Multi-scale finite element modeling of Eustachian tube function: influence of mucosal adhesion.

The inability to open the collapsible Eustachian tube (ET) leads to the development of chronic Otitis Media (OM). Although mucosal inflammation during OM leads to increased mucin gene expression and elevated adhesion forces within the ET lumen, it is not known how changes in mucosal adhesion alter the biomechanical mechanisms of ET function. In this study, we developed a novel multi-scale finite element model of ET function in adults that utilizes adhesion spring elements to simulate changes in mucosal adhesion. Models were created for six adult subjects, and dynamic patterns in muscle contraction were used to simulate the wave-like opening of the ET that occurs during swallowing. Results indicate that ET opening is highly sensitive to the level of mucosal adhesion and that exceeding a critical value of adhesion leads to rapid ET dysfunction. Parameter variation studies and sensitivity analysis indicate that increased mucosal adhesion alters the relative importance of several tissue biomechanical properties. For example, increases in mucosal adhesion reduced the sensitivity of ET function to tensor veli palatini muscle forces but did not alter the insensitivity of ET function to levator veli palatini muscle forces. Interestingly, although changes in cartilage stiffness did not significantly influence ET opening under low adhesion conditions, ET opening was highly sensitive to changes in cartilage stiffness under high adhesion conditions. Therefore, our multi-scale computational models indicate that changes in mucosal adhesion as would occur during inflammatory OM alter the biomechanical mechanisms of ET function. Copyright © 2016 John Wiley & Sons, Ltd.

Impaired viscosity of gastric secretion and its mucin content as potential contributing factors to the development of chronic constipation.

BACKGROUND: The alimentary tract mucosa continuously releases mucus-rich secretion. Mucin, the major component of mucus, determines its viscosity and provides lubrication for the luminal content of indigestible food particles. AIMS: To measure mucin secretion rate and its viscosity in patients with chronic constipation (CC) and in asymptomatic volunteers. METHODS: Nineteen patients with symptoms of CC and 19 controls were included in the study. Mucin secretion and viscosity were assessed in aspirated gastric juice in basal conditions and after stimulation with pentagastrin (1 h each). Mucin content was tested by PAS methodology. Viscosity was measured using cone/plate digital viscometer. RESULTS: Mucin secretion rates in basal and stimulated conditions in controls were 65 and 42 % higher than in patients with CC (P < 0.05 and P < 0.001, respectively). Basal viscosity in controls was 48 % higher than in CC (P < 0.05) at the lowest and 55 % higher (P < 0.05) at the middle velocities. Viscosity in pentagastrin-stimulated conditions in controls was 71 % higher than in CC (P < 0.01) at the lowest and 35 % higher (P < 0.05) at the middle velocities. CONCLUSIONS: (1) The significantly lower rate of soluble mucin secretion in patients with CC than in normal volunteers may reflect impairment in mucin-related lubrication. (2) Significantly lower viscosity of gastric secretion in patients with CC may result from the lower rate of mucin secretion and may also diminish lubrication within the alimentary tract. (3) This may potentially set the stage for the development of symptoms related to chronic constipation and open a new therapeutic avenue for this patient population.

Attraction of Brachyspira pilosicoli to mucin.

The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of various species, including humans. In the colon this spirochaete can penetrate the overlying mucus layer, attach by one cell end to the underlying enterocytes, and initiate localized colitis and diarrhoea. The aim of this study was to investigate whether, as part of the colonization process, B. pilosicoli is attracted to mucin. Fifteen B. pilosicoli strains isolated from humans, pigs, chickens and dogs, and a control strain of Brachyspira hyodysenteriae, were analysed for their ability to enter solutions of hog gastric mucin in an in vitro capillary tube assay. No significant attraction was detected with 1 % mucin, but some strains started to enter a 2 % solution, and attraction then increased with increasing concentrations to peak at around 6-8 % mucin. A similar increase was seen with B. hyodysenteriae, although this activity peaked at 6 % mucin and then declined, suggesting that the two species have different affinities for mucin. These mucin concentrations were much higher than those used in previous experimental studies with Brachyspira species. The viscosities of the 6-8 % mucin solutions were around 7-12 mPa s, which were similar to the measured viscosities of the mucus layer overlying the epithelium in the caecum and colon of experimental pigs. The strains varied in their motility, as assessed by their ability to enter tubes containing chemotaxis buffer, but there was no significant relationship between this motility and the extent of their ability to enter the mucin solutions. Different strains also had different propensities to enter the mucin solutions, but there were no consistent differences according to the host species of origin. B. pilosicoli strain 95/1000 was attracted towards a solution of d-serine, suggesting that chemotaxis was involved in the attraction to mucin; however, 95/1000 was also attracted to viscous solutions of polyvinylpyrrolidone (PVP), in a manner mirroring the response to mucin, and hence suggesting the involvement of viscotaxis in the attraction to mucin. B. hyodysenteriae B204 showed a similar viscotaxis to PVP. Further studies are required to determine whether the in vitro interaction of a given strain with mucin is a useful indicator of its in vivo colonization ability, and hence could be used as a potential marker for virulence.

Gastrointestinal microflora and mucins may play a critical role in the development of 5-Fluorouracil-induced gastrointestinal mucositis.

5-Fluorouracil (5-FU) is a commonly used chemotherapy agent in clinical oncology practice. Two of its major side effects are mucositis and diarrhoea. The structure of mucins offers mucosal protection, and allows maintenance of intestinal flora by providing attachment sites and preventing bacterial overgrowth and/or penetration. The aim of this study was to investigate changes in mucin secretion and microflora following treatment with 5-FU. Female DA rats were given a single 150 mg/ kg i.p. dose of 5-FU. Rats were killed at various time points after treatment. Control rats received no treatment. Jejunum, colon and faecal samples were collected. Standard microbiological culture techniques were used to identify bacteria, and real-time PCR was used to quantify bacteria in faecal samples. Goblet cells and cavitated goblet cells (having undergone mucus exocytosis) were also counted. Statistical analysis was carried out using Kruskal-Wallis test, a non-parametric method of testing equality of group medians. Following treatment with 5-FU, we showed decreases in Clostridium spp., Lactobacillus spp. and Streptococcus spp., and an increase in Escherichia spp. in the jejunum. In the colon, 5-FU caused decreases in Enterococcus spp., Lactobacillus spp. and Streptococcus spp. Real-time PCR of faecal samples showed decreasing trends in Lactobacillus spp. and Bacteroides spp., and an increasing trend in E. coli. Significant increases (P < 0.05) were seen in Clostridium spp. and Staphylococcus spp. at 24 h. Goblet cell numbers decreased significantly in the jejunum from 24-72 h, with a significant increase in the percentage of cavitated goblet cells. In conclusion, 5-FU treatment causes significant changes in intestinal flora and mucin secretion in rats. These changes could result in systemic effects and, in particular, may contribute to the development of chemotherapy-induced mucositis.

PFG-NMR diffusometry: a tool for investigating the structure and dynamics of noncommercial purified pig gastric mucin in a wide range of concentrations.

For the first time, Pulsed Field Gradient-Nuclear Magnetic Resonance, a powerful noninvasive tool for studying the dynamics and structure of complex gels, has been used to measure diffusion of probe molecules in aqueous solutions/gels of noncommercial purified pig gastric mucin (PGM), in a concentration range up to 5 wt %. Complementary data were obtained from rheology measurements. The combination of techniques revealed a strong pH dependency of the structure of the PGM samples while changes in concentration, ionic strength, and temperature appeared to induce less pronounced alterations. Viscosity was found to vary in a nonmonotonous way with pH, with the more viscous solutions found at intermediate pH. We propose that this finding is due to a reduced charge density at lower pH, which is expected to continuously increase the relative importance of hydrophobic associations. The results suggest a loose network of expanded fully charged PGM molecules with considerable mobility at neutral pH (pH 7.4). At intermediate pH (pH 4), a three-dimensional expanded network is favored. At pH 1, the charge density is low and microphase separation occurs since hydrophobic associations prevail. This leads to the formation of clusters concentrated in PGM molecules separated by regions depleted in PGM. The results obtained increase our knowledge about the gastric mucosal layer, which in vivo contains mucin in the same concentration range as that of the samples investigated here.

Viscosity and wettability of animal mucin solutions and human saliva.

OBJECTIVE: The purpose of this study was to compare viscosity and wettability between animal mucin solutions and human saliva. MATERIALS AND METHODS: Human whole and glandular saliva, porcine gastric mucin, bovine submaxillary mucin, and a mucin-based saliva substitute were used. Viscosity was measured with a cone-and-plate digital viscometer, while wettability on acrylic resin and Co-Cr alloy was determined by the contact angle. RESULTS: The viscosity of animal mucin solutions was proportional to mucin concentration, with the animal mucin solution of concentration 5.0 mg ml(-1) displaying similar viscosity to stimulated whole saliva. A decrease in contact angle was found with increasing animal mucin concentration. For the saliva samples tested, viscosity increased in the following order: stimulated parotid saliva, stimulated whole saliva, unstimulated whole saliva, stimulated submandibular-sublingual saliva. Contact angles of human saliva on the tested solid phases were inversely correlated with viscosity. Contact angles of human saliva on acrylic resin were much lower than those of animal mucin solutions and of those on Co-Cr alloy (P < 0.01). CONCLUSIONS: The effectiveness of animal mucin solutions in terms of their rheological properties was objectively confirmed, indicating a vital role for mucin in proper oral function as well as the development of effective salivary substitutes.

[Hydrochloric acid secretion in stomach--new facts]. / Wyzelnekwasu solnego w zoladku--nowe fakty.

UNLABELLED: Spectrophotometric studies suggest that in aqueous solutions, gastric mucins (mucus) are subject to hydrolysis with secretion of I-V ions. Increasing mucins concentration in the solutions reverses hydrolysis and traps H+ ions. On this basis the hypothesis is proposed that the mucins secreted by the cells of the stomach mucous membrane are the source of the Hions. MATERIAL AND METHODS: In order to test this hypothesis an expenment was conducted based on measuring the pH values of solutions containing 0, 5, 10, 15, 20, and 30 mg of mucins in 100 cm3. The mucins were dissolved in distilled water, 0.9% NaCl, 1.0% KCl, and in aqueous HCl solution of pH 2.3. RESULTS: The increase in mucins concentration in distilled water and in NaCl and KCl solutions causes a drop of pH: 7.0-->5.4; 7.5-->5.0 and 7.2-->5.4 respectively but in mucin-HGl solution pH value was constant. CONCLUSIONS: The results obtained in this experiment permit the assumption that gastric acid (HGI) is being produced by the mucus layer during the blood electrolyte flow from the blood vessels lumen to the gastric lumen. Na+ and K+ ions included in the blood electrolyte are being exchanged for H+ of the mucus when CL- ions are running through it easily. The ion exchanger properties of mucus allow protecting gastric epithelium--during the back diffusion of W ions mucus exchanges them for Na+ and K+ ions that makes the gastric epithelium safe.

Tumor differentiation phenotype in gastric differentiated-type tumors and its relation to tumor invasion and genetic alterations.

AIM: To clarify the relations between tumor differentiation phenotype and tumor invasion or genetic alterations in gastric differentiated-type tumors. METHODS: We examined the tumor differentiation phenotype, the presence of mutations in APC and p53, and the microsatellite instability (MSI) status in 48 gastric adenomas and 171 differentiated-type carcinomas. The tumor differentiation phenotype was determined by examining the expression of human gastric mucin (HGM), MUC6, MUC2 and CD10. The tumors were then classified into gastric- (G-), gastric and intestinal mixed- (GI-), or intestinal- (I-) phenotypes, according to the immunopositivity of the above markers. The presence of mutations in APC and p53 and the MSI status were also investigated in all the tumors. RESULTS: Gastric adenomas were significantly associated with CD10 expression, I-phenotype tumors and the presence of APC mutations, compared with carcinomas (66.7% vs 25.1%, P < 0.0001; 56.3% vs 14.6%, P < 0.0001; 39.6% vs 14.0%, P < 0.0001, respectively) and inversely associated with expressions of HGM and MUC6 and the presence of p53 mutations (10.4% vs 62.6%, P < 0.0001; 39.6% vs 64.3%, P = 0.003; 2.0% vs 26.3%, P = 0.001, respectively). The frequency of APC mutations was significantly higher in HGM-negative tumors, MUC6-negative tumors, CD10-positive tumors and I-phenotype tumors than in HGM-positive tumors, MUC6-positive tumors, CD10-negative tumors and G-phenotype tumors (32.7% vs 7.1%, P < 0.0001; 27.8% vs 14.0%, P = 0.0182; 37.3% vs 10.4%, P < 0.0001; and 38.5% vs 9.5%, P = 0.0017, respectively). The frequency of MSI was significantly higher in MUC6-positive tumors, CD10-negative tumors and G-phenotype tumors than in MUC6-negative tumors, CD10-positive tumors and I-phenotype tumors (24.8% vs 6.7%, P = 0.0009; 22.2% vs 8.0%, P = 0.0143; and 28.6% vs 9.6%, P = 0.0353, respectively). CONCLUSION: The tumor differentiation phenotype is closely related to tumor invasion and genetic alterations in gastric differentiated-type tumors.

Natural antibiotic function of a human gastric mucin against Helicobacter pylori infection.

Helicobacter pylori infects the stomachs of nearly a half the human population, yet most infected individuals remain asymptomatic, which suggests that there is a host defense against this bacterium. Because H. pylori is rarely found in deeper portions of the gastric mucosa, where O-glycans are expressed that have terminal alpha1,4-linked N-acetylglucosamine, we tested whether these O-glycans might affect H. pylori growth. Here, we report that these O-glycans have antimicrobial activity against H. pylori, inhibiting its biosynthesis of cholesteryl-alpha-D-glucopyranoside, a major cell wall component. Thus, the unique O-glycans in gastric mucin appeared to function as a natural antibiotic, protecting the host from H. pylori infection.

Transcriptional activation of the murine Muc5ac mucin gene in epithelial cancer cells by TGF-beta/Smad4 signalling pathway is potentiated by Sp1.

The nucleotide sequence of the pMS1 clone was submitted to the GenBank Nucleotide Sequence Database under accession number AF288076. Changes in the expression of mucin genes in gastrointestinal cancers is thought to contribute to the development of the disease. In our laboratory we have shown previously that MUC5AC is aberrantly expressed in rectosigmoid villous adenomas. However, the regulatory mechanisms underlying that altered profile of expression is unknown. In order to study its regulation at the transcriptional level, we have isolated and characterized 5.5 kb of the 5'-flanking region of the mouse Muc5ac mucin gene. The promoter is flanked by a TATA box and a transcriptional start site is located 22 bp downstream of the TATA box. Analysis of the sequence showed a high density of binding sites for Smad4, an essential factor in the signalling cascade activated by TGF-beta (transforming growth factor-beta), and Sp1, an important factor in the regulation of MUC5AC. This led us to study Muc5ac regulation by TGF-beta. We show that exogenous addition of TGF-beta to the cells induces Muc5ac endogenous expression, promoter activity and Smad4 binding to the promoter. By co-transfection studies we show that Smad4 is essential for Muc5ac promoter activation and that it does not synergize with Smad2 or Smad3. By gel-retardation and co-transfection assays, we identified Sp1 and Sp3 as important regulators of Muc5ac expression and showed that Smad4 and Sp1 act in a co-operative manner to transactivate Muc5ac promoter activity. Altogether these results bring new insights into the molecular mechanisms of TGF-beta-mediated up-regulation of Muc5ac and enhance our understanding as to how Muc5ac is regulated in certain pathologies of the gastrointestinal tract.

Mucoadhesive and cohesive properties of poly(acrylic acid)-cysteine conjugates with regard to their molecular mass.

The objective of this study was to evaluate the influence of the molecular mass and accordingly the polymer chain length on mucoadhesion and cohesion of thiolated polymers. Linear poly(acrylic acid)-cysteine (PAA-Cys) conjugates of 2-, 45-, 250- and 450 kDa (PAA(2)-Cys, PAA(45)-Cys, PAA(250)-Cys and PAA(450)-Cys) and polycarbophil-cysteine (PCP-Cys, 750-3000 kDa), all displaying on average 404.1+/-65.5 microMol thiol groups per gram polymer were compressed into tablets to perform disintegration tests, mucoadhesion studies and viscosity measurements. Moreover, the influence of free unbound cysteine on mucoadhesion was evaluated. Disintegration tests showed a stability of the tablets as following: PAA(2)-Cys

Inhibition of in vitro attachment of Giardia trophozoites by mucin.

An enzyme-linked immunoabsorbent assay was developed to quantify the number of Giardia sp. trophozoites attached to a substratum. Trophozoites were allowed to attach for 5 or 10 min to untreated or modified substratum, or allowed to attach in the presence of cytoskeletal inhibitors. Microtubule inhibitors, cochicine and nocodazole, were ineffective in blocking attachment, although the actin-disrupting agent cytochalasin B produced significant inhibition of attachment. Three different mucins were associated with significant inhibition of Giardia trophozoite attachment, which was reversed by treating the mucin-coated wells with 0.1% poly-L-arginine. Examination of mucin-treated substrata by high-resolution field emission scanning electron microscopy showed the presence of thin linear fibrils, whereas treatment of mucin-coated wells with poly-L-arginine revealed similar fibrils but of thicker dimensions. These data support the concept that mucin may inhibit Giardia sp. trophozoite attachment in vitro by electrostatic repulsion and that this can be eliminated by treatment of mucin-coated surfaces with polycationic agents, such as poly-L-arginine or poly-L-lysine.

Expression of a specific mucin type recognized by monoclonal antibodies in the rat gastric mucosa regenerating from acetic acid-induced ulcer.

BACKGROUND: Detailed research on the healing process of gastric mucosa from injury due to various necrotizing agents is important for regenerating medicine as well as for estimating the quality of ulcer healing. To elucidate this issue, we prepared monoclonal antibodies reacting with mucin molecules present in the specific region and layer of the gastrointestinal mucosa. METHODS: Acetic acid-induced gastric ulcers were prepared in 8-week-old male Wistar rats. Following 24-h fasting, the animals were killed at 20, 30 and 50 days after acid insult and their stomachs were removed immediately. Serial paraffin sections of the ulcer area were made and immunostained with three distinct monoclonal antibodies. RGM21 and HIK1083 react with mucins derived from the surface mucous cells of the corpus and the gland mucous cells of corpus and antrum, respectively. HCM31 stains sialomucin present in the small intestine and colonic mucosa of rat, but does not react with the intact gastric mucosa, except in the very narrow cardiac gland area. RESULTS: On the 20th day after ulcer preparation, the cells stained with RGM21 and HIK1083 were restricted only to the surface layer and the bottom layer of regenerating epithelia, respectively. HCN31 staining covered most of the regenerating epithelia. On the 30th day, the staining of HCM31 was enhanced in the regenerating area. Staining of RGM21 did not change, but the HIK1083 stained area increased in the lower part of the regenerating epithelia. On the 50th day, the staining with HCM31 weakened markedly in the lower part, and this area was occupied with HIK1083 positive cells. CONCLUSION: A notable but temporary expression of a kind of sialomucin specifically stained with HCM31 was observed in the regenerating epithelia during the healing stage of acetic acid-induced gastric damage.

Motility-regulated mucin association of Serpulina pilosicoli, the agent of colonic spirochetosis of humans and animals.

Colonic spirochetosis is a disease of humans and animals characterized by colonization of the colonic mucus gel and intimate attachment of Serpulina pilosicoli to the apical membrane of enterocytes. Motility-regulated mucin association plays a key role in colonic infection by the related spirochete Serpulina hyodysenteriae, the cause of swine dysentery. In this study the chemotaxis of Serpulina pilosicoli porcine isolate P43/6/78, human isolate SP16, and canine isolate 16242-94 was examined by anaerobic incubation of each spirochete in control medium or medium containing increasing concentrations of D-L serine or porcine gastric mucin (PGM). The porcine isolate had a chemotactic response towards 10 mM D-L serine, but not towards PGM. By contrast, the human and canine isolates were attracted towards 0.1% PGM, but not towards DL-serine. The composition of the growth medium appeared to modulate the chemotactic response of S. pilosicoli towards PGM; the loss of a chemotactic response of spirochetes grown in medium without pig fecal extract was restored by growing the spirochetes in medium containing 0.1% PGM. Serpulina pilosicoli displays a chemotactic response towards PGM which is modulated by the presence of certain substrate during the growth phase of the spirochete.

Anti-Helicobacter pylori activities of ebrotidine. A review of biochemical and animal experimental studies and data.

Infection with Helicobacter pylori (H. pylori) is now recognized as a major factor in the pathogenesis of gastric disease, and the successful therapy regimens require a combination of H2 blockers with gastroprotective and antimicrobial agents. Ebrotidine (N-[(E)-[[2-[[[2-[(diaminomethylene) amino]-4-thiazolyl] methyl]thio]ethyl]amino]methylene]-4-bromo-benzenesulfonamide, CAS 100981-43-9, FI-3542) is the only drug combining acid-suppressant activity with remarkable gastroprotective and anti-H. pylori properties. The drug not only displays a potent anti-H. pylori activity alone, but also exerts a strong potentiating effect on the efficacy of antimicrobial agents commonly used for H. pylori eradication, and the successful ulcer therapy with ebrotidine induces a significant (4-fold) increase in the H. pylori aggregation titer of gastric mucin. Moreover, the drug exhibits a strong inhibitory effect on H. pylori urease activity, the extent of which exceeds that of ranitidine, omeprazole and lansoprazole. Ebrotidine has also been demonstrated to exert a potent inhibitory action on the enzymatic activities directed towards mucus perimeter of gastric mucosal defense, causing a marked inhibition of H. pylori protease, lipase and phospholipase A2 activities. Another important property of ebrotidine is its ability to efficiently counteract the disruptive effects of H. pylori lipopolysaccharide on the integrity of gastric epithelium. This includes countering the interference by the lipopolysaccharide in mucosal integrin receptor interaction with proteins of extracellular matrix and the reversal of H. pylori disruptive effect on the binding of mucin to its gastric epithelial receptor. Furthermore, most recent data indicate that ebrotidine has the ability to reverse the impairment caused by H. pylori in feedback inhibition of gastrin release by somatostatin. This activity of ebrotidine apparently stems from the drug's ability to counter the untoward effect of H. pylori on the binding of somatostatin to its specific receptor on the gastric mucosal G-cells. The unique combination of acid suppressant, gastroprotective and anti-H. pylori activities makes ebrotidine a drug of choice in the treatment of gastric disease caused by H. pylori.

Mechanism of Helicobacter pylori pathogenesis: focus on mucus.

Although the clinical data provide increasingly convincing indications that Helicobacter pylori (H. pylori) is a causative factor in gastritis and peptic ulcer, the advances toward the clear understanding of this bacterium's pathogenic action are slow in coming. Having a niche bordering two major perimeters of gastric mucosal defense, H. pylori is capable of exerting detrimental effects on the mucus layer, as well as surface cells of the gastric epithelium. To cause such an effect, the bacteria must first, however, attach to the mucosa. Our findings indicate that this attachment involves specific structures on the epithelial cell surfaces, namely lactosylceramide sulfate and GM3 ganglioside. The analysis of the glycolipid distribution pattern in different regions of human stomach revealed that the antral mucosal content of GM3 and lactosylceramide sulfate are considerably higher than that of the fundus, which may account for the prevalence of H. pylori colonization of the antrum. We have also established that H. pylori causes considerable untoward changes in gastric mucus coat integrity. These changes are reflected in the loss of protective qualities of mucus due to the action of H. pylori-elaborated proteases and lipases. The result of H. pylori protease action is disintegration of the polymeric structure of mucin, whereas the elaborated lipases and phospholipase A2 in particular result in mucus lipid degradation, loss of mucosal surface, hydrophobicity, and lysophospholipid generation. The lytic activity of the resulting lysophospholipids is detrimental not only to mucus gel integrity, but even more so to the cell membrane of gastric epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)