An operative framework to model mucus clearance in silico by coupling cilia motion with the liquid environment.

Mucociliary clearance is the first defense mechanism of the respiratory tract against inhaled particles. This mechanism is based on the collective beating motion of cilia at the surface of epithelial cells. Impaired clearance, either caused by malfunctioning or absent cilia, or mucus defects, is a symptom of many respiratory diseases. Here, by exploiting the lattice Boltzmann particle dynamics technique, we develop a model to simulate the dynamics of multiciliated cells in a two-layer fluid. First, we tuned our model to reproduce the characteristic length- and time-scales of the cilia beating. We then check for the emergence of the metachronal wave as a consequence of hydrodynamic mediated correlations between beating cilia. Finally, we tune the viscosity of the top fluid layer to simulate the mucus flow upon cilia beating, and evaluate the pushing efficiency of a carpet of cilia. With this work, we build a realistic framework that can be used to explore several important physiological aspects of mucociliary clearance.

Colon mucus in colorectal neoplasia and beyond.

Little was known about mammalian colon mucus (CM) until the beginning of the 21st century. Since that time considerable progress has been made in basic research addressing CM structure and functions. Human CM is formed by two distinct layers composed of gel-forming glycosylated mucins that are permanently secreted by goblet cells of the colonic epithelium. The inner layer is dense and impenetrable for bacteria, whereas the loose outer layer provides a habitat for abundant commensal microbiota. Mucus barrier integrity is essential for preventing bacterial contact with the mucosal epithelium and maintaining homeostasis in the gut, but it can be impaired by a variety of factors, including CM-damaging switch of commensal bacteria to mucin glycan consumption due to dietary fiber deficiency. It is proven that impairments in CM structure and function can lead to colonic barrier deterioration that opens direct bacterial access to the epithelium. Bacteria-induced damage dysregulates epithelial proliferation and causes mucosal inflammatory responses that may expand to the loosened CM and eventually result in severe disorders, including colitis and neoplastic growth. Recently described formation of bacterial biofilms within the inner CM layer was shown to be associated with both inflammation and cancer. Although obvious gaps in our knowledge of human CM remain, its importance for the pathogenesis of major colorectal diseases, comprising inflammatory bowel disease and colorectal cancer, is already recognized. Continuing progress in CM exploration is likely to result in the development of a range of new useful clinical applications addressing colorectal disease diagnosis, prevention and therapy.

From Static to Dynamic: A Review on the Role of Mucus Heterogeneity in Particle and Microbial Transport.

Mucus layers (McLs) are on the front line of the human defense system that protect us from foreign abiotic/biotic particles (e.g., airborne virus SARS-CoV-2) and lubricates our organs. Recently, the impact of McLs on human health (e.g., nutrient absorption and drug delivery) and diseases (e.g., infections and cancers) has been studied extensively, yet their mechanisms are still not fully understood due to their high variety among organs and individuals. We characterize these variances as the heterogeneity of McLs, which lies in the thickness, composition, and physiology, making the systematic research on the roles of McLs in human health and diseases very challenging. To advance mucosal organoids and develop effective drug delivery systems, a comprehensive understanding of McLs' heterogeneity and how it impacts mucus physiology is urgently needed. When the role of airway mucus in the penetration and transmission of coronavirus (CoV) is considered, this understanding may also enable a better explanation and prediction of the CoV's behavior. Hence, in this Review, we summarize the variances of McLs among organs, health conditions, and experimental settings as well as recent advances in experimental measurements, data analysis, and model development for simulations.

NanI Sialidase Contributes to the Growth and Adherence of Clostridium perfringens Type F Strain F4969 in the Presence of Adherent Mucus.

Clostridium perfringens type F strains causing nonfoodborne human gastrointestinal diseases (NFD) typically produce NanI sialidase as their major secreted sialidase. Type F NFDs can persist for several weeks, indicating their pathogenesis involves intestinal colonization, including vegetative cell growth and adherence, with subsequent sporulation that fosters enterotoxin production and release. We previously reported that NanI contributes to type F NFD strain adherence and growth using Caco-2 cells. However, Caco-2 cells make minimal amounts of mucus, which is significant because the intestines are coated with adherent mucus. Therefore, it was important to assess if NanI contributes to the growth and adherence of type F NFD strains in the presence of adherent mucus. Consequently, the current study first demonstrated greater growth of nanI-carrying versus non-nanI-carrying type F strains in the presence of HT29-MTX-E12 cells, which produce an adherent mucus layer, versus their parental HT29 cells, which make minimal mucus. Demonstrating the specific importance of NanI for this effect, type F NFD strain F4969 or a complementing strain grew and adhered better than an isogenic nanI null mutant in the presence of HT29-MTX-E12 cells versus HT29 cells. Those effects involved mucus production by HT29-MTX-E12 cells since mucus reduction using N-acetyl cysteine reduced F4969 growth and adherence. Consistent with those in vitro results, NanI contributed to growth of F4969 in the mouse small intestine. By demonstrating a growth and adherence role for NanI in the presence of adherent mucus, these results further support NanI as a potential virulence factor during type F NFDs.

Oral Versus Gastrointestinal Mucosal Immune Niches in Homeostasis and Allostasis.

Different body systems (epidermis, respiratory tract, cornea, oral cavity, and gastrointestinal tract) are in continuous direct contact with innocuous and/or potentially harmful external agents, exhibiting dynamic and highly selective interaction throughout the epithelia, which function as both a physical and chemical protective barrier. Resident immune cells in the epithelia are constantly challenged and must distinguish among antigens that must be either tolerated or those to which a response must be mounted for. When such a decision begins to take place in lymphoid foci and/or mucosa-associated lymphoid tissues, the epithelia network of immune surveillance actively dominates both oral and gastrointestinal compartments, which are thought to operate in the same immune continuum. However, anatomical variations clearly differentiate immune processes in both the mouth and gastrointestinal tract that demonstrate a wide array of independent immune responses. From single vs. multiple epithelia cell layers, widespread cell-to-cell junction types, microbial-associated recognition receptors, dendritic cell function as well as related signaling, the objective of this review is to specifically contrast the current knowledge of oral versus gut immune niches in the context of epithelia/lymphoid foci/MALT local immunity and systemic output. Related differences in 1) anatomy 2) cell-to-cell communication 3) antigen capture/processing/presentation 4) signaling in regulatory vs. proinflammatory responses and 5) systemic output consequences and its relations to disease pathogenesis are discussed.

An intercrypt subpopulation of goblet cells is essential for colonic mucus barrier function.

The intestinal mucus layer, an important element of epithelial protection, is produced by goblet cells. Intestinal goblet cells are assumed to be a homogeneous cell type. In this study, however, we delineated their specific gene and protein expression profiles and identified several distinct goblet cell populations that form two differentiation trajectories. One distinct subtype, the intercrypt goblet cells (icGCs), located at the colonic luminal surface, produced mucus with properties that differed from the mucus secreted by crypt-residing goblet cells. Mice with defective icGCs had increased sensitivity to chemically induced colitis and manifested spontaneous colitis with age. Furthermore, alterations in mucus and reduced numbers of icGCs were observed in patients with both active and remissive ulcerative colitis, which highlights the importance of icGCs in maintaining functional protection of the epithelium.

TRIM34 attenuates colon inflammation and tumorigenesis by sustaining barrier integrity.

Loss of the colonic inner mucus layer leads to spontaneously severe colitis and colorectal cancer. However, key host factors that may control the generation of the inner mucus layer are rarely reported. Here, we identify a novel function of TRIM34 in goblet cells (GCs) in controlling inner mucus layer generation. Upon DSS treatment, TRIM34 deficiency led to a reduction in Muc2 secretion by GCs and subsequent defects in the inner mucus layer. This outcome rendered TRIM34-deficient mice more susceptible to DSS-induced colitis and colitis-associated colorectal cancer. Mechanistic experiments demonstrated that TRIM34 controlled TLR signaling-induced Nox/Duox-dependent ROS synthesis, thereby promoting the compound exocytosis of Muc2 by colonic GCs that were exposed to bacterial TLR ligands. Clinical analysis revealed that TRIM34 levels in patient samples were correlated with the outcome of ulcerative colitis (UC) and the prognosis of rectal adenocarcinoma. This study indicates that TRIM34 expression in GCs plays an essential role in generating the inner mucus layer and preventing excessive colon inflammation and tumorigenesis.

Mucus blocks probiotics but increases penetration of motile pathogens and induces TNF-α and IL-8 secretion.

The mucosal barrier in combination with innate immune system are the first line of defense against luminal bacteria at the intestinal mucosa. Dysfunction of the mucus layer and bacterial infiltration are linked to tissue inflammation and disease. To study host-bacterial interactions at the mucosal interface, we created an experimental model that contains luminal space, a mucus layer, an epithelial layer, and suspended immune cells. Reconstituted porcine small intestinal mucus formed an 880 ± 230 µm thick gel layer and had a porous structure. In the presence of mucus, sevenfold less probiotic and nonmotile VSL#3 bacteria transmigrated across the epithelial barrier compared to no mucus. The higher bacterial transmigration caused immune cell differentiation and increased the concentration of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α; p < .01). Surprisingly, the mucus layer increased transmigration of pathogenic Salmonella and increased secretion of TNF-α and IL-8 (p < .05). Nonmotile, flagella knockout Salmonella had lower transmigration and caused lower IL-8 and TNF-α secretion (p < .05). These results demonstrate that motility enables pathogenic bacteria to cross the mucus and epithelial layers, which could lead to infection. Using an in vitro coculture platform to understand the interactions of bacteria with the intestinal mucosa has the potential to improve the treatment of intestinal diseases.

Intravital microscopic optical coherence tomography imaging to assess mucus-mobilizing interventions for muco-obstructive lung disease in mice.

Airway mucus obstruction is a hallmark of chronic lung diseases such as cystic fibrosis, asthma, and COPD, and the development of more effective mucus-mobilizing therapies remains an important unmet need for patients with these muco-obstructive lung diseases. However, methods for sensitive visualization and quantitative assessment of immediate effects of therapeutic interventions on mucus clearance in vivo are lacking. In this study, we determined whether newly developed high-speed microscopic optical coherence tomography (mOCT) is sensitive to detect and compare in vivo effects of inhaled isotonic saline, hypertonic saline, and bicarbonate on mucus mobilization and clearance in Scnn1b-transgenic mice with muco-obstructive lung disease. In vivo mOCT imaging showed that inhaled isotonic saline-induced rapid mobilization of mucus that was mainly transported as chunks from the lower airways of Scnn1b-transgenic mice. Hypertonic saline mobilized a significantly greater amount of mucus that showed a more uniform distribution compared with isotonic saline. The addition of bicarbonate-to-isotonic saline had no effect on mucus mobilization, but also led to a more uniform mucus layer compared with treatment with isotonic saline alone. mOCT can detect differences in response to mucus-mobilizing interventions in vivo, and may thus support the development of more effective therapies for patients with muco-obstructive lung diseases.

Vertebrate mucus stimulates biofilm development and upregulates iron acquisition genes in Flavobacterium columnare.

Columnaris disease is responsible for substantial losses throughout the production of many freshwater fish species. One of the ways in which the bacterium Flavobacterium columnare is so effective in initiating disease is through the formation of biofilms on fish skin and gills. To further explore the interaction between host factors and bacterial cells, we assayed the ability of vertebrate mucus to enhance F. columnare biofilm development. Different concentrations of catfish, tilapia and pig mucus (5-60 µg/ml) increased biofilm growth at varying degrees among F. columnare isolates. Our data suggest that vertebrate mucus acts as a signalling molecule for the development of F. columnare biofilms; however, there are clear disparities in how individual isolates respond to different mucus fractions to stimulate biofilms. The expression of iron acquisition genes among two genomovar II isolates showed that ferroxidase, TonB receptor and the siderophore synthetase gene were all significantly upregulated among F. columnare biofilms. Interestingly, the siderophore acetyltransferase gene was only shown to be significantly upregulated in one of the genomovar II isolates. This work provides insight into our understanding of the interaction between F. columnare and vertebrate mucus, which likely contributes to the growth of planktonic cells and the transition into biofilms.

Montgomery T-tube for management of tracheomalacia: Impact on voice-related quality of life.

INTRODUCTION: Tracheostomy is a common procedure for management of tracheomalacia. However, the limitation to speak related to tracheostomy cannula could affect the quality of life. OBJECTIVES: we reported a new minimally invasive procedure to replace tracheostomy cannula with Montgomery T-tube to improve the ability of speaking. METHODS: This is a single center study including all consecutive patients undergoing the replacement of standard tracheostomy cannula with T-tube for management of tracheomalacia. The end-points were to evaluate (a) the changes in Voice-related quality of Life (V-RQOL) before and after T-tube placement; and (b) the complications related to T-tube. RESULTS: Eleven patients were included in the study. T-tube was placed using flexible bronchoscopy and laryngeal mask airway. A suture was inserted through the proximal end of T-tube. Once the stent was introduced with a clamp into the trachea, a traction was applied on the suture to facilitate the alignment of the upper end of the stent. The comparison of V-RQOL values before and after T-tube insertion showed a significant improvement in social/emotional (39.2 ± 6.1 vs 66.8 ± 1.9; P = .0001); physical functioning (21 ± 5.7 vs 56.4 ± 5.3; P = 0.0001) and total V-RQOL scores (33.9 + 5.4 vs 61.3 + 6.1; P = 0.0001). No complications were seen during the insertion of the stent. In two patients, T-tube was obstructed by mucus that resolved with aspiration using flexible bronchoscopy (mean follow-up: 18 ± 10 months). CONCLUSIONS: Our technique is simple and safe, not needing specific skills and/or cumbersome devices. The replacement of tracheostomy cannula with T-tube seems to improve the quality of voice without adding major complications.

Randomised study of the immunomodulatory effects of azithromycin in severely asthmatic horses.

Neutrophilic inflammation is believed to contribute to the airway obstruction and remodelling in equine asthma. Azithromycin, an antibiotic with immunomodulatory properties, reduces pulmonary neutrophilia and hyper-responsiveness in human asthmatics and decreases airway remodelling in rodent models of asthma. It was therefore hypothesised that azithromycin would improve lung function, mucus accumulation and central airway remodelling by decreasing luminal neutrophilia in severe equine asthma. The effects of a 10-day treatment with either azithromycin or ceftiofur, an antimicrobial without immune-modulating activity, were assessed using a blind, randomised, crossover design with six severe asthmatic horses in clinical exacerbation. Lung function, tracheal mucus accumulation, tracheal wash bacteriology, bronchial remodelling, airway neutrophilia and mRNA expression of proinflammatory cytokines (interleukin (IL)-8, IL-17A, IL-1ß, tumour necrosis factor-α) in bronchoalveolar lavage fluid were evaluated. Azithromycin decreased the expression of IL-8 (P=0.03, one-tailed) and IL-1ß (P=0.047, one-tailed) but failed to improve the other variables evaluated. Ceftiofur had no effect on any parameter. The reduction of neutrophilic chemoattractants (IL-8, IL-1ß) justifies further efforts to investigate the effects of a prolonged treatment with macrolides on airway neutrophilia and remodelling. The lack of efficacy of ceftiofur suggests that severe equine asthma should not be treated with antibiotics at first-line therapy.

Influence of frequency and amplitude on the mucus viscoelasticity of the novel mechano-acoustic Frequencer™.

BACKGROUND: Cystic fibrosis affects 1/3200 Caucasians. This genetic disease disturbs the ion and water homeostasis across epithelia, thus rendering mucus more viscous and harder to expel. Conventional treatments rely on the clapping method coupled with postural drainage. Despite the effectiveness of these procedures, they are invasive and enervating. METHODS: Here we study a new mechano-acoustic treatment device to help patients expectorate excess mucus, the Frequencer™. We test both normal and pathological synthetic mucin solutions (1 % and 4 % by weight) in vitro. We varied the frequency applied (from 20 Hz to 60 Hz) as well as the amplitude (from 50 % to 100 % intensity). Moreover, we assessed the effect of NaCl on mucus rehydration. RESULTS: A frequency of 40 Hz coupled with a 0.5 gL-1NaCl solution provokes partial mucus rehydration, regardless of the amplitude selected, as the work of adhesion measurements evidenced. CONCLUSIONS: Mechanical solicitation is fundamental to help patients affected by cystic fibrosis expectorate mucus. With an operating frequency of 20 Hz to 65 Hz, the Frequencer™ provides a gentler therapy than traditional methods (conventional chest physiotherapy). The Frequencer™ proved to be effective in the homogenization of synthetic mucin solutions in vitro in 20 min and elicited improved effectiveness in a mucin-rich environment.

Differences in the Intended Meaning of Congestion Between Patients and Clinicians.

Importance: Disagreement in the presumed meaning of common medical terms may impair communication between patients and caregivers. Objective: To clarify the intended meaning of the term congestion among otolaryngology clinic patients and to identify discrepancies in definitions between patients and otolaryngologists. Design, Setting, and Participants: In this cross-sectional survey study from an otolaryngology clinic at an academic center, a semantics-based questionnaire was provided to consecutive new patients during intake for a clinical encounter from December 2016 through February 2017, and to 31 otolaryngologists and 28 nonotolaryngologist physicians in February 2018. Respondent definitions for congestion were selected from a list of 16 proposed terms covering 4 general categories. Main Outcome and Measures: Symptom categories for term used to describe congestion by patients and clinicians. Results: Among 226 patient respondents (133 female [58.8%]; mean [SD] age, 54 [15.6] years), the most commonly selected definitions for congestion were from the obstructive (199; 88.1%) and mucus-related (196; 86.7%) symptom categories. More than 1 general category was selected by 208 patients (92.0%), whereas 11 patients (4.9%) described congestion only in terms of mucus-related symptoms. Definitions were limited to upper respiratory tract symptoms by 83 (36.7%) patients and lower respiratory tract symptoms by 2 (0.9%) patients. Among 31 otolaryngologists, congestion was most frequently defined in terms of obstructive symptoms (difference, 11.9%; 95% CI, 7.4%-16.5%). In contrast, patients more often described congestion in terms of pressure-related (difference, 38.8%; 95% CI, 7.5%-70.1%) or mucus-related (difference, 51.2%; 95% CI, 22.6%-79.9%) symptoms. A total of 22 otolaryngologists (71.0%) defined congestion using 1 to 4 symptoms, compared with only 69 patients (30.5%). Conclusions and Relevance: The definition of congestion appears to be highly variable and ambiguous for many patients. Moreover, the findings suggest that patients and otolaryngologists generally do not describe congestion using the same terms.

Fibroblasts Impact Goblet Cell Responses to Lactic Acid Bacteria After Exposure to Inflammatory Cytokines and Mucus Disruptors.

SCOPE: Mucus produced by goblet cells contributes to gut barrier function. Lactic acid bacteria (LAB) have been shown to impact mucus production. It is not completely known whether mucus production is influenced by the abundantly present fibroblasts in the intestine. METHODS AND RESULTS: The influence of fibroblasts on mucus-related genes including mucin-2 (MUC2), trefoil factor 3 (TFF3), resistin-like molecule ß (RETNLB), carbohydrate sulfotransferase 5 (CHST5), and galactose-3-O-sulfotransferase 2 (GAL3ST2) is examined after co-culture of LS174T-goblet cells and CCD-18Co colonic fibroblasts in the presence and absence of LAB-strains known to impact mucus function. This is also tested after exposure to TNF-α, IL-13, or the mucin synthesis inhibitor tunicamycin (Tm). Effects of fibroblasts are treatment duration- and bacterial species-dependent under homeostatic conditions. During TNF-α challenge, fibroblasts reverse Lactobacillus (L.) rhamnosus CCFM237-elicited declined TFF3 expression. After IL-13 exposure, L. rhamnosus CCFM237 and L. fermentum CCFM787 attenuate enhanced TFF3 and RETNLB expression, respectively, only in the presence of fibroblasts. LAB has no effects on Tm-induced decreased expression of goblet cell-related genes regardless of the presence of fibroblasts. CONCLUSION: It is demonstrated that goblet cell-fibroblast crosstalk impacts mucus synthesis and influences the effects of LAB on goblet cell-related genes. Effects are LAB-species and stressor dependent.

Mucus and mucins in diseases of the intestinal and respiratory tracts.

This review describes the organization and importance of mucus in the intestine and lungs in relation to the diseases cystic fibrosis (CF), ulcerative colitis and chronic obstructive pulmonary disease (COPD). The inner surfaces of the body are protected by mucus built around polymeric glycoproteins called mucins. In the disease CF, the small intestinal mucus is in contrast the normal attached to the epithelium, explaining the intestinal problems at this disease. The inner of the two mucus layers of colon is normally impenetrable to bacteria, keeping the commensals away from and protecting the epithelium. This impenetrable property is dependent on the bacterial composition and the host diet, observations that can explain the increased incidence of inflammatory bowel diseases in the western world as bacteria reach the epithelial cells in active ulcerative colitis. The respiratory tract is normally cleared by thick mucus bundles that moved by the cilia sweep the epithelial surface. In CF, the bundles are nonmoving already at birth. Cholinergic stimulations stop the bundle movement explaining some of the beneficial effect of anticholinergic treatment in COPD. In this disease as well as in more developed CF, an attached mucus layer is formed. This mucus has features similar to the protective inner colon mucus and is by this able to separate bacteria from the epithelial surface. When formed in healthy individuals this mucus can be coughed up, but in chronically diseased lungs, bacteria colonizing the mucus will remain in the lungs and the resulting inflammation contribute to the destruction of the lungs.

Helicobacter suis infection alters glycosylation and decreases the pathogen growth inhibiting effect and binding avidity of gastric mucins.

Helicobacter suis is the most prevalent non-Helicobacter pylori Helicobacter species in the human stomach and is associated with chronic gastritis, peptic ulcer disease, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. H. suis colonizes the gastric mucosa of 60-95% of pigs at slaughter age, and is associated with chronic gastritis, decreased weight gain, and ulcers. Here, we show that experimental H. suis infection changes the mucin composition and glycosylation, decreasing the amount of H. suis-binding glycan structures in the pig gastric mucus niche. Similarly, the H. suis-binding ability of mucins from H. pylori-infected humans is lower than that of noninfected individuals. Furthermore, the H. suis growth-inhibiting effect of mucins from both noninfected humans and pigs is replaced by a growth-enhancing effect by mucins from infected individuals/pigs. Thus, Helicobacter spp. infections impair the mucus barrier by decreasing the H. suis-binding ability of the mucins and by decreasing the antiprolific activity that mucins can have on H. suis. Inhibition of these mucus-based defenses creates a more stable and inhabitable niche for H. suis. This is likely of importance for long-term colonization and outcome of infection, and reversing these impairments may have therapeutic benefits.

N-Hydroxysulfosuccinimide Esters versus Thiomers: A Comparative Study Regarding Mucoadhesiveness.

The objective of the study was to compare poly(acrylic acid)- N-hydroxysulfosuccinimide reactive esters (PAA-Sulfo-NHS) and poly(acrylic acid)-cysteine conjugates (PAA-Cys) regarding their mucoadhesiveness. Polymer conjugates were synthesized in a water free environment and characterized by UV-vis spectroscopy and FTIR. Water uptake studies were performed, and the polymers were further examined for their mucoadhesive properties and cohesiveness using the rotating cylinder method. Tensile force measurements were conducted to define the strength of adhesion to porcine intestinal mucosa. Additionally, polymer-mucus mixtures were assessed for rheological synergism by measuring the increase in dynamic viscosity. Both modifications led to a prolonged adhesion time compared to unmodified PAA. Fast dissolution of PAA-Sulfo-NHS derivatives was monitored, whereas PAA-Cys tended to extensively swell while exhibiting high cohesive properties. Measurements of tensile force revealed up to 2.7-fold (PAA-Sulfo-NHS) and 2.3-fold (PAA-Cys) enhancement of the maximum detachment force and 7.6-fold (PAA-Sulfo-NHS) and 3.6-fold (PAA-Cys) increase in the total work of adhesion. Formation of a gel network between polymer and mucus was confirmed by a 10.8-fold (PAA-Sulfo-NHS) and 20.8-fold (PAA-Cys) increase in viscosity. Both types of polymers show high mucoadhesive properties due to the formation of covalent bonds with the mucus. As thiolated polymers are capable of forming stabilizing disulfide bonds within their polymeric network, they are advantageous over PAA-Sulfo-NHS.