Mucin 5AC Expression Research Articles

Alteration of Colonic Mucin Composition and Cytokine Expression in Acute Swine Dysentery.

Swine dysentery (SD) is an enteric disease associated with strongly β-hemolytic Brachyspira spp. that cause mucohemorrhagic diarrhea primarily in grower-finisher pigs. We characterized alteration of colonic mucin composition and local cytokine expression in the colon of pigs with acute SD after B. hyodysenteriae (Bhyo) infection and fed either a diet containing 30% distillers dried grains with solubles (DDGS) or a control diet. Colonic tissue samples from 9 noninoculated pigs (Control, N = 4; DDGS, N = 5) and 10 inoculated pigs experiencing acute SD (Bhyo, N = 4; Bhyo-DDGS, N = 6) were evaluated. At the apex of the spiral colon, histochemical staining with high-iron diamine-Alcian blue revealed increased sialomucin (P = .008) and decreased sulfomucin (P = .027) in Bhyo pigs relative to controls, with a dietary effect for sulfomucin. Noninoculated pigs fed DDGS had greater expression of sulfomucin (P = .002) compared to pigs fed the control diet. Immunohistochemically, there was de novo expression of mucin 5AC (MUC5AC) in the Bhyo group while mucin 2 (MUC2) expression was not significantly different between groups. RNA in situ hybridization to detect the pro-inflammatory cytokine IL-1β often showed increased expression in the Bhyo group although without statistical significance, and this was not correlated with MUC5AC or MUC2 expression, suggesting IL-1β is not a major regulator of their secretion in acute SD. Expression of the anti-inflammatory cytokine TGF-β1 was significantly suppressed in the Bhyo group compared to controls (P = .005). This study reveals mucin and cytokine alterations in the colon of pigs with experimentally induced SD and related dietary effects of DDGS.

Mucin 5AC is significantly upregulated in exosomes from the nasal lavage fluid and may promote the expression of COX-2, VEGF and MMP-9: an implication in nasal polyp pathogenesis

BACKGROUND: Exosomes are critical mediators of intercellular communication and could be involved in many human diseases; however, little is known about the role of exosomes in nasal polyps (NP). METHODS: Exosomes in nasal lavage fluids (NLF) were isolated by ultracentrifugation. Exosome identity was validated by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and specific exosomal markers. The exosome proteome was revealed by LC-MS/MS, and the expression of the candidate exosomal protein, mucin 5AC, was confirmed by Western blot analysis and immunohistochemistry (IHC). Cellular uptake of the exosomes was monitored by fluorescence confocal microscopy and the ensuing effects on COX-2, VEGF and MMP-2/MMP-9 were determined by Western blotting, ELISA and gelatin zymography, respectively. RESULTS: Mass spectrometry analysis and subsequent verification by Western blotting identified that mucin 5AC was significantly upregulated in exosomes from NLFs of NP patients. Moreover, the expression of mucin 5AC was increased in the tissue specimens of the NP patients. Functional assays suggest that the mucin 5 AC-enriched exosomes could be effectively taken up by chronic rhinosinusitis without NP (CRSsNP)-derived fibroblasts, the control cells, resulting in a significant increase in the expression of COX-2, VEGF and MMP-9. CONCLUSIONS: Mucin 5AC, the major airway mucin, cannot only be carried and transferred by nasal exosomes, but may also promote tissue remodeling and angiogenesis and thus could be a potential therapeutic target of NP.

Alnus hirsuta (Spach) Rupr. Attenuates Airway Inflammation and Mucus Overproduction in a Murine Model of Ovalbumin-Challenged Asthma

Alnus hirsuta (Spach) Rupr. (AH), a member of the Betulaceae family, is widely used in Eastern Asia of as a source of medicinal compounds for the treatment of hemorrhage, diarrhea, and alcoholism. In this study, we investigated the protective effects of a methanolic extract of AH branches against airway inflammation and mucus production in tumor necrosis factor (TNF)-α-stimulated NCI-H292 cells and in an ovalbumin (OVA)-challenged allergic asthma mouse model. Female BALB/c mice were injected with OVA (40 μg) and aluminum hydroxide (2 mg) on days 0 and 14 to induce allergic airway inflammation. The mice were then challenged with 1% OVA from days 21–23. Mice were treated with AH (50 and 100 mg/kg/day; 2% DMSO) or dexamethasone (positive control; 3 mg/kg/day) from days 18–23. AH treatment effectively attenuated airway resistance/hyperresponsiveness and reduced levels of T helper type 2 (Th2) cytokines, eotaxins, and number of inflammatory cells in bronchoalveolar lavage fluid, and immunoglobulin E in serums of OVA-challenged mice. In histological analysis, AH treatment significantly inhibited airway inflammation and mucus production in OVA-challenged mice. AH treatment downregulated the phosphorylation of I kappa B-alpha, p65 nuclear factor-kappa B (p65NF-κB), and mitogen-activated protein kinases with suppression of mucin 5AC (MUC5AC) in lung tissue. Moreover, AH treatment decreased the levels of pro-inflammatory cytokines and Th2 cytokines, as well as MUC5AC expression, and inhibited the phosphorylation of p65NF-κB in TNF-α-stimulated NCI-H292 cells. These results indicate that AH might represent a useful therapeutic agent for the treatment of allergic asthma.

Benzo(a)pyrene induces MUC5AC expression through the AhR/mitochondrial ROS/ERK pathway in airway epithelial cells

ObjectivesBenzo(a)pyrene (BaP) is a ubiquitous air pollutants, and BaP exposure leads to a risk of respiratory diseases. The oversecretion of airway mucus and high expression of mucin 5AC (MUC5AC) are associated with common respiratory disorders caused by air pollution. We aimed to investigate the effect of BaP on MUC5AC expression, especially the mechanisms by which BaP induces MUC5AC gene expression. MethodsThe human airway epithelial cell NCI-H292 was used to test the effects of BaP on the expression of MUC5AC in vitro. MUC5AC mRNA and protein expression were assessed with real-time quantitative PCR, immunochemistry, and western blotting. A luciferase assay was conducted to detect the activity of the promoter. The total cellular ROS and mitochondrial ROS were measured by corresponding probes. Small-interfering RNAs were used for gene silencing. AhR-overexpressing cell lines were constructed by transfection with AhR overexpression lentivirus. ResultsWe found that BaP stimulation upregulated the MUC5AC mRNA and protein levels and activated the ERK pathway. Suppressing ERK with U0126 (an ERK inhibitor) or knocking down ERK with siRNA decreased BaP-induced MUC5AC expression. The luciferase activity transfected with the MUC5AC promoter and cAMP-response element (CRE) was increased after BaP treatment, whereas CREB siRNA suppressed the BaP-induced overexpression of MUC5AC. In addition, BaP increased mitochondrial ROS production, and Mito-TEMP, a mitochondrial ROS inhibitor, inhibited BaP-induced MUC5AC expression and ERK activation. BaP increased the mRNA levels of CYP1A1 and CYP1B1, while Alizarin, a CYP1s inhibitor, suppressed the effects of BaP, including the MUC5AC overexpression, ERK activation and mitochondrial ROS generation. BaP induced the translocation of aryl hydrocarbon receptor (AhR) from the cytoplasm to the nucleus. SiRNA-mediated knockdown or chemical inhibition of AhR decreased the BaP-induced expression of MUC5AC, while the overexpression of AhR significantly enhanced the BaP-induced expression of MUC5AC. ITE, an endogenous AhR ligand, also upregulated the mRNA and protein expression of MUC5AC. Furthermore, resveratrol treatment inhibited the BaP-induced MUC5AC overexpression, AhR translocation, mitochondrial ROS production and ERK pathway activation. ConclusionHere, we highlighted the crucial role of AhR/mitochondrial ROS/ERK pathway activation in BaP-induced MUC5AC overexpression and identified resveratrol as a promising drug to reduce BaP-induced MUC5AC overexpression.

MUC5AC Expression in Various Tumor Types and Nonneoplastic Tissue: A Tissue Microarray Study on 10 399 Tissue Samples

Background: Mucin 5AC (MUC5AC) belongs to the glycoprotein family of secreted gel-forming mucins and is physiologically expressed in some epithelial cells. Studies have shown that MUC5AC is also expressed in several cancer types suggesting a potential utility for the distinction of tumor types and subtypes. Methods: To systematically determine MUC5AC expression in normal and cancerous tissues, a tissue microarray containing 10 399 samples from 111 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. Results: MUC5AC was expressed in normal mucus-producing cells of various organs. At least weak MUC5AC positivity was seen in 44 of 111 (40%) tumor entities. Of these 44 tumor entities, 28 included also tumors with strong positivity. MUC5AC immunostaining was most commonly seen in esophageal adenocarcinoma (72%), colon adenoma (62%), ductal adenocarcinoma of the pancreas (64%), mucinous carcinoma of the ovary (46%), diffuse gastric adenocarcinoma (44%), pancreatic ampullary adenocarcinoma (41%), intestinal gastric adenocarcinoma (39%), and bronchioloalveolar carcinoma (33%). Clinically relevant tumors with complete or almost complete absence of MUC5AC staining included small cell carcinoma of the lung (0% of 17), clear cell renal cell carcinoma (0% of 507), papillary thyroid carcinoma (0% of 359), breast cancer (2% of 1097), prostate cancer (2% of 228), soft tissue tumors (0.1% of 968), and hematological neoplasias (0% of 111). Conclusion: The highly standardized analysis of a broad range of cancers identified a ranking order of tumors according to their relative prevalence of MUC5AC expression.

Secretory Mucin 5AC Promotes Neoplastic Progression by Augmenting KLF4-Mediated Pancreatic Cancer Cell Stemness.

Secreted mucin 5AC (MUC5AC) is the most abundantly overexpressed member of the mucin family during early pancreatic intraepithelial neoplasia stage I (PanIN-I) of pancreatic cancer. To comprehend the contribution of Muc5ac in pancreatic cancer pathology, we genetically ablated it in an autochthonous murine model (KrasG12D; Pdx-1cre, KC), which mirrors the early stages of pancreatic cancer development. Neoplastic onset and the PanIN lesion progression were significantly delayed in Muc5ac knockout (KrasG12D; Pdx-1 cre; Muc5ac-/-, KCM) animals with a 50% reduction in PanIN-2 and 70% reduction in PanIN-3 lesions compared with KC at 50 weeks of age. High-throughput RNA-sequencing analysis from pancreatic tissues of KCM animals revealed a significant decrease in cancer stem cell (CSC) markers Aldh1a1, Klf4, EpCAM, and CD133. Furthermore, the silencing of MUC5AC in human pancreatic cancer cells reduced their tumorigenic propensity, as indicated by a significant decline in tumor formation frequency by limiting dilution assay upon subcutaneous administration. The contribution of MUC5AC in CSC maintenance was corroborated by a significant decrease in tumor burden upon orthotopic implantation of MUC5AC-depleted pancreatic cancer cells. Mechanistically, MUC5AC potentiated oncogenic signaling through integrin αvβ5, pSrc (Y416), and pSTAT3 (Y705). Phosphorylated STAT3, in turn, upregulated Klf4 expression, thereby enriching the self-renewing CSC population. A strong positive correlation of Muc5ac with Klf4 and pSTAT3 in the PanIN lesions of KC mouse pancreas reinforces the crucial involvement of MUC5AC in bolstering the CSC-associated tumorigenic properties of Kras-induced metaplastic cells, which leads to pancreatic cancer onset and progression. SIGNIFICANCE: This study elucidates that de novo expression of MUC5AC promotes cancer cell stemness during Kras-driven pancreatic tumorigenesis and can be targeted for development of a novel therapeutic regimen.

Telmisartan mitigates lipopolysaccharide (LPS)-induced production of mucin 5AC (MUC5AC) through increasing suppressor of cytokine signaling 1 (SOCS1)

ABSTRACT Acute lung injury (ALI) is a serious clinical pulmonary disease. The pathogenesis of ALI is related to the excessive release of inflammatory factors and upregulation of mucin 5AC (MUC5AC). Telmisartan is a novel antihypertension agent that exerts promising anti-inflammatory effects. The purpose of this study is to investigate whether Telmisartan has a protective role in lipopolysaccharide (LPS)-induced MUC5AC expression and to explore the underlying mechanism in human bronchial epithelial cells. Firstly, the decreased cell viability, elevated release of lactate dehydrogenase (LDH), and excessively released inflammatory factors tumor necrosis factor-α (TNF-α), interleukin- 6 (IL-6), and transforming growth factor-β (TGF)-β in bronchial BEAS-2B epithelial cells induced by stimulation with LPS were significantly reversed by the introduction of Telmisartan. Secondly, the upregulated MUC5AC and downregulated suppressor of cytokine signaling 1 (SOCS1) caused by stimulation with LPS were dramatically reversed by Telmisartan. Notably, treatment with Telmisartan attenuated LPS-induced activation of nuclear factor κ-B (NF-κB). Lastly, silencing of SOCS1 abolished the protective effects of Telmisartan against LPS-induced production of MUC5AC and the activation of NF-κB. Based on these findings, we conclude that Telmisartan displayed a protective effect against LPS by improving mitochondrial function, mitigating inflammatory response, and reducing the production of mucin 5AC by regulating the SOCS1/NF-κB axis in human bronchial epithelial cells.

Elevated MUC5AC expression is associated with mismatch repair deficiency and proximal tumor location but not with cancer progression in colon cancer

Mucin 5AC (MUC5AC) is a secreted gel-forming mucin expressed by several epithelia. In the colon, MUC5AC is expressed in scattered normal epithelial cells but can be abundant in colorectal cancers. To clarify the relationship of MUC5AC expression with parameters of tumor aggressiveness and mismatch repair deficiency (dMMR) in colorectal cancer, a tissue microarray containing 1812 colorectal cancers was analyzed by immunohistochemistry. MUC5AC expression was found in 261 (15.7%) of 1,667 analyzable colorectal cancers. MUC5AC expression strongly depended on the tumor location and gradually decreased from proximal (27.4% of cecum cancers) to distal (10.6% of rectal cancers; p < 0.0001). MUC5AC expression was also strongly linked to dMMR. dMMR was found in 21.3% of 169 cancers with MUC5AC positivity but in only 4.6% of 1051 cancers without detectable MUC5AC expression (p < 0.0001). A multivariate analysis showed that dMMR status and tumor localization predicted MUC5AC expression independently (p < 0.0001 each). MUC5AC expression was unrelated to pT and pN status. This also applied to the subgroups of 1136 proficient MMR (pMMR) and of 84 dMMR cancers. The results of our study show a strong association of MUC5AC expression with proximal and dMMR colorectal cancers. However, MUC5AC expression is unrelated to colon cancer aggressiveness.

CircARRDC3 contributes to interleukin‑13‑induced inflammatory cytokine and mucus production in nasal epithelial cells via the miR‑375/KLF4 axis.

Allergic rhinitis (AR) is a common inflammatory disorder of the nasal mucosa. It is a major risk factor for asthma development, and uncontrolled AR can lead to the worsening of asthma symptoms, which affects the quality of life and productivity of patients. Circular RNAs (circRNA) were reported to be involved in the pathogenesis of AR. The aim of the present study was to investigate the functional role of circRNA arrestin domain-containing 3 (circARRDC3) in AR progression. circARRDC3 knockdown suppressed the levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and eotaxin and mucin 5AC (MUC5AC) in IL-13-induced nasal epithelial cells. Moreover, circARRDC3 silencing promoted viability and suppressed apoptosis in IL-13-induced NECs. circARRDC3 targeted microRNA (miR)-375 and negatively regulated its expression. miR-375 inhibition reversed the effects of circARRDC3 knockdown on GM-CSF, eotaxin and MUC5AC expression levels, cell viability and cell apoptosis. In addition, miR-375 inhibited krueppel-like factor 4 (KLF4) expression through direct interaction, and miR-375 overexpression inhibited GM-CSF, eotaxin and MUC5AC expression levels, and cell apoptosis, which was abolished following KLF4 overexpression. In addition, circARRDC3, miR-375 and KLF4 were all dysregulated in the nasal mucosa of patients with AR. miR-375 expression was negatively correlated with circARRDC3 and KLF4 expression, and circARRDC3 expression was positively correlated with KLF4 expression. In conclusion, circARRDC3 contributed to the development of AR by regulating the miR-375/KLF4 axis. These findings may provide novel insights into the pathogenesis of AR.

Phloretin, an Apple Polyphenol, Inhibits Pathogen-Induced Mucin Overproduction.

Bacterial infection induces mucus overproduction, contributing to acute exacerbations and lung function decline in chronic respiratory diseases. A diet enriched in apples may provide protection from pulmonary disease development and progression. This study examined whether phloretin, an apple polyphenol, inhibits mucus synthesis and secretion induced by the predominant bacteria associated with chronic respiratory diseases. The expression of mucus constituent mucin 5AC (MUC5AC) in FVB/NJ mice and NCI-H292 epithelial cells is analyzed. Nontypeable Haemophilus influenzae (NTHi)-infected mice developed increased MUC5AC mRNA, which a diet containing phloretin inhibited. In NCI-H292 cells, NTHi, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa increased MUC5AC mRNA, which phloretin inhibited. Phloretin also diminished NTHi-induced MUC5AC protein secretion. NTHi-induced increased MUC5AC required toll-like receptor 4 (TLR4) and NADH oxidase 4 (NOX4) signaling and subsequent activation of the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) pathway. Phloretin inhibited NTHi-induced TLR4/NOX4 and EGFR/MAPK signaling, thereby preventing increased MUC5AC mRNA. EGFR activation can also result from increased EGFR ligand synthesis and subsequent ligand activation by matrix metalloproteinases (MMPs). In NCI-H292 cells, NTHi increased EGFR ligand and MMP1 and MMP13 mRNA, which phloretin inhibited. In summary, phloretin is a promising therapeutic candidate for preventing bacterial-induced mucus overproduction.

Molecular mechanism underlying miR‑130b‑Sp1 transcriptional regulation in LPS‑induced upregulation of MUC5AC in the bile duct epithelium.

Hepatolithiasis is a common disease that represents a serious health threat to the Chinese population. The pathological mechanism underlying hepatolithiasis is closely related to bacterial infections of the intrahepatic bile duct, followed by chronic inflammation and the overexpression of mucin 5AC (MUC5AC). However, the exact mechanism responsible for the lipopolysaccharide (LPS)-induced upregulation of MUC5AC has yet to be elucidated. Specificity protein 1 (Sp1) is a ubiquitous transcription factor that plays a vital role in the regulation of a number of genes that are responsible for normal cellular function. microRNA (miR/miRNA)-130b is a member of the miRNA family. miRNAs can bind to the 3′-untralsated region (3′-UTR) of a target gene and influence its expression levels. The present study found that LPS increases the expression of MUC5AC by influencing Sp1 secretion. Chromatin immunoprecipitation-quantitative PCR experiments further verified three Sp1 binding sites in the MUC5AC promoter sequence that can regulate the expression of MUC5AC. Further analysis demonstrated that Sp1 expression was regulated by miR-130b. Luciferase experiments identified one miR-130b binding site in the Sp1 3′-UTR region. In vivo experiments also confirmed the role of the miR-130b-Sp1-MUC5AC signaling pathway in the formation of biliary stones and indicated that this pathway may provide targeted therapeutic strategies for the treatment of intrahepatic bile duct stones.

Pepsin exposure in a non-acidic environment upregulates mucin 5AC (MUC5AC) expression via matrix metalloproteinase 9 (MMP9)/nuclear factor κB (NF-κB) in human airway epithelial cells.

Gastric reflux (GR) is a backflow of gastric content to the aerodigestive tract. GR was previously found to be associated with inflammatory airway diseases and a potential cause of airway remodeling. Chronic exposure to gastric content may induce damage from nose to lung, because digestive enzymes and acidity are toxic to airway epithelial cells. Recently, the toxicity of pepsin in a non-acidic environment was found to increase proinflammatory cytokines and receptors in the epithelium of the aerodigestive tract. However, the effect of pepsin in non-acidic conditions on mucin expression has not been investigated in human airway epithelial cells. The purpose of this study was to evaluate the effect of pepsin on mucin 5AC (MUC5AC) expression in upper and lower airway epithelial cells as an important potential factor of non-acidic GR-related airway inflammation. In NCI-H292 cells and human nasal epithelial cells (HNEpCs), the effects and signaling pathways of pepsin on MUC5AC expression were examined using reverse-transcription polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, zymography, Western blot, and immunofluorescence staining. Pepsin increased MUC5AC expression in non-acidic condition of NCI-H292 cells and HNEpCs. Further, pepsin activated matrix metalloproteinase 9 (MMP9) and phosphorylated nuclear factor κB (NF-κB). Moreover, inhibitors of MMP9 and NF-κB significantly attenuated pepsin-induced MUC5AC expression, and the knockdown of NF-κB by small interfering RNA (siRNA) significantly blocked pepsin-induced MUC5AC expression in human airway epithelial cells. These findings suggest that pepsin increased MUC5AC expression in non-acidic conditions via the activation of MMP9 and NF-κB in human airway epithelial cells.

Effects of Radix Angelicae Sinensis on airway mucus hypersecretion and the TNF-α/NF-κB signaling pathway in Yin-deficiency asthmatic mice

Objective: To investigate the therapeutic effects of Radix Angelicae Sinensis (RADA) on airway mucus hypersecretion and the tumor necrosis factor-α/ nuclear factor- κB (TNF-α/NF-κB) signaling pathway in Yin-deficiency asthma mice. Methods: KM mice were randomly divided into control group, model group, ambroxol group and RADA low, medium and high dose (2, 4 and 8 g/kg) group(n=12). Ovalbumin and the thyroid gland were used to replicate the model of Yin-deficiency asthma. Asthma symptoms in mice , immune globulin E (IgE) , TNF-α , and the expressions of Mucin 5ac (Muc5ac) and NF- κB in lung tissue were observed under the intervention of RADA. Results: RADA at the doses of 2,4 and 8 g/kg could alleviate the asthma symptoms of Yin-deficiency asthma mice significantly, reduce the levels of IgE in serum and TNF-α in bronchoalveolar lavage fluid (BALF), and inhibite the overexpressions of Muc5ac and NF- κB in lung tissue. Conclusion: RADA has significant anti-asthmatic effect. One of its mechanisms is to inhibit TNF-α/NF- κB signaling pathway and to alleviate airway mucus hypersecretion.

Protective action of Bacillus clausii probiotic strains in an in vitro model of Rotavirus infection

Rotavirus is the most common cause of acute gastroenteritis (AGE) in young children. Bacillus clausii (B. clausii) is a spore-forming probiotic that is able to colonize the gut. A mixture of four B. clausii strains (O/C, T, SIN and N/R) is commonly used for the treatment of AGE, and it has been demonstrated that it can reduce the duration and severity of diarrhea in children with AGE. Few studies have sought to characterize the mechanisms responsible for such beneficial effects. Intestinal effects of probiotics are likely to be strain-specific. We conducted a series of in vitro experiments investigating the activities of this mixture of B. clausii strains on biomarkers of mucosal barrier integrity and immune function in a cellular model of Rotavirus infection. B. clausii protected enterocytes against Rotavirus-induced decrease in trans-epithelial electrical resistance, and up-regulated expression of mucin 5AC and tight junction proteins (occludin and zonula occludens-1), all of which are important for effective mucosal barrier function. B. clausii also inhibited reactive oxygen species production and release of pro-inflammatory cytokines (interleukin-8 and interferon-β) in Rotavirus-infected cells, and down-regulated pro-inflammatory Toll-like receptor 3 pathway gene expression. Such mechanisms likely contributed to the observed protective effects of B. clausii against reduced cell proliferation and increased apoptosis in Rotavirus-infected enterocytes.

Qingfei oral liquid alleviates airway hyperresponsiveness and mucus hypersecretion via TRPV1 signaling in RSV-infected asthmatic mice

Pediatric asthma is exacerbated by Respiratory Syncytial Virus (RSV) infection, and Transient Receptor Potential Vanilloid 1 (TRPV1) promotes production of inflammatory cytokines and mucus hypersecretion in the pathology of this disease. Our previous research revealed that Qingfei oral liquid (QF) inhibited airway inflammation and mucus hypersecretion in RSV-infected asthmatic mice models and that this may be associated with the TRPV1-regulation of NF-κB and Mucin 5AC (MUC5AC) expression, but the exact mechanism is unknown. In the present study, LC–MS was used for analyzing the chemicals in QF, ovalbumin (OVA)-induced asthmatic mice inhaled RSV three consecutive times to create an RSV-infected asthmatic model. We found treatment from QF alleviated airway hyperresponsiveness (AHR) and reduced congestion, edema, and infiltration of inflammatory cells into pulmonary tissues. Additionally, QF was found to decrease expression of NF-κB and its downstream inflammatory cytokines IL-1β, IL-4, IL-5, and IL-13, as well as a decrease in MUC5AC and pro-inflammatory cytokines in PKC via a reduction in Protein Kinase C-dependent signaling. These findings suggest that QF can alleviate AHR and mucus hypersecretion caused by RSV infection in asthmatic mice, and its mechanism may be associated with the regulation of the TRPV1 signaling pathway.

Altered mucins and aquaporins indicate dry eye outcome in patients undergoing Vitreo-retinal surgery.

Vitreo-retinal (VR) surgeries induce conjunctival changes. However, there are no study reports regarding prevalence and severity of dry eye after these surgeries. This study evaluated dry eye outcome after VR surgery. Patients undergoing VR surgery classified as scleral buckle and microincision vitrectomy surgery (n = 44, mean age: 56.09±10.2 years) were recruited. Dry eye evaluation was done before and 8 weeks after surgery (2 weeks after omitting topical eye drops). Conjunctival imprint cytology for goblet cell count and tear Mucin 5AC (MUC5AC) protein estimation was done. Gene expressions of MUC5AC, MUC4, MUC16, Aquaporin 4 (AQP4) and AQP5 were analyzed in the conjunctival imprint cells by qPCR. None of the patients exhibited clinical signs of dry eye after VR surgery. But the conjunctival goblet cell density (GCD) was significantly lowered post-VR surgery (63% cases, **p = 0.012) with no alterations in the tear MUC5AC protein. Post-VR surgery, the conjunctival cell gene expression of MUC4, MUC16 and AQP4 were significantly increased (*p = 0.025, *p = 0.05 and *p = 0.02 respectively) and AQP5 was significantly lowered (*p = 0.037), with no change in MUC5AC expression. Tear cytokines were significantly increased post-VR surgery (anti-inflammatory: IL1RA, IL4, IL5, IL9, FGF; PDGFbb and pro-inflammatory: IL2, IL6, IL15, GMCSF and IFNg). Though clinical signs of dry eye were not observed after VR surgery, ocular surface changes in the form of reduced GCD, altered MUC5AC, MUC4, MUC16, AQP4, AQP5 and cytokines are suggestive of dry eye outcome at the molecular level especially inpatients aged above 51 years, especially female gender and those who are diabetic.

The pattern of mucin 5AC (MUC5AC) expression using immunohistochemistry and its prognostic significance in patients with pancreatic ductal adenocarcinoma.

e16756 Background: Mucin-5AC (MUC5AC) is a secreted form of mucin. Its expression correlates with poor outcome in uterine, ampullary adenocarcinoma and cholangiocarcinoma. Its prognostic significance in pancreatic ductal adenocarcinoma (PDAC) is not well established. Here, we explored the pattern of MUC5AC expression using immunohistochemistry (IHC) and its prognostic significance in patients with PDAC. Methods: This is a retrospective study conducted at the University of South Alabama/Mitchell Cancer Institute. Between 2015-2019, 218 patients with PDAC were identified. Among this cohort, only 45 patients had tissue available for MUC5AC IHC staining. Two pathologists in- dependently scored the expression of MUC5AC. Staining percentage was estimated in 10% increments. Unpaired t test and log-rank Wilcoxon tests were used for statistical analysis. Results: In our cohort, the median age was 65 years (42-85). Males represented 55%. Caucasians, African Americans and other ethnicities (e.g. Asians) represented 71%, 25% and 4% respectively. Twenty patients (44%) had metastatic stage IV disease and 25 patients (56%) had non-metastatic disease. Positive cytoplasmic and apical MUC5AC expression by IHC was seen in 82% and 80% of patients respectively. The median apical MUC5AC expression was higher in metastatic patients compared to non-metastatic patients (67.5 % vs 30%, p = 0.0187 ) while there was no difference in the median cytoplasmic MUC5AC expression between metastatic and non-metastatic patients (13.7 % vs 12.5%, p = 0.3328). In non-metastatic patients, compared to patients with positive apical MUC5AC expression, no expression (0%) was associated with worse overall survival (OS) (43 vs 13 m, p = 0.0322). In metastatic PDAC, there was no difference in OS between patients with positive or no apical MUC5AC expression (18 vs 21 m, p = 0.781). Compared to patients with positive cytoplasmic MUC5AC expression, patients with no cytoplasmic MUC5AC expression (0%) had no difference in OS in both non-metastatic (40 vs 32 m, p = 0.986) and metastatic (36 vs 12.5 m, p = 0.487) patients. Conclusions: MUC5AC expression assessment using IHC is feasible in patients with PDAC. In our cohort, apical, but not cytoplasmic, MUC5AC expression was different between metastatic and non-metatstaic patients and showed prognostic significance in non-metastatic patients. To our knowledge, this is the first study to show prognostic significance of MUC5AC expression in patients with PDAC using IHC.

Effects of Amniotic Membrane Extract on the Hyperplastic Response of the Middle Ear Mucosa in a Bacterially-Induced Otitis Media Rat Model: A Preliminary Study.

ObjectivesHuman amniotic membrane extract (AME) is known to contain numerous bioactive factors and anti-inflammatory substances. However, the anti-inflammatory effects of AME on the middle ear (ME) mucosa are unclear. This study assessed the effects of AME on the growth of the ME mucosa in response to bacterially-induced otitis media (OM).MethodsOM was induced by inoculating nontypeable Haemophilus influenzae (NTHi) into the ME cavity of rats. ME mucosal explants were cultured in AME concentrations of 0, 5, 10, or 50 μg/mL. The area of explant outgrowth was measured in culture and analyzed at 1, 3, 5, and 7 days after explantation. The expression of Ki-67, mucin 5AC (MUC5AC), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in the explants was also evaluated using quantitative polymerase chain reaction (PCR) and immunocytochemistry (ICC).ResultsThe NTHi-induced ME mucosa growth increased gradually over the 7-day culture period in all explants at different AME concentrations. There was a trend for mucosal growth inhibition at higher concentrations of AME, although the growth was not significantly different among the groups until day 5. The ME mucosal explants treated with the 50 μg/mL concentration of AME showed significantly suppressed growth on postexplantation day 7 compared with other explants on the same day. PCR and ICC staining revealed that the expression of Ki-67, MUC5AC, TNF-α, and IL-10 further decreased in the explants with higher concentrations of AME than in those with lower concentrations of AME.ConclusionOur results showed that higher concentrations of AME reduced the mucosal proliferative response in bacterial OM in rats. These findings provide evidence that AME has an influence on the inflammatory and proliferative responses to NTHi infection in ME mucosa.

Exposure to Traffic-Related Particulate Matter 2.5 Triggers Th2-Dominant Ocular Immune Response in a Murine Model.

Ambient particulate matter (PM), a major component of air pollution, aggravates ocular discomfort and inflammation, similarly to dry eye disease (DED) or allergies. However, the mechanism(s) by which PM induces the ocular inflammatory response is unknown. This study investigated the immunological response of traffic-related fine particulate matter (PM2.5) on the ocular surface in a murine model. C57BL/6 mice were exposed by topical application to PM2.5 or vehicle for 14 days to induce experimental environmental ocular disease. Corneal fluorescein staining and the number of ocular inflammatory cells were assessed in both groups. The expression of IL-1β, IL-6, tumor necrosis factor (TNF)-α, and mucin 5AC (MUC5AC) in the ocular surface were evaluated by real-time PCR. An immunohistochemical assay evaluated apoptosis and goblet cell density. ELISA was used to determine the levels of serum IgE and cytokines of Type 1 helper (Th1) and Type 2 helper (Th2) cells after in vitro stimulation of T cells in the draining lymph nodes (LNs). Exposure to traffic-related PM2.5 significantly increased corneal fluorescein staining and cellular toxicity in the corneal epithelium compared with the vehicle control. A significant increase in the number of CD11b+ cells on the central cornea and mast cells in the conjunctiva was observed in the PM2.5 group. Exposure to PM2.5 was associated with a significant increase in the corneal or conjunctival expression of IL-1β, IL-6, TNF, and MUC5AC compared to the vehicle, and increased maturation of dendric cells (DCs) (MHC-IIhighCD11c+) in draining LNs. In addition, PM2.5 exposure increased the level of serum IgE and Th2 cytokine production in draining LNs on day 14. In conclusion, exposure to traffic-related PM2.5 caused ocular surface damage and inflammation, which induced DC maturation and the Th2-cell-dominant allergic immune response in draining LNs.

Bifidobacterium infantis Relieves Allergic Asthma in Mice by Regulating Th1/Th2.

BackgroundBifidobacteria are among the probiotics used in treating intestinal diseases and are rarely used for allergic asthma treatment. The present study investigated the mechanism of B. infantis in treating allergic asthma in mice.Material/MethodsA total of 40 male Balb/c mice were randomized into control, ovalbumin (OVA), montelukast (Mon), and B. infantis (B10) groups, and allergic asthma was induced in the OVA, Mon, and B10 groups. Airway reactivity was measured on day 29 by methacholine at various doses. The numbers of total cells and inflammatory cells in bronchoalveolar lavage fluid (BALF) were counted by blood cell counter and Diff-Quik staining. Hematoxylin-eosin (HE) staining was performed to observe inflammatory cell infiltration in lung tissues. Total IgE and OVA-specific IgE in serum were measured by ELISA. Mucin 5AC expression was detected by Western blot to evaluate airway obstruction. The levels of Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5, IL-13) cytokines in BALF and tissues were detected by ELISA and qRT-PCR, respectively.ResultsThe mice in the OVA group had airway hyperreactivity, while the symptoms in the B10 group and Mon group were effectively relieved. B10 reduced the number of inflammatory cells in BALF as well as inflammatory cell infiltration in tissues. Moreover, the levels of total serum IgE, OVA-specific IgE, and Mucin 5AC were increased in the OVA group, but were reduced in the Mon group and B10 group. B. infantis increased the levels of Th1 cytokines and decreased those of Th2 cytokines.ConclusionsB. infantis can reduce the infiltration of inflammatory cells induced by OVA-specific antibodies in mice. B. infantis has therapeutic effects on allergic asthma by promoting Th1 and inhibiting Th2 immune responses.