Claes Wickström

Four modes of adhesion are used during Helicobacter pylori binding to human mucins in the oral and gastric niches.

Background:  Helicobacter pylori causes peptic ulcer disease and gastric cancer, and the oral cavity is likely to serve as a reservoir for this pathogen. We investigated the binding of H. pylori to the mucins covering the mucosal surfaces in the niches along the oral to gastric infection route and during gastric disease and modeled the outcome of these interactions.

New monoclonal antibodies to non-glycosylated domains of the secreted mucins MUC5B and MUC7.

The separation and characterization of salivary mucins is not straightforward because of their large size, heterogeneity, and molecular interactions. The MUC5B and MUC7 mucins are major glycoprotein components of saliva that are thought to play a vital role in maintaining oral health. MUC5B is also a major component of respiratory mucus and is produced by the tracheal and bronchial glands, while MUC7 has a more limited pattern of expression in the bronchial tree. MUC5B is a gel-forming mucin and thus confers viscosity, whereas MUC7 is much smaller. MUC7 has anti-fungal activity, and both mucins interact with bacteria. The aim of this work was to produce new monoclonal antibodies that can be used to quantify and characterize these mucins by standard laboratory procedures. Peptide sequences in non-conserved and non-glycosylated regions were selected and monoclonal antibodies produced by an efficient immunization and cloning strategy, and screening against purified mucins. Three new antibodies-EU-MUC5Ba and EU-MUC5Bb (against MUC5B) and EU-MUC7a (against MUC7)-were isolated that do not show cross-reactivity with other gel-forming mucins. All work on immunohistochemistry can be used for semi-quantitative immunoblotting after agarose gel electrophoresis. These reagents are valuable tools to study changes in these mucins in oral and respiratory disease, and unlike other monoclonal antibodies to these mucins they recognize epitopes that are not affected by glycosylation.

Salivary gel-forming mucin MUC5B--a nutrient for dental plaque bacteria.

INTRODUCTION Model systems with oral bacteria from dental plaque have demonstrated that the utilization of complex glycoproteins as a food source cannot be undertaken by single species but requires concerted degradation by a multi-species consortium, with each member contributing one or a few hydrolytic enzymes. Unlike previous studies, the aim of the present investigation was to explore the ability of fresh dental plaque to degrade salivary mucin, MUC5B, isolated by methods designed to retain intact the natural polymeric structure and physiological conformation, in an attempt to mimic the naturally occurring interaction between the oral microbiota and salivary mucins. METHODS Human salivary MUC5B was isolated from whole saliva by density-gradient centrifugation and incubated with freshly isolated supragingival dental plaque with samples subjected to fluorescent staining for viability and metabolic activity. In addition, the degradation of MUC5B oligosaccharide side chains was studied using a lectin assay, recognizing three different carbohydrate epitopes commonly found on mucin oligosaccharide side chains. RESULTS The addition of purified salivary MUC5B elicited a strong metabolic response from the biofilm cells, whereas individual strains of Streptococcus oralis and Streptococcus gordonii isolated from the same plaque were not able to utilize the MUC5B. The degradation of terminal saccharide moieties on the MUC5B was demonstrated by a marked decrease in both sialic acid and fucose reactivity. CONCLUSION These results have shown that dental plaque is capable of utilizing human salivary MUC5B as a nutrient source, a process possibly requiring the synergistic degradation of the molecule by a consortium of oral bacteria in the plaque community.

Proteolytic degradation of human salivary MUC5B by dental biofilms

The degradation of complex substrates, like salivary mucins, requires an arsenal of glycosidases and proteases to sequentially degrade the oligosaccharides and polypeptide backbone. The mucin MUC5B is a complex oligomeric glycoprotein, heterogeneous in molecular mass (14-40 x 10(6) Da), with a diverse repertoire of oligosaccharides, differing in composition and charge. The aim of this study was to investigate whether proteolytic degradation of the mucin polypeptide backbone could be identified and if cooperation of dental biofilm bacteria was required. Cooperative bacteria-mediated proteolysis of MUC5B was determined by comparing individual species and mixed consortia of strains isolated from supragingival plaque, and freshly harvested supragingival plaque. Proteolytic activity was analysed using fluorescent labelled substrate and by visualizing mucin degradation by SDS-PAGE. Dental plaque degraded the polypeptide backbone of the salivary MUC5B mucin. The mucin was also degraded by a specific consortium of isolated species from supragingival plaque, although individual species and other consortia did not. Certain bacteria in supragingival dental plaque therefore cooperate as a consortium to proteolyse human salivary MUC5B and hydrolyse glycosides.

Mucins MUC5B and MUC7 in minor salivary gland secretion of children and adults

OBJECTIVE The study was designed to investigate the relative amount of MUC5B and MUC7 in minor salivary glands in children and adults, in order to test the hypothesis that secretion of salivary mucins changes between childhood and adulthood. METHODS Ninety individuals in the age-groups 3-year-olds, 14-year-olds, and young adults 20-25 year-olds were recruited. Sialopapers were applied on the labial and the buccal mucosa and then placed in the Periotron 8,000 (Proflow ) for calculation of the amount of saliva. The assessment of MUC5B and MUC7 was carried out in an ELISA using the LUM5B-2 and the LUM7-1 antiserum, respectively. RESULTS MUC5B and MUC7 were detected in the labial minor gland saliva in all age groups. In buccal gland saliva, only a few individuals in each age group showed detectable amounts of the mucins. In the labial area, a significantly lower level of MUC7 was noted in 3-year-olds compared with adults. CONCLUSION The results indicate a site-dependent difference in minor gland mucin secretion and an age-related difference in the labial gland secretion of MUC7.

Role of (p)ppGpp in Biofilm Formation by Enterococcus faecalis

ABSTRACT Enterococcus faecalis strain OG1RF and its (p)ppGpp-deficient ΔrelA, ΔrelQ, and ΔrelA ΔrelQ mutants were grown in biofilms and evaluated for growth profiles, biofilm morphology, cell viability, and proteolytic activity. E. faecalis lacking (p)ppGpp had a diminished capacity to sustain biofilm formation over an extended period of time and expressed abundant proteolytic activity.

A systematic review of methods to diagnose oral dryness and salivary gland function

BackgroundThe most advocated clinical method for diagnosing salivary dysfunction is to quantitate unstimulated and stimulated whole saliva (sialometry). Since there is an expected and wide variation in salivary flow rates among individuals, the assessment of dysfunction can be difficult. The aim of this systematic review is to evaluate the quality of the evidence for the efficacy of diagnostic methods used to identify oral dryness.MethodsA literature search, with specific indexing terms and a hand search, was conducted for publications that described a method to diagnose oral dryness. The electronic databases of PubMed, Cochrane Library, and Web of Science were used as data sources. Four reviewers selected publications on the basis of predetermined inclusion and exclusion criteria. Data were extracted from the selected publications using a protocol. Original studies were interpreted with the aid of Quality Assessment of Diagnostic Accuracy Studies (QUADAS) tool.ResultsThe database searches resulted in 224 titles and abstracts. Of these abstracts, 80 publications were judged to meet the inclusion criteria and read in full. A total of 18 original studies were judged relevant and interpreted for this review. In all studies, the results of the test method were compared to those of a reference method.Based on the interpretation (with the aid of the QUADAS tool) it can be reported that the patient selection criteria were not clearly described and the test or reference methods were not described in sufficient detail for it to be reproduced. None of the included studies reported information on uninterpretable/intermediate results nor data on observer or instrument variation. Seven of the studies presented their results as a percentage of correct diagnoses.ConclusionsThe evidence for the efficacy of clinical methods to assess oral dryness is sparse and it can be stated that improved standards for the reporting of diagnostic accuracy are needed in order to assure the methodological quality of studies. There is need for effective diagnostic criteria and functional tests in order to detect those individuals with oral dryness who may require oral treatment, such as alleviation of discomfort and/or prevention of diseases.

Elevated levels of salivary lactoferrin, a marker for chronic periodontitis?

BACKGROUND AND OBJECTIVE Whole saliva is a complex mixture of fluids essential for the well-being of the oral hard and soft tissues. Saliva contains numerous antimicrobial proteins that help protect the oral ecosystem from infectious agents. Chronic periodontitis is an infectious chronic inflammatory condition that affects the tooth-supporting structures and leads to their destruction. The aim of the present study was to investigate differences in concentrations of salivary lactoferrin in subjects with and without periodontal disease and correlate these values with clinical variables associated with periodontal disease. MATERIAL AND METHODS Stimulated whole saliva was collected from 17 subjects with chronic periodontitis and 17 periodontally healthy control subjects. Data relating to bleeding on probing, probing pocket depth and horizontal bone loss were registered. Concentrations of lactoferrin, lysozyme and IgA in stimulated whole saliva were quantified using ELISA. RESULTS Subjects with chronic periodontits showed higher concentrations of lactoferrin in stimulated whole saliva compared with periodontally healthy control subjects (p < 0.05). Salivary concentrations of lactoferrin were positively correlated with bleeding on probing (p < 0.001) and the number of sites with probing pocket depth ≥ 6 mm (p < 0.001). CONCLUSION Lactoferrin is raised in stimulated whole saliva in subjects with chronic periodontitis and is correlated with probing pocket depth ≥ 6 mm.

Differential metabolic activity by dental plaque bacteria in association with two preparations of MUC5B mucins in solution and in biofilms

Salivary mucin, MUC5B, is an oligomeric glycoprotein, heterogeneous in size and with a diverse repertoire of oligosaccharides, which differ in composition and charge. Since complex salivary glycoproteins are considered to be the major source of nutrients for the oral supragingival microbiota, the major aim of the current study was to determine whether different preparations of non-denatured MUC5B could be isolated exhibiting different biological properties in relation to the microflora associated with the surfaces of the oral cavity. Two preparations, solMUC5B and gelMUC5B, were isolated by density-gradient centrifugation and were shown to have different buoyant densities, carbohydrate content and surface-adsorbing characteristics. To ascertain differences in biological activity, the two mucin preparations, both in solution and adsorbed to a model surface, were incubated with freshly isolated dental plaque and assayed for metabolic (dehydrogenase) activity with the fluoresecent substrate CTC (5-cyano-2,3-ditolyl tetrazolium chloride). The plaque bacteria exhibited higher metabolism with the solMUC5B preparation in solution, with 79.4 % active plaque cells compared to the controls without mucin (9.6 %), while gelMUC5B showed 48.2 % active cells with the same plaque population. In contrast, the same mucins adhered to a surface elicited a significantly lower metabolic response, with surface-associated plaque cells showing only 12.1 % active cells with solMUC5B and 29.2 % with gelMUC5B. These results suggested that the metabolism by the plaque cells adsorbed to surface-associated mucins was downregulated compared to the same cells suspended in mucin solution. This was confirmed in an experiment where active dispersed plaque/solMUC5B suspensions were shown to lose significant metabolic activity (e.g. 74.9 to 19.3 %) when allowed to interact with gelMUC5B adsorbed to a surface. Clearly, the solMUC5B and gelMUC5B preparations exhibited different biological activity when assayed with freshly plaque bacteria in suspension and in a biofilm.

Gel-forming and cell-associated mucins: preparation for structural and functional studies.

Secreted and transmembrane mucins are important components of innate defence at the bodys mucosal surfaces. The secreted mucins are large, polymeric glycoproteins, which are largely responsible for the gel-like properties of mucus secretions. The cell-tethered mucins, however, are monomeric but are typically composed of two subunits, a larger extracellular subunit which is heavily glycosylated while the smaller more sparsely glycosylated subunit has a short extracellular region, a single-pass transmembrane domain, and a cytoplasmic tail. These two families of mucins represent high-molecular-weight glycoproteins containing serine and threonine-rich domains that are the attachment sites for large numbers of O-glycans. The high-M ( r ) and high sugar content have been exploited for the separation of mucins from the majority of components in mucus secretions. In this chapter, we describe current and well-established methods (caesium chloride density-gradient centrifugation, gel-filtration and anion-exchange chromatography, and agarose gel electrophoresis) for the extraction and purification of gel-forming and cell-surface mucins which can subsequently be used for a variety of structural and functional studies.