The rheological and structural characteristics of gastric (MUC5AC) and duodenal (MUC2) mucin solutions, the structural basis of the adherent mucus layer in the two organs, and their interactions with polyphenols, the phytochemicals which are linked with a number of health benefits, were investigated using particle tracking microrheology and scattering techniques. We used biochemically well characterised porcine mucins as models for human mucins to characterise their viscoelasticity, structure and dynamics as a function of concentration and pH. Additionally, the mesoscopic forces that mediate the integrity of the network were investigated using reducing (dithiothreitol) and chaotropic agents (guanidinium chloride and urea). Mucins in solution were found to be flexible and three distinct viscoelastic regimes were identify ed. At neutral pH, both types of mucin were found to form flexible self-assembled semi-dilute networks above a critical concentration (c*) where the viscosity scales as c 0.53+-0.08 and c 0.53 +-0.06 for MUC5AC and MUC2 respectively. Above a second critical concentration, the entanglement concentration (Ce), the peptide backbones reptate and entangle and there is a sharp increase in viscosity, c 3.92+- 0.38 for MUC5AC and c 5.1 0+-0.08 for MUC2. At low pH, both types of mucin solution undergo a sol-gel transition, forming pH-switchable gels. The addition of tea-derived polyphenols and tea extracts to the mucin solutions has revealed the strong interaction of galloylated phenolic molecules with mucins, which eventually leads to the gelation of the solution. Cross-linking of mucins by galloylated polyphenols is thus expected to have an impact on the physicochemical environment of the stomach and small intestine; the alteration of the organisation of the mucin polymer network is expected to modulate the barrier properties of the two adherent mucus layers which will affect nutrient absorption and the viscoelastic microenvironment of intestinal bacteria.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24523号 / 医博第4965号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 藤田 恭之, 教授 波多野 悦朗 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Optimiser le diagnostic paraclinique prédictif et non invasif du déficit en cellules souches limbiques (DCSL) : in vitro après empreinte cytologique (EC) par la recherche immunohistochimique (IH) de marqueurs pertinents et par reverse-transcription polymerase chain reaction (RT-PCR), technique de haute sensibilité ; in vivo par microscopie confocale (MCIV). Matériel et Méthodes: La preuve diagnostique de DCSL est la présence de cellules conjonctivales au sein de la cornée, la persistance de cellules cornéennes signant un DCSL partiel. L'EC a été standardisée. La spécificité de chacun des marqueurs cornéens ou conjonctivaux sélectionnés a été recherchée sur des tissus normaux avant d'aborder une étude prospective monocentrique, sur 22 yeux de 18 patients cliniquement suspects de DCSL. Les cellules épithéliales de la cornée centrale étaient recueillies par EC puis transférées sur lame. L'expression des marqueurs de différentiation conjonctivale (K7, K13, K19, MUC5AC) et cornéenne (K12) a été recherchée par IH sur les 22 EC et celle de la MUC5AC par RT-PCR sur 4 d'entre elles. Enfin, la cornée et le limbe de 7 patients ont étés analysés par MCIV. La concordance entre les techniques est évaluée. Conclusion: Ces techniques complémentaires dépendent de l'équipement du service et de l'accessibilité à un laboratoire compétent. Un organigramme décisionnel a été établi pour permettre de faire un diagnostic fiable de DCSL, les examens paracliniques étant inutiles dans certains cas. La recherche IH de kératines conjonctivales K7/K13 et/ou la détection de MUC5AC par RT-PCR sur cellules cornéennes recueillies par EC sont des techniques diagnostiques de haute valeur scientifique, tandis que l'IH K12/MUC5AC doit être abandonnée pour manque de sensibilité et celle de K3/K19 pour manque de spécificité. La MCIV, quand elle est réalisable, est une technique hautement sensible qui permet une quantification du degré de sévérité de la maladie avec une forte concordance avec les autres examens / Purpose: to optimize minimally invasive techniques for diagnosing limbal stem cell deficiency: in vitro after impression cytology (IC) by means of immunocytochemical detection of relevant markers and reverse-transcription polymerase chain reaction (RT PCR), an highly sensitive method; in vivo by confocal microscopy (IVCM). Material and Methods: Presence of conjunctival cells in central cornea was diagnosis proof of LSCD, whereas corneal epithelial cells’ remaining traduces partial LSCD. IC was labeled. Corneal or conjunctival specificity of each marker was previously assessed on healthy tissues. In a prospective case control observational study, 22 eyes of 18 patients clinically suspected of LSCD were enrolled. Epithelial cells from central cornea were collected by IC. Conjunctival (K7, K13, K19, MUC5AC) and corneal (K12) differentiation markers were assessed by immunocytochemistry on each of 22 eyes and MUC5AC RT-PCR was assessed for 4 of them. Cornea and limbus of 7 eyes were assessed by IVCM. The inter-examination agreement was determined. Conclusion: These techniques require skilled technicians and laboratory facilities. We propose a decision tree model to provide unfailing LSCD diagnosis, complementary examinations being sometimes useless. Clinical examination can lead to LSCD misdiagnosis. Immunostaining of conjunctival keratins K7 and K13 as well as MUC5AC detection by RT-PCR are highly effective methods whereas MUC5AC/K12 immuno staining are not sensible and both K3 and K19 are not specific. IVCM of great sensitivity if realizable allows quantification of LSCD severity. Combining both methods provides in every case unfailing diagnosis of LSCD and evaluation of the extent of the disease with high agreement

In healthy airways, mucus forms part of the innate immune response protecting the respiratory epithelium from damage by pathogens and environmental debris (Rose and Voynow, 2006). Conversely, in many respiratory diseases, mucus becomes part of the airway disease pathology. Mucus hypersecretion along with reduced clearance can cause blockage of the small airways, impairing gas exchange, promoting inflammation and becoming a culture medium for bacterial colonisation (Thornton et al., 2008). Recurrent airway obstruction (RAO) is a common yet poorly understood equine chronic respiratory disease where such altered mucus properties and clearance have been identified as major factors in the disease pathology (Davis and Rush, 2002; Gerber et al., 2000; Kaup et al., 1990; Robinson, 2001). The gel-forming mucins are largely responsible for the transport properties of mucus. The major equine airway gel-forming mucin in health is Muc5b and to a lesser extent Muc5ac; produced in specialised respiratory epithelial goblet cells and sub-mucosal glands (Rousseau et al., 2011b). Changes in mucin relative and net amounts and their macromolecular properties and interactions have been attributed to the altered physical properties of airway mucus in airways disease (Groneberg et al., 2002a; Jefcoat et al., 2001; Kirkham et al., 2002; Robinson et al., 2003; Sheehan et al., 1995).The project investigates the biochemical properties of mucins present in mucus from healthy horses and horses with RAO. This project identifies the anatomical presence of mucin-producing goblet cells and glands in fixed tissues from the respiratory tracts of healthy horses and subsequently examines mucin-production sites in respiratory tracts from horses with RAO. Finally the project investigates a methodology for the study of mucin production in airway cells harvested from live horses suffering from RAO.Our investigations confirmed that horses with RAO have more endotracheal mucus than healthy controls, and that Muc5b is the predominant mucin with Muc5ac also present in RAO horse mucus, both during symptomatic disease and when horses are asymptomatic. Mucins are produced in epithelial goblet cells and sub-mucosal glands dispersed throughout the length and circumference of the equine trachea and bronchi. Goblet cell hyperplasia occurs in symptomatic exposed RAO horse airways, although goblet cells are smaller than in asymptomatic RAO horse airways. Exposure to a dusty stable environment is associated with more goblet cells per length of bronchial compared to tracheal epithelium in all horses. RAO horses have larger sub-mucosal glands containing more mucin than control horses. Primary epithelial cell cultures grown at an air liquid interface are an alternative approach to study equine airway mucus, although the use of this culture system is in its early stages. We have developed novel ways to harvest equine airway epithelial cells (tracheal brushing) and shown it is possible to freeze cells collected via tracheal epithelial brushing in 20 % FBS and then culture to ALI at a later date.

Helicobacter pylori (H. pylori) is a human-specific gastric pathogen which is responsible for a spectrum of diseases ranging from superficial gastritis to gastric and duodenal ulceration, and which is also highly associated with gastric cancer. The pathogenesis of severe gastric disorders caused by H. pylori is multifactorial and involves complex interactions between the microbe and the gastric mucosa. H. pylori expresses several adhesion proteins. These molecules have important roles in the establishment of persistent infection and chronic inflammation, which cause tissue damage. The aim of this thesis was to study the attachment of this bacterium to human gastric epithelium, mediated by blood group antigens in both health and disease. One of the bestcharacterized H. pylori adhesins is the histo-blood group antigen binding adhesin (BabA), which binds specifically to the Lewis b antigen (Leb) in the gastric mucosa. A protective mucus layer lines the stomach. The mucosal glycosylation patterns (GPs) vary between different cell lineages, different locations along the gastrointestinal (GI) tract and different developmental stages. In addition, GPs undergo changes during malignant transformation. MUC5AC is a mucin molecule produced by the surface epithelium. Three distinctly different types of human gastrointestinal tissue were studied by bacterial adherence analysis in situ. MUC5AC is the most important carrier of Leb and the new results demonstrate that it forms major receptors for H. pylori adherence. By analysing an H. pylori babA-deletion mutant, a novel adhesin-receptor binding mode was found. Surprisingly, the mutant bound efficiently to both human gastric mucosa and to gastric mucosa of Leb transgenic mice. The sialylated and fucosylated blood group antigen, sialyl-dimeric-Lewis x (sdiLex), was structurally identified as the new receptor. A positive correlation was found between adherence of H. pylori to sialyl-Lewis x (sLex) and elevated levels of inflammation response in the human gastric mucosa. These results were supported by detailed analysis of sialylated and fucosylated blood group antigen glycosylation patterns and, in addition, in situ bacterial adherence to gastric mucosa of experimentally challenged Rhesus monkey. The cognate sialic acid-binding adhesin (SabA) was purified by the retagging technique, and the corresponding sabA-gene was identified. H. pylori lipopolysaccharide (LPS) contains various Lewis blood group antigens such as Lewis x (Lex) and Lewis y (Ley). Additional bacterial adherence modes, which are independent of the BabA and/or SabA adhesins, could possibly be mediated by Lex interactions. Adherence of a clinical isolate and its corresponding Lex mutant to human gastric mucosa with various gastric pathologies was studied in situ. The results suggest that H. pylori LPS plays a distinct but minor role in promotion of bacterial adhesion. Taken together, the results suggest mechanisms for continuous selection of H. pylori strains, involving capacity to adapt to changes in the local environment such as shifts in cell differentiation and associated glycosylation patterns. Adherence of H. pylori is dependent on both the BabA and the SabA adhesin. Multi-step dependent attachment mechanisms may direct the microbes to distinct ecological niches during persistent infections, driving the chronic inflammation processes further toward the development of peptic ulcer disease and/or malignant transformation. Key words: H. pylori, BabA, adhesin, Lewis b, MUC5AC, sialyl-dimeric-Lewis x, chronic inflammation, SabA, Lewis x, LPS.

The polymeric gel-forming mucins provide the structural framework of saliva and the mucus barriers that cover the mucosal surfaces of the alimentary canal. Dietary compounds may influence the barrier properties of these protective layers. The effects of green tea polyphenols, which have many health benefits but have low bioavailability and contribute to the astringency of green tea, on the structural properties of the mucins in the alimentary canal are investigated here. Using well characterised, highly purified salivary mucins MUC5B and MUC7, and porcine gastric mucins, the effects of the green tea polyphenol epigallocatechin-3-gallate (EGCG) on mucins were studied here. Using rate-zonal centrifugation coupled to agarose gel electrophoresis, atomic force microscopy and particle tracking microrheology, EGCG, at concentrations found in a cup of green tea, caused increased aggregation of MUC5B in human whole saliva, and increased aggregation and viscosity of purified MUC5B. It was revealed using recombinant proteins of the N- and C-terminal regions of MUC5B that EGCG had these effects by aggregating the terminal globular protein domains of MUC5B. In contrast, MUC5B trypsin-resistant high molecular weight glycopeptides were not aggregated by EGCG, demonstrating that the oligosaccharide-rich, highly-glycosylated regions of mucins are not involved in the EGCG-induced aggregation of mucins. EGCG also caused the majority of MUC7 in human whole saliva to aggregate, and purified MUC7 also showed substantial aggregation in the presence of EGCG.Porcine gastric mucins were also used in order to model human gastric mucins. First, the identity of the porcine gastric mucins was explored using tandem mass spectrometry and immunohistochemistry. This revealed that Muc5ac was expressed by the surface epithelium and was the prominent mucin in porcine gastric mucus. Muc6 was expressed by gastric submucosal glands, but was not a major component of the secreted mucus barrier. Porcine Muc5ac and Muc6 were shown to be aggregated by EGCG. These data demonstrate that mucins from both saliva and the stomach are substantially altered by EGCG. This may contribute to the astringency and low bioavailability of EGCG. In contrast, the green tea polyphenol epicatechin (EC) did not cause aggregation of salivary mucins or porcine gastric mucins, suggesting that the galloyl ring of EGCG (which is absent in EC) is important for its aggregation of mucins, and that EC has different mechanisms of astringency. The structure of the mucins in the alimentary canal was studied using Raman spectroscopy, Raman optical activity (ROA) and Tip-enhanced Raman spectroscopy (TERS). The secondary structure of the oligosaccharide-rich regions of mucins was shown to be largely disordered, with some contribution of poly-proline II helix. The N- and C-terminal regions of MUC5B were largely β-sheet in structure, with some disordered structure also present in the C-terminal region. Raman spectroscopy could reliably distinguish between MUC5B glycoforms, demonstrating the sensitivity of this technique to mucin glycosylation and secondary structure. The first TERS spectra along the length of a MUC5B chain are reported, and suggest that patterns may exist in the glycosylation of MUC5B. Therefore, Raman spectroscopies are novel tools that shed new light on mucin structure and in future may be useful for studying the changes to mucin structure during interactions, such as those with polyphenols.