compared to the DSS group, RGal also protected the colon epithelium and promoted the maintenance of mucosal enterocytes and mucus secreting goblet cells, in addition to conserving collagen homeostasis and increasing cell proliferation. In an in vitro barrier function assay, RGal reduced the cellular permeability after exposure to IL-1ß, while decreasing IL-8 secretion and claudin-1 expression and preserving the distribution of occludin. Furthermore, we also observed that RGal accelerated the wound healing in Caco-2 epithelial cell line. In conclusion, RGal ameliorates intestinal barrier function in vivo and in vitro and may represent an attractive and promising molecule for the therapeutic management of ulcerative colitis.

Abnormal Responses to Local Esophageal Food Allergen Injections in Adult Patients With Eosinophilic Esophagitis.

Skin tests and measurement of serum levels of immunoglobulin E do not accurately identify foods for elimination from the diets of patients with eosinophilic esophagitis (EoE). We investigated whether an esophageal prick test, in which the esophageal mucosa is challenged by local injection of allergen extracts, could identify individuals with esophageal sensitization. During endoscopy, 6 allergens were injected in the esophagus of 8 patients with EoE and 3 patients without EoE (controls). A second endoscopy was performed after 24 hours to evaluate delayed responses. Five of the 8 patients with EoE had evidence for an acute response (luminal obstruction and mucosal blanching); 2 other patients had a delayed wheal or flare reaction. No responses were observed in controls. We conclude that esophageal mucosal food allergen injections induce acute and/or delayed responses in patients with EoE but not controls. The esophageal prick test deserves further exploration because it may guide elimination diets.

Intestinal Fungal Dysbiosis Is Associated With Visceral Hypersensitivity in Patients With Irritable Bowel Syndrome and Rats.

BACKGROUND & AIMS: Visceral hypersensitivity is one feature of irritable bowel syndrome (IBS). Bacterial dysbiosis might be involved in the activation of nociceptive sensory pathways, but there have been few studies of the role of the mycobiome (the fungal microbiome) in the development of IBS. We analyzed intestinal mycobiomes of patients with IBS and a rat model of visceral hypersensitivity. METHODS: We used internal transcribed spacer 1-based metabarcoding to compare fecal mycobiomes of 18 healthy volunteers with those of 39 patients with IBS (with visceral hypersensitivity or normal levels of sensitivity). We also compared the mycobiomes of Long-Evans rats separated from their mothers (hypersensitive) with non-handled (normally sensitive) rats. We investigated whether fungi can cause visceral hypersensitivity using rats exposed to fungicide (fluconazole and nystatin). The functional relevance of the gut mycobiome was confirmed in fecal transplantation experiments: adult maternally separated rats were subjected to water avoidance stress (to induce visceral hypersensitivity), then given fungicide and donor cecum content via oral gavage. Other rats subjected to water avoidance stress were given soluble ß-glucans, which antagonize C-type lectin domain family 7 member A (CLEC7A or DECTIN1) signaling via spleen-associated tyrosine kinase (SYK), a SYK inhibitor to reduce visceral hypersensitivity, or vehicle (control). The sensitivity of mast cells to fungi was tested with mesenteric windows (ex vivo) and the human mast cell line HMC-1. RESULTS: α diversity (Shannon index) and mycobiome signature (stability selection) of both groups of IBS patients differed from healthy volunteers, and the mycobiome signature of hypersensitive patients differed from that of normally sensitive patients. We observed mycobiome dysbiosis in rats that had been separated from their mothers compared with non-handled rats. Administration of fungicide to hypersensitive rats reduced their visceral hypersensitivity to normal levels of sensitivity. Administration of cecal mycobiomes from rats that had been separated from their mothers (but not non-handled mycobiome) restored hypersensitivity to distension. Administration of soluble ß-glucans or a SYK inhibitor reduced visceral hypersensitivity, compared with controls. Particulate ß-glucan (a DECTIN-1 agonist) induced mast cell degranulation in mesenteric windows and HMC-1 cells responded to fungal antigens by release of histamine. CONCLUSIONS: In an analysis of patients with IBS and controls, we associated fungal dysbiosis with IBS. In studies of rats, we found fungi to promote visceral hypersensitivity, which could be reduced by administration of fungicides, soluble ß-glucans, or a SYK inhibitor. The intestinal fungi might therefore be manipulated for treatment of IBS-related visceral hypersensitivity.

Acetylcholine-producing T cells in the intestine regulate antimicrobial peptide expression and microbial diversity.

The cholinergic anti-inflammatory pathway reduces systemic tumor necrosis factor (TNF) via acetylcholine-producing memory T cells in the spleen. These choline acetyltransferase (ChAT)-expressing T cells are also found in the intestine, where their function is unclear. We aimed to characterize these cells in mouse and human intestine and delineate their function. We made use of the ChAT-enhanced green fluorescent protein (eGFP) reporter mice. CD4Cre mice were crossed to ChATfl/fl mice to achieve specific deletion of ChAT in CD4+ T cells. We observed that the majority of ChAT-expressing T cells in the human and mouse intestine have characteristics of Th17 cells and coexpress IL17A, IL22, and RORC The generation of ChAT-expressing T cells was skewed by dendritic cells after activation of their adrenergic receptor ß2 To evaluate ChAT T cell function, we generated CD4-specific ChAT-deficient mice. CD4ChAT-/- mice showed a reduced level of epithelial antimicrobial peptides lysozyme, defensin A, and ang4, which was associated with an enhanced bacterial diversity and richness in the small intestinal lumen in CD4ChAT-/- mice. We conclude that ChAT-expressing T cells in the gut are stimulated by adrenergic receptor activation on dendritic cells. ChAT-expressing T cells may function to mediate the host AMP secretion, microbial growth and expansion.

Mucosal integrity and sensitivity to acid in the proximal esophagus in patients with gastroesophageal reflux disease.

Acid reflux episodes that extend to the proximal esophagus are more likely to be perceived. This suggests that the proximal esophagus is more sensitive to acid than the distal esophagus, which could be caused by impaired mucosal integrity in the proximal esophagus. Our aim was to explore sensitivity to acid and mucosal integrity in different segments of the esophagus. We used a prospective observational study, including 12 patients with gastroesophageal reflux disease (GERD). After stopping acid secretion-inhibiting medication, two procedures were performed: an acid perfusion test and an upper endoscopy with electrical tissue impedance spectroscopy and esophageal biopsies. Proximal and distal sensitivity to acid and tissue impedance were measured in vivo, and mucosal permeability and epithelial intercellular spaces at different esophageal levels were measured in vitro. Mean lag time to heartburn perception was much shorter after proximal acid perfusion (0.8 min) than after distal acid perfusion (3.9 min) (P = 0.02). Median in vivo tissue impedance was significantly lower in the distal esophagus (4,563 Ω·m) compared with the proximal esophagus (8,170 Ω·m) (P = 0.002). Transepithelial permeability, as measured by the median fluorescein flux was significantly higher in the distal (2,051 nmol·cm(-2)·h(-1)) than in the proximal segment (368 nmol·cm(-2)·h(-1)) (P = 0.033). Intercellular space ratio and maximum heartburn intensity were not significantly different between the proximal and distal esophagus. In GERD patients off acid secretion-inhibiting medication, acid exposure in the proximal segment of the esophagus provokes symptoms earlier than acid exposure in the distal esophagus, whereas mucosal integrity is impaired more in the distal esophagus. These findings indicate that the enhanced sensitivity to proximal reflux episodes is not explained by increased mucosal permeability.

Absence of diurnal variation in visceromotor response to colorectal distention in normal Long Evans rats.

BACKGROUND: Enhanced colorectal sensitivity (i.e. visceral hypersensitivity) is thought to be a pathophysiological mechanism in irritable bowel syndrome (IBS). In healthy men a circadian variation in rectal perception to colonic distention was described. Disturbed day and night rhythms, which occur in shift work and trans meridian flights, are associated with the prevalence of IBS. This raises the question whether disruptions of circadian control are responsible for the observed pathology in IBS. Prior to investigating altered rhythmicity in relation to visceral hypersensitivity in a rat model for IBS, it is relevant to establish whether normal rats display circadian variation similar to healthy men.  METHODOLOGY AND FINDINGS: In rodents colorectal distension leads to reproducible contractions of abdominal musculature. We used quantification of this so called visceromotor response (VMR) by electromyography (EMG) to assess visceral sensitivity in rats. We assessed the VMR in normal male Long Evans rats at different time points of the light/dark cycle. Although a control experiment with male maternal separated rats confirmed that intentionally inflicted (i.e. stress induced) changes in VMR can be detected, normal male Long Evans rats showed no variation in VMR along the light/dark cycle in response to colorectal distension. CONCLUSIONS: In the absence of a daily rhythm of colorectal sensitivity in normal control rats it is not possible to investigate possible aberrancies in our rat model for IBS.

Histological Response to Fluticasone Propionate in Patients With Eosinophilic Esophagitis Is Associated With Improved Functional Esophageal Mucosal Integrity.

OBJECTIVES: The esophageal mucosal integrity is impaired in patients with eosinophilic esophagitis (EoE). We aimed to evaluate the effect of fluticasone propionate on inflammation and functional and structural markers of esophageal mucosal barrier integrity in adult patients with EoE. METHODS: In this prospective study, we included 15 EoE patients (median age (IQR), 43 (30-45) years). Patients underwent upper endoscopy before and after an 8-week course of swallowed fluticasone propionate 500 µg BID. Several parameters of esophageal mucosal barrier integrity were evaluated: esophageal electrical tissue impedance in vivo during endoscopy, transepithelial electrical resistance (TER) and transepithelial molecule flux in Ussing chambers using esophageal biopsy specimens, and intercellular spaces as a structural marker of permeability using electron microscopy. Esophageal eosinophils and mast cells were counted, and expression of inflammatory cytokines and barrier integrity proteins was investigated using qPCR. Esophageal symptoms and signs were also assessed. RESULTS: Peak eosinophil and mast cell counts decreased significantly after fluticasone propionate treatment. The esophageal mucosal integrity increased substantially during treatment, as shown by increased extracellular impedance and TER (both P<0.01) and decreased transepithelial molecule flux in Ussing chambers (P<0.05). Whereas expression of genes encoding for inflammatory cytokines (IL5, IL13, eotaxin-3, periostin, TSLP) decreased after treatment, expression of genes encoding for barrier integrity proteins (filaggrin and desmoglein-1) increased. CONCLUSIONS: Fluticasone propionate treatment decreases eosinophilic inflammation and improves the esophageal mucosal barrier integrity in adult EoE patients. Improvement of the mucosal barrier integrity correlates with normalization of expression of desmoglein-1 and filaggrin marker genes.

Dietary Marine n-3 PUFAs Do Not Affect Stress-Induced Visceral Hypersensitivity in a Rat Maternal Separation Model.

BACKGROUND: Although never evaluated for efficacy, n-3 (ω-3) long-chain polyunsaturated fatty acids (LCPUFAs) are commercially offered as treatment for irritable bowel syndrome (IBS). OBJECTIVE: This study was designed to investigate, in a mast cell-dependent model for visceral hypersensitivity, whether this pathophysiologic mechanism can be reversed by dietary LCPUFA treatment via peroxisome proliferator-activated receptor γ (PPARG) activation. METHODS: Maternally separated rats were subjected to hypersensitivity-inducing acute stress at adult age. Reversal was attempted by protocols with tuna oil-supplemented diets [4% soy oil (SO) and 3% tuna oil (SO-T3) or 3% SO and 7% tuna oil (SO-T7)] and compared with control SO diets (7% or 10% SO) 4 wk after stress. The PPARG agonist rosiglitazone was evaluated in a 1 wk preventive protocol (30 mg · kg⁻¹ · d⁻¹). Erythrocytes were assessed to confirm LCPUFA uptake and tissue expression of lipoprotein lipase and glycerol kinase as indicators of PPARG activation. Colonic mast cell degranulation was evaluated by toluidine blue staining. In vitro, human mast cell line 1 (HMC-1) cells were pretreated with rosiglitazone, eicosapentaenoic acid, or docosahexaenoic acid, stimulated with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore or compound 48/80 and evaluated for tumor necrosis factor α (TNF-α) and ß-hexosaminidase release. RESULTS: Stress led to visceral hypersensitivity in all groups. Hypersensitivity was not reversed by SO-T3 or control treatment [prestress vs. 24 h poststress vs. posttreatment area under the curve; 76 ± 4 vs. 128 ± 12 (P < 0.05) vs. 115 ± 14 and 82 ± 5 vs. 127 ± 16 (P < 0.01) vs. 113 ± 19, respectively]. Comparison of SO-T7 with its control showed similar results [74 ± 6 vs. 103 ± 13 (P < 0.05) vs. 115 ± 17 and 66 ± 3 vs. 103 ± 10 (P < 0.05) vs. 117 ± 11, respectively]. Erythrocytes showed significant LCPUFA uptake in the absence of colonic PPARG activation. Rosiglitazone induced increased PPARG target gene expression, but did not prevent hypersensitivity. Mast cell degranulation never differed between groups. Rosiglitazone and LCPUFAs significantly reduced PMA/calcium ionophore-induced TNF-α release but not degranulation of HMC-1 cells. CONCLUSION: Dietary LCPUFAs did not reverse stress-induced visceral hypersensitivity in maternally separated rats. Although further research is needed, claims concerning LCPUFAs as a treatment option in IBS cannot be confirmed at this point and should be regarded with caution.

Proton pump inhibitors partially restore mucosal integrity in patients with proton pump inhibitor-responsive esophageal eosinophilia but not eosinophilic esophagitis.

BACKGROUND & AIMS: Histologic analysis is used to distinguish patients with proton pump inhibitor-responsive eosinophilia (PPI-REE) from those with eosinophilic esophagitis (EoE). It is not clear whether these entities have different etiologies. Exposure to acid reflux can impair the integrity of the esophageal mucosal. We proposed that patients with EoE and PPI-REE might have reflux-induced esophageal mucosal damage that promotes transepithelial flux of allergens. We therefore assessed the integrity of the esophageal mucosal in these patients at baseline and after PPI. METHODS: We performed a prospective study of 16 patients with suspected EoE and 11 controls. Patients had dysphagia, endoscopic signs of EoE, and esophageal eosinophilia (>15 eosinophils/high-power field [eos/hpf]). All subjects underwent endoscopy at baseline; endoscopy was performed again on patients after 8 weeks of treatment with high-dose esomeprazole. After PPI treatment, patients were diagnosed with EoE (>10 eos/hpf; n = 8) or PPI-REE (≤10 eos/hpf; n = 8). We evaluated the structure (intercellular spaces) and function (electrical tissue impedance, transepithelial electrical resistance, transepithelial molecule flux) of the esophageal mucosal barrier. RESULTS: Compared with controls, electrical tissue impedance and transepithelial electrical resistance were reduced in patients with EoE (P < .001 and P < .001, respectively) and PPI-REE (P = .01 and P = .06, respectively), enabling transepithelial small-molecule flux. PPI therapy partially restored these changes in integrity and inflammation in patients with PPI-REE, but not in those with EoE. CONCLUSIONS: The integrity of the esophageal mucosa is impaired in patients with EoE and PPI-REE, allowing transepithelial transport of small molecules. PPI therapy partially restores mucosal integrity in patients with PPI-REE, but not in those with EoE. Acid reflux might contribute to transepithelial allergen flux in patients with PPI-REE. Trialregister.nl number: NTR3480.

Stress-induced visceral hypersensitivity in maternally separated rats can be reversed by peripherally restricted histamine-1-receptor antagonists.

BACKGROUND: The histamine-1 receptor (H1R) antagonist ketotifen increased the threshold of discomfort in hypersensitive IBS patients. The use of peripherally restricted and more selective H1R antagonists may further improve treatment possibilities. We examined the use of fexofenadine and ebastine to reverse post-stress visceral hypersensitivity in maternally separated rats. METHODS: The visceromotor response to colonic distension was assessed in adult maternally separated and nonhandled rats pre- and 24 hours post water avoidance. Subsequently rats were treated with vehicle alone or different dosages of fexofenadine (1.8 and 18 mg/kg) or ebastine (0.1 and 1.0 mg/kg) and re-evaluated. Colonic tissue was collected to assess relative RMCP-2 and occludin expression levels by Western blot and histamine-1 receptor by RT-qPCR. ß-hexosaminidase release by RBL-2H3 cells was used to establish possible mast cell stabilizing properties of the antagonists. KEY RESULTS: Water avoidance only induced enhanced response to distension in maternally separated rats. This response was reversed by 1.8 and 18 mg/kg fexofenadine. Reversal was also obtained by 1.0 but not 0.1 mg/kg ebastine. RMCP-2 expression levels were comparable in these two ebastine treatment groups but occludin was significantly higher in 1.0 mg/kg treated rats. There were no differences in histamine-1 receptor expression between nonhandled and maternally separated rats. Fexofenadine but not ebastine showed mast cell stabilizing quality. CONCLUSIONS: Our results indicate that the peripherally restricted 2(nd) generation H1-receptor antagonists fexofenadine and ebastine are capable of reversing post stress visceral hypersensitivity in rat. These data justify future IBS patient trials with these well tolerated compounds.

Mucosal immune cell numbers and visceral sensitivity in patients with irritable bowel syndrome: is there any relationship?

OBJECTIVES: Repeated exposure to stress leads to mast cell degranulation, microscopic inflammation, and subsequent visceral hypersensitivity in animal models. To what extent this pathophysiological pathway has a role in patients with the irritable bowel syndrome (IBS) has not been properly investigated. The objective of this study was to assess the relationship between visceral hypersensitivity, microscopic inflammation, and the stress response in IBS. METHODS: Microscopic inflammation of the colonic mucosa was evaluated by immunohistochemistry in 66 IBS patients and 20 healthy volunteers (HV). Rectal sensitivity was assessed by a barostat study using an intermittent pressure-controlled distension protocol. Salivary cortisol to a psychological stress was measured to assess the stress response. RESULTS: Compared with HV, mast cells, T cells, and macrophages were decreased in IBS patients. Similarly, λ-free light chain (FLC)-positive mast cells were decreased but not immunoglobulin E (IgE)- and IgG-positive mast cells. There were no differences between hypersensitive and normosensitive IBS patients. No relation was found between any of the immune cells studied and the thresholds of discomfort, urge, first sensation, or IBS symptoms (e.g., abdominal pain, stool-related complaints, bloating). Finally, stress-related symptoms and the hypothalamic-pituitary-adrenal-axis response to stress were not correlated with the number of mast cells or the presence of visceral hypersensitivity. CONCLUSIONS: Although the number of mast cells, macrophages, T cells, and λFLC-positive mast cells is decreased in IBS compared with HV, this is not associated with the presence of visceral hypersensitivity or abnormal stress response. Our data question the role of microscopic inflammation as an underlying mechanism of visceral hypersensitivity, but rather suggest dysregulation of the mucosal immune system in IBS.