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The intestinal barrier is essential in human health and constitutes the interface between the outside and the internal milieu of the body. A functional intestinal barrier allows absorption of nutrients and fluids but simultaneously prevents harmful substances like toxins and bacteria from crossing the intestinal epithelium and reaching the body. An altered intestinal permeability, a sign of a perturbed barrier function, has during the last decade been associated with several chronic conditions, including diseases originating in the gastrointestinal tract but also diseases such as Alzheimer and Parkinson disease. This has led to an intensified interest from researchers with diverse backgrounds to perform functional studies of the intestinal barrier in different conditions. Intestinal permeability is defined as the passage of a solute through a simple membrane and can be measured by recording the passage of permeability markers over the epithelium via the paracellular or the transcellular route. The methodological tools to investigate the gut barrier function are rapidly expanding and new methodological approaches are being developed. Here we outline and discuss, in vivo, in vitro and ex vivo techniques and how these methods can be utilized for thorough investigation of the intestinal barrier.

The human gut relies on several cellular and molecular mechanisms to allow for an intact and dynamical intestinal barrier. Normally, only small amounts of luminal content pass the mucosa, however, if the control is broken it can lead to enhanced passage, which might damage the mucosa, leading to pathological conditions, such as inflammatory bowel disease (IBD). It is well established that genetic, environmental, and immunological factors all contribute in the pathogenesis of IBD, and a disturbed intestinal barrier function has become a hallmark of the disease. Genetical studies support the involvement of intestinal barrier as several susceptibility genes for IBD encode proteins with key functions in gut barrier and homeostasis. IBD patients are associated with loss in bacterial diversity and shifts in the microbiota, with a possible link to local inflammation. Furthermore, alterations of immune cells and several neuro-immune signaling pathways in the lamina propria have been demonstrated. An inappropriate immune activation might lead to mucosal inflammation, with elevated secretion of pro-inflammatory cytokines that can affect the epithelium and promote a leakier barrier. This review will focus on the main cells and molecular mechanisms in IBD and how these can be targeted in order to improve intestinal barrier function and reduce inflammation.

Background The relationship between intestinal epithelial integrity and the development of intestinal disease is of increasing interest. A reduction in mucosal integrity has been associated with ulcerative colitis, Crohn’s disease and potentially could have links with colorectal cancer development. The Ussing chamber system can be utilised as a valuable tool for measuring gut integrity. Here we describe step-by-step methodology required to measure intestinal permeability of both mouse and human colonic tissue samples ex vivo, using the latest equipment and software. This system can be modified to accommodate other tissues. Methods An Ussing chamber was constructed and adapted to support both mouse and human tissue to measure intestinal permeability, using paracellular flux and electrical measurements. Two mouse models of intestinal inflammation (dextran sodium sulphate treatment and T regulatory cell depletion using C57BL/6-FoxP3 DTR mice) were used to validate the system along with human colonic biopsy samples. Results Distinct regional differences in permeability were consistently identified within mouse and healthy human colon. In particular, mice showed increased permeability in the mid colonic region. In humans the left colon is more permeable than the right. Furthermore, inflammatory conditions induced chemically or due to autoimmunity reduced intestinal integrity, validating the use of the system. Conclusions The Ussing chamber has been used for many years to measure barrier function. However, a clear and informative methods paper describing the setup of modern equipment and step-by-step procedure to measure mouse and human intestinal permeability isn’t available. The Ussing chamber system methodology we describe provides such detail to guide investigation of gut integrity.

Peyer's patches (PPs) are collections of lymphoid follicles in the small intestine, responsible for scanning the intestinal content for foreign antigens such as soluble molecules, particulate matter as well as intact bacteria and viruses. The immune cells of the patch are separated from the intestinal lumen by a single layer of epithelial cells, the follicle-associated epithelium (FAE). This epithelium covers the dome of the follicle and contains enterocyte-like cells and M cells, which are particularly specialized in taking up antigens from the gut. However, the presence and number of goblet cells as well as the presence of mucus on top of the FAE is controversial. When mouse ileal PPs were mounted in a horizontal Ussing-type chamber, we could observe a continuous mucus layer at mounting and new, easily removable mucus was released from the villi on the patch upon stimulation. Confocal imaging using fluorescent beads revealed a penetrable mucus layer covering the domes. Furthermore, immunostaining of FAE from mice, rats and humans with a specific antibody against the main component of intestinal mucus, the MUC2 mucin, clearly identify mucin-containing goblet cells. Transmission electron micrographs further support the identification of mucus releasing goblet cells on the domes of PPs in these species.

Background The T cell cytokine profile is a key prognostic indicator of post-surgical outcome for colorectal cancer (CRC). Whilst T H 1 (IFN-γ + ) cell-mediated responses generated in CRC are well documented and are associated with improved survival, antigen-specific T H 17 (IL-17A + ) responses have not been similarly measured. Methods We sought to determine the cytokine profile of circulating tumour antigen-(5T4/CEA) specific T cells of 34 CRC patients to address whether antigen-specific IL-17A responses were detectable and whether these were distinct to IFN-γ responses. Results As with IFN-γ-producing T cells, anti-5T4/CEA T H 17 responses were detectable predominantly in early stage (TNM I/II) CRC patients. Moreover, whilst IL-17A was always produced in association with IFN-γ, this release was mainly from two distinct T cell populations rather than by ‘dual producing’ T cells. Patients mounting both tumour-specific T H 1 + /T H 17 + responses exhibited prolonged relapse-free survival. Conclusions Tumour antigen-specific T H 17 responses play a beneficial role in preventing post-operative colorectal tumour recurrence.

Food-grade titanium dioxide (fgTiO 2 ) is a bio-persistent particle under intense regulatory scrutiny. Yet paradoxically, the only known cell reservoirs for fgTiO 2 are graveyard intestinal pigment cells which are metabolically and immunologically quiescent. Here we identify immunocompetent cell targets of fgTiO 2 in humans, most notably in the subepithelial dome region of intestinal Peyer’s patches. Using multimodal microscopies with single-particle detection and per-cell / vesicle image analysis we achieve correlative dosimetry, quantitatively recapitulating human cellular exposures in the ileum of mice fed a fgTiO 2 -containing diet. Epithelial microfold cells selectively funnel fgTiO 2 into LysoMac and LysoDC cells with ensuing accumulation. Notwithstanding, proximity extension analyses for 92 protein targets reveal no measureable perturbation of cell signalling pathways. When chased with oral ΔaroA - Salmonella , pro-inflammatory signalling is confirmed, but no augmentation by fgTiO 2 is revealed despite marked same-cell loading. Interestingly, Salmonella causes the fgTiO 2 -recipient cells to migrate within the patch and, sporadically, to be identified in the lamina propria, thereby fully recreating the intestinal tissue distribution of fgTiO 2 in humans. Immunocompetent cells that accumulate fgTiO 2 in vivo are now identified and we demonstrate a mouse model that finally enables human-relevant risk assessments of ingested, bio-persistent (nano)particles. Food-grade titanium dioxide (fgTiO 2 ) is a biopersistent particle, but neither the target cells nor the risks of fgTiO 2 are well understood. Here, the authors identify immunocompetent cell targets of fgTiO 2 in humans, most notably in the subepithelial dome region of intestinal Peyer’s patches, and demonstrate a mouse model allowing human-relevant risk assessments of ingested, bio-persistent (nano)particles.

Vasoactive intestinal polypeptide (VIP) is a neuroendocrine peptide distributed throughout the human body, including the CNS, where it is particularly abundant in brain regions associated with anxiety and depression. Based on earlier studies indicating that peripheral VIP may cross through the blood–brain barrier, we hypothesized plasma VIP levels to be associated with symptoms of anxiety and depression, as well as brain volume and resting-state functional connectivity in the amygdala, hippocampus, parahippocampus, and orbitofrontal cortex. Plasma VIP concentrations and anxiety/depression symptoms were measured in 37 healthy females. Functional and structural magnetic resonance imaging were used to evaluate functional connectivity and brain volume respectively, and their associations with VIP concentrations within brain regions associated with anxiety and depression. Negative correlations were found between VIP levels and symptoms of anxiety ( r  = − 0.44, p  = 0.002) and depression ( r  = − 0.50, p  = 0.001). Functional connectivity demonstrated significant VIP-dependent positive associations between the amygdala seed region with both the right parahippocampus ( t (33)  = 3.1 , p FDR  = 0.02) and right lateral orbitofrontal cortex (OFC; t (33)  = 2.9 , p FDR  = 0.02). Moreover, VIP concentrations were significantly, positively correlated with brain volume in the left amygdala ( r  = 0.28, p  = 0.007) and left lateral OFC ( r  = 0.29, p  = 0.004). The present findings highlight a potential role for VIP in the neurobiology of affective symptoms.

Improved biomarkers are needed for pediatric inflammatory bowel disease. Here we identify a diagnostic lipidomic signature for pediatric inflammatory bowel disease by analyzing blood samples from a discovery cohort of incident treatment-naïve pediatric patients and validating findings in an independent inception cohort. The lipidomic signature comprising of only lactosyl ceramide (d18:1/16:0) and phosphatidylcholine (18:0p/22:6) improves the diagnostic prediction compared with high-sensitivity C-reactive protein. Adding high-sensitivity C-reactive protein to the signature does not improve its performance. In patients providing a stool sample, the diagnostic performance of the lipidomic signature and fecal calprotectin, a marker of gastrointestinal inflammation, does not substantially differ. Upon investigation in a third pediatric cohort, the findings of increased lactosyl ceramide (d18:1/16:0) and decreased phosphatidylcholine (18:0p/22:6) absolute concentrations are confirmed. Translation of the lipidomic signature into a scalable diagnostic blood test for pediatric inflammatory bowel disease has the potential to support clinical decision making. Diagnostic blood-based biomarkers of pediatric IBD are limited. Here, the authors demonstrate a diagnostic lipidomic signature, comprising only of two molecular lipids. Translation of this signature into a scalable test has the potential to support clinical decision making.

Background Despite the substantial number of older adults suffering from gastrointestinal (GI) symptoms little is known regarding the character of these complaints and whether they are associated with an altered intestinal barrier function and psychological distress. Our aim was to explore the relationship between self-reported gut health, intestinal permeability and psychological distress among older adults. Methods Three study populations were included: 1) older adults with GI symptoms ( n  = 24), 2) a group of older adults representing the general elderly population in Sweden ( n  = 22) and 3) senior orienteering athletes as a potential model of healthy ageing ( n  = 27). Questionnaire data on gut-health, psychological distress and level of physical activity were collected. Intestinal permeability was measured by quantifying zonulin in plasma. The level of systemic and local inflammation was monitored by measuring C-reactive protein (CRP), hydrogen peroxide in plasma and calprotectin in stool samples. The relationship between biomarkers and questionnaire data in the different study populations was illustrated using a Principal Component Analysis (PCA). Results Older adults with GI symptoms displayed significantly higher levels of both zonulin and psychological distress than both general older adults and senior orienteering athletes. The PCA analysis revealed a separation between senior orienteering athletes and older adults with GI symptoms and showed an association between GI symptoms, psychological distress and zonulin. Conclusions Older adults with GI symptoms express increased plasma levels of zonulin, which might reflect an augmented intestinal permeability. In addition, this group suffer from higher psychological distress compared to general older adults and senior orienteering athletes. This relationship was further confirmed by a PCA plot, which illustrated an association between GI symptoms, psychological distress and intestinal permeability.

Irritable bowel syndrome (IBS) is a functional gastrointestinal (GI) disorder characterized by recurring abdominal pain and disturbed bowel habits. The aetiology of IBS is unknown but there is evidence that genetic, environmental and immunological factors together contribute to the development of the disease. Current treatment of IBS includes lifestyle and dietary interventions, laxatives or antimotility drugs, probiotics, antispasmodics and antidepressant medication. The gut–brain axis comprises the central nervous system, the hypothalamic pituitary axis, the autonomic nervous system and the enteric nervous system. Within the intestinal mucosa there are close connections between immune cells and nerve fibres of the enteric nervous system, and signalling between, for example, mast cells and nerves has shown to be of great importance during GI disorders such as IBS. Communication between the gut and the brain is most importantly routed via the vagus nerve, where signals are transmitted by neuropeptides. It is evident that IBS is a disease of a gut–brain axis dysregulation, involving altered signalling between immune cells and neurotransmitters. In this review, we analyse the most novel and distinct neuro-immune interactions within the IBS mucosa in association with already existing and potential therapeutic targets.