We normally live in symbiosis with approximately 10(13) bacteria present in the colon. Among the several mechanisms maintaining the bacteria/host balance, there is limited understanding of the structure, function, and properties of intestinal mucus. We now demonstrate that the mouse colonic mucus consists of two layers extending 150 mum above the epithelial cells. Proteomics revealed that both of these layers have similar protein composition, with the large gel-forming mucin Muc2 as the major structural component. The inner layer is densely packed, firmly attached to the epithelium, and devoid of bacteria. In contrast, the outer layer is movable, has an expanded volume due to proteolytic cleavages of the Muc2 mucin, and is colonized by bacteria. Muc2(-/-) mice have bacteria in direct contact with the epithelial cells and far down in the crypts, explaining the inflammation and cancer development observed in these animals. These findings show that the Muc2 mucin can build a mucus barrier that separates bacteria from the colon epithelia and suggest that defects in this mucus can cause colon inflammation.

https://www.pnas.org/content/pnas/105/39/15064.full.pdf

The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria

The different compartments of the gastrointestinal tract are inhabited by populations of micro-organisms. By far the most important predominant populations are in the colon where a true symbiosis with the host exists that is a key for well-being and health. For such a microbiota, 'normobiosis' characterises a composition of the gut 'ecosystem' in which micro-organisms with potential health benefits predominate in number over potentially harmful ones, in contrast to 'dysbiosis', in which one or a few potentially harmful micro-organisms are dominant, thus creating a disease-prone situation. The present document has been written by a group of both academic and industry experts (in the ILSI Europe Prebiotic Expert Group and Prebiotic Task Force, respectively). It does not aim to propose a new definition of a prebiotic nor to identify which food products are classified as prebiotic but rather to validate and expand the original idea of the prebiotic concept (that can be translated in 'prebiotic effects'), defined as: 'The selective stimulation of growth and/or activity(ies) of one or a limited number of microbial genus(era)/species in the gut microbiota that confer(s) health benefits to the host.' Thanks to the methodological and fundamental research of microbiologists, immense progress has very recently been made in our understanding of the gut microbiota. A large number of human intervention studies have been performed that have demonstrated that dietary consumption of certain food products can result in statistically significant changes in the composition of the gut microbiota in line with the prebiotic concept. Thus the prebiotic effect is now a well-established scientific fact. The more data are accumulating, the more it will be recognised that such changes in the microbiota's composition, especially increase in bifidobacteria, can be regarded as a marker of intestinal health. The review is divided in chapters that cover the major areas of nutrition research where a prebiotic effect has tentatively been investigated for potential health benefits. The prebiotic effect has been shown to associate with modulation of biomarkers and activity(ies) of the immune system. Confirming the studies in adults, it has been demonstrated that, in infant nutrition, the prebiotic effect includes a significant change of gut microbiota composition, especially an increase of faecal concentrations of bifidobacteria. This concomitantly improves stool quality (pH, SCFA, frequency and consistency), reduces the risk of gastroenteritis and infections, improves general well-being and reduces the incidence of allergic symptoms such as atopic eczema. Changes in the gut microbiota composition are classically considered as one of the many factors involved in the pathogenesis of either inflammatory bowel disease or irritable bowel syndrome. The use of particular food products with a prebiotic effect has thus been tested in clinical trials with the objective to improve the clinical activity and well-being of patients with such disorders. Promising beneficial effects have been demonstrated in some preliminary studies, including changes in gut microbiota composition (especially increase in bifidobacteria concentration). Often associated with toxic load and/or miscellaneous risk factors, colon cancer is another pathology for which a possible role of gut microbiota composition has been hypothesised. Numerous experimental studies have reported reduction in incidence of tumours and cancers after feeding specific food products with a prebiotic effect. Some of these studies (including one human trial) have also reported that, in such conditions, gut microbiota composition was modified (especially due to increased concentration of bifidobacteria). Dietary intake of particular food products with a prebiotic effect has been shown, especially in adolescents, but also tentatively in postmenopausal women, to increase Ca absorption as well as bone Ca accretion and bone mineral density. Recent data, both from experimental models and from human studies, support the beneficial effects of particular food products with prebiotic properties on energy homaeostasis, satiety regulation and body weight gain. Together, with data in obese animals and patients, these studies support the hypothesis that gut microbiota composition (especially the number of bifidobacteria) may contribute to modulate metabolic processes associated with syndrome X, especially obesity and diabetes type 2. It is plausible, even though not exclusive, that these effects are linked to the microbiota-induced changes and it is feasible to conclude that their mechanisms fit into the prebiotic effect. However, the role of such changes in these health benefits remains to be definitively proven. As a result of the research activity that followed the publication of the prebiotic concept 15 years ago, it has become clear that products that cause a selective modification in the gut microbiota's composition and/or activity(ies) and thus strengthens normobiosis could either induce beneficial physiological effects in the colon and also in extra-intestinal compartments or contribute towards reducing the risk of dysbiosis and associated intestinal and systemic pathologies.

/pdf/prebiotic-effects-metabolic-and-health-benefits-3ioljfudp0.pdf

Prebiotic effects: metabolic and health benefits.

https://orbilu.uni.lu/bitstream/10993/37916/1/Desai%282016%29Cell.pdf

A Dietary Fiber-Deprived Gut Microbiota Degrades the Colonic Mucus Barrier and Enhances Pathogen Susceptibility

Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues.

The structural and mechanical properties of gels formed from biopolymers are discussed both in terms of the techniques used to characterise these systems, and in terms of the systems themselves The techniques included are spectroscopic, chiroptical and scattering methods, optical and electron microscopy, thermodynamic and kinetic methods and rheological characterisation The systems considered are presented in order of increasing complexity of secondary, tertiary and quaternary structure, starting with gels which arise from essentially ‘disordered’ biopolymers via formation of ‘quasicrystalline’ junction zones (eg gelatin, carrageenans, agarose, alginates etc), and extending to networks derived from globular and rod-like species (fibrin, globular proteins, caseins, myosin) by a variety of crosslinking mechanisms Throughout the text, efforts are made to pursue the link (both from experiment and from theory) between the structural methods and mechanical measurements As far as we are aware this is the first major Review of this area since that of J D Ferry in 1948 — The interest shown by polymer physicists in more complex biochemical systems, and the multi-disciplinary approaches now being applied in this area, make the format adopted here, in our opinion, the most logical and appropriate

Structural and mechanical properties of biopolymer gels

Divergent results from in vitro studies on the thickness and appearance of the gastrointestinal mucus layer have previously been reported. With an in vivo model, we studied mucus gel thickness over time from stomach to colon. The gastrointestinal tissues of Inactin-anesthetized rats were mounted luminal side up for intravital microscopy. Mucus thickness was measured with a micropipette before and after mucus removal by suction. The mucus layer was translucent and continuous; it was thickest in the colon (∼830 μm) and thinnest in the jejunum (∼123 μm). On mucus removal, a continuous, firmly adherent mucus layer remained attached to the epithelial surface in the corpus (∼80 μm), antrum (∼154 μm), and colon (∼116 μm). In the small intestine, this layer was very thin (∼20 μm) or absent. After mucus removal, there was a continuous increase in mucus thickness with the highest rate in the colon and the lowest rate in the stomach. In conclusion, the adherent gastrointestinal mucus gel in vivo is continuous and can be divided into two layers: a loosely adherent layer removable by suction and a layer firmly attached to the mucosa.

The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo

Secretion of bicarbonate into the adherent layer of mucus gel creates a pH gradient with a near-neutral pH at the epithelial surfaces in stomach and duodenum, providing the first line of mucosal protection against luminal acid. The continuous adherent mucus layer is also a barrier to luminal pepsin, thereby protecting the underlying mucosa from proteolytic digestion. In this article we review the present state of the gastroduodenal mucus bicarbonate barrier two decades after the first supporting experimental evidence appeared. The primary function of the adherent mucus gel layer is a structural one to create a stable, unstirred layer to support surface neutralization of acid and act as a protective physical barrier against luminal pepsin. Therefore, the emphasis on mucus in this review is on the form and role of the adherent mucus gel layer. The primary function of the mucosal bicarbonate secretion is to neutralize acid diffusing into the mucus gel layer and to be quantitatively sufficient to maintain a near-neutral pH at the mucus-mucosal surface interface. The emphasis on mucosal bicarbonate in this review is on the mechanisms and control of its secretion and the establishment of a surface pH gradient. Evidence suggests that under normal physiological conditions, the mucus bicarbonate barrier is sufficient for protection of the gastric mucosa against acid and pepsin and is even more so for the duodenum.

https://www.physiology.org/doi/pdf/10.1152/ajpcell.00102.2004

Gastroduodenal mucus bicarbonate barrier: Protection against acid and pepsin

The barrier that protects the undamaged gastroduodenal mucosa from autodigestion by gastric juice is a dynamic multicomponent system. The major elements of this barrier are the adherent mucus gel layer, which is percolated by the HCO3- secretion from the underlying epithelial cells; the epithelial layer itself, which provides a permeability barrier and can rapidly repair superficial damage by a process of cell migration referred to as reepithelization or restitution; and a specially adapted vasculature, which provides a supply of HCO3- for transcellular transport and/or diffusion into the mucus layer. Passive diffusion of intestinal HCO3- into the lumen is particularly important when there is superficial damage resulting in increased leakiness of the mucosal epithelium. The process of reepithelization occurs by the migration of performed cells from gastric pits or duodenal crypts. This process is quite distinct from the wound healing and associated inflammatory response that accompany more severe injury or chronic damage. The adherent mucus gel acts as a physical barrier against luminal pepsin and provides a stable unstirred layer that supports surface neutralization of acid by mucosal HCO3-. Surface neutralization by mucosal HCO3- provides a major mechanism of protection against acid in the proximal duodenum. In the stomach, where luminal acidity can fall to around pH 1, other mechanisms of protection must exist, since the surface pH gradient is reported to collapse when luminal H+ exceeds approximately 10 mM. This collapse of the surface pH gradients may reflect, at least in part, that such studies have been mostly performed on non-acid-secreting mucosa where the supply of HCO3- to the interstitium from the parietal cells will be reduced. However, because the gastric mucosa can withstand prolonged exposure to acid without apparent damage, this implies an intrinsic resistance of the epithelial apical surface. This is amply illustrated within the gastric glands that do not secrete mucus and HCO3- yet are exposed to undiluted pepsin and an isotonic solution of HCl. Bicarbonate and mucus secretions together with mucosal blood flow are under paracrine, endocrine, and neural control. The rate of reepithelialization will depend on local chemotactic factors, adhesion mechanisms, and the creation of an acid/pepsin/irritant-free environment under a protective gelatinous or mucoid cap. If optimal conditions are met, then the rate of reepithelialization appears to depend primarily on the intrinsic properties of the migrating cells themselves rather than control by exogenous mediators.(ABSTRACT TRUNCATED AT 400 WORDS)

Gastroduodenal mucosal protection

The thickness of adherent mucus gel on the surface of colonic mucosa was measured in surgically resected specimens from 46 'control' patients most of whom had carcinoma of the colon; 12 were from right colon, 17 left colon, and 21 from rectum. In addition specimens were examined from 17 patients with ulcerative colitis and 15 patients with Crohn's disease. In controls a continuous layer of mucus was readily seen on specially prepared sections viewed by phase contrast illumination. Mean values for right and left colon and rectum were 107 (48), 134 (68), and 155 (54) microns respectively with a significant difference between right colon and rectum (p = 0.015). Values in ulcerative colitis showed greater variation and in those areas with acute inflammation mucosa was denuded of the mucus layer. In contrast, values for Crohn's disease were normal or greater than normal in thickness--right colon 190 (83) microns compared with 107 48 microns, p = 0.0093. A series of validation experiments are described for the method used to measure mucus thickness. The possible role of mucus in the pathogenesis of inflammatory bowel disease is discussed.

https://gut.bmj.com/content/gutjnl/35/3/353.full.pdf

Thickness of adherent mucus gel on colonic mucosa in humans and its relevance to colitis.

of trypsin without EDTA. A monolayer of radioactively labelled BHK cells was incubated at 37°C for2min in 0.25 % trypsin; one half of the cells in suspension were harvested, washed and assayed for adhesion, and the other half of the cells were incubated for a further 15min in 0.25% trypsin before harvesting, washing and assaying for adhesion. Cells incubated in trypsin for only 2min did not show a Mn2+-stimulated adhesion that was much greater than the CaZ+/Mg2+-stimulated adhesion. In contrast, BHK cells incubated for 15min in trypsin showed an Mn'+-stimulated adhesion that was twice that for the Ca2+/Mg2+-stimulated adhesion. These cells completely excluded Trypan Blue. When such strongly trypsin-treated '4C-labelled BHK cells were preincubated in complete growth medium for up to 60min before assaying for adhesion, this difference between adhesion in Mn2+ buffer and Ca2+/MgZ+ buffer decreased. Thus the CaZ+/Mg2+-stimulated adhesion was 51 and 86% respectively of the MnZ+stimulated adhesion for BHK cells that had been freshly treated with trypsin and for the same cells that had been allowed to recover for 60min in growth medium. MnZ+ is considered to affect the cytoskeleton of the cell, possibly by its release of membrane-bound Ca2+ (Rabinowitch & DeStefano, 1973; Gwynn et al., 1976). Our experiments show, firstly, that Mn2+ causes abnormal spreading and does not replace the requirement for CaZ+/Mg2+ and secondly, that changes that occur with cell transformation or proteolysis for trypsin potentiate this atypical MnZ+ effect.

Adrian Allen

Papers

The adherent gastrointestinal mucus gel layer: thickness and physical state in vivo

Gastroduodenal mucus bicarbonate barrier: Protection against acid and pepsin

Gastroduodenal mucosal protection

Thickness of adherent mucus gel on colonic mucosa in humans and its relevance to colitis.

A colorimetric assay for glycoproteins based on the periodic acid/Schiff stain [proceedings].