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Aim: Airway mucins in sputum are promising respiratory disease biomarkers, despite posing substantial analytical challenges due to their physicochemical properties and rare and heterogenous nature of the matrix. We aimed to identify a suitable sputum collection and processing method, and qualify a bioanalytical method for MUC5AC and MUC5B quantification in clinical samples., Method: Mucins were quantified in induced and spontaneous sputum collected from the same COPD patients, following various sample processing procedures. LC-MS/MS method used truncated recombinant mucins as surrogate analytes in surrogate matrix., Results: Frozen spontaneous sputum was found to be a suitable and convenient matrix for mucin quantification and fit-for-purpose method qualification was performed., Conclusion: Our methodology provides accurate and reliable MUC5AC and MUC5B quantification and facilitates multi-site clinical sputum collection.

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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

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 FEV 1 , forced vital capacity (FVC), and forced expiratory flow, midexpiratory phase (FEF 25-75% ). 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 FEV 1 and FEF 25-75% , 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 FEV 1 , FEV 1 /FVC, FEF 25-75% , 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., Competing Interests: Declaration of interests RGB reports grants from the National Institutes of Health (NIH), Foundation for the NIH (FNIH), and the COPD Foundation, during the study, and grants from NIH outside the submitted work. ERB has undertaken clinical trials through his employer, Wake Forest School of Medicine and University of Arizona, for AstraZeneca, MedImmune, Boehringer Ingelheim, Genentech, Johnson & Johnson (Janssen), Novartis, Regeneron, and Sanofi Genzyme. ERB has also served as a paid consultant for AstraZeneca, MedImmune, Boehringer Ingelheim, GlaxoSmithKline, Novartis, Regeneron, and Sanofi Genzyme, outside the submitted work. SAC reports personal fees from AstraZeneca, GlaxoSmithKline, Amgen, Glenmark, Sunovion, Genentech, and UpToDate, outside the submitted work. CBC reports grants from NIH/National Heart, Lung, and Blood Institute (NHLBI), FNIH, and COPD Foundation, during the conduct of the study. CBC reports personal fees from PulmonX, Nuvaira, and MGC Diagnostics, and is a Global Medical Expert for GlaxoSmithKline, outside the submitted work. MKH reports grants from NHLBI during the conduct of the study, personal fees from GlaxoSmithKline, Boehringer Ingelheim, AstraZeneca, Merck, Mylan, Teva, and Verona, and research support from Sanofi, Sunovion, and Novartis, outside the submitted work. NNH reports grants from NIH and COPD Foundation, grants and personal fees from AstraZeneca, GlaxoSmithKline, and Boehringer Ingelheim, and personal fees from Mylan, outside the submitted work. ATH reports grants from NHLBI and FNIH during the conduct of the study. EAH reports grants from NIH during the conduct of the study. EAH is a founder and shareholder of VIDA Diagnostics, outside the submitted work. FJM reports personal fees from GlaxoSmithKline, AstraZeneca, Boehringer Ingelheim, and Raziel, during the conduct of the study. FJM reports personal fees and non-financial support from AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Miller Communications, National Society for Continuing Education, PeerView Communications, Chiesi, Sunovion, Physicians Education Resource, Canadian Respiratory Network, Teva, CSL Behring, and Sanofi/Regeneron; non-financial support from ProterixBio, Gilead, Nitto, Zambon, and twoXAR; personal fees, non-financial support, and non-personnel travel support from Genentech; personal fees from MD Magazine, Methodist Hospital Brooklyn, New York University, UpToDate, WebMD/MedScape, Patara/Respivant, Bayer, American Thoracic Society, Rockpointe, CME Outfitters, Dartmouth University, DevPro, Gala, Integritas, IQVIA, Projects in Knowledge, Vindico, and Academy for Continuing Healthcare Learning; IPF Study Steering Committee for Afferent/Merck, Biogen, Veracyte, Prometic, and Bayer; IPF Advisor for Bridge Biotherapeutics; IPF teleconference with AbbVie; and grants from NIH, Rare Disease Healthcare Communications, and Promedior/Roche, outside the submitted work. RP reports grants from NHLBI and COPD Foundation, during the conduct of the study. RP reports grants from the Department of Veterans Affairs, and personal fees from Partner Therapeutics, outside the submitted work. PGW reports personal fees from Sanofi, Regeneron, Glenmark Pharmaceuticals, Theravance, GlaxoSmithKline, and NGM Pharma, outside the submitted work. WKO'N reports grants from NIH/NHLBI, during the conduct of the study. RCB reports grants from NIH, during the conduct of the study; and personal fees from Parion Sciences, outside the submitted work. MK reports grants from NIH, during the conduct of the study; contracts from Genentech, Gala Therapeutics, AstraZeneca, Ionis Pharmaceuticals, and personal fees from Boehringer Ingelheim and Amgen, outside the submitted work. In addition, MK has a patent (methods for diagnosing or predicting chronic bronchitis) pending. All other authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (© 2021 Wiley-VCH GmbH.)

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.

Aflatoxin M1 (AFM1), ochratoxin A (OTA), and zearalenone (ZEA) are mycotoxins commonly found in milk. Mycotoxin contamination has caused food safety concerns worldwide since most of the toxic effects in humans are serious. The combined toxic effects of these mycotoxins on intestinal epithelial cells have not been reported. Herein, we investigated the combined effects of AFM1, OTA, and ZEA on intestinal integrity and define the underlying mechanisms(s) of their effects in Caco-2/HT29-MTX co-cultures. Our results showed that the mixtures of AFM1 + OTA, AFM1 + ZEA, and AFM1 + ZEA + OTA significantly increased epithelial permeability. Immunofluorescence analysis and transmission electron microscopy revealed that mycotoxins altered TJ proteins morphology and disrupted their structures. Also, the present study showed that mixtures of mycotoxins significantly modulated MUC5AC and MUC5B mRNA levels and protein secretion. This study demonstrated that the effects of mixtures of mycotoxins on intestinal barrier function were more significant than AFM1 alone. More importantly, the damage of intestinal integrity caused by mycotoxins was correlated to the change of the TJ proteins location and the decrease of mucin secretion. Mixtures of AFM1, OTA, and ZEA in food might pose a health risk to consumers, particularly in children, and toxin risks should be considered., (Copyright © 2019. Published by Elsevier B.V.)

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.

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.).

The identification of body fluids at a crime scene is an important aspect of forensic casework analysis, being a source for investigative leads and contributing to case evidence. Yet, current methods for the forensic identification of body fluids suffer from several limitations, ranging from poor sensitivity and specificity, to sample destruction and interference with subsequent DNA analysis. Moreover, current identification assays target only one body fluid at the time. Besides being inefficient in terms of time, money and sample consumption, poor identification methods can also negatively influence the outcome of a (court) case. In this study, eleven potential protein biomarkers and antibodies were selected and assessed on their suitability for serving as identification markers, as a first step towards the development of a new multiplex protein-based body fluid identification assay relying on antigen-antibody interactions. Performing antibody-based dot blot assays, the specificity of the biomarkers for their target body fluids was evaluated, and biomarker detection was studied in diluted, mixed, aged and simulated casework samples. Hereby, nine out of eleven markers were identified as promising biomarkers to identify blood, semen, saliva, urine and sweat. With the identification of these targets and detection antibodies, a major step forward has been taken towards the development of a highly sensitive and specific, fast and non-labour-intensive protein-based body fluid identification assay, suitable for on-site analysis and able to test for multiple body fluids in a single reaction., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Alveolar epithelia play an essential role in maintaining the integrity and homeostasis of lungs, in which alveolar epithelial type II cells (AECII) are a cell type with stem cell potential for epithelial injury repair and regeneration. However, mechanisms behind the physiological and pathological roles of alveolar epithelia in human lungs remain largely unknown, partially owing to the difficulty of isolation and culture of primary human AECII cells. In the present study, we aimed to characterize alveolar epithelia generated from A549 lung adenocarcinoma cells that were cultured in an air-liquid interface (ALI) state. Morphological analysis demonstrated that A549 cells could reconstitute epithelial layers in ALI cultures as evaluated by histochemistry staining and electronic microscopy. Immunofluorescent staining further revealed an expression of alveolar epithelial type I cell (AECI) markers aquaporin-5 protein (AQP-5), and AECII cell marker surfactant protein C (SPC) in subpopulations of ALI cultured cells. Importantly, molecular analysis further revealed the expression of AQP-5, SPC, thyroid transcription factor-1, zonula occludens-1 and Mucin 5B in A549 ALI cultures as determined by both immunoblotting and quantitative RT-PCR assay. These results suggest that the ALI culture of A549 cells can partially mimic the property of alveolar epithelia, which may be a feasible and alternative model for investigating roles and mechanisms of alveolar epithelia in vitro.

Oral lichen planus (OLP) is among the most common oral diseases. Its etiopathogenesis has yet to be clearly identified. OLP patients complain of mouth dryness. This study aimed to assess the level of Mucin 5B in OLP patients with xerostomia. This study was conducted on 30 OLP patients and 30 healthy individuals. In addition to patient complaint of mouth dryness, xerostomia was assessed by tongue blade and lipstick tests. Stimulated and unstimulated saliva were collected in plastic vials by spitting method. Level of Mucin 5B was measured by ELISA. Unstimulated saliva flow was significantly lower in OLP patients (P = 0.0001). Stimulated saliva flow was not significantly different between the two groups (P > 0.05). Level of Mucin 5B in unstimulated saliva was significantly lower in OLP group (P = 0.0001) while it was not significantly different in stimulated saliva of the two groups (P > 0.05). Level of Mucin 5B was significantly higher in serum of OLP patients (P = 0.016). Both saliva flow and level of Mucin 5B decrease in OLP patients. Since Mucin 5B is effective for wetting and lubrication of the oral cavity, this result can suggest a possible reason for mouth dryness in OLP patients.

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.).

Background: Acute otitis media, an infection of the middle ear, can become chronic after multiple episodes. Microbial influence on chronic otitis media remains unclear. It has been reported that mucin glycoproteins are required for middle ear immune defense against pathogens. We aim to characterize the middle ear effusion (MEE) microbiome using high-throughput sequencing and assess potential associations in microbiome diversity with the presence of the secretory mucins MUC5B and MUC5AC. We hypothesize that MEEs containing MUC5B will exhibit a microbiome largely devoid of typical acute otitis media bacteria., Methods: Fifty-five MEEs from children undergoing myringotomy at Children's National Health System were recovered. Mucin was semiquantitatively determined through Western blot analysis. DNA was subjected to 16S rRNA amplicon sequencing using the Illumina MiSeq platform. Raw data were processed in mothur (SILVA reference database). Alpha- and beta-diversity metrics were determined. Abundance differences between sample groups were estimated., Results: MUC5B was present in 94.5% and MUC5AC in 65.5% of MEEs. Sequencing revealed 39 genera with a relative abundance ≥0.1%. Haemophilus (22.54%), Moraxella (11.11%) and Turicella (7.84%) were the most abundant. Turicella and Pseudomonas proportions were greater in patients older than 24 months of age. In patients with hearing loss, Haemophilus was more abundant, while Turicella and Actinobacteria were less abundant. Haemophilus was also more abundant in samples containing both secretory mucins., Conclusions: The microbiome of MEEs from children with chronic otitis media differs according to specific clinical features, such as mucin content, age and presence of hearing loss. These associations provide novel pathophysiologic insights across the spectrum of otitis media progression.

Objective: To observe the therapeutic effect of simple 3.0% saline nasal irrigation and combined treatment of 3.0% saline nasal irrigation and budesonide nasal spray for vasomotor rhinitis (VMR), and explore the long-term effect for VMR. Through examination of levels of substance P (SP) and mucin (MUC)5B in nasal lavage fluid, the mechanisms of nasal irrigation treatment for VMR was discussed. Methods: One hundred and one patients from Department of Otorhinolaryngology Head and Neck Surgery, Huashan Hospital of Fudan University with VMR were randomly divided into 4 groups. The number of patients was 24 in control group, 25 in budesonide nasal spray treatment group (budesonide group), 25 in nasal irrigation treatment group (nasal irrigation group) and 27 in budesonide nasal spray + nasal irrigation group (combined treatment group). Control patients were left untreated. Budesonide group was under budesonide nasal spray treatment, nasal irrigation group was treated using 3.0% saline with a temperature of 40℃ and combined treatment group was given both treatments. The duration of the intervention period was 3 months (90 days). Visual Analog Scale (VAS) was used to evaluate nasal symptoms, and the health-related quality of life was assessed using the 12-item Short Form Health Survey version 2.0 (SF-12v2). Enzyme-linked immunosorbent assay (ELISA) was used to assess the contents of SP and MUC5B in nasal lavage fluid before and after 3-month treatments in budesonide and nasal irrigation group in the study. MUC5B in nasal lavage fluid after the SP challenge and anticholinergic drug intervention in control group were also evaluated with ELISA. Results: Nighty out of 101 patients completed the study. In the budesonide and combined treatment group after relevant interventions, the total VAS score of nasal symptoms decreased (5.91±0.21 vs 3.82±0.15, 6.18±0.17 vs 3.92±0.15, t value was 8.193, 10.060, respectively, all P <0.05) and SF-12v2 score increased (146.00±1.23 vs 152.30±0.97, 146.00±1.08 vs 155.40±0.90, t value was 3.982, 6.697, respectively, all P <0.05), with both scores showed no significant differences in the nasal irrigation group (5.96±0.17 vs 5.72±0.15, 146.10±1.17 vs 147.00±0.94, t value was 1.038, 0.607, respectively, all P >0.05) after the first month. In the budesonide and combined treatment group after relevant interventions, the total VAS score of nasal symptoms decreased (5.91±0.21 vs 5.05±0.15, 6.18±0.17 vs 5.10±0.12, t value was 3.374, 5.351, respectively, all P <0.05) and SF-12v2 score increased (146.00±1.23 vs 150.90±0.76, 146.00±1.08 vs 153.60±0.94, t value was 3.373, 5.343, respectively, all P <0.05), with both scores showed no significant differences in the nasal irrigation group (5.96±0.17 vs 5.78±0.17, 146.10±1.17 vs 148.10±0.80, t value was 0.716, 1.438, respectively, all P >0.05) after the second month. By the end of the third month, in nasal irrigation and combined treatment group, the VAS score was diminished (5.96±0.17 vs 4.80±0.12, 6.18±0.17 vs 4.44±0.13, t value was 5.485, 8.264, respectively, all P <0.05) and SF-12v2 score was elevated (146.10±1.17 vs 150.80±0.96, 146.00±1.08 vs 152.90±0.85, t value was 3.163, 5.008, respectively, all P <0.05), but there were no significant differences in budesonide group (5.91±0.21 vs 5.68±0.18, 146.00±1.23 vs 148.40±0.85, t value was 0.819, 1.587, respectively, all P >0.05). Additionally, SP in nasal lavage fluid decreased and MUC5B showed no statistical changes in budesonide group after three months, however, SP showed no any changes and MUC5B reduced significantly in nasal lavage fluid in nasal irrigation group. Furthermore, the anticholinergic drug could not decrease the concentration of MUC5B after the SP challenge in nasal cavity in control group. Conclusions: The therapeutic effect of simple nasal irrigation with 3.0% saline or combined treatment of 3.0% saline nasal irrigation and nasal corticosteroids is superior to simple nasal corticosteroids. Nasal corticosteroids plays a role in the inhibition of sensory nerve endings in nasal mucosa, but neurotransmitter plays a limited role in the pathogenesis of VMR.

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., Competing Interests: The authors have declared that no competing interest exists.

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., (Copyright © 2016 The American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

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., (© 2015 John Wiley & Sons Ltd.)

Objectives: To determine the use of the mucin proteins MUC5B and MUC5AC as prognosis markers for non-small cell lung cancer (NSCLC) carrying epidermal growth factor receptor (EGFR) mutations., Setting: Patients who underwent surgical resection at Nagasaki University Hospital and related facilities in Japan between June 1996 and March 2013., Participant: 159 Japanese patients (male: n=103; female: n=56) with NSCLC, who underwent surgical resection (EGFR-mutant type: n=78, EGFR wild type: n=81)., Results: Patients whose tumours expressed MUC5B had significantly longer overall survival and relapse-free survival compared to the MUC5B-negative patients with EGFR mutant NSCLC (p=0.0098 and p=0.0187, respectively). In patients with EGFR wild-type NSCLC, there was no association with MUC5B expression. MUC5AC expression was not different between EGFR mutant and wild-type NSCLC., Conclusions: Present findings indicate that MUC5B, but not MUC5AC, is a novel prognostic biomarker for patients with NSCLC carrying EGFR mutations but not for patients with NSCLC carrying wild-type EGFR., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

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., (© 2015 Eur J Oral Sci.)

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., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The pathogenesis of mucoinfective lung disease in cystic fibrosis (CF) patients likely involves poor mucus clearance. A recent model of mucus clearance predicts that mucus flow depends on the relative mucin concentration of the mucus layer compared with that of the periciliary layer; however, mucin concentrations have been difficult to measure in CF secretions. Here, we have shown that the concentration of mucin in CF sputum is low when measured by immunologically based techniques, and mass spectrometric analyses of CF mucins revealed mucin cleavage at antibody recognition sites. Using physical size exclusion chromatography/differential refractometry (SEC/dRI) techniques, we determined that mucin concentrations in CF secretions were higher than those in normal secretions. Measurements of partial osmotic pressures revealed that the partial osmotic pressure of CF sputum and the retained mucus in excised CF lungs were substantially greater than the partial osmotic pressure of normal secretions. Our data reveal that mucin concentration cannot be accurately measured immunologically in proteolytically active CF secretions; mucins are hyperconcentrated in CF secretions; and CF secretion osmotic pressures predict mucus layer-dependent osmotic compression of the periciliary liquid layer in CF lungs. Consequently, mucin hypersecretion likely produces mucus stasis, which contributes to key infectious and inflammatory components of CF lung disease.

The mucosal pellicle is defined as the protein film adsorbed onto oral mucosa. This study aimed at characterizing the ultrastructure of human epithelial buccal cells and localizing salivary mucins MUC5B, a major constituent of the mucosal pellicle. Cells were sampled from the buccal surface and prepared for Transmission Electron Microscopy using high-pressure freezing/cryosubstitution followed by immunogold labelling of MUC5B. Morphologically, cells were visualized as typical cells of the superficial layer of a squamous nonkeratinized epithelium with a partly degraded plasma membrane. The outer surface of the plasma membrane was lined with a biological material of medium electron density. MUC5B were detected in the extracellular space, and particularly in the vicinity of the plasma membrane, sometimes onto fibrils protruding from the membrane. This area was, therefore, considered as constituting the mucosal pellicle, which appeared as a mixed film of both salivary and epithelial components. The distribution of gold particles suggested that the surface of the pellicle was not uniform, and that the film thickness could reach up to 100 nm. This work showed the feasibility of visualizing and characterizing the mucosal pellicle directly on human epithelial buccal cells sampled in a noninvasive manner., (© 2014 Wiley Periodicals, Inc.)

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.

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., (© 2014 S. Karger AG, Basel.)

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.

Conclusion: MUC5B immunohistochemical staining was a valuable method for identifying fungi in fungal ball sinusitis, especially in distinguishing aspergillus and mucor. Combined infection with Aspergillus and Mucor fungi was the most common pattern in fungal ball sinusitis., Objective: To assess the value of MUC5B immunohistochemical staining in identifying fungi in fungal ball sinusitis by comparing it with conventional Grocott methenamine silver (GMS) staining., Methods: GMS staining and MUC5B immunohistochemical staining were used to identify fungi in mucopurulent cheesy or clay-like tissues from sinuses in 180 fungal ball sinusitis patients, and the examination results were compared., Results: In 180 samples of fungal ball sinusitis, GMS staining showed Aspergillus in 130, Mucor in 88, and Candida albicans in 6, while MUC5B immunohistochemical staining identified Aspergillus in 166, Mucor in 172, and Candida albicans in 16. The fungal detection rate for MUC5B immunohistochemical staining was markedly greater than that for GMS staining (p < 0.01). This implies that MUC5B immunohistochemical staining was more sensitive than GMS staining in identifying fungi in fungal ball sinusitis. Mixed infection of Aspergillus and Mucor was present in 146 of 180 patients (81.1%).

Objectives: Factors influencing xerostomia during intensity-modulated radiation therapy (IMRT) were assessed., Methods: A 6-week study of 32 head and neck cancer (HNC) patients was performed. Subjects completed the Xerostomia Inventory (XI) and provided stimulated saliva (SS) at baseline, week 2 and at end of IMRT. Influence of SS flow rate (SSFR), calcium and mucin 5b (MUC5b) concentrations and radiation dose on xerostomia was determined., Results: HNC subjects experienced mean SSFR decline of 36% by visit 2 (N = 27; P = .012) and 57% by visit 3 (N = 20; P = .0004). Concentrations of calcium and MUC5b increased, but not significantly during IMRT (P > .05). Xerostomia correlated most with decreasing salivary flow rate as determined by Spearman correlations (P < .04) and linear mixed models (P < .0001)., Conclusions: Although IMRT is sparing to the parotid glands, it has an early effect on SSFR and the constituents in saliva in a manner that is associated with the perception of xerostomia., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: The salivary mucin MUC5B, present in (sero)mucous secretions including submandibular gland (SMG) saliva, plays an important role in the lubrication of the oral mucosa and is thought to be related to the feeling of dry mouth. We investigated if MUC5B levels in SMG saliva could distinguish between the presence or absence of severe dry mouth complaints 12  months after radiotherapy (RT) for head-and-neck cancer (HNC)., Findings: Twenty-nine HNC patients with a residual stimulated SMG secretion rate of ≥ 0.2 ml/10 min at 12  months after RT were analyzed. MUC5B (in U; normalized to 1) and total protein levels (mg/ml) were measured in SMG saliva at baseline and 12 months after RT using ELISA and BCA protein assay, respectively. Overall, median MUC5B levels decreased after RT from 0.12 to 0.03 U (p = 0.47). Patients were dichotomized into none/mild xerostomia (n = 12) and severe xerostomia (n = 17) based on a questionnaire completed at 12 months. SMG and whole saliva flow rates decreased after RT but were comparable in both groups. The median MUC5B level was higher in patients with no or mild xerostomia compared to patients with severe xerostomia (0.14 vs 0.01 U, p = 0.22). Half of the patients with severe xerostomia had no detectable MUC5B at 12 months after RT. No differences in total protein levels were observed., Conclusions: Qualitative saliva parameters like MUC5B need further investigation in RT-induced xerostomia. This pilot study showed a trend towards lower MUC5B levels in the SMG saliva of patients with severe xerostomia 12 months after RT for HNC.

In recent years, minor salivary glands, due to their involvement in the health and homeostasis of the oral cavity, have been the focus of several research investigations. Despite the fact that a considerable amount of data has been collected, many aspects of their functional features, including the secretory components they produce, remain to be ascertained. In this study we have analyzed the ultrastructural distribution of the MUC5B mucin in human palatal and buccal glands by means of post-embedding immunoelectron microscopy. Thin sections of normal human buccal and palatal glands obtained at surgery, were treated with polyclonal antibodies to human salivary MUC5B. Intense MUC5B reactivity was observed in the secretory granules of mucous cells of all glands examined. The present results provide new data regarding the secretory pattern of MUC5B in human buccal and palatal glands, indicating their significant contribution to the maintenance of the mucous biofilm that protects buccal and palatal mucosal areas., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Mucus accumulation is a feature of inflammatory airway disease in the horse and has been associated with reduced performance in racehorses. In this study, we have analysed the two major airways gel-forming mucins Muc5b and Muc5ac in respect of their site of synthesis, their biochemical properties, and their amounts in mucus from healthy horses and from horses with signs of airway mucus accumulation. Polyclonal antisera directed against equine Muc5b and Muc5ac were raised and characterised. Immunohistochemical staining of normal equine trachea showed that Muc5ac and Muc5b are produced by cells in the submucosal glands, as well as surface epithelial goblet cells. Western blotting after agarose gel electrophoresis of airway mucus from healthy horses, and horses with mucus accumulation, was used to determine the amounts of these two mucins in tracheal wash samples. The results showed that in healthy horses Muc5b was the predominant mucin with small amounts of Muc5ac. The amounts of Muc5b and Muc5ac were both dramatically increased in samples collected from horses with high mucus scores as determined visually at the time of endoscopy and that this increase also correlated with increase number of bacteria present in the sample. The change in amount of Muc5b and Muc5ac indicates that Muc5b remains the most abundant mucin in mucus. In summary, we have developed mucin specific polyclonal antibodies, which have allowed us to show that there is a significant increase in Muc5b and Muc5ac in mucus accumulated in equine airways and these increases correlated with the numbers of bacteria.

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.

Oral homeostasis depends largely on proteins and mucins present in saliva that coat all oral surfaces. The present study compared the protein composition of residual fluid on mucosal surfaces in subjects with normal salivary flow with that of patients with dry mouth caused by salivary hypofunction. Samples of residual mucosal fluid were collected using paper strips and then analysed by protein electrophoresis and immunoblotting. In both patients and controls, residual fluids on mucosal surfaces (except the anterior tongue in control subjects) had higher protein concentrations than unstimulated whole-mouth saliva. High-molecular-weight mucin (MUC5B) was present in greater amounts on the anterior tongue than on other surfaces in control subjects. In dry mouth patients who were unable to provide a measurable saliva sample, MUC5B was often still present on all mucosal surfaces but in reduced amounts on the anterior tongue. The membrane-bound mucin, MUC1, was prominent on buccal and labial surfaces in patients and controls. Statherin was still present on surfaces that were dried to remove salivary fluid, suggesting that it may be adsorbed as a protein pellicle. It is concluded that oral mucosal surfaces in dry mouth patients can retain MUC5B and other salivary proteins, although the functional integrity of these proteins is uncertain.

As a result of their presence throughout the mouth in the submucosa or between muscle fibers, minor salivary glands secrete directly and continuously into the oral cavity, providing mucosal surfaces with highly glycosylated proteins that are active in bacterial aggregation and in oral tissue lubrication. In this study, we investigated the ultrastructural localization of the MUC5B and MUC7 mucins in human labial glands by means of a postembedding immunogold technique. Thin sections of normal human labial glands, obtained during surgery, were incubated with polyclonal antibodies to human salivary mucins MUC5B and MUC7, and then with gold-labeled secondary antibodies. Specific MUC5B reactivity was found in the secretory granules of mucous cells of all glands examined, and was associated with the luminal membrane of duct cells. MUC7 labeling was observed in the granules of both mucous and seromucous secretory cells of the glandular parenchyma. Quantitative analyses demonstrated that seromucous granules have higher immunogold labeling densities for MUC7 than mucous granules. Our immunohistochemical data extend the results of previous light microscopic studies of MUC5B and MUC7 localizations, pointing out the significant contribution of human labial glands in the secretion process of these two mucins.

This study examined the expression, in oral keratinocytes and in the major and minor salivary glands, of Trefoil factor family 3 (TFF3) peptide. Trefoil factor family 3 messenger RNA (mRNA) and peptide were detected in cultures of normal oral keratinocytes by quantitative real-time polymerase chain reaction (PCR) and western blotting, respectively. Trefoil factor family 3 was found, by immunohistochemical analyses, to be expressed in the basal layers of the oral mucosal epithelium. In salivary glands, immunohistochemical staining showed that TFF3 peptide expression was strongest in the mucous acini of the submandibular and the small salivary glands. Serous cells in the same glands showed weak staining. In the parotid gland, many serous acini showed weak positive staining, while other areas did not. In all glands examined, the intercalated, striated, and collecting ducts were moderately TFF3-positive. Double immunostaining confirmed that mucous (MUC5B positive) cells were moderately or strongly positive for TFF3 and that some serous (MUC7 positive) cells showed restricted TFF3 expression, mostly in a granular pattern. The prevalence of the TFF3 peptide in the salivary secretions of healthy volunteers was detected by western blotting of saliva from minor salivary glands (four of five) and the parotid gland (one of five) and of mixed submandibular/sublingual saliva (five of five). In conclusion, the submandibular and small salivary glands appear to be the major producers of oral TFF3, but duct cells of all glands and keratinocytes of the oral mucosa may also contribute as sources of TFF3 in the oral cavity.

Objective: The aim of the present study was to assess the salivary levels of MUC5B and MUC7 in individuals with dyspeptic disease and Helicobacter pylori (H. pylori) in the stomach, compared to individuals without dyspeptic disease., Methods: 30 individuals with dyspeptic disease, who underwent endoscopy for upper gastrointestinal complaints at Hospital Pedro Ernesto-RJ, Brasil and tested positive for H. pylori, and 23 controls with no dyspeptic disease, with mean age 53.5+/-4.4 years, were included in the study. Saliva samples and 3 antral biopsy were taken for PCR analysis and histologic examination. In addition, saliva samples were tested by ELISA with F2 monoclonal antibody and EU7A antibody against MUC7, to determine MUC5B and MUC7 levels, prior to endoscopic examination. The expression pattern of the proteins was quantified by comparison to a pooled saliva sample of 19 healthy volunteers., Results: MUC5B and MUC7 salivary levels were higher in the individuals with dyspeptic disease than in controls (p<0.0001). 33.3% (9/30) of the dyspeptic individuals and 0% of the controls had H. pylori in the oral cavity., Conclusions: Individuals with gastric diseases, with H. pylori in the stomach, showed higher levels of salivary H. pylori receptors-MUC5B and MUC7-than individuals without gastric diseases. These results suggest that higher levels of specific salivary mucins could be useful as risk indicators for infection by H. pylori.

Salivary mucins MG1 and MG2 have been found in the oral cavity where they perform several functions such as the formation of the mucous layer covering the oral mucosa and teeth. Recent studies have demonstrated their presence in other organs and tissues. The aim of this study was to determine their expression in human bulbourethral (Cowper's) glands. Normal bulbourethral glands were obtained at surgery and fixed in a mixture of 1% paraformaldehyde-1.25% glutaraldehyde in 0.1 M cacodylate buffer and embedded in Epon resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells were intensely reactive with anti-MG1, whereas no labeling was detected for MG2. These results indicate that MG1 is not exclusively a salivary component and furthermore show that bulbourethral glands represent a significant source of the MG1 detected in human seminal plasma.

Rationale: Overproduction of mucus is a contributory factor in the progression of chronic obstructive pulmonary disease (COPD). The polymeric mucins are major macromolecules in the secretion. Therefore, we hypothesized that the polymeric mucin composition or properties may be different in the sputum from individuals with COPD and smokers without airflow obstruction., Objectives: To determine the major polymeric mucins in COPD sputum and whether these are different in the sputum from individuals with COPD compared with that from smokers without airflow obstruction., Methods: The polymeric mucin composition of sputum from patients with COPD and smokers without airflow obstruction was analyzed by Western blotting analysis. The tissue localization of the mucins was determined by immunohistochemistry, and their size distribution was analyzed by rate-zonal centrifugation., Measurements and Main Results: MUC5AC and MUC5B were the major mucins. MUC5AC was the predominant mucin in the smoker group, whereas MUC5B was more abundant from the patients with COPD, with a significant difference in the ratio of MUC5B to MUC5AC (P = 0.004); this ratio was correlated with FEV(1) in the COPD group (r = 0.63; P = 0.01). The lower-charged glycosylated form of MUC5B was more predominant in COPD (P = 0.012). No significant associations were observed with respect to sex, age, or pack-year history. In both groups, MUC5AC was produced by surface epithelial cells and MUC5B by submucosal gland cells. Finally, there was a shift toward smaller mucins in the COPD group., Conclusions: Our data indicate that there are differences in mucin amounts and properties between smokers with and without COPD. Further studies are needed to examine how this may impact disease progression.

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.