Mucus threads from surface goblet cells clear particles from the airways.

BACKGROUND: The mucociliary clearance system driven by beating cilia protects the airways from inhaled microbes and particles. Large particles are cleared by mucus bundles made in submucosal glands by parallel linear polymers of the MUC5B mucins. However, the structural organization and function of the mucus generated in surface goblet cells are poorly understood. METHODS: The origin and characteristics of different mucus structures were studied on live tissue explants from newborn wild-type (WT), cystic fibrosis transmembrane conductance regulator (CFTR) deficient (CF) piglets and weaned pig airways using video microscopy, Airyscan imaging and electron microscopy. Bronchoscopy was performed in juvenile pigs in vivo. RESULTS: We have identified a distinct mucus formation secreted from the surface goblet cells with a diameter less than two micrometer. This type of mucus was named mucus threads. With time mucus threads gathered into larger mucus assemblies, efficiently collecting particles. The previously observed Alcian blue stained mucus bundles were around 10 times thicker than the threads. Together the mucus bundles, mucus assemblies and mucus threads cleared the pig trachea from particles. CONCLUSIONS: These results demonstrate that normal airway mucus is more complex and has a more variable structural organization and function than was previously understood. These observations emphasize the importance of studying young objects to understand the function of a non-compromised lung.

Variations in mucous cell numbers in gills of Atlantic salmon (Salmo salar) presmolt in commercial freshwater farms in Norway.

Fish gills are heavily exposed to the external milieu and may react against irritants with different cellular responses. We describe variations in mucous cell counts in gills from healthy Atlantic salmon (Salmo salar) presmolts in five recirculating aquaculture system (RAS) farms and one flow-through farm. Based on certain criteria, mucous cells were histologically quantified in a defined lamellar region of the gills and the counts were analysed. Immunohistochemistry (IHC) was used to investigate epithelial responses. The median number of total mucous cells in the defined region was 59 per fish. Between the farms, the medians varied from 31 to 101 with the lowest in the flow-through farm. A regression model was fitted with "total mucous cells" as the dependent variable and with "fish length" and "fish farm" as independent variables. The proportion of variation in mucous cell counts explained by the model was twice as high when "fish farm" was included compared to only "fish length." IHC revealed proliferative responses in coherence with high mucous cell numbers. Conclusively, the variation in mucous cell counts depends on combined farm-related factors. Establishing a baseline for mucous cell counts is fundamental in the development of high-throughput monitoring programmes of gill health in farmed fish.

Using 3D gastrointestinal tract in vitro models with microfold cells and mucus secreting ability to assess the hazard of copper oxide nanomaterials.

BACKGROUND: Copper oxide nanomaterials (CuO NMs) are exploited in many products including inks, cosmetics, textiles, wood preservatives and food contact materials. Their incorporation into these products may enhance oral exposure in consumer, environmental and occupational settings. Undifferentiated and differentiated monocultures of Caco-2 cells are commonly used to assess NM toxicity to the intestine in vitro. However, the integration of other cell types into Caco-2 in vitro models increases their physiological relevance. Therefore, the aim of this study is to evaluate the toxicity of CuO NMs and copper sulphate (CuSO4) to intestinal microfold (M) cell (Caco-2/Raji B) and mucus secreting (Caco-2/HT29-MTX) co-culture in vitro models via assessment of their impact on barrier integrity, viability and interleukin (IL)-8 secretion. The translocation of CuO NMs and CuSO4 across the intestinal barrier was also investigated in vitro. RESULTS: CuO NMs and CuSO4 impaired the function of the intestinal barrier in the co-culture models [as indicated by a reduction in transepithelial electrical resistance (TEER) and Zonular occludens (ZO-1) staining intensity]. Cu translocation was observed in both models but was greatest in the Caco-2/Raji B co-culture. CuO NMs and CuSO4 stimulated an increase in IL-8 secretion, which was greatest in the Caco-2/HT29-MTX co-culture model. CuO NMs and CuSO4 did not stimulate a loss of cell viability, when assessed using light microscopy, nuclei counts and scanning electron microscopy. CuO NMs demonstrated a relatively similar level of toxicity to CuO4 in both Caco-2/Raji B and Caco-2/HT29-MTX co- culture models. CONCLUSIONS: The Caco-2/Raji B co-culture model was more sensitive to CuO NM and CuSO4 toxicity than the Caco-2/HT29-MTX co-culture model. However, both co-culture models were less sensitive to CuO NM and CuSO4 toxicity than simple monocultures of undifferentiated and differentiated Caco-2 cells, which are more routinely used to investigate NM toxicity to the intestine. Obtained data can therefore feed into the design of future studies which assess the toxicity of substances (e.g. NMs) and pathogens to the intestine (e.g. by informing model and endpoint selection). However, more testing with a wider panel of NMs would be beneficial in order to help select which in vitro models and endpoints to prioritise when screening the safety of ingested NMs. Comparisons with in vivo findings will also be essential to identify the most suitable in vitro model to screen the safety of ingested NMs.

Histochemical distribution of four types of enzymes and mucous cells in the intestine of koi carp (Cyprinus carpio var. koi).

The main purpose of this study was to investigate the distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non-specific esterase (NSE), peroxidase (POD), and mucous cells in the intestine of the koi carp Cyprinus carpio var. koi. ACP activity was located in the striated border, enterocytes, and lamina propria of the anterior and middle intestines. The ACP activity in the anterior intestine was higher than that in the middle and posterior intestines. ALP existed in the striated border of enterocytes and lamina propria, serosa, muscular layer, and the junction between muscular layer and submucosa layer of the intestine. The ALP activity in the anterior intestine was higher than that in the middle and posterior intestines. NSE activity was localized in the cytoplasm of enterocytes in the whole intestine, and the middle intestine showed the lower NSE activity than the anterior and posterior intestines. POD activity was localized in the blood cells of the lamina propria and cytoplasm of enterocytes in all intestinal segments. The POD activity among the anterior, middle, and posterior intestines was non-significantly different. Alcian blue periodic acid-Schiff histochemical results revealed three types of mucous cells in the intestine. The total number of mucous cells and percentage of type I cells among the anterior, middle, and posterior intestines were non-significantly different. The percentage of the type II cells was the highest in the posterior intestine, while the lowest in the anterior intestine. The percentage of the type III cells was the highest in the anterior intestine, while the lowest in the posterior intestine.

A new index for characterizing micro-bead motion in a flow induced by ciliary beating: Part I, experimental analysis.

Mucociliary clearance is one of the major lines of defense of the respiratory system. The mucus layer coating the pulmonary airways is moved along and out of the lung by the activity of motile cilia, thus expelling the particles trapped in it. Here we compare ex vivo measurements of a Newtonian flow induced by cilia beating (using micro-beads as tracers) and a mathematical model of this fluid flow, presented in greater detail in a second companion article. Samples of nasal epithelial cells placed in water are recorded by high-speed video-microscopy and ciliary beat pattern is inferred. Automatic tracking of micro-beads, used as markers of the flow generated by cilia motion, enables us also to assess the velocity profile as a function of the distance above the cilia. This profile is shown to be essentially parabolic. The obtained experimental data are used to feed a 2D mathematical and numerical model of the coupling between cilia, fluid, and micro-bead motion. From the model and the experimental measurements, the shear stress exerted by the cilia is deduced. Finally, this shear stress, which can easily be measured in the clinical setting, is proposed as a new index for characterizing the efficiency of ciliary beating.

The composition of the gut microbiota shapes the colon mucus barrier.

Two C57BL/6 mice colonies maintained in two rooms of the same specific pathogen-free (SPF) facility were found to have different gut microbiota and a mucus phenotype that was specific for each colony. The thickness and growth of the colon mucus were similar in the two colonies. However, one colony had mucus that was impenetrable to bacteria or beads the size of bacteria-which is comparable to what we observed in free-living wild mice-whereas the other colony had an inner mucus layer penetrable to bacteria and beads. The different properties of the mucus depended on the microbiota, as they were transmissible by transfer of caecal microbiota to germ-free mice. Mice with an impenetrable mucus layer had increased amounts of Erysipelotrichi, whereas mice with a penetrable mucus layer had higher levels of Proteobacteria and TM7 bacteria in the distal colon mucus. Thus, our study shows that bacteria and their community structure affect mucus barrier properties in ways that can have implications for health and disease. It also highlights that genetically identical animals housed in the same facility can have rather distinct microbiotas and barrier structures.

Assessment of cytology and mucin 2 in colorectal mucus collected from patients with inflammatory bowel disease: Results of a pilot trial.

BACKGROUND AND AIM: Non-invasive diagnosis of colorectal disease remains problematic, fecal biomarkers presenting the only current option. Colorectal mucus is the diagnostically informative element of stool samples, but its separation from stool is difficult. We aimed to: (i) test a novel method of non-invasive colorectal mucus sampling in a pilot clinical trial; (ii) evaluate sampling method acceptance by study participants; (iii) characterize the collected material cytologically; and (iv) assess feasibility of quantitative protein analysis in the samples. METHODS: A total of 141 patients with IBD (58), IBS (50), and healthy controls (33) participated in the study. Samples rich in colorectal mucus were self-collected by swabbing the anal area immediately following defecation. Collected samples were examined cytologically and subjected to quantitative analysis for total protein and mucin 2 (MUC2). RESULTS: The novel sampling technique was assessed as "good" or "adequate" by 96% of study participants. A total of 55% of the collected samples were free of fecal contamination. Cytology showed large numbers of well preserved inflammatory cells in IBD cases. Total protein values varied in all groups, being affected by fecal contamination. MUC2 levels were similar among all IBD-free individuals (control and IBS groups) and elevated in IBD patients (p < 0.001). MUC2 measurement applied as a test for IBD detection provided sensitivity = 72.4% and specificity = 86.7%. CONCLUSIONS: A novel non-invasive method for collecting human colorectal mucus has been successfully tested. The method was very well accepted by trial participants. The results have proven high quality of collected samples for both cytological investigation and diagnostic biomarker analysis.

[Non-specific bronchial hyper-responsiveness and polymorphysm of xenobiotics biotransformation GSTM1 and GSTT1 genes under neutrophilic bronchial asthma in children].

With a view to study the effect of genes GSTT1 and GSTM1 deletion on the non-specific bronchial hyperresponsiveness in children with neutrophilic bronchial asthma (BA) 46 school age children having neutrophilic BA (1st clinical group) and their 48 coevals with eosinophilic phenotype of the disease (2nd clinical group) were subjected to a complex examination at the pulmo-allergologic department of the regional child clinical hospital of Chernivtsi. The study proved that genotype T1+M1del was more frequently registered in patients with the neutrophilic phenotype of the disease, and genotype T1delM1del was equifrequent in patients with different types of the inflammation of the respiratory ways. In patients with neutrophilic BA and deletion polymorphism of genes GSTT1 and GSTM1, there was a tendency to decreasing of the bronchial lability index through the decrease of bronchodilation, and bronchial response to histamine occurred to be higher than in children with the absence of polymorphism of the referred genes of the xenobiotics biotransformation system.

Human airway ciliary dynamics.

Airway cilia depend on precise changes in shape to transport the mucus gel overlying mucosal surfaces. The ciliary motion can be recorded in several planes using video microscopy. However, cilia are densely packed, and automated computerized systems are not available to convert these ciliary shape changes into forms that are useful for testing theoretical models of ciliary function. We developed a system for converting planar ciliary motions recorded by video microscopy into an empirical quantitative model, which is easy to use in validating mathematical models, or in examining ciliary function, e.g., in primary ciliary dyskinesia (PCD). The system we developed allows the manipulation of a model cilium superimposed over a video of beating cilia. Data were analyzed to determine shear angles and velocity vectors of points along the cilium. Extracted waveforms were used to construct a composite waveform, which could be used as a standard. Variability was measured as the mean difference in position of points on individual waveforms and the standard. The shapes analyzed were the end-recovery, end-effective, and fastest moving effective and recovery with mean (± SE) differences of 0.31(0.04), 0.25(0.06), 0.50(0.12), 0.50(0.10), µm, respectively. In contrast, the same measures for three different PCD waveforms had values far outside this range.

Eosinophils and mast cells: a comparison of nasal mucosa histology and cytology to markers in nasal discharge in patients with chronic sino-nasal diseases.

Allergic rhinitis (AR), nasal polyps (NP) as well as chronic rhinosinusitis (CRS) are all known to be associated with eosinophilic infiltration and elevated numbers of mast cells (MC) within the mucosa. Both cell types and their markers eosinophilic cationic protein (ECP) and tryptase are utilized in the diagnosis and management of chronic sino-nasal diseases. Mucosal cytology samples were gathered by cytobrush, histological samples were obtained from the inferior turbinate. In both sample sets, the number of eosinophils and MC was determined. Their corresponding markers ECP and tryptase were quantified from nasal discharge. Patients were grouped with reference to their main diagnosis: AR (n = 34), NP (n = 25), CRS (n = 27) and controls (n = 34). Eosinophil counts from cytobrush and ECP levels were significantly elevated in NP compared to all other groups-31- and 13-fold over control, respectively. However, histologic review did not reveal any difference in eosinophil count among groups. Tryptase was significantly elevated threefold in AR versus CRS and controls. No correlation to cytological and histological MC counts could be found. ECP levels in nasal discharge as well as eosinophil counts can provide useful information with regard to the diagnosis. Likewise, tryptase concentrations can do. The presented data show that the measurement of markers in nasal discharge is superior in differentiating among diagnosis groups. Given that the collection of nasal secretions is more comfortable for patients than the more invasive techniques, we recommend first line ECP and tryptase testing performed on nasal secretions.

Role of nicotinic receptors and acetylcholine in mucous cell metaplasia, hyperplasia, and airway mucus formation in vitro and in vivo.

BACKGROUND: Airway mucus hypersecretion is a key pathophysiologic feature in a number of lung diseases. Cigarette smoke/nicotine and allergens are strong stimulators of airway mucus; however, the mechanism of mucus modulation is unclear. OBJECTIVES: We sought to characterize the pathway by which cigarette smoke/nicotine regulates airway mucus and identify agents that decrease airway mucus. METHODS: IL-13 and γ-aminobutyric acid type A receptors (GABA(A)Rs) are implicated in airway mucus. We examined the role of IL-13 and GABA(A)Rs in nicotine-induced mucus formation in normal human bronchial epithelial (NHBE) and A549 cells and secondhand cigarette smoke-induced, ovalbumin-induced, or both mucus formation in vivo. RESULTS: Nicotine promotes mucus formation in NHBE cells; however, the nicotine-induced mucus formation is independent of IL-13 but sensitive to the GABA(A)R antagonist picrotoxin. Airway epithelial cells express α7-, α9-, and α10-nicotinic acetylcholine receptors (nAChRs), and specific inhibition or knockdown of α7- but not α9/α10-nAChRs abrogates mucus formation in response to nicotine and IL-13. Moreover, addition of acetylcholine or inhibition of its degradation increases mucus in NHBE cells. Nicotinic but not muscarinic receptor antagonists block allergen- or nicotine/cigarette smoke-induced airway mucus formation in NHBE cells, murine airways, or both. CONCLUSIONS: Nicotine-induced airway mucus formation is independent of IL-13, and α7-nAChRs are critical in airway mucous cell metaplasia/hyperplasia and mucus production in response to various promucoid agents, including IL-13. In the absence of nicotine, acetylcholine might be the biological ligand for α7-nAChRs to trigger airway mucus formation. α7-nAChRs are downstream of IL-13 but upstream of GABA(A)Rα2 in the MUC5AC pathway. Acetylcholine and α7-nAChRs might serve as therapeutic targets to control airway mucus.

Coarse-grained modeling of mucus barrier properties.

We designed a simple coarse-grained model of the glycocalyx layer, or adhesive mucus layer (AML), covered by mucus gel (luminal mucus layer) using a polymer lattice model and stochastic sampling (replica exchange Monte Carlo) for canonical ensemble simulations. We assumed that mucin MUC16 is responsible for the structural properties of the AML. Other mucins that are much smaller in size and less relevant for layer structure formation were not included. We further assumed that the system was in quasi-equilibrium. For systems with surface coverage and concentrations of model mucins mimicking physiological conditions, we determined the equilibrium distribution of inert nanoparticles within the mucus layers using an efficient replica exchange Monte Carlo sampling procedure. The results show that the two mucus layers penetrate each other only marginally, and the bilayer imposes a strong barrier for nanoparticles, with the AML layer playing a crucial role in the mucus barrier.

An ex vivo method for studying mucus formation, properties, and thickness in human colonic biopsies and mouse small and large intestinal explants.

The colon mucus layers minimize the contact between the luminal flora and the epithelial cells, and defects in this barrier may lead to colonic inflammation. We now describe an ex vivo method for analysis of mucus properties in human colon and mouse small and large intestine. Intestinal explants were mounted in horizontal perfusion chambers. The mucus surface was visualized by adding charcoal particles on the apical side, and mucus thickness was measured using a micropipette. Mucus thickness, adhesion, and growth rate were recorded for 1 h. In mouse and human colon, the ability of the mucus to act as a barrier to beads the size of bacteria was also evaluated. Tissue viability was monitored by transepithelial potential difference. In mouse ileum, the mucus could be removed by gentle aspiration, whereas in colon ∼40 µm of the mucus remained attached to the epithelial surface. Both mouse and human colon had an inner mucus layer that was not penetrated by the fluorescent beads. Spontaneous mucus growth was observed in human (240 µm/h) and mouse (100 µm/h) colon but not in mouse ileum. In contrast, stimulation with carbachol induced a higher mucus secretion in ileum than colon (mouse ileum: Δ200 µm, mouse colon: Δ130 µm, human colon: Δ140 µm). In conclusion, while retaining key properties from the mucus system in vivo, this setup also allows for studies of the highly dynamic mucus system under well-controlled conditions.

Effects of dietary Ergosan on cutaneous mucosal immune response in rainbow trout (Oncorhynchus mykiss).

The effects of dietary Ergosan on the growth performance and mucosal immunity in rainbow trout skin were investigated. 60 rainbow trout (100-110 g) were randomly assigned to 2 groups in triplicates and fed one of the experimental diet formulated with 5 g kg⁻¹ Ergosan or control diet for 50 days. Results showed that on the 45th day of feeding trial, Ergosan supplementation significantly enhanced the growth performance compared to control group. Various enzyme activities, namely lysozyme, protease, alkaline phosphatase and esterase in treatment group were also enhanced on the 45th and 50th day. Skin mucus in Ergosan-fed fish showed the agglutination of erythrocytes while in control group, no visible agglutination was shown. In addition, skin mucus in treatment group showed strong antibacterial activity against Yersinia ruckeri. In conclusion, the major immune components of rainbow trout mucus that are involved in the non-specific immunity were enhanced by administration of Ergosan in 5 g kg⁻¹.

Comparative effects of four feed types on white spot disease susceptibility and skin immune parameters in rainbow trout, Oncorhynchus mykiss (Walbaum).

The effects on rainbow trout, Oncorhynchus mykiss (Walbaum), immune parameters by differently formulated fish feed types containing immunostimulants have been tested in a double-blind, duplicated and controlled study performed over 50 days. A total of 800 rainbow trout (10-12 g) were kept in eight duplicate fish tanks (each containing 100 fish) and fed at a daily feeding rate of 1.5% of the biomass. The feed types were (1) control feed (C) without additives, (2) feed containing beta-glucan, nucleotides, manno-oligosaccharides (MOS), vitamins C and E (GNMCE), (3) feed containing probiotic bacteria and plant extracts (PP) and (4) feed with nucleotides, manno-oligosaccharides, vitamins C and E (NMCE). Plasma lysozyme activity was increased in fish fed two feed types (GNMCE and NMCE) but slightly depressed in fish fed PP. A non-significant trend for a higher mucous cell density at days 30 and 50 was shown in all fish receiving feeds with additives compared to the control group. All fish became infected with Ichthyophthirius multifiliis when exposed, but fish fed GNMCE showed a significantly lower infection both at days 30 and 50. Expression of genes encoding C3 and MHCII was significantly up-regulated in fish fed GNMCE for 50 days, and the expression of genes coding Hepcidin was significantly down-regulated in fish fed NMCE for 50 days. Beta-glucan was the single component, when used in combination with other feed ingredients, which was found associated with increased parasite resistance, increased lysozyme and immune gene up-regulation.

[Methodological approaches to the complex study of the functional morphology of the tracheal epithelial lining in the experiment].

An algorithm is described for the functional and morphological analysis of the trachea in small laboratory animals, including the consecutive in vivo study of the velocity of mucus movement and in vitro analysis of motor activity of the ciliary apparatus in combination with the histological study of the organ. The proposed methodological approaches permits, during a short period of time (20-30 min per one animal) to supravitally register and document in digital format video files with their subsequent visual estimation and mathematical analysis of the parameters studied. These techniques provide high reproducibility of the results with a maximum efficiency and information value and permit to undertake a comprehensive morpho-functional analysis of the investigated object.

Effects of iron on rainbow trout gill cells in primary culture.

This study investigated the effects of iron in the form of iron sulphate (FeSO(4)·7H(2)O), over the range 0.01-1 mM on rainbow trout primary gill cells cultured on semi-permeable membranes. The endpoints measured were cell proliferation, mucous cell numbers, area of mucus in mucous cells, ultrastructural analysis and transepithelial resistance. Regardless of the concentration, FeSO(4) did not modify the apical surface of pavement cells (microridge) and mucous cells. However, at 1 mM, this metal reduced cell numbers, by inhibiting cell proliferation and causing cell death, and induced a decrease in transepithelial resistance. It is interesting to note that cell numbers were also reduced in the presence of 0.5 mM iron salt, although this reduction did not modify transepithelial resistance. FeSO(4) reduced mucous cell number but did not change mucus area in mucous cells suggesting that this metal could induce a discharge of mucous cells, but mucus secretion would be total and not partial. In conclusion, our in vitro model has allowed to study some toxic effect but also resistance of gill epithelium in presence of iron.

The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria.

We normally live in symbiosis with approximately 10(13) bacteria present in the colon. Among the several mechanisms maintaining the bacteria/host balance, there is limited understanding of the structure, function, and properties of intestinal mucus. We now demonstrate that the mouse colonic mucus consists of two layers extending 150 mum above the epithelial cells. Proteomics revealed that both of these layers have similar protein composition, with the large gel-forming mucin Muc2 as the major structural component. The inner layer is densely packed, firmly attached to the epithelium, and devoid of bacteria. In contrast, the outer layer is movable, has an expanded volume due to proteolytic cleavages of the Muc2 mucin, and is colonized by bacteria. Muc2(-/-) mice have bacteria in direct contact with the epithelial cells and far down in the crypts, explaining the inflammation and cancer development observed in these animals. These findings show that the Muc2 mucin can build a mucus barrier that separates bacteria from the colon epithelia and suggest that defects in this mucus can cause colon inflammation.

Distribution of mucous cells on the body surface of Japanese flounder Paralichthys olivaceus.

The distribution of mucous cells was examined in the skin on the ocular and blind sides of Japanese flounder Paralichthys olivaceus. Observations were performed on both body sides at the following regions: cheek, lower jaw (blind side), gill cover (ocular side), dorsal side, lateral line, belly and caudal peduncle. The mucous cells observed were elliptic and positively stained for periodic acid Schiff reaction and Mayer's mucicarmine and showed a higher density and larger size on the ocular side compared to the blind side. Low densities of mucous cells were observed on the lower jaw compared with other regions of the body. The depth of the crack located between scales was deeper on the ocular side than the blind side, which might reflect total epidermis area and total number of mucous cells. Bacterial infection elucidated some information on the effect on the density and size of mucous cells, where the density and size decreased slightly after infection. Only the lower jaw, however, showed an increased number of mucous cells. The results show that the potential of skin to secrete mucus is higher on the ocular than on the blind side and bacterial infection decreases mucous secretion.