Mucin Expression Research Articles

Expression and localisation of MUC1 modified with sialylated core-2 O-glycans in mucoepidermoid carcinoma

Mucoepidermoid carcinoma (MEC) is the most frequent of the rare salivary gland malignancies. We previously reported high expression of Mucin 1 (MUC1) modified with sialylated core-2 O-glycans in MEC by using tissue homogenates. In this study, we characterised glycan structures of MEC and identified the localisation of cells expressing these distinctive glycans on MUC1. Mucins were extracted from the frozen tissues of three patients with MEC, and normal salivary glands (NSGs) extracted from seven patients, separated by supported molecular matrix electrophoresis (SMME) and the membranes stained with various lectins. In addition, formalin-fixed, paraffin-embedded sections from three patients with MEC were subjected to immunohistochemistry (IHC) with various monoclonal antibodies and analysed for C2GnT-1 expression by in situ hybridisation (ISH). Lectin blotting of the SMME membranes revealed that glycans on MUC1 from MEC samples contained α2,3-linked sialic acid. In IHC, MUC1 was diffusely detected at MEC-affected regions but was specifically detected at apical membranes in NSGs. ISH showed that C2GnT-1 was expressed at the MUC1-positive in MEC-affected regions but not in the NSG. MEC cells produced MUC1 modified with α2,3-linked sialic acid-containing core-2 O-glycans. MUC1 containing these glycans deserves further study as a new potential diagnostic marker of MEC.

Pogostemon cablin (Blanco) Benth granule revealed a positive effect on improving intestinal barrier function and fecal microbiota in mice with irinotecan-induced intestinal mucositis

Pogostemon cablin (Blanco) Benth (PCB), a medicinal and edible homologous Chinese herb, has a protective effect on the structure and function of intestine. In this study, we aimed to investigate the effect of PCB granule (PCBG) on the improvement of irinotecan-induced intestinal mucositis and the regulation of intestinal microorganisms in mice. Our results demonstrated that PCBG supplementation significantly improved diarrhea symptoms caused by irinotecan, as evidenced by inhibiting weight loss, reversing intestinal atrophy, protecting against splenomegaly and balancing oxidative stress. Furthermore, compared with the model group, PCBG restored the intestinal morphology and improved intestinal barrier dysfunction by promoting the expression of tight junction proteins and mucin. Moreover, high-throughput sequencing analysis revealed that PCBG improved the flora disorder caused by irinotecan and regulated microbial community structure, such as decreasing the relative abundance of Bacteroides as well as increasing the relative abundance of Lactobacillus. Meanwhile, the disordered microbial functions in intestinal mucositis mice were recovered more closely to the controls by PCBG. Finally, we found that a robust correlation between the specific microbiota and intestinal mucositis-related index. In summary, these findings revealed the beneficial effects of PCBG on the intestinal barrier and gut microbiota of irinotecan-induced intestinal mucositis, which may be one of the potential strategies to reduce the clinical side effects of irinotecan.

Meclofenamate Suppresses MUC5AC Mucin Gene Expression by Regulating the NF-kB Signaling Pathway in Human Pulmonary Mucoepidermoid NCI-H292 Cells.

The current study aimed to reveal the potential effect of meclofenamate, a nonsteroidal anti-inflammatory drug, on the gene expression of airway MUC5AC mucin. Human pulmonary mucoepidermoid NCI-H292 cells were pretreated with meclofenamate for 30 min and stimulated with phorbol 12-myristate 13-acetate (PMA) for 24 h. Thereafter, the effect of meclofenamate on the PMA-induced nuclear factor kappa B (NF-kB) signaling pathway was assessed. Meclofenamate inhibited glycoprotein production and mRNA expression of MUC5AC mucins induced by PMA by inhibiting the degradation of inhibitory kappa Bα (IkBα) and NF-kB p65 nuclear translocation. These results suggest meclofenamate suppresses mucin gene expression by regulating NF-kB signaling pathway in human pulmonary epithelial cells.

Abstract 1779: Fourth-generation chimeric antigen receptor T cells targeting mucin 1 against breast cancer

Abstract Breast cancer (BC) is the most common cause of deaths worldwide, which is required effective treatment strategies. The second-generation chimeric antigen receptor (CAR) T cell therapy was approved by U.S. Food and Drug Administration for hematological malignancies, exhibiting impressive efficacy. However, CAR T cell therapy has not been approved in solid tumors and it has intensively been the subjects of research and development. To develop CAR T cell therapy, it is crucial to identify target antigen specifically expressed on tumor cells. Mucin 1 (MUC1) was previously reported to be overexpressed in several tumors. In this study, we examined MUC1 expression in BC tissues and cultured cell lines, and to improve CAR T cell efficiency, we generated fourth-generation anti-MUC1 CAR T cells (αM.CAR4 T cells), containing anti-MUC1 single-chain variable fragment (scFv), three costimulatory molecules (CD28, CD137, CD27) and CD3ζ. High levels of MUC1 expression were observed in the tissues and cell lines. Comparing to second- and third-generations (αM.CAR2 T and αM.CAR3 T cells), the αM.CAR4 T cells showed greater proliferation in the CAR T cell production process. The anti-tumor functions of all CAR T cells were specific to MUC1 expression since their killing activities were illustrated against MUC1-positive but not MUC1-negative BC cells. Interestingly, at low effector to target ratios, the αM.CAR4 T cells exhibited higher tumor cell lysis than those of αM.CAR2 T and αM.CAR3 T cells. Furthermore, after exposure to MUC1-positive BC cells, the αM.CAR4 T cells expressed lower programmed cell death protein 1 (PD-1) level. Taken together, the αM.CAR4 T cells are potential to be further developed for treatments of MUC1-positive BC and other cancers. Citation Format: Kamonlapat Supimon, Thanich Sangsuwannukul, Mutita Junking, Pa-thai Yenchitsomanus. Fourth-generation chimeric antigen receptor T cells targeting mucin 1 against breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1779.

Abstract 1750: Removal of mucin glycans by Bifidobacterium bifidum enhances chemotherapy-mediated cell death in mucinous adenocarcinomas

Abstract Background: Mucinous colorectal adenocarcinoma, a subtype of colorectal cancer, is characterized by mucin production and increased resistance to several chemotherapeutic interventions; resulting in a poorer prognosis compared to non-mucus secreting cancers. Advances in treatment are stymied by the limited number of efficacious chemotherapeutic agents. The most common drug used to treat colorectal cancer is 5-Fluorouracil (5-FU), but less than 10% of mucinous tumors respond to this drug. The production of mucus has been theorized to limit the uptake of chemotherapeutic agents via charge repulsion and size exclusion, thereby promoting chemo-resistance and driving poor outcomes. Hypothesis: We predict that mucin-degrading microbes secrete glycosyl hydrolase enzymes which cleave mucin glycans and promote the uptake of chemotherapeutic agents and enhance cell death. Methods & Results: We queried the human gut microbiota and identified multiple diverse microbes which encode enzymes to degrade mucin glycans. Among the mucin-degrading bacteria, we found that Bifidobacterium bifidum harbored one of the most extensive repertoires of mucin-degrading enzymes. B. bifidum was grown in a chemically defined medium with or without porcine intestinal mucus and the secreted products were then added to mucus-producing adenocarcinoma cell lines: HT29-MTX and T84 cells. Supernatant from cultures with mucus removed sialic acid residues and enhanced 5-FU uptake after 48 hrs in both cell lines. Supernatant without mucus also promoted 5-FU mediated cell death; although to a lesser degree than supernatant from cultures incubated with mucus. Gene expression analysis and immunostaining revealed decreased MUC13 gene expression and protein in B. bifidum metabolite treated cell lines compared to media controls. In contrast, we found that secreted products from B. dentium, which is unable to degrade mucus, had no effect on 5-FU induced cell death. Conclusions: Our data suggests that B. bifidum elicits chemo-sensitivity via the production of mucin-degrading enzymes, which cleave negatively charged sialic acid, increasing the activity of 5-FU in these cancers as well as by lowering the expression of adherent mucins. This work points to a novel role of mucin-degrading microbes to promote 5-FU penetration of mucinous colorectal adenocarcinoma. This strategy has the potential to provide unique treatment strategies for patients with mucinous colorectal cancer. Citation Format: Leah K. Stripe, Amy C. Engevik, Mindy A. Engevik. Removal of mucin glycans by Bifidobacterium bifidum enhances chemotherapy-mediated cell death in mucinous adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1750.

Profile of MUC5AC and MUC5B mucins expression in asthma patients under cold exposure

Introduction. Cold airway hyperresponsiveness (CAH) is a common condition in patients with asthma, which worsens the clinical course of the disease and the patients’ quality of life. MUC5AC and MUC5B are the main secreted mucins in the respiratory tract, which are involved in normal mucociliary clearance, but also capable of provoking the development of pathological changes in case of dysregulation of their balanced production.Aim. The aim of this study was to determine the dynamics of MUC5AC and MUC5B expression during experimental cooling in patients with asthma depending on the status of CAH.Materials and methods. The study enrolled 98 subjects including 26 patients with chronic non-obstructive bronchitis without exacerbation (control group) and 72 patients with asthma. The expression of MUC5AC, MUC5B and TRPM8 was determined in the upper respiratory tract by quantitative reverse transcription PCR. The production of MUC5AC and MUC5B was also measured in sputum by ELISA. All patients underwent a bronchoprovocation test with isocapnic cold air hyperventilation to detect CAH, and a similar nasal challenge was performed to assess the effect of cooling on the expression of the studied genes.Results. Patients with asthma had 4.22-fold increase in the expression of MUC5AC (p=0.02) in the nasal epithelium as compared with the control group. CAH was associated with an initial 7.33-fold upregulation of MUC5AC (p=0.008) as well as with further increase in MUC5AC expression but a decrease in MUC5B in response to cooling, which was not observed in asthma patients without CAH. Basal TRPM8 expression was associated with baseline level of MUC5AC (ρ=0.41, p=0.04), MUC5B (ρ=0.55, p<0.001) and amount of sputum produced after the cold bronchoprovocation.Conclusion. Asthma patients with CAH demonstrate a more pronounced imbalance in the production of mucins, which is aggravated by cold exposure. This, in turn, can lead to a number of pathological disorders associated with a more severe course of the disease.

BMAL1 regulates MUC1 overexpression in ovalbumin-induced asthma.

Asthma often presents with a daily rhythm; however, the underlying mechanisms remain unclear. Circadian rhythm genes have been proposed to regulate inflammation and mucin expression. Here, ovalbumin (OVA)-induced mice and serum shock human bronchial epidermal cells (16HBE) were used in in vivo and in vitro models, respectively. We constructed a brain and muscle ARNT-like 1 (BMAL1) knockdown 16HBE cell line to analyze the effects of rhythmic fluctuations on mucin expression. Serum immunoglobulin E (IgE) and circadian rhythm genes in asthmatic mice showed rhythmic fluctuation amplitude. Mucin (MUC) 1 and MUC5AC expression was increased in the lung tissue of the asthmatic mice. MUC1 expression was negatively correlated with that of the circadian rhythm genes, particularly BMAL1 (r=-0.546, P=0.006). There was also a negative correlation between BMAL1 and MUC1 expression (r=-0.507, P=0.002) in the serum shock 16HBE cells. BMAL1 knockdown negated the rhythmic fluctuation amplitude of MUC1 expression and upregulated MUC1 expression in the 16HBE cells. These results indicate that the key circadian rhythm gene, BMAL1, causes periodic changes in airway MUC1 expression in OVA-induced asthmatic mice. Targeting BMAL1 to regulate periodic changes in MUC1 expression may, therefore, improve asthma treatments.

Low Expression of Mucin 2, High Expression of Mucin 13, and High Expression of Nuclear Factor Kappa-Light-Enhancer of Activated B Cells were Significant Pathways in Colorectal Cancer Development.

To prove the role of MUC2, MUC13, and NFκB protein expression as significant carcinogenesis pathways in colorectal cancer development. This is a nested case-control study conducted at the Udayana University, Sanglah General hospital, from September 2020 to 2021. All eligible subjects who visited the Digestive Surgery outpatient clinic with a colorectal surgery plan 2021 were included. The subjects were classified as case group (cancerous colonic mucosa) and control group (normal colonic mucosa), proved by histopathology examination. The parameters in this study were the expression of MUC2, MUC13, and NFκB by immunohistochemistry analysis. The data in this study will be collected and tabulated in SPSS 25.0 (Chicago, Illinois, USA). A total of 36 subjects with colorectal cancer (case group) and 36 subjects with normal colonic mucosa (control group) were analyzed in this study. The cancerous colonic mucosa significantly had a lower MUC2, higher MUC13, and higher NFκB expression. After multivariate analysis for controlling the age variable, the result showed that only MUC2, MUC13, and NFκB expressions were still significant with p<0.05. The effect from MUC2, MUC13, and NFκB expression totally could assess up to 85.4% of the risk of developing colorectal cancer. There was a significantly lower MUC2, higher MUC13, and higher NFκB expression in the carcinogenesis of colorectal cancer, representing the influence of the inflammatory pathway and the abnormality of the protective barrier. Therefore, in the future, this result could remark a future early prediction or scoring system to assess colorectal cancer in clinical application.

Dietary nucleotides influences intestinal barrier function, immune responses and microbiota in 3-day-old weaned piglets.

Nucleotides (NTs) play a pivotal role in the growth and development of the intestine. This study aimed to evaluate the effects of nucleotides supplementation on the intestinal barrier function, immune responses and microbiota in 3-day-old weaned piglets. Ninety-six piglets weaned at 3-days after birth were randomly assigned to 2 treatments (6 replicates/treatment, 8 piglets/replicate) according to the average body weight. The dietary treatments consisted of the control (CON; fed a basal artificial milk) and nucleotides groups (NT; fed a basal artificial milk with 0.035% nucleotides, the contents of CMP, UMP, AMP, GMP, and IMP were 1:1:1:1:1, respectively). Diarrhea rates were recorded, and blood and intestinal samples were collected on day 35 of the piglets. The current study showed that NTs supplementation tended to decrease the diarrhea rate of weaned piglets (P<0.10). NTs increased villus height and the villus height-to-crypt depth (V/C) ratio in the ileum (P<0.05). Dietary NTs up-regulated protein expression of ZO-1 in ileal mucosa (P<0.05), and the protein expression of Occludin tended to increase. Furthermore, NTs up-regulated the mRNA expression of Mucin (MUC)2, while the mRNA expression of MUC4 was down-regulated in the ileal mucosa (P<0.05). Besides, supplementation with NTs increased the ileal mucosa genes expression of IL-21, INF-γ, IL-10, IL-4, IL-6 and TNF-α (P<0.05). Furthermore, dietary NTs increased the protein expression of NF-κB, IL-6 and TNF-α (P<0.05), and the proteins expression of Occludin and p-NF-κB tended to be up-regulated in the ileal mucosa (P<0.10). Furthermore, NTs supplementation increased short chain fatty acid in the colonic (P<0.05). And NTs supplementation reduced the Firmicutes/Bacteroidota ratio in the colon, at the genus level, NTs enriched the relative abundance of Prevotella, Faecalibacterium and Olsenella (P<0.05). These data indicate that NTs could increase the villus height, increase the V/C, regulate the expression of tight junction protein and mucin, improve the intestinal barrier of piglets, regulate the secretion of cytokines, improve the biological immunity, increase the abundance of beneficial bacteria, and thus reduce the diarrhea of piglets.

Impact of polystyrene microplastic exposure on gilthead seabream (Sparus aurata Linnaeus, 1758): Differential inflammatory and immune response between anterior and posterior intestine

Plastics are the most widely discharged waste into the aquatic ecosystems, where they break down into microplastics (MPs) and nanoplastics (NPs). MPs are ingested by several marine organisms, including benthic and pelagic fish species, contributing to organ damage and bioaccumulation.This study aimed to assess the effects of MPs ingestion on gut innate immunity and barrier integrity in gilthead seabreams (Sparus aurataLinnaeus, 1758) fed for 21 days with a diet enriched with polystyrene (PS-MPs; 1–20 μm; 0, 25 or 250 mg /kg b.w./die).Physiological fish growth and health status were not impacted by PS-MPs treatments at the end of experimental period. Inflammation and immune alterations were revealed by molecular analyses in both anterior (AI) and posterior intestine (PI) and were confirmed by histological evaluation. PS-MPs triggered TLR-Myd88 signaling pathway with following impairment of cytokines release. Specifically, PS-MPs increased pro-inflammatory cytokines gene expression (i.e., IL-1β, IL-6 and COX-2) and decreased anti-inflammatory ones (i.e., IL-10). Moreover, PS-MPs also induced an increase in other immune-associated genes, such as Lys, CSF1R and ALP. TLR-Myd88 signaling pathway may also lead to the mitogen-activated protein kinases (MAPK) signaling pathway activation. Here, MAPK (i.e., p38 and ERK) were activated by PS-MPs in PI, following the disruption of intestinal epithelial integrity, as evidenced by reduced gene expression of tight junctions (i.e. ZO-1, Cldn15, Occludin, and Tricellulin), integrins (i.e., Itgb6) and mucins (i.e., Muc2-like and Muc13-like). Thus, all the obtained results suggest that the subchronic oral exposure to PS-MPs induces inflammatory and immune alterations as well as an impact on intestinal functional integrity in gilthead seabream, with a more evident effect in PI.

Establishment and characterization of a multi-drug resistant cell line for canine mammary tumors.

Canine mammary tumors are the most common tumor disease of female dogs, and adjuvant chemotherapy often results in multi-drug resistance. Currently, the mechanisms underlying the development of tumor multi-drug resistance are unclear. The translation of research applications that can be used to effectively overcome tumor resistance is similarly hampered. Therefore, it is urgent to construct multi-drug resistance models of canine mammary tumors that can be used for research, to explore the mechanisms and means of overcoming resistance. In this study, the canine triple negative breast cancer cell line CMT-7364 was induced to develop multidrug resistance using doxorubicin by high-dose drug pulse method. The drug resistance and the expression of drug transport pumps of the cells was verified by CCK8 assay, immunoblotting, qPCR and immunofluorescence. Next, we used scratch assay and Transwell invasion assay to compare the migration and invasion abilities of the two cell lines and examined the expression of EMT-related proteins in both using immunoblotting. The differences of transcriptome between parental and drug-resistant cell lines were detected by RNA-seq sequencing. Finally, mouse xenograft models of drug-resistant and parental cell lines were constructed to evaluate the tumorigenic ability. After more than 50 generations of continuous passages stimulated by high-dose drug pulse method, the morphology of drug-resistant cell line CMT-7364/R tended to be mesenchymal-like and heterogeneous under light microscopy compared with the parental cell line CMT-7364/S, and developed resistance to doxorubicin and other commonly used chemotherapeutic drugs. In CMT-7364/R, BCRP was expressed at higher levels at both transcriptional and protein levels, while P-glycoprotein was not significantly different. Secondly, the migration and invasion ability of CMT-7364/R was significantly enhanced, with decreased expression of E-cadherin and increased expression of vimentin and mucin 1-N terminus. Finally, mouse xenograft models were constructed, while there was no significant difference in the volume of masses formed at 21 days. In summary, by using the canine mammary tumor cell line CMT-7364/S as the parental cell line, we successfully constructed a multidrug-resistant CMT-7364/R with high-dose drug pulse methods. Compared to its parental cell line, CMT-7364/R has decreased growth rate, overexpression of BCRP and increased migration and invasion ability due to EMT. The results of this study showed that CMT-7364/R might serve as a model for future studies on tumor drug resistance.

The Antioxidant Properties of Lavandula multifida Extract Contribute to Its Beneficial Effects in High-Fat Diet-Induced Obesity in Mice.

Obesity is a worldwide public health problem whose prevalence rate has increased steadily over the last few years. Therefore, it is urgent to improve the management of obesity and its comorbidities, and plant-based treatments are receiving increasing attention worldwide. In this regard, the present study aimed to investigate a well-characterized extract of Lavandula multifida (LME) in an experimental model of obesity in mice and explore the underlying mechanisms. Interestingly, the daily administration of LME reduced weight gain as well as improved insulin sensitivity and glucose tolerance. Additionally, LME ameliorated the inflammatory state in both liver and adipose tissue by decreasing the expression of various proinflammatory mediators (Il-6, Tnf-α, Il-1β, Jnk-1, Pparα, Pparγ, and Ampk) and prevented increased gut permeability by regulating the expression of mucins (Muc-1, Muc-2, and Muc-3) and proteins implicated in epithelial barrier integrity maintenance (Ocln, Tjp1, and Tff-3). In addition, LME showed the ability to reduce oxidative stress by inhibiting nitrite production on macrophages and lipid peroxidation. These results suggest that LME may represent a promising complementary approach for the management of obesity and its comorbidities.

Autologous Serum Eye Drops (ASEDs) as dry eye disease treatment option

Background: Nowadays, there are a lot of treatment options available for dry eye syndrome (DES) or disease (DED). Autologous Serum Eye Drops (ASEDs) is one of the options that offer a potential advantage over conventional therapy. Biochemical elements in ASEDs resemble real tears and act as a lacrimal substitute to provide lubrication. As a severe dry eye treatment, using ASEDs has grown in favor. Aim: This review aims to deliver knowledge from the current literature on applying ASEDs for dry eye disease in the medical worker. Method: The method used was a literature review. A comprehensive search was performed on Pubmed and Google Scholar databases in December 2022 using each of the following keywords “ASEDs”, “serum”, “eyedrop”, “DED” and “DES”. Conclusion: Dry eye disease is incurable and symptom management is the focus of therapy. Studies have shown that ASEDs reduced dry eye symptoms in over 60% of patients by inhibiting the release of inflammatory cytokines and increasing the number of goblet cells and mucin expression in the conjunctiva. The serum eye drop is typically well tolerated and can be one of the best treatment options for severe DED due to its close resemblance to tears, preservatives free and minimum side effects.

Pulmonary epithelial markers in phenotypes of chronic lung allograft dysfunction

Airway epithelial injury is thought to be a key event in the pathogenesis of chronic lung allograft dysfunction (CLAD). We investigated whether markers of epithelial activity and injury in bronchoalveolar lavage fluid (BAL) correlate with CLAD diagnosis and major CLAD phenotypes: bronchiolitis obliterans syndrome (BOS) vs. restrictive allograft syndrome (RAS)-related phenotypes (including RAS, mixed phenotype, and all other patients with RAS-like opacities). CLAD status and phenotypes were retrospectively determined in a cohort of all consecutive adult, first, bilateral lung transplants performed 2010-2015, with available BAL samples. All patients with RAS-related phenotypes were included and 1:1 matched with BOS patients based on the time from transplant to CLAD-onset. Subjects who were CLAD-free for a minimum of 3 years post-transplant were 1:1 matched to CLAD patients and included as controls. Proteins that maintain the barrier function of the airway epithelial mucosa (club cell secretory protein (CCSP), surfactant protein-D (SP-D) and epithelial mucins: MUC1, MUC5AC, MUC5B, MUC16), as well as epithelial cell death markers (M30&M65 representing epithelial cell apoptosis and overall death, respectively), were measured in BAL obtained within 6-months post CLAD onset using a double-sandwich ELISA or a multiplex bead assay. Protein levels were compared using Mann-Whitney-U-test. Association between protein levels and graft survival was assessed using Cox proportional hazards models, adjusted for CMV serology mismatch status and CLAD phenotype. Fifty-four CLAD (27 BOS, 11 RAS, 7 mixed, 9 others with RAS-like opacities) patients and 23 CLAD-free controls were included. Median BAL levels were significantly higher in patients with CLAD compared to CLAD-free controls for M30 (124.5 vs. 88.7 U/L), MUC1 (6.8 vs. 3.2 pg/mL), and MUC16 (121.0 vs. 30.1 pg/mL). When comparing CLAD phenotypes, M30 was significantly higher in patients with RAS-related phenotypes than BOS (160.9 vs. 114.6 U/L). In multivariable models, higher M30 and MUC5B levels were associated with decreased allograft survival after CLAD onset independent of phenotype (P<0.05 for all). Airway epithelial mucins and cell death markers are enhanced in the BAL of patients with CLAD and can assist in differentiating between CLAD phenotypes and post-CLAD outcomes. Abnormal airway mucin expression and epithelial cell death may be involved in the pathogenesis of CLAD, and therefore their detection may aid in future selection of targeted therapies.

Antimicrobial peptide cathelicidin LL-37 preserves intestinal barrier and organ function in rats with heat stroke

Global warming increases the incidence of heat stroke (HS) and HS causes the reduction of visceral blood flow during hyperthermia, leading to intestinal barrier disruption, microbial translocation, systemic inflammation and multiple organ failure. Cathelicidin LL-37 exhibits antimicrobial activities, helps innate immunity within the gut to maintain intestinal homeostasis, and augments intestinal wound healing and barrier function. Thus, we evaluated the effects and possible mechanisms of cathelicidin LL-37 on HS. Wistar rats were placed in a heating-chamber of 42 ̊C to induce HS. Changes in rectal temperature, hemodynamic parameters, and survival rate were measured during the experimental period. Blood samples and ilea were collected to analyze the effects of LL-37 on systemic inflammation, multiple organ dysfunction, and intestinal injury. Furthermore, LS174T and HT-29 cells were used to assess the underlying mechanisms. Our data showed cathelicidin LL-37 ameliorated the damage of intestinal cells induced by HS. Intestinal injury, systemic inflammation, and nitrosative stress (high nitric oxide level) caused by continuous hyperthermia were attenuated in HS rats treated with cathelicidin LL-37, and hence, improved multiple organ dysfunction, coagulopathy, and survival rate. These beneficial effects of cathelicidin LL-37 were attributed to the protection of intestinal goblet cells (by increasing transepithelial resistance, mucin-2 and Nrf2 expression) and the improvement of intestinal barrier function (less cyclooxygenase-2 expression and FITC-dextran translocation). Interestingly, high cathelicidin expression in the ileal samples of inflammatory bowel disease patients was associated with better clinical outcome. These results suggest that cathelicidin LL-37 could prevent heat stress-induced intestinal damage and heat-related illnesses.

The postbiotic of hawthorn-probiotic ameliorating constipation caused by loperamide in elderly mice by regulating intestinal microecology.

Constipation is common gastrointestinal disorder with high prevalence and recurrence, making people suffering. However, the treatment for constipation remains ineffectual. We aimed to the study the effects and mechanisms of postbiotic of hawthorn-probiotic on loperamide modeled old KM mice. Constipated mice were grouped and treated with 10% lactulose (Y), hawthorn group (S), probiotic group (F) and postbiotic of hawthorn-probiotic (FS). Fecal changes were observed. AQP3 and Enac-γ were measured by RT-qPCR and Western blotting, intestinal barrier by H&E and immunofluorescence staining, cell proliferation and apoptosis by CCK8 and flow cytometry. Gut microbiota was further determined by 16 s rRNA sequence of feces. Postbiotic of hawthorn-probiotic improved intestinal movement and pathomorphology, elevated AQP3, Enac-γ and mucin-2 expression, accompanied by decreased serum TNF-α and cell apoptosis, but increased proliferation. Furthermore, it modified the gut microbiota of constipated mice, featured by upregulation of Lactobacillaceae. Postbiotic of hawthorn-probiotic relieved constipation by combined effects of regulating intestinal water and sodium metabolism, maintain intestinal barrier and gut microflora.Graphical Abstract.

Eliciting effective tumor immunity against ovarian cancer by cancer stem cell vaccination

Advanced ovarian cancer (OC) patients have limited benefit from current relevant cytotoxic and targeted therapies following debulking surgery. Therefore, new therapeutic strategies are in urgent need. Immunotherapy has shown great potential in tumor treatment, especially in tumor vaccine development. The study objective was to evaluate the immune effects of cancer stem cells (CSCs) vaccines on OC. The CD44+CD117+CSCs were isolated from human OC HO8910 and SKOV3 cells using the magnetic cell sorting system; the cancer stem-like cells were selected from murine OC ID8 cell by no-serum formed sphere culture. The CSC vaccines were prepared by freezing and thawing these CSCs, which were then injected into mice followed by challenging the different OC cells. The in vivo antitumor efficacy of CSC immunization revealed the vaccines were capable of significantly provoking immune responses to autologous tumor antigens in vaccinated mice as the mice were found to have markedly inhibited tumor growth, prolonged survival, and decreased CSC counts in OC tissues when compared to mice without the CSC vaccination. The in vitro cytotoxicities of immunocytes toward SKOV3, HO8910 and ID8 cells indicated a significant killing efficacy compared with the controls. However, the antitumor efficacy was remarkably reduced whilst the mucin-1 expression in CSC vaccines was down-regulated by small interfering RNA. Overall, findings from this study provided the evidence that has deepened our understanding of CSC vaccine immunogenicity and anti-OC efficacy, particularly for the role of dominant antigen mucin-1. It is possible to turn the CSC vaccine into an immunotherapeutic approach against ovarian cancer.

Effects of Pendrin Protein in Nasal Epithelial Cells on Mucin Production in the Context of Type 2 Inflammation.

Chronic rhinosinusitis (CRS) is a heterogeneous disease. The pathogenesis of chronic sinusitis is still unclear; however, the nasal cavity and paranasal sinuses are commonly affected by type 2 inflammation, which is caused by Th2 cytokines such as interleukin (IL)-5, IL-4, and IL-13. Previous studies have shown that pendrin promotes local infiltration of neutrophils through the production of human neutrophil elastase (HNE), which is essential for the secretion of mucin 5AC (MUC5AC) in chronic inflammatory diseases of the lower respiratory tract. This study investigated pendrin expression and its relationship to mucin in type 2 inflammation. A total of 40 patients (10 CRS patients with nasal polyps,10 CRS patients without nasal polyps, and 20 nasal septum deviation patients) were included in this study and were divided into the CRS group and the NC group. A normal nasal mucosa tissue culture model was established in vitro. IL-13 was used to stimulate primary cultures of human nasal epithelial cells (HNECs). Western blot (WB), enzyme-linked immunosorbent assay (ELISA), and quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect the expression of pendrin, MUC5AC, and MUC5B. After transfecting HNECs with siRNA pendrin or negative control (NC), EGF receptor (EGFR), HNE, MUC5AC, and MUC5B expression were analyzed using qRT-PCR, WB, or ELISA in terms of their relationships with pendrin. Pendrin expression in the tissue was also analyzed. After IL-13 stimulation, pendrin, MUC5AC, and MUC5B expression levels were upregulated; the optimal concentration of IL-13 was 50 ng/mL. The expression levels of HNE, EGFR, MUC5AC, and MUC5B were downregulated after transfection with siRNA pendrin-1650. Pendrin expression in the NC group was lower than in the CRS group. IL-13 is implicated in the inflammation of nasal mucosa, and pendrin is closely related to the excessive secretion of mucin. The expression of mucin is downregulated after transfection with siRNA pendrin. There is a positive relationship between pendrin and EFGR/HNE. Moreover, pendrin plays an important role in type 2 inflammation.

Mucin expression in liver cancer

Increasing incidence, difficulties in early diagnosis, and a high mortality rate in liver cancer (LC) determine the relevance of studying the mechanisms of its development. The aim of the work is to evaluate the expression of high molecular weight glycoproteins MUC-1, MUC-13 in liver cancer. The object of study is LC tissue samples of 65 patients from the archives and 34 blood serum samples from patients with morphologically confirmed LC. The age of subjects was 26– 97 years. The level of antibodies to MUC-1 and MUC-13 was studied by ELISA. The reference value ranges of MUC-1 (0.250 ± 0.10 ng/ml) and MUC-13 (0.321 ± 0.13 ng/ml) in the blood serum of healthy individuals were established. The concentration of antibodies to MUC-1 and MUC-13 in the blood serum in RP was significantly higher than that in practically healthy individuals. The concentration of MUC-1 and MUC-13 in the LC tumor tissue was higher than that in the blood serum of apparently healthy individuals and LC patients. With a confirmed LC diagnosis, the level of antibodies to MUC-1 in the blood serum, which exceeds 0.373 ng/ml, and the level of antibodies to MUC-13, which is more than 0.939 ng/ml, may indicate a high risk of a tumor process.

GATA4 Forms a Positive Feedback Loop with CDX2 to Transactivate MUC2 in Bile Acids-Induced Gastric Intestinal Metaplasia.

Gastric intestinal metaplasia (GIM), a common precancerous lesion of gastric cancer, can be caused by bile acid reflux. GATA binding protein 4 (GATA4) is an intestinal transcription factor involved in the progression of gastric cancer. However, the expression and regulation of GATA4 in GIM has not been clarified. The expression of GATA4 in bile acid-induced cell models and human specimens was examined. The transcriptional regulation of GATA4 was investigated by chromatin immunoprecipitation and luciferase reporter gene analysis. An animal model of duodenogastric reflux was used to confirm the regulation of GATA4 and its target genes by bile acids. GATA4 expression was elevated in bile acid-induced GIM and human specimens. GATA4 bound to the promoter of mucin 2 (MUC2) and stimulate its transcription. GATA4 and MUC2 expression was positively correlated in GIM tissues. Nuclear transcription factor-κB activation was required for the upregulation of GATA4 and MUC2 in bile acid-induced GIM cell models. GATA4 and caudal-related homeobox 2 (CDX2) reciprocally transactivated each other to drive the transcription of MUC2. In chenodeoxycholic acid-treated mice, MUC2, CDX2, GATA4, p50, and p65 expression levels were increased in the gastric mucosa. GATA4 is upregulated and can form a positive feedback loop with CDX2 to transactivate MUC2 in GIM. NF-κB signaling is involved in the upregulation of GATA4 by chenodeoxycholic acid.