Expression Of CLDN4 Research Articles

Intestinal adaptations to energy source of milk replacer in neonatal dairy calves

Most milk replacers (MR) contain more lactose compared with whole milk, which, when fed at a large meal size, could influence gut barrier function in calves. This study evaluated how replacing lactose in MR with fat (on a wt/wt basis) affects intestinal histomorphology and permeability in neonatal dairy calves. Thirty-four Holstein-Friesian bull calves were blocked by dam parity and randomly assigned to 1 of 2 treatments (n = 17): a high-lactose (46.1% lactose, 18.0% crude fat, and 23.9% crude protein of dry matter) or a high-fat MR (HF; 39.9% lactose, 24.6% crude fat, and 24.0% crude protein of dry matter). Calves were individually housed and fed pooled colostrum at 1.5 h and 12 h postnatally, at 18 and 9% of metabolic body weight (BW0.75), respectively. From 24 h postnatally until the end of the study (d 7), calves were transitioned to be fed MR (prepared at 15% solids) at 18% of BW0.75 twice daily at 0700 and 1900 h. During postprandial sampling on d 6, intestinal permeability was assessed by mixing lactulose (1.03 g/kg of BW0.75) and d-mannitol (0.31 g/kg of BW0.75) into the morning meal without altering total meal volume. Sequential blood samples were collected via jugular catheter, and total urine was collected for 12 h to measure the marker content. Calves were euthanized 3 h after the morning meal on d 7, and gastrointestinal tract tissues and digesta were collected for analysis of histomorphology, digesta osmolality, and gene expression. The empty gastrointestinal tracts of HF calves were heavier, although length did not differ and differences in histomorphology were minor. Digesta osmolality changed along the tract without differences between treatments. Plasma lactulose was greater in HF, although plasma d-mannitol and the recovery of both markers in urine were unaffected. No significant differences were detected in gene expression, although HF calves tended to have lower expression of TJP1 and CLDN2 and higher expression of proinflammatory cytokine IL1B in ileum tissue. In conclusion, partially replacing lactose in MR with fat resulted in a heavier and more permeable gut, with minor histomorphological differences.

Tight junction protein claudin-2 promotes cell entry of Bombyx mori cypovirus.

Claudin-2 is a major component of tight junctions (TJs), which play an important role in reovirus entry into host cells. The Bombyx mori cytoplasmic polyhedosis virus (BmCPV) relates to the cypovirus strain of the reovirus family. So far, the role of claudin-2 in the process of BmCPV infection is not known. In the present study, it was observed that increasing expression of the claudin-2 gene (CLDN2) may concomitantly elevate BmCPV infection. Contrarily, knockdown of CLDN2 expression by siRNAs can reduce BmCPV infection. Similarly, antibody-based blockage of claudin-2 could also decrease BmCPV cell entry. These results suggest that claudin-2 can promote BmCPV infection in vitro. Moreover, immunofluorescence (IF) assays showed that claudin-2 can interact with BmCPV during viral infection. Specifically, co-immunoprecipitation experiments indicated that claudin-2 binds the BmCPV VP7 (instead of VP3 proteins). The interaction between VP7 and claudin-2 was further confirmed by bimolecular fluorescence complementation (BIFC). Altogether, our results suggest that BmCPV cell entry can be promoted upon interaction of VP7 with claudin-2. These findings provide new mechanistic insights related to BmCPV infection. KEY POINTS: •Claudin-2 could promote BmCPV infection of cells. •Claudin-2 interacted with BmCPV during BmCPV infection. •Claudin-2 could interact with BmCPV VP7 protein, but not with VP3 proteins.

Claudin-1/4 as directly target gene of HIF-1α can feedback regulating HIF-1α by PI3K-AKT-mTOR and impact the proliferation of esophageal squamous cell though Rho GTPase and p-JNK pathway.

Immunohistochemical microarray comprising 80 patients with esophageal squamous cell carcinoma (ESCC) and discovered that the expression of CLDN1 and CLDN4 were significantly higher in cancer tissues compared to para-cancerous tissues. Furthermore, CLDN4 significantly affected the overall survival of cancer patients. When two ESCC cell lines (TE1, KYSE410) were exposed to hypoxia (0.1% O2), CLDN1/4 was shown to influence the occurrence and development of esophageal cancer. Compared with the control culture group, the cancer cells cultured under hypoxic conditions exhibited obvious changes in CLDN1 and CLDN4 expression at both the mRNA and protein levels. Through genetic intervention and Chip, we found that HIF-1α could directly regulate the expression of CLDN1 and CLDN4 in cancer cells. Hypoxia can affect the proliferation and apoptosis of cancer cells by regulating the PI3K-Akt-mTOR pathway. Molecular analysis further revealed that CLDN1 and CLDN4 can participate in the regulation process and had a feedback regulatory effect on HIF-1α expression in cancer cells. In vitro cellular experiments and vivo experiments in nude mice further revealed that changes in CLDN4 expression in cancer cells could affect the proliferation of cancer cells via regulation of Rho GTP and p-JNK pathway. Whether CLDN4 can be target for the treatment of ESCC needs further research.

Abstract 1204: SO-N102, a novel CLDN18.2-targeting antibody-drug conjugate with strong anti-tumor effect in various solid tumors expressing low target levels

Abstract Claudin (CLDN) 18.2, a member of a large family of transmembrane proteins with distinct functions, has been shown to have a high prevalence, predominantly in gastric and pancreatic cancer. In addition, ectopic expression of CLDN18.2 was described for other cancer types including ovarian, lung, liver and colon, whereas healthy tissue expression is restricted to the stomach. SO-N102 represents a CLDN18.2 targeting antibody-drug conjugate based on a novel proprietary highly specific monoclonal antibody conjugated to a derivative of PNU-159682 using SMACTM technology for the therapy of patients with various CLDN18.2-positive solid tumors, mainly of gastric and pancreatic origin. The CLDN18.2 protein sequence is highly conserved in mammalians with 100% identity in the targeted extracellular loop among rodents, cynomolgus monkey and human. SO-N102 showed excellent specificity for CLDN18.2, strong binding to the target followed by efficient tumor cell killing. Preferential binding to selected patient-derived tumor tissues was observed ex vivo when compared to healthy stomach tissues from mice and cynomolgus monkey. Single-agent therapeutic activity of SO-N102 was demonstrated in 10 patient-derived mouse xenografts models (gastric, pancreatic, liver, colon and lung adenocarcinomas). Complete responses were observed in all models, independent of CLDN18.2 expression levels, ranging from low (IHC1+) to high (IHC3+), with minimum efficacious doses between 0.2 mg/kg and 0.6 mg/kg. An acceptable tolerability profile was observed in preliminary toxicity studies at 10 mg/kg (mouse), 6 mg/kg (rat) and 1 mg/kg (cynomolgus monkey). SO-N102 demonstrated favorable pharmacokinetic properties with half-lives in the range of 8 days and 13 days in cynomolgus monkey and rat, respectively. Stability of SO-N102 without any significant loss of the payload was demonstrated in vitro and in animals. Further toxicology studies in rats and cynomolgus monkeys were initiated, paralleled by process development and manufacturing activities with the plan to initiate the first clinical study with SO-N102 in patients in the first half of 2022. Citation Format: Lenka Kyrych Sadilkova, Iva Valentova, Simona Hoskova, Pavel Vopalensky, Lukas Bammert, Roger Beerli, Ulrich Moebius, Radek Spisek. SO-N102, a novel CLDN18.2-targeting antibody-drug conjugate with strong anti-tumor effect in various solid tumors expressing low target levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1204.

The Potential of 2-aza-8-Oxohypoxanthine as a Cosmetic Ingredient

In this study, we verified the effects of 2-aza-8-oxohypoxanthine (AOH) on human epidermal cell proliferation by performing DNA microarray analysis. Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, which measures mitochondrial respiration in normal human epidermal keratinocyte (NHEK) cells. Gene expression levels were determined by DNA microarray analysis of 177 genes involved in skin aging and disease. AOH showed a significant increase in cell viability at concentrations between 7.8 and 31.3 μg/mL and a significant decrease at concentrations above 250 μg/mL. DNA microarray analysis showed that AOH significantly increased the gene expression of CLDN1, DSC1, DSG1, and CDH1 (E-cadherin), which are involved in intercellular adhesion and skin barrier functioning. AOH also up-regulated the expression of KLK5, KLK7, and SPIMK5, which are proteases involved in stratum corneum detachment. Furthermore, AOH significantly stimulated the expression of KRT1, KRT10, TGM1, and IVL, which are considered general differentiation indicators, and that of SPRR1B, a cornified envelope component protein. AOH exerted a cell activation effect on human epidermal cells. Since AOH did not cause cytotoxicity, it was considered that the compound had no adverse effects on the skin. In addition, it was found that AOH stimulated the expression levels of genes involved in skin barrier functioning by DNA microarray analysis. Therefore, AOH has the potential for practical use as a cosmetic ingredient. This is the first report of efficacy evaluation tests performed for AOH.

IL-6/STAT3 signaling pathway regulates the proliferation and damage of intestinal epithelial cells in patients with ulcerative colitis via H3K27ac.

The aim of the present study was to investigate the effect of the IL-6/STAT3 signaling pathway on intestinal epithelial barrier injury in patients with ulcerative colitis (UC). Fifty-two patients with UC and 21 healthy subjects were recruited. The expression level of IL-6 in plasma was determined by ELISA. Normal human colon mucosal epithelial NCM460 cells were treated with IL-6 or plasma from the patients with UC. Then, the transepithelial electrical resistance value, fluorescein yellow permeability and zonulin release were evaluated. Using reverse transcription-quantitative (q)PCR and western blotting, claudin (CLDN) 1 and CLDN2 expression levels were analyzed. Furthermore, western blotting was used to detect phosphorylation of STAT3. Chromatin immunoprecipitation-qPCR was performed to investigate the enrichment of H3K27ac in the promoter regions of CLDN1 and CLDN2. The present study revealed that IL-6 content was elevated in the plasma from patients with UC and increased with the progression of the disease. IL-6 was also observed to induce intestinal epithelial cell barrier injury and regulate barrier function by influencing the expression of tight junction-related proteins, as well as STAT3. The IL-6/STAT3 signaling pathway regulated transcription of CLDN1 and CLDN2 by affecting the enrichment of histone H3K27ac in their promoter regions. Thus, the significantly increased expression level of IL-6 in the peripheral blood of patients with UC indicates a positive association with the development of UC. Furthermore, the IL-6/STAT3 signaling pathway influences the function of the intestinal barrier by affecting the H3K27ac level in intestinal epithelial cells.

The LINC00922 aggravates ovarian cancer progression via sponging miR-361-3p

BackgroundLong noncoding RNA (lncRNA) LINC00922 has been reported to promote tumorigenesis of lung and breast cancer. However, the functions and mechanisms of LINC00922 in ovarian cancer (OC) remain unclarified. The current study aims to clarify the detailed functions and underlying mechanisms of LINC00922 in the progression of OC.MethodsLINC00922 expression in OC tissues and cells was identified by a comprehensive strategy of data miming, computational biology and quantitative real-time polymerase chain reaction (RT-qPCR) experiment. In vitro CCK-8, wound healing, transwell invasion, western blotting and in vivo tumorigenesis assays LINC00922 were conducted to evaluate the functions of LINC00992. Subsequently, bioinformatics technology and dual luciferase reporter assay were performed to confirm the between miR-361-3p and LINC00922 or CLDN1. Finally, rescue experiments were performed to confirm whether LINC00922 effect functions of OC cells through regulation of miR-361-3p.ResultsLINC00922 was significantly upregulated in OC tissues and cell lines, which is significantly positively corelated with the poor prognosis of patients with OC. LINC00922 knockdown inhibited proliferation and tumorigenesis of OC cells in vitro and vivo. In addition, LINC00922 knockdown suppressed migration, invasion, and EMT of OC cells in vitro. Mechanically, LINC00922 could competitively bind with miR-361-3p to relieve the repressive effect of miR-361-3p on its target gene CLDN1 in OC cells. In addition, silencing miR-361-3p promoted OC cell proliferation, migration, invasion, EMT and Wnt/β-catenin signaling, while LINC00922 knockdown inhibited Wnt/β-catenin signaling by upregulating miR-361-3p. Rescue experiments revealed that LINC00922 knockdown inhibited OC cell proliferation, migration, invasion and EMT by regulating miR-361-3p.ConclusionThis study suggested that LINC00922 could competitively bind with miR-361-3p to promote the CLDN1 expression and activate Wnt/β-catenin signaling in OC progression, which providing a promising therapeutically target for OC.

Colonic Handling of Calcium Appears To Occur Via Different Pathways in Mouse Models of Infants and Adults

ObjectivesCalcium (Ca2+) is a vital micronutrient for many physiological functions with the greatest rate of accumulation occurring during the critical period of infancy. Previous work has demonstrated that molecular mechanisms of intestinal Ca2+ absorption across the small intestine are significantly different in animal models of infants and adults to permit greater absorption early in life. The colon contributes to overall Ca2+ balance in adults via transcellular, TRPV6 mediated and paracellular claudin-2 and -12 mediated pathways. Whether these same colonic pathways contribute to overall Ca2+ absorption in infants is not known. Here we aimed to investigate the molecular details of Ca2+ absorption across the large intestine in murine models of infancy relative to older mice. MethodsMice at 14 days (P14) were used as a model of suckling infants and mice at 2 months were employed to represent adult physiology. Wildtype and mutant mice with a non-functioning Trpv6 or deletions of Cldn2 or Cldn12 were used. Net 45Ca2+ flux (JCa) and Ca2+ permeability (PCa) were measured in Ussing chambers. Gene expression was determined by real-time PCR. ResultsJCa indicates net absorption across the colon at both P14 and 2 months. While gene expression of Trpv6 and S100g suggest greater cellular uptake of Ca2+ into colonocytes at P14, net JCain vitro was not different than at 2 months. In contrast to previous work in mice at 2 months, TRPV6 does not mediate JCa at P14. PCa was 20% greater at P14 than 2 months, suggesting greater capacity for bidirectional diffusion of Ca2+ down an electrochemical gradient in younger mice. In contrast to previous work in mice at 2 months, claudin-2 and claudin-12 do not mediate PCa at P14 and, expression of Cldn2 and Cldn12 were significantly reduced in younger mice. ConclusionsThese results improve our understanding of intestinal Ca2+ handling during a critical age early in life. Future work is required to delineate molecular details under in vivo conditions of colonic Ca2+ transport in infants. Funding SourcesThis work was funded by grants from the Women and Children’s Health Research Institute, which is supported by the Stollery Children’s Hospital Foundation, and the National Sciences and Engineering Research Council to RTA, who is the Canada Research Chair in Renal Epithelial Transport Physiology.

Luteolin alleviates ulcerative colitis through SHP-1/STAT3 pathway.

Previous studies have demonstrated that Luteolin has a positive effect on epithelial barrier integrity by promoting the function of tight protein, however, little is known about the underline mechanism of Luteolin. In this study, we constructed Caco-2 cell monolayer to explore the effects and the regulation mechanism of Luteolin in intestinal epithelial barrier integrity. Caco-2 cells were co-treated with TNF-α, Interferon-γ (IFN-γ) and Luteolin for 24h. Overexpression or knockdown of SHP-1 was applied to study the effects of protein phosphoserine phosphatase-1 (SHP-1) on epithelial barrier integrity. Cell viability was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Barrier function was detected by trans-epithelial electrical resistance (TEER) and FITC-dextran assay. The expression levels of SHP-1, phosphorylation signal transducer and activator of transcription 3 (p-STAT3), STAT3 and tight junction proteins were measured by qRT-PCR or western blot. In vivo model of ulcerative colitis was established to detect the function of Luteolin in ulcerative colitis. We clarified that Luteolin protected intestinal epithelial barrier function of Caco-2 monolayers by increasing the resistance values and tight junction (TJ) protein expression. The expression of OCLN, CLDN1, and ZO1 was increased by Luteolin, while the expression of CLDN2 was decreased. Furthermore, Luteolin significantly alleviated the symptom of ulcerative colitis in DSS-induced mice. The in vitro cell model proved that overexpression of SHP-1 promotes the epithelial barrier function and knockdown of SHP-1 or STAT3 activation destroyed the protective effects of Luteolin on the expression of TJ proteins. We found that the treatment of Luteolin promoted epithelial barrier function and Luteolin might preserve intestinal epithelial barrier function through suppression of STAT3 signaling pathway by SHP-1.

Effects of histone deacetylase inhibitors Tricostatin A and Quisinostat on tight junction proteins of human lung adenocarcinoma A549 cells and normal lung epithelial cells.

Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for non-small cell lung cancer (NSCLC). However, more preclinical studies of HDAC inhibitors in NSCLC and normal lung epithelial cells are required to evaluate their antitumor activities and mechanisms. The bicellular tight junction molecule claudin-2 (CLDN-2) is highly expressed in lung adenocarcinoma tissues and increase the proliferation of adenocarcinoma cells. Downregulation of the tricellular tight junction molecule angulin-1/LSR induces malignancy via EGF-dependent CLDN-2 and TGF-β-dependent cellular metabolism in human lung adenocarcinoma cells. In the present study, to investigate the detailed mechanisms of the antitumor activities of HDAC inhibitors in lung adenocarcinoma, human lung adenocarcinoma A549 cells and normal lung epithelial cells were treated with the HDAC inibitors Trichostatin A (TSA) and Quisinostat (JNJ-2648158) with or without TGF-β. Both HDAC inhibitors increased anguin-1/LSR, decrease CLDN-2, promoted G1 arrest and prevented the migration of A549 cells. Furthermore, TSA but not Quisinostat with or without TGF-β induced cellular metabolism indicated as the mitochondrial respiration measured using the oxygen consumption rate. In normal human lung epithelial cells, treatment with TSA and Quisinostat increased expression of LSR and CLDN-2 and decreased that of CLDN-1 with or without TGF-β in 2D culture. Quisinostat but not TSA with TGF-β increased CLDN-7 expression in 2D culture. Both HDAC inhibitors prevented disruption of the epithelial barrier measured as the permeability of FD-4 induced by TGF-β in 2.5D culture. TSA and Quisinostat have potential for use in therapy for lung adenocarcinoma via changes in the expression of angulin-1/LSR and CLDN-2.

Ginsenosides repair UVB-induced skin barrier damage in BALB/c hairless mice and HaCaT keratinocytes

BackgroundGinsenosides (GS) have potential value as cosmetic additives for prevention of skin photoaging. However, their protective mechanisms against skin barrier damage and their active monomeric constituents are unknown. MethodsGS monomer types and their relative proportions were identified. A UVB-irradiated BALB/c hairless mouse model was used to assess protective effects of GS components on skin epidermal thickness and transepidermal water loss (TEWL). Skin barrier function, reflected by filaggrin (FLG), involucrin (IVL), claudin-1 (Cldn-1), and aquaporin 3 (AQP3) levels and MAPK phosphorylation patterns, were analyzed in UVB-irradiated hairless mice or HaCaT cells. ResultsTotal GS monomeric content detected by UPLC was 85.45% and was largely attributed to 17 main monomers that included Re (16.73%), Rd (13.36%), and Rg1 (13.38%). In hairless mice, GS ameliorated UVB-induced epidermal barrier dysfunction manifesting as increased epidermal thickness, increased TEWL, and decreased stratum corneum water content without weight change. Furthermore, GS treatment of UVB-irradiated mice restored protein expression levels and epidermal tissue distributions of FLG, IVL, Cldn-1, and AQP3, with consistent mRNA and protein expression results obtained in UVB-irradiated HaCaT cells (except for unchanging Cldn-1 expression). Mechanistically, GS inhibited JNK, p38, and ERK phosphorylation in UVB-irradiated HaCaT cells, with a mixture of Rg2, Rg3, Rk3, F2, Rd, and Rb3 providing the same protective MAPK pathway inhibition-associated upregulation of IVL and AQP3 expression as provided by intact GS treatment. ConclusionGS protection against UVB-irradiated skin barrier damage depends on activities of six ginsenoside monomeric constituents that inhibit the MAPK signaling pathway.

E74-like factor 3 suppresses microRNA-485-5p transcription to trigger growth and metastasis of ovarian cancer cells with the involvement of CLDN4/Wnt/β-catenin axis.

Ovarian cancer (OC) is one of the most prevailing gynecological malignancies with high mortality rate, while E74 like ETS transcription factor 3 (ELF3) is reported to be associated with tumorigenesis. This work aims to analyze the role of ELF3 on the suppression of miR-485-5p transcription in OC. Expression of ELF3 in OC and its correlation with overall survival were predicted on a bioinformation system GEPIA. Then, the level of ELF3 in OC tissues and cells and in normal ones was evaluated. Binding relationships between ELF3 and microRNA (miR)-485-5p, and between miR-485-5p and claudin-4 (CLND4) were predicted through Bioinformatics tools. Altered expression of ELF3, miR-485-5p and CLND4 was introduced alone or jointly to probe their influences on OC cell growth. ELF3 was suggested to be highly expressed in OC, which was linked to poor prognosis in patients. Abundant expression of ELF3 was identified in OC tissues and cell lines as relative to the normal ones. ELF3 inhibition suppressed growth and metastasis of OC cells. ELF3 transcriptionally suppressed miR-485-5p expression to further enhance CLDN4 expression. Overexpression of miR-485-5p led to similar trends as ELF3 inhibition did. Importantly, upregulation of CLDN4 was found to block the roles of ELF3 inhibition in OC cells. In addition, the Wnt/signaling pathway suppressed by miR-485-5p mimic was reactivated following CLDN4 overexpression. This study evidenced that ELF3 suppresses miR-485-5p transcription to enhance CLDN4 expression, leading to Wnt/β-catenin activation and promoting OC cell growth and metastasis. This work may provide new ideas for gene-based therapies for OC.

Direct assessment of individual skin barrier components by electrical impedance spectroscopy.

Expression of the tight junction proteins Cldn1 and 4 is altered in skin diseases such as atopic dermatitis, and Cldn1 deficiency affects skin barrier formation. Impedance spectroscopy (IS) has been proven to allow detection of alterations in the skin barrier but is currently unable to separate effects on viable epidermis (VE) and stratum corneum (SC). Effects of siRNA-mediated Cldn1 and 4 knockdown in reconstructed human epidermis (RHE) on VE and SC barrier function were investigated with Ussing chamber-based IS. Barrier components were sequentially altered, employing iron oxide nanoparticles and EGTA, to identify their contribution to the impedance spectrum. Resistance changes due to apically applied hyperosmolar electrolyte were used to identify barrier defects non-invasively. IS of RHE yielded two relaxation frequencies, representing the barrier properties of the SC (~1000Hz) and VE (~100Hz). As proof of concept, it was shown that the Cldn1 knockdown-induced resistance drop arises from the impairment of both SC and VE, indicated by a shift of both relaxation frequencies. Hyperosmolar electrolyte penetration allowed non-invasive detection of Cldn1 knockdown via time-dependent frequency shifts. The absence of Cldn4 knockdown-induced changes revealed the weaknesses of transepithelial electrical resistance analysis. In conclusion, the present technique allows to separately measure the barrier properties of SC and VE and further evaluate the Cldn1 and 4 knockdown impact on the skin barrier. As the measurement with agarose-embedded electrolyte allowed non-invasive identification of the Cldn1 knockdown, this opens the way to detailed in vivo skin barrier assessment.

87P Epigenetics of the epigenetics: Impact of Let-7a expression restoration in CD8+ cells of HER2+ breast cancer patients

HER-2+ breast cancer (BC) remains challenging as the use of Trastuzumab is ineffective in some cases. Let-7a is considered as a well-known tumor suppressor microRNA. It is epigenetically regulated by hypermethylation and suppressed by lncRNA, lin28 in many cancer such as BC. Here, we aim at restoring the expression of Let-7a through epigenetic modulation to study the impact of Let-7a on PD-1, PD-L1 and CLDN1 expression levels in CD8+ cells of HER2+ BC patients.

Lack of peptide YY signaling in mice disturbs gut microbiome composition in response to high-fat diet.

Peptide YY (PYY), produced by endocrine L cells in the gut, is known for its critical role in regulating gastrointestinal functions as well as satiety. However, how these processes are integrated with maintaining a healthy gut microbiome composition is unknown. Here, we show that lack of PYY in mice leads to distinct changes in gut microbiome composition that are diet‐dependent. While under chow diet only slight differences in gut microbiome composition could be observed, high‐fat diet (HFD) aggravated these differences. Specifically an increased abundance of the Bacteroidetes phylum with a corresponding decrease of the Firmicutes/Bacteroidetes ratio could be detected in Pyy‐knockout (KO) mice in response to HFD. Detailed analysis of the Bacteroidetes phylum further revealed that the Alistipes genus belonging to the Rikenellaceae family, the Parabacteroides belonging to the Tannerellaceae family, as well as Muribaculum were increased in Pyy‐KO mice. In order to investigate whether these changes are associated with changed markers of gut barrier and immunity, we analyzed the colonic expression of various pro‐inflammatory cytokines, as well as tight junction proteins and mucin 2, and identified increased mRNA expression of the tight junction proteins Cldn2 and Ocel1 in Pyy‐KO mice, while pro‐inflammatory cytokine expression was not significantly altered. Together these results highlight a critical gene‐environment interaction between diet and the gut microbiome and its impact on homeostasis of the intestinal epithelium under conditions of reduced PYY signaling which is commonly seen under obese conditions.

Differential effects of intense exercise and pollution on the airways in a murine model

BackgroundExercise-induced bronchoconstriction (EIB) is a transient airway narrowing, occurring during or shortly after intensive exercise. It is highly prevalent in non-asthmatic outdoor endurance athletes suggesting an important contribution of air pollution in the development of EIB. Therefore, more research is necessary to investigate the combination of exercise and pollutants on the airways.MethodsBalbc/ByJ mice were intranasally challenged 5 days a week for 3 weeks with saline or 0.2 mg/ml diesel exhaust particles (DEP), prior to a daily incremental running session or non-exercise session. Once a week, the early ventilatory response was measured and lung function was determined at day 24. Airway inflammation and cytokine levels were evaluated in bronchoalveolar lavage fluid. Furthermore, innate lymphoid cells, dendritic cells and tight junction mRNA expression were determined in lung tissue.ResultsSubmaximal exercise resulted in acute alterations of the breathing pattern and significantly improved FEV0.1 at day 24. DEP exposure induced neutrophilic airway inflammation, accompanied with increased percentages of CD11b+ DC in lung tissue and pro-inflammatory cytokines, such as IL-13, MCP-1, GM-CSF and KC. Occludin and claudin-1(Cldn-1) expression were respectively increased and decreased by DEP exposure. Whereas, exercise increased Cldn-3 and Cldn-18 expression. Combining exercise and DEP exposure resulted in significantly increased SP-D levels in the airways.ConclusionDEP exposure induced typical airway neutrophilia, DC recruitment and pro-inflammatory cytokine production. Whereas, intensive exercise induced changes of the breathing pattern. The combination of both triggers resulted in a dysregulation of tight junction expression, suggesting that intensive exercise in polluted environments can induce important changes in the airway physiology and integrity.

Comprehensive analysis of expression and prognostic value of the claudin family in human breast cancer

Claudins (CLDN) are structural components of tight junctions that function in paracellular transport and maintain the epithelial barrier function. Altered expression and distribution of members of the claudin family have been implicated in several cancers including breast cancer (BC). We performed a comprehensive analysis of the expression and prognostic value of claudins in BC using various online databases. Compared with normal tissues, CLDN3, 4, 6, 7, 9, and 14 were upregulated in BC tissues, whereas CLDN2, 5, 8, 10, 11, 15, 19, and 20 were downregulated. A high expression of CLDN2, 5, 6, 9, 10, 11, and 14–20 was associated with better relapse-free survival (RFS), whereas a high CLDN3 expression correlated with poor RFS. In addition, a high expression of CLDN3, 4, 14, and 20 was associated with poor overall survival (OS), whereas that of CLDN5 and CLDN11 was linked to a better OS. Although METABRIC and TCGA datasets revealed 22% and 27% gene alterations, respectively, in the members of the claudin family, these were not associated with survival. These findings suggest CLDN3, 5, and 11 could be promising therapeutic targets for patients with BC.

Sex-dependent modulation of immune development in mice by secretory IgA–coated Lactobacillus reuteri isolated from breast milk

Lactobacilli, commonly present in human breast milk, appear to colonize the neonatal gut and provide protection to infants against various infections, thereby promoting immune development. This study examined the potential probiotic role of breast milk-derived Lactobacillus reuteri FN041 in immune development in mice. The FN041 were gavaged either to BALB/c dams (n = 6/group) during the lactation period or to their offspring (n = 6/sex per intervention) after weaning separately (cointervention). All interventions induced increased intestinal barriers in 5-wk-old offspring, especially in the females. Immunoglobulin A plasmocytes in ileal tissue and secretory IgA (sIgA) in ileal contents increased in all 5-wk-old offspring of cointervention. The activation of mRNA expression of 17 genes was sex-dependent, especially in 5-wk-old offspring. Broader genes were regulated in female mice. The effect of cointervention on the Shannon index of total microbiota is sex-related. The Shannon index of sIgA-coated microbiota increased in both sexes. The sIgA-coated microbiota showed intergroup differences according to β diversity, especially in female mice that showed an increase in Bifidobacterium of Actinobacteria. The sIgA-coated Bifidobacterium was positively correlated with mRNA expression of Tlr9. The sIgA-coated Lactobacillus in male offspring was negatively correlated with mRNA expression of Cldn2. In conclusion, L. reuteri FN041 promoted the production of intestinal sIgA and the expression of genes related to antimicrobial peptides in the offspring and enhanced the function of the mucosal barrier, depending on sex and treatment manner.

Corticosteroid enhances epithelial barrier function in intestinal organoids derived from patients with Crohn\u2019s disease

Corticosteroids (CS), first-line therapeutics for Crohn’s disease (CD) with moderate or severe disease activity, were found to restore intestinal permeability in CD patients, whereas the underlying molecular events are still largely unknown. This study aimed to investigate the effect and mechanisms of CS prednisolone on epithelial barrier using CD patient-derived intestinal organoids. 3D intestinal organoids were generated from colon biopsies of inactive CD patients. To mimic the inflammatory microenvironment, a mixture of cytokines containing TNF-α, IFN-γ, and IL-1β were added to the organoid culture with or without pre-incubation of prednisolone or mifepristone. Epithelial permeability of the organoids was assessed by FITC-D4 flux from the basal to luminal compartment using confocal microscopy. Expression of junctional components were analyzed by qRT-PCR, immunofluorescence staining, and western blot. Activity of signaling pathways were analyzed using western blot. Exposure of the cytokines significantly disrupted epithelial barrier of the intestinal organoids, which was partially restored by prednisolone. On the molecular level, the cytokine mixture resulted in a significant reduction in E-cadherin and ILDR-1, an increase in CLDN-2, MLCK, and STAT1 phosphorylation, whereas prednisolone ameliorated the abovementioned effects induced by the cytokine mixture. This study demonstrates that prednisolone confers a direct effect in tightening the epithelial barrier, identifies novel junctional targets regulated by prednisolone, and underscores intestinal barrier restoration as a potential mechanism that contributes to the clinical efficacy of prednisolone in CD patients.Key messagesPrednisolone confers a direct preventive effect against cytokine-induced barrier dysfunction.Prednisolone regulates the expression of CLDN-2, E-cadherin, and ILDR-1.The effect of prednisolone is GR-, MLCK-, and STAT1-dependent.

Phase II study of zolbetuximab plus pembrolizumab in claudin 18.2: Positive locally advanced or metastatic gastric or gastroesophageal junction adenocarcinoma (G/GEJ)—ILUSTRO Cohort 3.

TPS260 Background: Five-year survival with advanced G/GEJ is poor, and limited biomarkers exist to inform optimal treatment selection. Pembrolizumab, an anti–programmed death-1 receptor (PD-1) antibody, is approved for advanced/metastatic PD-ligand 1–positive (PD-L1+) G/GEJ that progressed after ≥2 lines of therapy. The transmembrane tight junction protein claudin 18.2 (CLDN18.2) is normally confined to gastric mucosa but is often overexpressed in G/GEJ with roughly one-third of patients (pts) having high expression (≥75%). Zolbetuximab, a chimeric IgG1 monoclonal antibody, binds to CLDN18.2 and mediates cancer cell death through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity. Phase 2 (NCT01630083) results showed prolonged survival with zolbetuximab + epirubicin, oxaliplatin, and capecitabine (EOX) vs EOX alone in G/GEJ. Results of nonclinical studies showed enhanced antitumor activity with zolbetuximab + anti-murine PD-1 antibody, and it was hypothesized that a combination with pembrolizumab (new Cohort 3) might augment ADCC and antitumor immune response in CLDN18.2 overexpressing G/GEJ. Methods: This phase 2 open-label study (NCT03505320) will enroll ~112 adult pts from 22 sites in 5 countries into 3 cohorts; this abstract describes Cohort 3 (~62 pts). Key eligibility criteria are advanced/metastatic G/GEJ, measurable disease (RECIST v1.1), adequate organ function and performance status, and high/intermediate (Cohort 3A) or high (Cohort 3B) expression of CLDN18.2. Central testing of tumor tissue will determine CLDN18.2 expression; pts are considered CLDN18.2 positive (CLDN18.2+) if ≥75% (high) or ≥50% to < 75% (intermediate) of tumor cells demonstrate moderate-to-strong membranous IHC staining. Patients in Cohort 3B are required to be PD-L1+, defined as a combined positive score ≥1 (IHC staining per the Dako 22C3 PD-L1 assay). Patients will receive zolbetuximab + pembrolizumab in the third/later line in Cohort 3A and third line in Cohort 3B. In Cohort 3A (safety cohort), zolbetuximab will be administered at a loading dose of 800 mg/m2 IV on Day 1 Cycle 1 followed by 600 mg/m2 IV every 3 weeks; a reduction from 600 mg/m2 every 3 weeks is permitted. Pembrolizumab 200 mg IV will be administered on Day 1 of each 21-day cycle. Cohort 3B (expansion cohort) zolbetuximab dose is determined from results of Cohort 3A. Imaging will occur every 6 weeks for 24 weeks and every 12 weeks thereafter. The primary endpoint is objective response rate; additional endpoints include duration of response, disease control rate, and progression-free survival by independent review committee and investigator assessment. Pharmacokinetics, safety/tolerability, quality of life, and immunogenicity will be assessed. The study is currently recruiting pts. Clinical trial information: NCT03505320.