Salivary film thickness and MUC5B levels at various intra-oral surfaces.

OBJECTIVES: In this study, we investigated the salivary film thickness and the MUC5B levels at various intra-oral locations in healthy volunteers, with a focus on the palate. Besides, measurements of the palatal surface area were included to explore the possible relationships between the palatal surface area and the palatal salivary film and MUC5B levels. MATERIALS AND METHODS: The salivary film thickness was determined using filter strips, which were pressed to the mucosal surfaces of five different intra-oral locations; conductance was then analysed using a Periotron. After elution of the strips, the MUC5B levels at various intra-oral locations were determined using ELISA. The palatal surface area was measured using an intra-oral scanner. The surface area was subsequently calculated using the software. RESULTS: The anterior tongue had the thickest salivary film and also the highest levels of MUC5B, while the anterior palate had the thinnest salivary film and lowest MUC5B levels. There was no association between the palatal surface area and the salivary film thickness of the palate. CONCLUSION: The salivary film and MUC5B levels are unequally distributed over the intra-oral regions of the soft tissues. The lack of association between the palatal surface area and the salivary film thickness indicates that a larger surface area is not associated with a relative thinner palatal salivary film. CLINICAL RELEVANCE: The results of the current study increase our understanding of saliva distribution in the oral cavity and could be used as reference values for future studies.

Comparison of proteins with anti-influenza virus effects in parotid and submandibular-sublingual saliva in humans.

BACKGROUND: Saliva possesses antiviral activity, with submandibular-sublingual (SMSL) saliva having higher antiviral activity than parotid saliva. Various salivary proteins have inactivating effects on influenza A virus (IAV), but the detailed relationship between antiviral proteins and salivary anti-IAV activities in the parotid and SMSL glands is unknown. Here, to identify salivary proteins with anti-IAV activity, salivary proteins from parotid and SMSL glands were identified, quantified, and compared using liquid chromatography-mass spectrometry. METHODS: Twelve healthy male volunteers participated in the study. Parotid and SMSL saliva was collected by suction and collection devices. We assessed anti-IAV activities, protein concentrations, and protein-bound sialic acid concentrations in parotid and SMSL saliva. RESULTS: SMSL had significantly higher anti-IAV activity than parotid saliva. SMSL also had higher concentrations of glycoproteins, such as mucin 5B and mucin 7, protein-bound sialic acid, cystatins, and lysozyme C, compared with parotid saliva. Salivary mucin 5B and mucin 7 concentrations significantly positively correlated with the salivary protein-bound sialic acid concentration. Salivary anti-IAV activity significantly positively correlated with protein-bound sialic acid, mucin 5B, mucin 7, cystatin-C, -S, and -SN concentrations. CONCLUSION: Salivary mucins, cystatins, and lysozyme C contribute to the high anti-IAV activity of SMSL saliva.

MUC5B Promoter Variant and Rheumatoid Arthritis with Interstitial Lung Disease.

BACKGROUND: Given the phenotypic similarities between rheumatoid arthritis (RA)-associated interstitial lung disease (ILD) (hereafter, RA-ILD) and idiopathic pulmonary fibrosis, we hypothesized that the strongest risk factor for the development of idiopathic pulmonary fibrosis, the gain-of-function MUC5B promoter variant rs35705950, would also contribute to the risk of ILD among patients with RA. METHODS: Using a discovery population and multiple validation populations, we tested the association of the MUC5B promoter variant rs35705950 in 620 patients with RA-ILD, 614 patients with RA without ILD, and 5448 unaffected controls. RESULTS: Analysis of the discovery population revealed an association of the minor allele of the MUC5B promoter variant with RA-ILD when patients with RA-ILD were compared with unaffected controls (adjusted odds ratio, 3.8; 95% confidence interval [CI], 2.8 to 5.2; P=9.7×10-17). The MUC5B promoter variant was also significantly overrepresented among patients with RA-ILD, as compared with unaffected controls, in an analysis of the multiethnic case series (adjusted odds ratio, 5.5; 95% CI, 4.2 to 7.3; P=4.7×10-35) and in a combined analysis of the discovery population and the multiethnic case series (adjusted odds ratio, 4.7; 95% CI, 3.9 to 5.8; P=1.3×10-49). In addition, the MUC5B promoter variant was associated with an increased risk of ILD among patients with RA (adjusted odds ratio in combined analysis, 3.1; 95% CI, 1.8 to 5.4; P=7.4×10-5), particularly among those with evidence of usual interstitial pneumonia on high-resolution computed tomography (adjusted odds ratio in combined analysis, 6.1; 95% CI, 2.9 to 13.1; P=2.5×10-6). However, no significant association with the MUC5B promoter variant was observed for the diagnosis of RA alone. CONCLUSIONS: We found that the MUC5B promoter variant was associated with RA-ILD and more specifically associated with evidence of usual interstitial pneumonia on imaging. (Funded by Société Française de Rhumatologie and others.).

Airway Mucin Concentration as a Marker of Chronic Bronchitis.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by chronic bronchitic and emphysematous components. In one biophysical model, the concentration of mucin on the airway surfaces is hypothesized to be a key variable that controls mucus transport in healthy persons versus cessation of transport in persons with muco-obstructive lung diseases. Under this model, it is postulated that a high mucin concentration produces the sputum and disease progression that are characteristic of chronic bronchitis. METHODS: We characterized the COPD status of 917 participants from the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) using questionnaires administered to participants, chest tomography, spirometry, and examination of induced sputum. Total mucin concentrations in sputum were measured with the use of size-exclusion chromatography and refractometry. In 148 of these participants, the respiratory secreted mucins MUC5AC and MUC5B were quantitated by means of mass spectrometry. Data from chronic-bronchitis questionnaires and data on total mucin concentrations in sputum were also analyzed in an independent 94-participant cohort. RESULTS: Mean (±SE) total mucin concentrations were higher in current or former smokers with severe COPD than in controls who had never smoked (3166±402 vs. 1515±152 µg per milliliter) and were higher in participants with two or more respiratory exacerbations per year than in those with zero exacerbations (4194±878 vs. 2458±113 µg per milliliter). The absolute concentrations of MUC5B and MUC5AC in current or former smokers with severe COPD were approximately 3 times as high and 10 times as high, respectively, as in controls who had never smoked. Receiver-operating-characteristic curve analysis of the association between total mucin concentration and a diagnosis of chronic bronchitis yielded areas under the curve of 0.72 (95% confidence interval [CI], 0.65 to 0.79) for the SPIROMICS cohort and 0.82 (95% CI, 0.73 to 0.92) for the independent cohort. CONCLUSIONS: Airway mucin concentrations may quantitate a key component of the chronic bronchitis pathophysiologic cascade that produces sputum and mediates disease severity. Studies designed to explore total mucin concentrations in sputum as a diagnostic biomarker and therapeutic target for chronic bronchitis appear to be warranted. (Funded by the National Heart, Lung, and Blood Institute and others.).

Localization of Secretory Mucins MUC5AC and MUC5B in Normal/Healthy Human Airways.

RATIONALE: MUC5AC and MUC5B are the predominant gel-forming mucins in the mucus layer of human airways. Each mucin has distinct functions and site-specific expression. However, the regional distribution of expression and cell types that secrete each mucin in normal/healthy human airways are not fully understood. OBJECTIVES: To characterize the regional distribution of MUC5B and MUC5AC in normal/healthy human airways and assess which cell types produce these mucins, referenced to the club cell secretory protein (CCSP). METHODS: Multiple airway regions from 16 nonsmoker lungs without a history of lung disease were studied. MUC5AC, MUC5B, and CCSP expression/colocalization were assessed by RNA in situ hybridization and immunohistochemistry in five lungs with histologically healthy airways. Droplet digital PCR and cell cultures were performed for absolute quantification of MUC5AC/5B ratios and protein secretion, respectively. MEASUREMENTS AND MAIN RESULTS: Submucosal glands expressed MUC5B, but not MUC5AC. However, MUC5B was also extensively expressed in superficial epithelia throughout the airways except for the terminal bronchioles. Morphometric calculations revealed that the distal airway superficial epithelium was the predominant site for MUC5B expression, whereas MUC5AC expression was concentrated in proximal, cartilaginous airways. RNA in situ hybridization revealed MUC5AC and MUC5B were colocalized with CCSP-positive secretory cells in proximal superficial epithelia, whereas MUC5B and CCSP-copositive cells dominated distal regions. CONCLUSIONS: In normal/healthy human airways, MUC5B is the dominant secretory mucin in the superficial epithelium and glands, with distal airways being a major site of expression. MUC5B and MUC5AC expression is a property of CCSP-positive secretory cells in superficial airway epithelia.

Muc5b is mainly expressed and sialylated in the nasal olfactory epithelium whereas Muc5ac is exclusively expressed and fucosylated in the nasal respiratory epithelium.

The nose is a complex organ that filters and warms breathing airflow. The nasal epithelium is the first barrier between the host and the external environment and is covered by a mucus gel that is poorly documented. Mucins are large, heavily O-glycosylated polymeric molecules secreted in the nose lumen by specialized cells, and they are responsible for the biochemical properties of the mucus gel. The mucus traps particles and clears them, and it also bathes microbiota, host molecules, and receptors that are all essential for odor perception in the olfactory epithelium. We used histology and immunohistochemistry to study the expression of the two main airway polymeric mucins, Muc5ac and Muc5b, in wild-type, green fluorescent protein-reporter Muc5b, and in genetically Muc5b-deficient mice. We report that Muc5ac is produced by goblet cells at the cell surface in the respiratory epithelium but is not expressed in the olfactory epithelium, whereas Muc5b is secreted by Bowman's glands situated in the lamina propria beneath the olfactory epithelium and also by goblet cells in the distal part of the respiratory epithelium. We also observed that Muc5b-deficient mice exhibited depletion of Bowman's glands. Using lectins, we found that terminally O-glycosylated chains of Muc5b were sialylated but not fucosylated, whereas Muc5ac was fucosylated but not sialylated. Specific localization and specific terminal glycosylation of the two mucins suggest different functions of the mucins.

Physical and chemical characteristics of mucin secreted by pseudomyxoma peritonei (PMP).

Background: Pseudomyxoma peritonei (PMP) is a rare disease with excess intraperitoneal mucin secretion. Treatment involves laparotomy, cytoreduction and chemotherapy that is very invasive with patients often acquiring numerous compromises. Hence a mucolytic comprising of bromelain and N-acetyl cystein has been developed to solubilise mucin in situ for removal by catherization. Owing to differences in mucin appearance and hardness, dissolution varies. Therefore the current study investigates the inter-mucin physical and chemical characteristics, in order to reformulate an effective mucolytic for all mucin. Method: PMP mucin, from the three categories (soft, semi hard and hard mucin) was solubilised and then various physical characteristics such as turbidity, density, kinematic viscosity were measured. The water content and the density of solid mucin were also determined. This was followed by the determination of sialic acid, glucose, lipid, Thiol (S-S and S-H) content of the samples. Lastly, the distribution of MUC2, MUC5B and MUC5AC was determined using western blot technique. Results: Both turbidity and kinematic viscosity and sialic acid content increased linearly as the hardness of mucin increased. However, density, hydration, protein, glucose, lipid and sulfhydryl and disulphide content decreased linearly as hardness of mucin increased. The distribution ratio of mucins (MUC2:MUC5B:MUC5AC) in soft mucin is 2.25:1.5:1.0, semi hard mucin is 1:1:1 and hard mucin is 3:2:1. Conclusion: The difference in texture and hardness of mucin may be due to cellular content, hydration, glucose, protein, lipids, thiol and MUC distribution. Soft mucin is solely made of glycoprotein whilst the others contained cellular materials.

Sialendoscopy of Salivary Glands Affected by Sjögren Syndrome: A Randomized Controlled Pilot Study.

PURPOSE: Sialendoscopy of the major salivary glands could alleviate the oral symptoms of Sjögren syndrome (SS) and restore salivary function. The aim of this pilot study was to evaluate the effect of sialendoscopy of the major salivary glands on salivary flow, saliva composition, and mouthfeel in patients with SS and to collect data for sample size analysis for a larger clinical trial. MATERIALS AND METHODS: Twenty patients diagnosed with SS were randomly assigned to a nonintervention control group or a sialendoscopy group. Unstimulated whole saliva flow, stimulated whole saliva flow, Clinical Oral Dryness Score, Xerostomia Inventory score, and EULAR Sjögren's Syndrome Patient Reported Index score were obtained 1 week before (T0), 1 week after (T2), and 8 weeks after (T3) sialendoscopy. Unstimulated whole saliva was analyzed for amylase concentration, activity, and mucin 5B concentration. Amylase and mucin 5B output were calculated. RESULTS: In the sialendoscopy group, unstimulated and stimulated whole saliva flows were numerically higher at T2 and T3 compared with T0. Xerostomia Inventory score was significantly lower in the sialendoscopy group at T2 compared with T0 (P = .03). Unstimulated and stimulated whole saliva flows were higher in the sialendoscopy group compared with the control group at T2 and T3 (not meaningful). Significant differences were found between groups for the EULAR Sjögren's Syndrome Patient Reported Index score at T2 (P = .03) and T3 (P = .001). Xerostomia Inventory score and Clinical Oral Dryness Score in the sialendoscopy group were lower compared with the control group at T2 (P = .02) and at T3 (P = .04), indicating less oral dryness. CONCLUSION: This pilot study indicates a positive effect of sialendoscopy on some parameters, but it cannot yet be concluded that it has a positive effect on salivary flow in patients with SS. These preliminary results need to be verified in a randomized controlled trial with a larger sample and longer follow-up period.

MUC5AC and MUC5B enhance the characterization of mucinous adenocarcinomas of the lung and predict poor prognosis.

AIMS: From the viewpoint of histogenesis, lung adenocarcinoma can be subdivided into two groups: terminal respiratory unit (TRU) and non-TRU types. We recently reported a non-TRU type adenocarcinoma designated as ciliated adenocarcinoma (we now prefer central type adenocarcinoma). We suggest reasons that mucinous adenocarcinoma should encompass central type adenocarcinoma to represent its biological characteristics as non-TRU type adenocarcinoma. METHODS AND RESULTS: Mucin (MUC)5AC and MUC5B were expressed more significantly in non-TRU type adenocarcinoma (P < 0.01). Thirty-five (76.1%) and 45 cases (97.8%) of 46 non-TRU type adenocarcinoma showed positivity for MUC5AC and MUC5B. Twelve (7.6%) and eight (5.1%) cases of 157 TRU type adenocarainoma showed positivity for MUC5B and MUC5AC. NKX2-1 gene expression was measured with quantitative reverse transcription-polymerase chain reaction (qRT-PCR). ΔΔCt of NKX2-1 gene expression was 6.79 for TRU type adenocarcinoma and 0.6 for non-TRU type adenocarcinoma. Overall survival and disease-free survival were poorer in non-TRU type adenocarcinoma (P = 0.02 and P = 0.03). A multivariate test also showed that non-TRU type adenocarcinoma is an independent prognostic factor (P = 0.04). CONCLUSION: MUC5AC and MUC5B were specific makers for non-TRU adenocarcinoma, including both central type adenocarcinoma and mucinous adenocarcinoma. We suggest that non-TRU type adenocarcinoma presents a poorer prognosis, so it should be regarded separately from TRU type adenocarcinoma.

Interindividual variation, correlations, and sex-related differences in the salivary biochemistry of young healthy adults.

A cross-sectional observational study was conducted to evaluate interindividual biochemical variation in unstimulated whole saliva in a population of 268 systemically healthy young students, 18-30 yr of age, with no apparent caries lesions or periodontal disease. Salivary flow rate, protein content, pH, buffering capacity, mucins MUC5B and MUC7, albumin, secretory IgA, cystatin S, lactoferrin, chitinase, amylase, lysozyme, and proteases were measured using ELISAs and enzymatic activity assays. Significant differences were found between male and female subjects. Salivary pH, buffering capacity, protein content, MUC5B, secretory IgA, and chitinase activity were all lower in female subjects compared with male subjects, whereas MUC7 and lysozyme activity were higher in female subjects. There was no significant difference between sexes in salivary flow rate, albumin, cystatin S, amylase, and protease activity. Principal component analysis (PCA) and spectral clustering (SC) were used to assess intervariable relationships within the data set and to identify subgroups. Spectral clustering identified two clusters of participants, which were subsequently described. This study provides a comprehensive overview of the distribution and inter-relations of a set of important salivary biochemical variables in a systemically healthy young adult population, free of apparent caries lesions and periodontal disease. It highlights significant gender differences in salivary biochemistry.

Mucin levels in saliva of adolescents with dental caries.

BACKGROUND: Human saliva, a complex secretion that contains a mixture of inorganic and organic molecules, plays an essential role in the maintenance of oral health. Mucins are the major macromolecular component of the secretion and are considered the first line of defense for epithelial tissues. The aim of this study was to compare levels of mucins (MUC5B, MUC7, and MUC1) in saliva of young subjects with dental caries. MATERIAL AND METHODS: All patients had DMF (decay/missing/filled) higher than value 0. Eight subjects with DMF=3 (control group) and 27 adolescents with DMF >11 (research group) were recruited for this study. Clinical evaluation procedures were oral examination, including tooth, periodontal, oral mucosal status, and collection of saliva samples. Saliva was collected for mucin assay. Enzyme-linked immunosorbent assay was used to quantitate MUC5B, MUC7, and MUC1. RESULTS: Our results indicate that adolescents with very high intensity of dental caries disease had increased levels of MUC1 and MUC5B. The membrane mucin MUC1 protein levels in the group with DMF>11 (research group) were higher compared to the group with DMF=3 (control group), and the increase was statistically significant (p=0.011). Similarly, secreted mucin MUC5B protein levels were higher (p=0.06) in the group with DMF>11 (research group). Although MUC7 protein levels were slightly reduced in symptomatic subjects, the decrease was statistically insignificant (p=0.918). CONCLUSIONS: Our data suggest links between the production of mucins, especially MUC1 and MUC5B in saliva, and dental caries disease.

Concentration of salivary protective proteins within the bound oral mucosal pellicle.

OBJECTIVES: To study which salivary proteins form the protective bound mucosal pellicle and to determine the role of transglutaminase in pellicle development. MATERIALS AND METHODS: Oral epithelial cells were collected and underwent washes of different strengths, followed by homogenisation. SDS-PAGE, western blotting, IgA ELISAs and amylase activity assays were completed on cell homogenates and compared to saliva samples to confirm which salivary proteins were bound to cell surfaces. RESULTS: Salivary mucins, MUC5B and MUC7, were strongly retained on the oral epithelial cell surface. Other bound proteins including cystatin S, carbonic anhydrase VI, secretory component and IgA could be washed off. IgA was present in concentrated levels in the bound mucosal pellicle compared to amounts in saliva. Amylase, one of the most abundant proteins present in saliva, showed minimal levels of binding. Transglutaminase 3 presence was confirmed, but proteins that it catalyses cross-links between, statherin and proline-rich proteins, showed minimal presence. CONCLUSION: Some protective salivary proteins including mucins and IgA become concentrated on oral surfaces in the bound mucosal pellicle, through specific interactions. Concentration of mucins would contribute to lubrication to prevent abrasion damage to soft tissues, whilst increased IgA could create an 'immune reservoir' against mucosal infection.

Composition of enamel pellicle from dental erosion patients.

Oral health is dependent upon a thin mobile film of saliva on soft and hard tissues. Salivary proteins adhere to teeth to form the acquired enamel pellicle which is believed to protect teeth from acid erosion. This study investigated whether patients suffering diet-induced dental erosion had altered enamel pellicles. Thirty patients suffering erosion were compared to healthy age-matched controls. Subjects wore a maxillary splint holding hydroxyapatite and human enamel blocks for 1 h. The acquired enamel pellicle was removed from the blocks and compared to the natural incisor pellicle. Basic Erosive Wear Examination scores confirmed that dental erosion was present in erosion patients and absent from healthy age-matched controls. Erosion patients had half the amount of proteins (BCA assay) within the acquired pellicle forming on splint blocks compared to normal controls (p < 0.05). In particular, statherin, a calcium-binding protein, was 35% less abundant (p < 0.05). Calcium concentration within the acquired pellicle was also reduced by 50% in erosion patients (p < 0.001). In contrast, the natural pellicle on the incisor had similar amounts of total protein in erosion patients and healthy controls. In summary, the formation of new acquired pellicles on surfaces was reduced in erosion patients, which may explain their greater susceptibility to acid erosion of teeth.

Expression of MUC2, MUC5AC, MUC5B, and MUC6 mucins in colorectal cancers and their association with the CpG island methylator phenotype.

Mucinous differentiation is associated with both CpG island methylator phenotype and microsatellite instability in colorectal cancer. The mucinous phenotype derives from abundant expression of the colonic goblet cell mucin, MUC2, and de novo expression of gastric foveolar mucin, MUC5AC. We, therefore, investigated the protein expression levels of MUC2 and MUC5AC, as well as MUC5B and MUC6, in molecular subtypes of colorectal cancer. Seven-hundred and twenty-two incident colorectal carcinomas occurring in 702 participants of the Melbourne Collaborative Cohort Study were characterized for methylator status, MLH1 methylation, somatic BRAF and KRAS mutations, microsatellite-instability status, MLH1, MSH2, MSH6, and PMS2 mismatch repair, and p53 protein expression, and their histopathology was reviewed. Protein expression levels of MUC2, MUC5AC, MUC5B, MUC6, and the putative mucin regulator CDX2 were compared with molecular and clinicopathological features of colorectal cancers using odds ratios and corresponding 95% confidence intervals. MUC2 overexpression (>25% positive tumor cells) was observed in 33% colorectal cancers, MUC5B expression in 53%, and de novo MUC5AC and MUC6 expression in 50% and 39%, respectively. Co-expression of two or more of the mucins was commonly observed. Expression of MUC2, MUC5AC and MUC6 was strongly associated with features associated with tumorigenesis via the serrated neoplasia pathway, including methylator positivity, somatic BRAF p.V600E mutation, and mismatch repair deficiency, as well as proximal location, poor differentiation, lymphocytic response, and increased T stage (all P<0.001). Overexpression was observed in tumors with and without mucinous differentiation. There were inverse associations between expression of all four mucins and p53 overexpression. CDX2 expression was inversely associated with MUC2, MUC5AC and MUC6 expression. Our results suggest that, in methylator-positive tumors, mucin genes on chromosome 11p15.5 region undergo increased expression via mechanisms other than direct regulation by CDX2.

Liquid chromatography-tandem mass spectrometry approach for quantification of mucins from sputum using 13C,15N-labeled peptides as internal standards.

Mucins are of great interest owing to their involvement in physiological and pathological processes in the airways. A method that allows accurate quantification of such proteins in sputum samples may be helpful for research in this field. A liquid chromatographic selected reaction monitoring (SRM) method was developed for the quantification of two mucins, MUC5AC and MUC5B, in induced sputum samples. Sample preparation for the assay included solubilization, reduction, and alkylation prior to tryptic digestion. Solid phase extraction using C18 sorbent was used for sample cleanup prior to the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A cysteine-containing peptide was selected for quantification of MUC5AC protein, whereas a non-cysteine peptide was used for the quantification of MUC5B protein. Stable isotope-labeled synthetic peptides were used as internal standards, and linear calibration curves were constructed in the range of 0.3 to 40 pmol/L. Both mucins could be determined with a precision of 6 to 19% and an accuracy of 98 to 114%. The method is transferable to robotics and is suitable to be run in a 96-well format.

Airway mucin MUC5AC and MUC5B concentrations and the initiation and progression of chronic obstructive pulmonary disease: an analysis of the SPIROMICS cohort.

BACKGROUND: We previously described the contributions of increased total airway mucin concentrations to the pathogenesis and diagnosis of the chronic bronchitic component of chronic obstructive pulmonary disease (COPD). Here, we investigated the relative contribution of each of the major airway gel-forming mucins, MUC5AC and MUC5B, to the initiation, progression, and early diagnosis of airways disease in COPD. METHODS: SPIROMICS was a multicentre, observational study in patients aged 40-80 years recruited from six clinical sites and additional subsites in the USA. In this analysis, MUC5AC and MUC5B were quantitated by stable isotope-labelled mass spectrometry in induced sputum samples from healthy never-smokers, ever-smokers at risk for COPD, and ever-smokers with COPD. Participants were extensively characterised using results from questionnaires, such as the COPD assessment test (CAT) and St George's Respiratory Questionnaire; quantitative CT, such as residual volume/total lung capacity ratio (RV/TLC) and parametric response mapping-functional small airway disease (PRM-fSAD); and pulmonary function tests, such as FEV1, forced vital capacity (FVC), and forced expiratory flow, midexpiratory phase (FEF25-75%). Absolute concentrations of both MUC5AC and MUC5B were related to cross-sectional (baseline, initial visit) and 3-year follow-up longitudinal data, including lung function, small airways obstruction, prospective acute exacerbations, and smoking status as primary outcomes. This study is registered with ClinicalTrials.gov (NCT01969344). FINDINGS: This analysis included 331 participants (mean age 63 years [SEM 9·40]), of whom 40 were healthy never-smokers, 90 were at-risk ever-smokers, and 201 were ever-smokers with COPD. Increased MUC5AC concentrations were more reliably associated with manifestations of COPD than were MUC5B concentrations, including decreased FEV1 and FEF25-75%, and increased prospective exacerbation frequency, RV/TLC, PRM-fSAD, and COPD assessment scores. MUC5AC concentrations were more reactive to cigarette smoke exposure than were MUC5B concentrations. Longitudinal data from 3-year follow-up visits generated a multivariate-adjusted odds ratio for two or more exacerbations of 1·24 (95% CI 1·04-1·47, p=0·015) for individuals with high baseline MUC5AC concentration. Increased MUC5AC, but not MUC5B, concentration at baseline was a significant predictor of FEV1, FEV1/FVC, FEF25-75%, and CAT score decline during the 3-year follow-up. Moreover, current smokers in the at-risk group showed raised MUC5AC concentrations at initial visits and decreased lung function over 3 years. By contrast, former smokers in the at-risk group showed normal MUC5AC concentrations at the initial visit and preserved lung function over 3 years. INTERPRETATION: These data indicate that increased MUC5AC concentration in the airways might contribute to COPD initiation, progression, exacerbation risk, and overall pathogenesis. Compared with MUC5B, greater relative changes in MUC5AC concentrations were observed as a function of COPD severity, and MUC5AC concentration seems to be an objective biomarker to detect disease in at-risk and pre-COPD individuals. These data suggest that MUC5AC-producing pathways could be potential targets for future therapeutic strategies. Thus, MUC5AC could be a novel biomarker for COPD prognosis and for testing the efficacy of therapeutic agents. FUNDING: National Institutes of Health; National Heart, Lung, and Blood Institute.

Comparing endocervical mucus proteome of humans and rhesus macaques.

PURPOSE: Endocervical mucus changes play a key role in regulating fertility throughout the menstrual cycle and in response to hormonal contraceptives. Non-human primates (NHP) provide the most translational animal model for reproductive tract studies, as they have hormonally-regulated menstrual cycles and mucus changes, similar to women. EXPERIMENTAL DESIGN: We used TMT labelling and LC-LC/MS to compare the proteins found in the mucus of the rhesus macaque to the mucus of the human endocervix. Data are available via ProteomeXchange with identifier PXD021710. RESULTS: We found 3048 total proteins present in both rhesus mucus and human mucus, and of these, 57% showed a similar expression pattern. An even higher similarity occurred in the top 500 most prevalent proteins, with overlap in 341 (68%) proteins. Mucin MUC5B was the most highly expressed mucin protein (top 10 expressed proteins in both) but other key proteins related to mucus structure were present in both samples. CONCLUSIONS AND CLINICAL RELEVANCE: We find that the mucus proteome of the endocervical mucus is highly conserved in NHP and women. This supports use of the NHP model system for studies of the endocervix and trials of novel fertility treatments targeting the cervix.

MUC5B Is the predominant mucin glycoprotein in chronic otitis media fluid.

Chronic otitis media (COM), e.g. "glue" ear is characterized by middle ear effusion and conductive hearing loss. Although mucous glycoproteins (mucins), which contribute to increased effusion viscosity, have been analyzed in ear tissue specimens, no studies have been reported that characterize the molecular identity of secreted mucin proteins present in actual middle ear fluid. For this study, effusions from children with COM undergoing myringotomy at Children's National Medical Center, Washington, DC were collected. These were solubilized and gel fractionated, and the protein content was identified using a liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics approach. Western blot analyses with mucin specific antibodies and densitometry were performed to validate the mass spectrometry findings. LC-MS/MS results identified mucin MUC5B by >26 unique peptides in six of six middle ear effusion samples, whereas mucin MUC5AC was only identified in one of six middle ear effusions. These findings were validated by Western blot performed on the same six and on an additional 11 separate samples where densitometry revealed on average a 6.4-fold increased signal in MUC5B when compared with MUC5AC (p = 0.0009). In summary, although both MUC5AC and MUC5B mucins are detected in middle ear effusions, MUC5B seems to be predominant mucin present in COM secretions.