Mucin Secretion Research Articles

Gastroprotective effects of hydrogen sulfide, carbon monoxide and nitric oxide on an experimental ulcer model in rats

Background/Aim: Gastric mucosal injury induced by several agents such as ethanol, stress or nonsteroidal anti-inflammatory drugs (NSAIDs) is a common severe disorder. Hydrogen sulfide (H2S), carbon monoxide (CO) and nitric oxide (NO) are gaseous autacoids that are endogenously produced in mammalian tissues. Recently, several studies confirmed that H2S, CO and NO play a role in gastroprotection. Our work aimed to evaluate and compared the gastroprotective effects of H2S, CO and NO on ethanol-, indomethacin- and stress-induced rat ulcer models. Methods: The effects of NaHS (5 mg/kg), CORM-2 (5 mg/kg) and L-arginine (100 mg/kg) were investigated on gastric ulcer models induced by ethanol (1 ml 96% i.g.), stress (cold+immobility) and indomethacin (40 mg/kg i.g.). The ulcer index, gastric mucus secretion, free and total acidity, and levels of TNF-α, PGE2, MDA GSH, COX-1, COX-2 were measured. Results: NaHS and CORM-2 decreased the increased TNF-α and MDA levels in ethanol-induced ulcer. L-arginine reduced mucin secretion, TNF-α and GSH levels in stress-induced gastric ulcer. Conclusion: The present study showed that H2S and CO may have gastroprotective activity against ethanol-induced ulcers and NO may be gastroprotective against stress-induced ulcers.

Evaluation of oral mucin secretion and pH neutralization by tablet application "Oral pH 7 Rebalancer"

Evaluation of oral mucin secretion and pH neutralization by tablet application "Oral pH 7 Rebalancer"

Characterisation of Gel-Forming Mucins Produced In Vivo and In Ex Vivo Conjunctival Explant Cultures.

One key element to the health of the ocular surface encompasses the presence of gel-forming mucins in the pre-ocular tear film. Conjunctival goblet cells are specialized epithelial cells that secrete mucins necessary for tear film stability and general homeostasis. Their dysfunction can be linked to a range of ocular surface inflammation disorders and chronic injuries. To obtain new perspectives and angles to tackle mucin deficiency, the need for an accurate evaluation of their presence and corresponding mucin secretion in ex vivo conjunctival cultures has become a requisite. In vitro, goblet cells show a significant decrease in the production and secretion of gel-forming mucins, accompanied by signs of dedifferentiation or transdifferentiation. Explant cultures on laminin-treated CLP-PEG hydrogels can, however, support the production of gel-forming mucins. Together, we challenge the current paradigm to evaluate the presence of cultured goblet cells solely based on their general mucin (MUC) content through imaging analyses, showing the need for additional techniques to assess the functionality of goblet cells. In addition, we broadened the gel-forming mucin profile of in vivo goblet cells with MUC5B and MUC6, while MUC2 and MUC6 is added to the profile of cultured goblet cells.

Dupilumab-induced ocular surface disease: A systematic review

Dupilumab-induced ocular surface disease: A systematic review

Proteinase-activated receptors in GtoPdb v.2021.3

Proteinase-activated receptors (PARs, nomenclature as agreed by the NC-IUPHAR Subcommittee on Proteinase-activated Receptors [39]) are unique members of the GPCR superfamily activated by proteolytic cleavage of their amino terminal exodomains. Agonist proteinase-induced hydrolysis unmasks a tethered ligand (TL) at the exposed amino terminus, which acts intramolecularly at the binding site in the body of the receptor to effect transmembrane signalling. TL sequences at human PAR1-4 are SFLLRN-NH2, SLIGKV-NH2, TFRGAP-NH2 and GYPGQV-NH2, respectively. With the exception of PAR3, synthetic peptides with these sequences (as carboxyl terminal amides) are able to act as agonists at their respective receptors. Several proteinases, including neutrophil elastase, cathepsin G and chymotrypsin can have inhibitory effects at PAR1 and PAR2 such that they cleave the exodomain of the receptor without inducing activation of Gαq-coupled calcium signalling, thereby preventing activation by activating proteinases but not by agonist peptides. Neutrophil elastase (NE) cleavage of PAR1 and PAR2 can however activate MAP kinase signaling by exposing a TL that is different from the one revealed by trypsin [82]. PAR2 activation by NE regulates inflammation and pain responses [111, 72] and triggers mucin secretion from airway epithelial cells [112].

The effect of ingredients commonly used in nasal and inhaled solutions on the secretion of mucus in vitro

Hypersecretion of mucus is associated with impaired mucociliary clearance that can influence the retention of active pharmaceutical ingredients in the airway but is also linked with recurrent airway disease. Therefore, the effect on mucin secretion of a range of ingredients used in solutions delivered to the nose and lung was studied. Mucin secretion from explants of ovine epithelium was quantified using an enzyme-linked lectin assay (ELLA) or sandwich ELLA depending on the compatibility of the ingredients with the assay. Benzalkonium chloride (0.015% w/w), Methocel™ E50 premium LV (1.0% w/w), propylene glycol (1.5% w/w), potassium sorbate + propylene glycol (0.3% w/w + 1.5% w/w) and polysorbate 80 (0.025% w/w), used at common working concentrations, all increased the secretion of mucin from the explants (P < 0.05). Ethylenediamine tetraacetic acid-disodium salt (EDTA) (0.015% w/w), Avicel® RC591 (1.5% w/w), fluticasone furoate (0.0004% w/w, concentration in solution) and dimethyl sulfoxide (DMSO) (0.2% w/w) did not affect mucin secretion. Compounds increasing mucin secretion could alter the rate of mucociliary clearance and the mucus could provide a barrier to drug absorption. This could predispose patients to disease and affect the activity of delivered drugs, decreasing or increasing their clinical efficacy.

Bordetella Adenylate Cyclase Toxin Elicits Airway Mucin Secretion through Activation of the cAMP Response Element Binding Protein.

The mucus layer protects airway epithelia from damage by noxious agents. Intriguingly, Bordetella pertussis bacteria provoke massive mucus production by nasopharyngeal epithelia during the initial coryza-like catarrhal stage of human pertussis and the pathogen transmits in mucus-containing aerosol droplets expelled by sneezing and post-nasal drip-triggered cough. We investigated the role of the cAMP-elevating adenylate cyclase (CyaA) and pertussis (PT) toxins in the upregulation of mucin production in B. pertussis-infected airway epithelia. Using human pseudostratified airway epithelial cell layers cultured at air–liquid interface (ALI), we show that purified CyaA and PT toxins (100 ng/mL) can trigger production of the major airway mucins Muc5AC and Muc5B. Upregulation of mucin secretion involved activation of the cAMP response element binding protein (CREB) and was blocked by the 666-15-Calbiochem inhibitor of CREB-mediated gene transcription. Intriguingly, a B. pertussis mutant strain secreting only active PT and producing the enzymatically inactive CyaA-AC– toxoid failed to trigger any important mucus production in infected epithelial cell layers in vitro or in vivo in the tracheal epithelia of intranasally infected mice. In contrast, the PT– toxoid-producing B. pertussis mutant secreting the active CyaA toxin elicited a comparable mucin production as infection of epithelial cell layers or tracheal epithelia of infected mice by the wild-type B. pertussis secreting both PT and CyaA toxins. Hence, the cAMP-elevating activity of B. pertussis-secreted CyaA was alone sufficient for activation of mucin production through a CREB-dependent mechanism in B. pertussis-infected airway epithelia in vivo.

FILIP1L Loss Is a Driver of Aggressive Mucinous Colorectal Adenocarcinoma and Mediates Cytokinesis Defects through PFDN1.

Aneuploid mucinous colorectal adenocarcinoma (MAC) is an aggressive subtype of colorectal cancer with poor prognosis. The tumorigenic mechanisms in aneuploid MAC are currently unknown. Here we show that downregulation of Filamin A-interacting protein 1-like (FILIP1L) is a driver of MAC. Loss of FILIP1L increased xenograft growth, and, in colon-specific knockout mice, induced colonic epithelial hyperplasia and mucin secretion. The molecular chaperone prefoldin 1 (PFDN1) was identified as a novel binding partner of FILIP1L at the centrosomes throughout mitosis. FILIP1L was required for proper centrosomal localization of PFDN1 and regulated proteasome-dependent degradation of PFDN1. Importantly, increased PFDN1, caused by downregulation of FILIP1L, drove multinucleation and cytokinesis defects in vitro and in vivo, which were confirmed by time-lapse imaging and 3D cultures of normal epithelial cells. Overall, these findings suggest that downregulation of FILIP1L and subsequent upregulation of PFDN1 is a driver of the unique neoplastic characteristics in aggressive aneuploid MAC. SIGNIFICANCE: This study identifies FILIP1L as a tumor suppressor in mucinous colon cancer and demonstrates that FILIP1L loss results in aberrant stabilization of a centrosome-associated chaperone protein to drive aneuploidy and disease progression.

Morphological and histochemical features of the digestive tract of Leiarius marmoratus (Gill, 1870).

Leiarius marmoratus, a freshwater catfish from Pimelodidae family, shows great biological and commercial relevance because of its geographic distribution and adaptation to fish-farm. The knowledge of the morphological characteristics of the digestive tract is fundamental to the understanding of fish physiology and nutrition, which helps in the planning of diets to provide better management and success in fish farming. Thus, this work described the morphology and histochemistry of the digestive tract of L. marmoratus adults. After euthanasia, the animals were dissected for analysis of the digestive tract. The oesophagus is a short and distensive organ with longitudinal folds that allow the passage of large food, e.g., other fishes. Oesophageal mucosa layer shows a stratified epithelium with goblet cells and club cells. The secretion of goblet cells is composed of neutral and acidic mucins that are anchored in the epithelium luminal face by epithelial cells fingerprint-like microridges, lubricating the surface to facilitate the food sliding. Club cells have protein secretion that can be involved in alarm signals when epithelium is damaged and in immunological defence. The saccular stomach is highly distensible to store large food. Gastric mucosa layer is composed of epithelial cells with intense secretion of neutral mucin to protect against self-digestion of gastric juice. Cardiac and fundic regions of stomach show well-developed gastric glands composed of oxynticopeptic cells. These cells have numerous mitochondria, highlighting their intense activity in the synthesis of acid and enzymes. The intestine is divided into three regions: anterior, middle and posterior. Although it is a short tube, intestine shows longitudinal folds and microvilli of enterocytes to increase the contact surface. These folds are higher in the anterior region of the intestine, highlighting their function in digestion and absorption. Intestinal goblet cells have acidic and neutral mucins that lubricate the epithelium and aid in digestive processes. These cells increase in number towards aboral, and they are related to the protection and lubrication to expulsion of faecal bolus.

Cystic Fibrosis Clinical Isolates of Aspergillus fumigatus Induce Similar Muco-inflammatory Responses in Primary Airway Epithelial Cells.

Aspergillus is increasingly associated with lung inflammation and mucus plugging in early cystic fibrosis (CF) disease during which conidia burden is low and strains appear to be highly diverse. It is unknown whether clinical Aspergillus strains vary in their capacity to induce epithelial inflammation and mucus production. We tested the hypothesis that individual colonising strains of Aspergillus fumigatus would induce different responses. Ten paediatric CF Aspergillus isolates were compared along with two systemically invasive clinical isolates and an ATCC reference strain. Isolates were first characterised by ITS gene sequencing and screened for antifungal susceptibility. Three clusters (A−C) of Aspergillus isolates were identified by ITS. Antifungal susceptibility was variable, particularly for itraconazole. Submerged CF and non-CF monolayers as well as differentiated primary airway epithelial cell cultures were incubated with conidia for 24 h to allow germination. None of the clinical isolates were found to significantly differ from one another in either IL-6 or IL-8 release or gene expression of secretory mucins. Clinical Aspergillus isolates appear to be largely homogenous in their mucostimulatory and immunostimulatory capacities and, therefore, only the antifungal resistance characteristics are likely to be clinically important.

Evaluation of the toxicity of sodium dodecyl sulphate (SDS) in the MucilAir™ human airway model in vitro

The aim of the study was to use multiple in vitro assays to assess the effects of a model irritant, sodium dodecyl sulphate (SDS) (≤10 mM (0.29 %, w/v)), on an in vitro model of the airway, MucilAir™. The use of MucilAir™ in recovery studies was also explored.A 24 h exposure increased IL-8 release at an SDS concentration ≥0.63 mM (0.018 %, w/v). Mucin secretion increased and transepithelial electrical resistance (TEER) decreased at SDS concentrations ≥1.25 mM (0.04 %, w/v). Cytotoxicity (lactate dehydrogenase (LDH) release into basolateral chamber) was observed at SDS concentrations of ≥2.5 mM (0.07 %, w/v). The sensitivity of the assays was IL-8 release > TEER = mucin secretion > LDH release.After 7 days, full or partial recovery was observed for intermediate concentrations of SDS using all assays but not at 5 and 10 mM SDS. Morphologically, erosion and cell loss were observed at these concentrations. Resazurin metabolism at 7 days tended to decrease in a dose-dependent manner at SDS concentrations above 2.5 mM (0.07 %, w/v).Together, these data support a No Observable Effect Level of 0.31 mM (0.009 % w/v) SDS and the use of MucilAir™ as a relevant model for airway toxicity studies.

A comparison of three mucus-secreting airway cell lines (Calu-3, SPOC1 and UNCN3T) for use as biopharmaceutical models of the nose and lung

The aim of this work was to compare three existing mucus-secreting airway cell lines for use as models of the airways to study drug transport in the presence of mucus.Each cell line secreted mature, glycosylated mucins, evidenced by the enzyme-linked lectin assay. The secretagogue, adenylyl-imidodiphosphate, increased mucin secretion in SPOC1 (3.5-fold) and UNCN3T (1.5-fold) cells but not in Calu-3 cells. In a novel mucus-depleted (MD) model the amount of mucus in the non-depleted wells was 3-, 8- and 4-fold higher than in the mucus-depleted wells of the Calu-3, SPOC1 and UNCN3T cells respectively. The permeability of 'high mucus’ cells to testosterone was significantly less in SPOC1 and UNCN3T cells (P < 0.05) but not Calu-3 cells.Mucin secretion and cytokine release were investigated as indicators of drug irritancy in the SPOC1 and UNCN3T cell lines. A number of inhaled drugs significantly increased mucin secretion at high concentrations and the release of IL-6 and IL-8 from SPOC1 or UNCN3T cells (P < 0.05).SPOC1 and UNCN3T cell lines are better able to model the effect of mucus on drug absorption than the Calu-3 cell line and are proposed for use in assessing drug-mucus interactions in inhaled drug and formulation development.

Neutrophil Elastase and Chronic Lung Disease

Neutrophil elastase (NE) is a major inflammatory protease released by neutrophils and is present in the airways of patients with cystic fibrosis (CF), chronic obstructive pulmonary disease, non-CF bronchiectasis, and bronchopulmonary dysplasia. Although NE facilitates leukocyte transmigration to the site of infection and is required for clearance of Gram-negative bacteria, it also activates inflammation when released into the airway milieu in chronic inflammatory airway diseases. NE exposure induces airway remodeling with increased mucin expression and secretion and impaired ciliary motility. NE interrupts epithelial repair by promoting cellular apoptosis and senescence and it activates inflammation directly by increasing cytokine expression and release, and indirectly by triggering extracellular trap release and exosome release, which magnify protease activity and inflammation in the airway. NE inhibits innate immune function by digesting opsonins and opsonin receptors, degrading innate immune proteins such as lactoferrin, and inhibiting macrophage phagocytosis. Importantly, NE-directed therapies have not yet been effective in preventing the pathologic sequelae of NE exposure, but new therapies are being developed that offer both direct antiprotease activity and multifunctional anti-inflammatory properties.

Mechanobiology of conjunctival epithelial cells exposed to wall shear stresses.

The human conjunctival epithelial cells (HCEC) line the inner sides of the eyelids and the anterior part of the sclera. They include goblet cells that secret mucus into the tear film that protects the ocular surface. The conjunctival epithelium is subjected to mechano-physical stimuli due to eyelid movement during blinking, during wiping and rubbing the eyes, and when exposed to wind and air currents. We cultured primary HCEC under air-liquid interface (ALI) conditions in custom-designed wells that can be disassembled for installation of the in vitro model in a flow chamber. We exposed the HCEC after ALI culture of 8-10days to steady and oscillatory airflows. The in vitro model of HCEC was exposed to steady wall shear stresses (sWSS) of 0.5 and 1.0 dyne/cm2 for lengths of 30 and 60min and to oscillatory wall shear stresses (oWSS) of 0.5 and 0.77 dyne/cm2 amplitudes for a length of 10min. Cytoskeletal alterations and MUC5AC mucin secretion in response to WSS were investigated using immunohistochemically fluorescent staining and enzyme-linked lectin assay (ELLA), respectively. The results revealed that both exposure times and sWSS values increased the polymerization of F-actin filaments while mucin secretion decreased. However, after a recovery of 24h in the incubator we observed a decrease of F-actin fibers and mucin secretion only for exposure of 30min. The length of exposure was more influential on cytoskeletal alterations than the level of sWSS. The very small effect of sWSS on mucin secretion is most likely related to the much smaller amount of goblet cell than in other mucus-secreting tissue. The results for both oWSS amplitudes revealed similar trends regarding F-actin and mucin secretion. Immediately post-exposure we observed an increase in polymerization of F-actin filaments while mucin secretion decreased. However, after 24-h recovery we observed that both F-actin and mucin secretion returned to the same values as for unexposed cultures. The results of this study suggest that WSS should be considered while exploring the physiological characteristics of HCEC.

Role of Goblet Cells in Intestinal Barrier and Mucosal Immunity.

Goblet cells and the mucus they secrete serve as an important barrier, preventing pathogens from invading the mucosa to cause intestinal inflammation. The perspective regarding goblet cells and mucus has changed, with current evidence suggesting that they are not passive but play a positive role in maintaining intestinal tract immunity and mucosal homeostasis. Goblet cells could obtain luminal antigens, presenting them to the underlying antigen-presenting cells (APCs) that induces adaptive immune responses. Various immunomodulatory factors can promote the differentiation and maturation of goblet cells, and the secretion of mucin. The abnormal proliferation and differentiation of goblet cells, as well as the deficiency synthesis and secretion of mucins, result in intestinal mucosal barrier dysfunction. This review provides an extensive outline of the signaling pathways that regulate goblet cell proliferation and differentiation and control mucins synthesis and secretion to elucidate how altering these pathways affects goblet functionality. Furthermore, the interaction between mucins and goblet cells in intestinal mucosal immunology is described. Therefore, the contribution of goblet cells and mucus in promoting gut defense and homeostasis is illustrated, while clarifying the regulatory mechanisms involved may allow the development of new therapeutic strategies for intestinal disorders.

Mixed neuroendocrine-non neuroendocrine neoplasm of the colon

Mixed neuroendocrine non-neuroendocrine neoplasm (MiNEN) is a rare diagnosis in which neuroendocrine component is combined with a non-neuroendocrine component, each of which is morphologically and immunohistochemically recognisable as a discrete component and constitute ≥30% of the neoplasm. This case study reports two elderly patients with MiNEN in their colon, the first presented with caecal tumour on colonoscopy and the second had an obstructing mass in hepatic flexure region. They underwent hemicolectomy which revealed similar morphological findings which showed high grade adenocarcinoma in the superficial part and poorly differentiated components with signet rings and extracellular mucin in the deeper aspects. Areas of rhabdoid differentiation is also present in the first patient. Grade 3 neuroendocrine component is well defined within the deep aspect and constitute about 30% of the tumour. These components are positive for synaptophysin and focally for chromogranin with high Ki67. Perineural and lymphovascular invasion is identified in both cases along with metastasis to regional lymph nodes. MiNEN has an aggressive biological behaviour, mostly driven by its (often high-grade) neuroendocrine component, and a dismal prognosis. In summary, we are discussing two cases of MiNEN with poorly differentiated mucin secreting adenocarcinoma and grade 3 neuroendocrine tumour. 1.Frizziero M, Chakrabarty B, Nagy B, et al. Mixed neuroendocrine non-neuroendocrine neoplasms: a systematic review of a controversial and underestimated diagnosis. J Clin Med 2020; 9: 273.2.WHO Classification of Tumours Editorial Board. WHO Classification of Tumours: Digestive System Tumours. 5th ed. Lyon: IARC, 2019.3.de Mestier L, Cros J, Neuzillet C, et al. Digestive system mixed neuroendocrine-non-neuroendocrine neoplasms. Neuroendocrinology 2017; 105: 412–25.

MUC5B regulates the airway inflammation induced by atmospheric PM2.5 in rats and A549 cells

Atmospheric PM2.5 can induce airway inflammation and mucin secretion. MUC5B is required for airway defense. However, the research on the role of MUC5B in airway inflammation induced by atmospheric PM2.5 remains limited. This study was designed to explore the role of MUC5B in airway inflammation induced by atmospheric PM2.5. In vivo, Wistar rats were exposed to 0, 1.5, 7.5, 37.5 mg/ kg PM2.5 saline suspension via intratracheal instillation. HE staining and AB-PAS staining were used to observe the airway inflammation and goblet cell hyperplasia. In vitro, normal A549 cells and MUC5B-knockdown A549 cells were exposed to 0, 100, 200 and 400 μg/mL PM2.5 for 6 h, 12 h, 24 h and 48 h. ELISA was used to measure the levels of TNF-α and IL-1β in serum and bronchoalveolar lavage fluid of rats and in cell culture. Real time-PCR and ELISA were used to quantify the mRNA and protein levels of MUC5B in trachea and lung of rats and in A549 cells. PM2.5 could cause the infiltration of inflammatory cells and increase the mucus secretions and goblet cell metaplasia. MUC5B is related to rats’ airway inflammation induced by PM2.5. A549 cells exposed to PM2.5 in higher concentration and longer time, the protein level of MUC5B was significantly increased, while the levels of IL-1β, TNF-α and MUC5B mRNA were significantly decreased. Compared with normal A549 cells, the levels of IL-1β and TNF-α were significantly higher in Muc5b-knockdown cells. Atmospheric PM2.5 can induce airway inflammation and mucin secretion. MUC5B played a critical role in controlling the inflammatory response induced by PM2.5.

Autophagy of mucin granules contributes to resolution of airway mucous metaplasia

Exacerbations of muco-obstructive airway diseases such as COPD and asthma are associated with epithelial changes termed mucous metaplasia (MM). Many molecular pathways triggering MM have been identified; however, the factors that regulate resolution are less well understood. We hypothesized that the autophagy pathway is required for resolution of MM by eliminating excess non-secreted intracellular mucin granules. We found increased intracellular levels of mucins Muc5ac and Muc5b in mice deficient in autophagy regulatory protein, Atg16L1, and that this difference was not due to defects in the known baseline or stimulated mucin secretion pathways. Instead, we found that, in mucous secretory cells, Lc3/Lamp1 vesicles colocalized with mucin granules particularly adjacent to the nucleus, suggesting that some granules were being eliminated in the autophagy pathway rather than secreted. Using a mouse model of MM resolution, we found increased lysosomal proteolytic activity that peaked in the days after mucin production began to decline. In purified lysosomal fractions, Atg16L1-deficient mice had reduced proteolytic degradation of Lc3 and Sqstm1 and persistent accumulation of mucin granules associated with impaired resolution of mucous metaplasia. In normal and COPD derived human airway epithelial cells (AECs), activation of autophagy by mTOR inhibition led to a reduction of intracellular mucin granules in AECs. Our findings indicate that during peak and resolution phases of MM, autophagy activity rather than secretion is required for elimination of some remaining mucin granules. Manipulation of autophagy activation offers a therapeutic target to speed resolution of MM in airway disease exacerbations.

It Takes Two to Tango: How a Dysregulation of the Innate Immunity, Coupled With Candida Virulence, Triggers VVC Onset.

Vulvovaginal candidiasis (VVC) is a symptomatic inflammation of the vagina mainly caused by C. albicans. Other species, such as C. parapsilosis, C. glabrata, C. tropicalis, and C. krusei, are mainly associated to the recurrent form of the disease (RVVC), although with a lower frequency. In its yeast form, C. albicans is tolerated by the vaginal epithelium, but switching to the invasive hyphal form, co-regulated with the expression of genes encoding virulence factors such as secreted aspartyl proteases (Sap) and candidalysin, allows for tissue damage. Vaginal epithelial cells play an important role by impairing C. albicans tissue invasion through several mechanisms such as epithelial shedding, secretion of mucin and strong interepithelial cell connections. However, morphotype switching coupled to increasing of the fungal burden can overcome the tolerance threshold and trigger an intense inflammatory response. Pathological inflammation is believed to be facilitated by an altered vaginal microbiome, i.e., Lactobacillus dysbiosis. Notwithstanding the damage caused by the fungus itself, the host response to the fungus plays an important role in the onset of VVC, exacerbating fungal-mediated damage. This response can be triggered by host PRR-fungal PAMP interaction and other more complex mechanisms (i.e., Sap-mediated NLRP3 activation and candidalysin), ultimately leading to strong neutrophil recruitment. However, recruited neutrophils appear to be ineffective at reducing fungal burden and invasion; therefore, they seem to contribute more to the symptoms associated with vaginitis than to protection against the disease. Recently, two aspects of the vulvovaginal environment have been found to associate with VVC and induce neutrophil anergy in vitro: perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) and heparan sulfate. Interestingly, CAGTA antibodies have also been found with higher frequency in VVC as compared to asymptomatic colonized women. This review highlights and discusses recent advances on understanding the VVC pathogenesis mechanisms as well as the role of host defenses during the disease.

Genetic toxicity testing using human in vitro organotypic airway cultures: Assessing DNA damage with the CometChip and mutagenesis by Duplex Sequencing.

The organotypic human air‐liquid‐interface (ALI) airway tissue model has been used as an in vitro cell culture system for evaluating the toxicity of inhaled substances. ALI airway cultures are highly differentiated, which has made it challenging to evaluate genetic toxicology endpoints. In the current study, we assayed DNA damage with the high‐throughput CometChip assay and quantified mutagenesis with Duplex Sequencing, an error‐corrected next‐generation sequencing method capable of detecting a single mutation per 107 base pairs. Fully differentiated human ALI airway cultures were treated from the basolateral side with 6.25 to 100 μg/mL ethyl methanesulfonate (EMS) over a period of 28 days. CometChip assays were conducted after 3 and 28 days of treatment, and Duplex Sequencing after 28 days of treatment. Treating the airway cultures with EMS resulted in time‐ and concentration‐dependent increases in DNA damage and a concentration‐dependent increase in mutant frequency. The mutations observed in the EMS‐treated cultures were predominantly C → T transitions and exhibited a unique trinucleotide signature relative to the negative control. Measurement of physiological endpoints indicated that the EMS treatments had no effect on anti‐p63‐positive basal cell frequency, but produced concentration‐responsive increases in cytotoxicity and perturbations in cell morphology, along with concentration‐responsive decreases in culture viability, goblet cell and anti‐Ki67‐positive proliferating cell frequency, cilia beating frequency, and mucin secretion. The results indicate that a unified 28‐day study can be used to measure several important safety endpoints in physiologically relevant human in vitro ALI airway cultures, including DNA damage, mutagenicity, and tissue‐specific general toxicity.