Expression Of CLDN4 Research Articles

Abstract LB284: CLAUDIN-1 targeting antibody ALE.C04 drives single activity and restores anti-PD1 efficacy in solid tumors

Abstract Claudin 1 (CLDN1) is a protein confined within the normal epithelial tight junctions of different tissues. Upon malignant transformation, CLDN1 is overxpressed and epitopes become exposed outside the tight junctions (non-Junctional CLDN1). ALE.C04 is a highly specific humanized monoclonal antibody that recognizes a unique CLDN1 exposed epitope in different solid tumors. Herein we show that non-Junctional CLDN1 (NJ-CLDN1) is frequently overexpressed in solid tumors. Pre-clinically, ALE.C04 drives tumor growth inhibition in different CDXs and PDX in vivo tumor models. Notably, CLDN1 expression on tumor cells positively correlates with T cell exclusion, a mechanism described to hinder the efficacy of Checkpoint inhibitors (CPIs). On this line, the overexpression of Cldn1 in mouse tumor cells promoted T-cell exclusion and resistance to anti-PD1 treatment. Importantly, ALE.C04 restored both T-cell infiltration and anti-PD1 efficacy in tumors. Mechanistically, NJ-CLDN1 interacts with different components involved in extracellular matrix remodeling, thus establishing a physical barrier that exclude immune cells from the tumor nest. ALE.C04 perturbs the interface between CLDN1+ tumor cells and the stroma, thus restoring immune cell infiltration. Our pre-clinical data showed that: i) NJ-CLDN1 is a novel and druggable target in solid tumors ii) ALE.C04 is a selective anti-CLDN1 antibody capable of driving anti-tumor activity as both single agent and in combination with anti-PD1. Citation Format: Alberto Toso, Geoffrey Teixeira, Tina Zimmermann, Stefanie Gill Gill, Daniela Schmitter, Markus Meyer, Marion Muller, Laurent Mailly, Thomas Baumert, Luigi Manenti, Roberto Iacone. CLAUDIN-1 targeting antibody ALE.C04 drives single activity and restores anti-PD1 efficacy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB284.

Effects of dietary polyherbal mixtures on growth performance, antioxidant capacity, immune function and jejunal health of yellow-feathered broilers

This study aimed to investigate the effects of polyherbal mixtures (PHM) on growth performance, antioxidant capacities, immune function, and intestinal health in yellow-feathered broilers. PHM is composed of five traditional Chinese medicine herbs (Portulaca oleracea L., Radix Sophora flavescens, Thalictrum glandulosissimum, Terra flava usta, and Pogostemon cablin). A total of 270 one-day-old yellow-feathered broilers were randomly allotted into 3 treatments for a 42-d feeding trial, each with 6 replicates of 15 birds. The dietary treatments consisted of a basal diet (CON), a basal diet supplemented with 50 mg/kg chlortetracycline (CTC), and a basal diet supplemented with 1000 mg/kg PHM. The results showed that dietary PHM supplementation increased body weight, ADG, and decreased F/G compared to the CON. PHM also increased spleen index and mRNA expression of IL-4 (d 21), and thymus index, serum IgA (d 42) and IgG, IL-4 and sIgA in jejunal mucosa (d 21 and 42), but decreased serum IFN-γ and mRNA expression of IFN-γ (d 21 and 42). In addition, PHM increased serum SOD, GSH-Px (d 21 and 42) and T-AOC (d 42), but decreased the content of serum MDA (d 21), the up-regulated mRNA expression of GSH-Px, CAT (d 21), SOD and CAT (d 42). Furthermore, PHM also improved the intestinal epithelial barrier indicators by the up-regulated mRNA expression of CLDN-1, OCLN (d 21 and 42) and ZO-1 (d 21), and the increased of villus height and villus height to crypt depth in jejunum (d 42). The high-throughput sequencing results showed that dietary PHM supplementation increased the alpha diversity and relative abundance of Oscillospira and Ruminococcus (d 21) and Lactobacillus (d 42), whereas decreasing that of Enterococcus (d 21) compared with CON. PICRUSt analysis revealed that metabolic pathways of carbohydrate, energy, lipid, cofactors, and vitamins were significantly enriched in the PHM group. Spearman's correlation analysis revealed that the genera Lactobacillus, Enterococcus, Ruminococcus, Oscillospira, and Faecalibacterium were related to growth performance, intestinal integrity, immune-related factors, antioxidant indices, and tight junction proteins. In conclusion, the results indicated that dietary PHM supplementation improved growth performance and immune status of yellow-feathered broilers by enhancing antioxidant capacities, barrier function, and modulated jejunal microbial communities. PHM used in our study has the potential to replace prophylactic antibiotic use in poultry production systems.

Agaricus subrufescens fermented rye affects the development of intestinal microbiota, local intestinal and innate immunity in suckling-to-nursery pigs

BackgroundAgaricus subrufescens is considered as one of the most important culinary-medicinal mushrooms around the world. It has been widely suggested to be used for the development of functional food ingredients to promote human health ascribed to the various properties (e.g., anti-inflammatory, antioxidant, and immunomodulatory activities). In this context, the interest in A. subrufescens based feed ingredients as alternatives for antibiotics has also been fuelled during an era of reduced/banned antibiotics use. This study aimed to investigate the effects of a fermented feed additive -rye overgrown with mycelium (ROM) of A. subrufescens—on pig intestinal microbiota, mucosal gene expression and local and systemic immunity during early life. Piglets received ROM or a tap water placebo (Ctrl) perorally every other day from day 2 after birth until 2 weeks post-weaning. Eight animals per treatment were euthanized and dissected on days 27, 44 and 70.ResultsThe results showed ROM piglets had a lower inter-individual variation of faecal microbiota composition before weaning and a lower relative abundance of proteobacterial genera in jejunum (Undibacterium and Solobacterium) and caecum (Intestinibacter and Succinivibrionaceae_UCG_001) on day 70, as compared to Ctrl piglets. ROM supplementation also influenced gut mucosal gene expression in both ileum and caecum on day 44. In ileum, ROM pigs showed increased expression of TJP1/ZO1 but decreased expression of CLDN3, CLDN5 and MUC2 than Ctrl pigs. Genes involved in TLR signalling (e.g., TICAM2, IRAK4 and LY96) were more expressed but MYD88 and TOLLIP were less expressed in ROM pigs than Ctrl animals. NOS2 and HIF1A involved in redox signalling were either decreased or increased in ROM pigs, respectively. In caecum, differentially expressed genes between two groups were mainly shown as increased expression (e.g., MUC2, PDGFRB, TOLLIP, TNFAIP3 and MYD88) in ROM pigs. Moreover, ROM animals showed higher NK cell activation in blood and enhanced IL-10 production in ex vivo stimulated MLN cells before weaning.ConclusionsCollectively, these results suggest that ROM supplementation in early life modulates gut microbiota and (local) immune system development. Consequently, ROM supplementation may contribute to improving health of pigs during the weaning transition period and reducing antibiotics use.

CLDN4 as a Novel Diagnostic and Prognostic Biomarker and Its Association with Immune Infiltrates in Ovarian Cancer.

Ovarian cancer (OC) is the seventh most prevalent type of cancer in women and the second most common cause of cancer-related deaths in women worldwide. Because of the high rates of relapse, there is an immediate and pressing need for the discovery of innovative sensitive biomarkers for OC patients. Using TCGA and GSE26712 datasets, we were able to identify 17 survival-related DEGs in OC that had differential expression. CLDN4 was the gene that caught our attention the most out of the 17 important genes since its expression was much higher in OC samples than in nontumor samples. The findings of the ROC assays then confirmed the diagnostic utility of the test in screening OC specimens to differentiate them from nontumor specimens. Patients with high CLDN4 expression predicted a shorter overall survival (OS) and disease-specific survival (DSS) than those with low CLDN4 expression, according to clinical research. Patients with low CLDN4 expression predicted longer OS and DSS. Analysis using both univariate and multivariate techniques revealed that CLDN4 expression was an independent factor associated with a poor prognosis for OS and DSS. Based on multivariate analysis, the C-indexes and calibration plots of the nomogram suggested an effective predictive performance for OC patients. After that, we investigated whether or not there was a link between the infiltration of immune cells and the expression of the CLDN4 gene. We found that the expression of CLDN4 was positively associated with Th17 cells, NK CD56bright cells, while negatively associated with Th2 cells, pDC, and T helper cells. In the end, we carried out RT-PCR on our cohort and confirmed that the level of CLDN4 expression was noticeably elevated in OC specimens in comparison to nontumor tissues. The diagnostic usefulness of CLDN4 expression for OC was also validated by the findings of ROC tests. Thus, our findings revealed that CLDN4 may serve as a predictive biomarker in OC to assess both the clinical outcome of OC patients and the level of immune infiltration.

Proteomic Analysis Reveals Changes in Tight Junctions in the Small Intestinal Epithelium of Mice Fed a High-Fat Diet.

The impact of a high-fat diet (HFD) on intestinal permeability has been well established. When bacteria and their metabolites from the intestinal tract flow into the portal vein, inflammation in the liver is triggered. However, the exact mechanism behind the development of a leaky gut caused by an HFD is unclear. In this study, we investigated the mechanism underlying the leaky gut related to an HFD. C57BL/6J mice were fed an HFD or control diet for 24 weeks, and their small intestine epithelial cells (IECs) were analyzed using deep quantitative proteomics. A significant increase in fat accumulation in the liver and a trend toward increased intestinal permeability were observed in the HFD group compared to the control group. Proteomics analysis of the upper small intestine epithelial cells identified 3684 proteins, of which 1032 were differentially expressed proteins (DEPs). Functional analysis of DEPs showed significant enrichment of proteins related to endocytosis, protein transport, and tight junctions (TJ). Expression of Cldn7 was inversely correlated with intestinal barrier function and strongly correlated with that of Epcam. This study will make important foundational contributions by providing a comprehensive depiction of protein expression in IECs affected by HFD, including an indication that the Epcam/Cldn7 complex plays a role in leaky gut.

Autism-like behavior of murine offspring induced by prenatal exposure to progestin is associated with gastrointestinal dysfunction due to claudin-1 suppression.

Autism spectrum disorders (ASD) are associated with the contribution of many prenatal risk factors; in particular, the sex hormone progestin and vitamin D receptor (VDR) are associated with gastrointestinal (GI) symptoms in ASD development, although the related mechanism remains unclear. We investigated the possible role and mechanism of progestin 17-hydroxyprogesterone caproate (17-OHPC) exposure-induced GI dysfunction and autism-like behaviours (ALB) in mouse offspring. An intestine-specific VDR-deficient mouse model was established for prenatal treatment, while transplantation of haematopoietic stem cells (HSCT) with related gene manipulation was used for postnatal treatment for 17-OHPC exposure-induced GI dysfunction and ALB in mouse offspring. The in vivo mouse experiments found that VDR deficiency mimics prenatal 17-OHPC exposure-mediated GI dysfunction, but has no effect on 17-OHPC-mediated autism-like behaviours (ALB) in mouse offspring. Furthermore, prenatal 17-OHPC exposure induces CLDN1 suppression in intestine epithelial cells, and transplantation of HSCT with CLDN1 expression ameliorates prenatal 17-OHPC exposure-mediated GI dysfunction, but has no effect on 17-OHPC-mediated ALB in offspring. In conclusion, prenatal 17-OHPC exposure triggers GI dysfunction in autism-like mouse offspring via CLDN1 suppression, providing a possible explanation for the involvement of CLDN1 and VDR in prenatal 17-OHPC exposure-mediated GI dysfunction with ASD.

Visfatin increases the invasive potential of ovarian granulosa tumor spheroids by reprogramming glucose metabolism.

Visfatin is an adipokine with nicotinamide phosphoribosyltransferase (NAMPT) activity, the concentration of which is higher in ascitic fluid than in serum, and is associated with ovarian cancer peritoneal dissemination. Furthermore, potentially important effects of visfatin on glucose metabolism have been previously reported. However, the mechanism underlying the effects of visfatin on ovarian cancer cell invasion, and whether this involves altered glucose metabolism, has not been elucidated. Here we tested the hypothesis that visfatin, which can reprogram cancer metabolism, promotes invasion by ovarian cancer spheroids. Visfatin increased glucose transporter (GLUT)1 expression and glucose uptake in adult granulosa cell tumor-derived spheroid cells (KGN), and also increased the activities of hexokinase 2 and lactate dehydrogenase. We showed a visfatin-induced increase in glycolysis in KGN cells. Moreover, visfatin increased the potential invasiveness of KGN spheroid cells by upregulating MMP2 (matrix metalloproteinase 2) and downregulating CLDN3 and CLDN4 (claudin 3 and 4) gene expression. Interestingly, an inhibitor of GLUT1 (STF-31) and lactate dehydrogenase (LDHA) abolished the stimulatory effect of visfatin on the potential invasiveness of KGN cells. More importantly, silencing expression of the NAMPT gene in KGN cells demonstrated its important effect on glycolysis and invasiveness in adult granulosa cell tumor cells. In summary, visfatin appears to increase adult granulosa cell tumor invasiveness through effects on glucose metabolism, and to be an important regulator of glucose metabolism in these cells.

Adverse effect of assisted reproductive technology-related hyperoestrogensim on the secretion and absorption of uterine fluid in superovulating mice during the peri-implantation period.

This study aimed to investigate the potential mechanism of hyperoestrogensim elicited by ovulation induction affects endometrial receptivity and leads to embryo implantation abnormality or failure. Establishment of ovulation induction mouse model. Changes in mouse body weight, ovarian weight, serum E2 level and oestrous cycle were observed. During the peri-implantation period, morphological changes in the mouse uterus and implantation sites and the localization and protein levels of oestrogen receptors ERα and ERβ, the tight junction factors CLDN3 and OCLN, the aquaporins AQP3, AQP4 and AQP8, and the sodium channel proteins SCNN1α, SCNN1β and SCNN1γ were observed. The expression and cellular localization of ERα, CLDN3, AQP8 and SCNN1 β in RL95-2 cell line were also detected by western blotting and immunofluorescence. Ovarian and body weights were significantly higher in the 5 IU and 10 IU groups than in the CON group. The E2 level was significantly higher in the 10 IU group than in the CON group. The mice in the 10 IU group had a disordered oestrous cycle and were in oestrus for a long time. At 5.5 dpc, significantly fewer implantation sites were observed in the 10 IU group than in the CON (p<0.001) and 5 IU (p<0.05) groups. The probability of abnormal implantation and abortion was higher in the 10 IU group than in the CON and 5 IU groups. CLDN3, OCLN, AQP8 and SCNN1β in the mouse endometrium were localized on the luminal epithelium and glandular epithelium and expression levels were lower in the 10 IU group than in the CON group. The protein expression level of ERα was increased by 50% in the 10 IU group compared to the CON group. The expressions of CLDN3, AQP8, SCNN1β in RL95-2 cell line were significantly depressed by the superphysiological E2, ERα agonist or ERβ agonist, which could be reversed by the oestrogen receptor antagonist. ART-induced hyperoestrogenism reduces CLDN3, AQP8 and SCNN1β expression through ERα, thereby destroying tight junctions and water and sodium channels in the endometrial cavity epithelium, which may cause abnormal implantation due to abnormal uterine fluid secretion and absorption.

Antioxidant Effect of Jatropha dioica Extract on Immunoreactivity of Claudin 2 in the Kidney of Rats with Induced Diabetes

Introduction Diabetes is related to different complications, such as nephropathy. In kidneys, the tight junctions (TJ) regulate the paracellular transport of solutes and water. The claudins are the most significant component in the TJ and are expressed throughout the epithelium and modulated permeability. Objective To evaluate the antioxidant effect of Jatropha dioica (JD) extract and claudin 2 (CLDN 2) in the kidney of rats with induced diabetes. Methods It is an experimental, explanatory, prospective, longitudinal, and analytical study. The effect of hydro-alcoholic extract from JD root on the presence of CLDN 2 in rats with induced diabetes was evaluated. The experiment lasting 21 days, treated 40 rats with and without induced diabetes by an intraperitoneal (IP) injection of alloxan. The study groups were exposed to JD extract at 25, 50, and 100 mg/kg. There was no significant morphological difference in the renal tissue morphology of the treated and control groups. Conclusion These results demonstrate that JD extract at 50 mg/kg ameliorated the tubular damage induced by diabetes, and the mechanism might be related to the CLDN2 expression. Finally, JD could also increase total antioxidant capacity to protect the kidney from oxidative stress.

The Role of Tight Junctions in Atopic Dermatitis: A Systematic Review.

Tight junctions are transmembrane proteins that regulate the permeability of water, solutes including ions, and water-soluble molecules. The objective of this systematic review is to focus on the current knowledge regarding the role of tight junctions in atopic dermatitis and the possible impact on their therapeutic potential. A literature search was performed in PubMed, Google Scholar, and Cochrane library between 2009 and 2022. After evaluation of the literature and taking into consideration their content, 55 articles were finally included. TJs' role in atopic dermatitis extends from a microscopic scale to having macroscopic effects, such as increased susceptibility to pathogens and infections and worsening of atopic dermatitis features. Impaired TJ barrier function and skin permeability in AD lesions is correlated with cldn-1 levels. Th2 inflammation inhibits the expression of cldn-1 and cldn-23. Scratching has also been reported to decrease cldn-1 expression. Dysfunctional TJs' interaction with Langerhans cells could increase allergen penetration. Susceptibility to cutaneous infections in AD patients could also be affected by TJ cohesion. Dysfunction of TJs and their components, especially claudins, have a significant role in the pathogenesis and vicious circle of inflammation in AD. Discovering more basic science data regarding TJ functionality may be the key for the use of specific/targeted therapies in order to improve epidermal barrier function in AD.

The role of claudin 18.2 and HER-2 in pancreatic cancer outcomes.

To examine the prognostic value of claudin 18.2 (CLDN18.2) and human epidermal growth factor receptor-2 (HER-2) expression in patients with resected pancreatic ductal adenocarcinoma (PDAC). This study enrolled patients who underwent surgery and were diagnosed with PDAC at Suleyman Demirel University Hospital, Turkey between 2015 and 2019. Sixty-eight patients with resected PDAC treated at a medical oncology clinic were assessed. All patients were over the age of 18 years, underwent follow-up and treatment in our unit, and had pathology slides that we could access. Clinicopathological data were obtained from medical files, including the patients' age, sex, pathological parameters, and clinical stage according to the Eighth International Union against Cancer/American Joint Committee on Cancer. Patient survival and the period from the date of diagnosis to death were assessed in the follow-up data. There was no statistically significant difference between CLDN18.2 and HER-2 expression scores for samples and patient clinicopathological characteristics. No HER-2 expression scores of ≥2 were found in the samples. Only 25% (n = 17) of the samples had HER-2 expression scores of +1. CLDN18.2 expression was detected in 54.4% (n = 37) of the patient samples. CLDN18.2 expression scores were +1 in 30.8% (n = 21) of the patient samples, +2 in 16.2% (n = 11), and +3 in 7.4% (n = 5). When CLDN18.2 and HER-2 expression were compared, a statistically significant difference and moderate positive correlation were observed. No significant relationship between HER-2 expression and survival was observed in the survival analysis of PDAC patients; however, high CLDN18.2 expression was related to longer overall survival. Our study is the third to research CLDN18.2 expression in PDAC. HER-2 expression is low and CLDN18.2 expression is high in patients with PDAC. HER-2 expression is not related to overall survival but CLDN18.2 is related and may be used as a prognostic marker in patients with PDAC.

Ozoile Reduces the LPS-Induced Inflammatory Response in Colonic Epithelial Cells and THP-1 Monocytes

Inappropriate activation of immune functions in intestinal epithelial cells can lead to inflammation that is characterized also by infiltration into intestinal tissue of monocytes/macrophages. Current therapies for intestinal inflammation include anti-inflammatory, immunosuppressive and biological drugs. Ozoile (stable ozonides) has been reported to exert anti-inflammatory effects. However, ozonated oil has been used mainly for topical applications and no data are available about its effects on intestinal cells or immune cells. In this study, we evaluated Ozoile effects on human HT-29 colonic cells and THP-1 monocytic cells stimulated with LPS to induce inflammation. HT-29 and THP-1 cells were treated with LPS in the presence/absence of Ozoile for 4 h. Biomarkers of inflammation, some members of tight junctions and the adhesion molecule ICAM were assessed by qRT-PCR. Protein expression was analyzed by Western blotting. The release of TNF-α and IL-1β was measured by ELISA. In HT-29, Ozoile inhibited LPS-induced expression of TNF-α, IL-1β, ZO-1, CLDN1, NOS2 and MMP-2 and increased the expression of Nrf2 and SOD2 antioxidant proteins. In THP-1 cells, the LPS induction of TNF-α, IL-1β and ICAM was counteracted by Ozoile treatment. Our in vitro results demonstrate the effectiveness of Ozoile in reducing the inflammatory response in intestinal and monocytic cells. Further in vivo studies are necessary to confirm its possible use for intestinal inflammatory conditions.

Laxative effect of Wenyang Yiqi Decoction on loperamide-induced astriction model mice.

Although a common disease, astriction is difficult to treat and severely affects quality of life. Wenyang Yiqi Decoction (WYD) is a kind of traditional Chinese medicine (TCM) that is used to treat astriction; however, the mechanism remains unclear. Therefore, this work assessed the laxative effect of WYD on loperamide-induced astriction (LIA) model mice. We replicated a constipation model in mice and detected changes in fecal parameters such as feces quantity and water content, intestinal transit function, and histopathological changes in the constipated mice. After five days of WYD intervention, mouse tissues were taken out for detection. We also measured the levels of gastrin (Gas), substance P (SP), acetylcholinesterase (AChE), and vasoactive intestinal peptide (VIP) in the mice's serum. Additionally, we used quantitative real-time polymerase chain reaction (qRT-PCR) and Western Blot to detect c-Kit and stem cell factor (SCF), and examined the effects of WYD on the tight junction (TJ) proteins occludin (Ocln), zonula occludens-1 (ZO-1), and claudin-1 (Cldn-1) in the mice's intestines. Through histopathological changes, we observed less destruction of epithelial cells and greater integrity of goblet and epithelial cells in WYD-treated mice than in mice in the loperamide group. qRT-PCR and western blot analysis of c-Kit and SCF showed that WYD could boost the levels of c-Kit and SCF. The qRT-PCR and immunohistochemical (IHC) analyses of enteral tight occludin (Ocln), occludenas-1 (ZO-1), and cldin-1 (Cldn-1) showed that WYD could boost the level of ZO-1 and decrease the level of Cldn-1. The study also investigated the effect of WYD treatment on the enteral barrier function of astriction model mice and found that the TJ proteins (ZO-1, Cldn-1) in the colon of the astriction model mice had significant changes compared to the normal group, and WYD intervention was found to increase the expression of ZO-1, and decrease the expression of Cldn-1. WYD alleviates LIA by regulating enteral hormones, boosting the number of interstitial cells of Cajal (ICCs), or adjusting enteral block action.

A phase 1, open-label, dose escalation and expansion, multicenter study of claudin 18.2-targeted chimeric antigen receptor T-cells in patients with unresectable, locally advanced, or metastatic gastric, gastroesophageal junction, esophageal, or pancreatic adenocarcinoma.

TPS480 Background: Locally advanced and metastatic gastric, esophageal, gastroesophageal junction, and pancreatic adenocarcinomas (GC, EC, GEJC, and PC) have poor overall survival, and more effective therapies are needed. Claudin 18.2 (CLDN18.2), a tight junction protein, is commonly expressed in these cancers. Monoclonal antibody and CAR-T cell therapies targeting CLDN18.2 have shown promising antitumor activity (Türeci O et al, 2019; Qi C et al, 2022). This study aims to assess the safety and preliminary efficacy of a CLDN18.2 targeted CAR-T, LB1908, in subjects with GC, EC, GEJC, and PC. Methods: This is a phase 1, open-label, multicenter, dose-escalation and expansion study of LB1908 that aims to identify the recommended phase 2 dose (NCT05539430; open to enrollment early 2023). During prescreening, that may occur as early as at initial diagnosis, we will screen subjects for tumor expression of CLDN18.2 by IHC of ≥ 1+ in ≥ 50% of tumor cells. Inclusion criteria include: must be ≥18 and ≤75 years old; histologically/cytologically confirmed unresectable, locally advanced, or metastatic adenocarcinoma of the GC, EC, GEJC, and PC; progressed on at least standard 1st line therapy; ECOG PS 0 or 1; life expectancy &gt; 4 months per investigator judgment; and adequate organ function per protocol. The study has 2 parts. In Part A (dose escalation), 12 to 21 subjects with GC, EC, or GEJC will be enrolled in up to 3 planned dose level cohorts (0.5X106, 1.5X106, and 3.0X106 CAR+ viable T cells/kg), to determine the recommended dose for expansion (RDE). In Part B (dose expansion) the RDE will be administered to up to 35 subjects with GC, EC, and GEJC in one expansion cohort and PC in another cohort (n=11-17 for GC/EC/GEJ, to total 23 subjects when pooling with RDE-treated subjects from part A; n=18 for PC). LB1908 will be manufactured from autologous T cells collected via PMBC apheresis. Subjects may receive optional bridging therapy followed by lymphodepleting chemotherapy with fludarabine 30 mg/m2/day and cyclophosphamide 300 mg/m2/day for 3 days. One infusion of LB1908 will then be administered, and subjects will be followed post-infusion for safety, laboratory, and disease assessments. Primary endpoints are incidence, duration, and severity of AEs and laboratory abnormalities (Parts A and B); and frequency of DLTs at each dose level (Part A). Secondary and exploratory endpoints include: objective response rate, disease control rate, duration of response, progression-free survival, characterization of PK by CAR-positive T cell counts, CAR transgene level in blood, effusions, and target tissues, and evaluation of immunogenicity by presence of anti-LB1908 antibodies. Acknowledgments: This study is funded by Legend Biotech USA Inc. Clinical trial information: NCT05539430 .

Acute Stress Regulates Sex-Related Molecular Responses in the Human Jejunal Mucosa: Implications for Irritable Bowel Syndrome.

Irritable bowel syndrome (IBS) is a prevalent gastrointestinal disorder linked to intestinal barrier dysfunction and life stress. We have previously reported that female sex per se determines an increased susceptibility to intestinal barrier dysfunction after cold pain stress (CPS). We aimed to identify sex-related molecular differences in response to CPS in healthy subjects to understand the origin of sex bias predominance in IBS. In 13 healthy males and 21 females, two consecutive jejunal biopsies were obtained using Watson's capsule, at baseline, and ninety minutes after CPS. Total mucosal RNA and protein were isolated from jejunal biopsies. Expression of genes related to epithelial barrier (CLDN1, CLDN2, OCLN, ZO-1, and ZO-3), mast cell (MC) activation (TPSAB1, SERPINA1), and the glucocorticoid receptor (NR3C1) were analyzed using RT-qPCR. NR3C1, ZO-1 and OCLN protein expression were evaluated through immunohistochemistry and western blot, and mucosal inflammation through MC, lymphocyte, and eosinophil numbering. Autonomic, hormonal, and psychological responses to CPS were monitored. We found an increase in jejunal MCs, a reduced CLDN1 and OCLN expression, and an increased CLDN2 and SERPINA1 expression 90 min after CPS. We also found a significant decrease in ZO-1, OCLN, and NR3C1 gene expression, and a decrease in OCLN protein expression only in females, when compared to males. CPS induced a significant increase in blood pressure, plasma cortisol and ACTH, and subjective stress perception in all participants. Specific and independent sex-related molecular responses in epithelial barrier regulation are unraveled by acute stress in the jejunum of healthy subjects and may partially explain female predominance in IBS.

Impacts of sodium butyrate on intestinal mucosal barrier and intestinal microbial community in a weaned piglet model.

Butyrate is thought to enhance intestinal mucosal homeostasis, but the detailed mechanism remains unclear. Therefore, further investigation on the mechanism of butyrate regulation of intestinal mucosal homeostasis was performed. This study used weaned piglets with similar intestinal metabolic function to humans as a research model. The dietary supplemented 0.2% sodium butyrate group (0.2% S) and negative control group (CON) were established to detect the effects of butyrate on growth performance, intestinal tissue morphology, mucosal barrier function, and intestinal microbial community structure in weaned piglets. There was an increase in average daily gain (ADG) during three different experimental periods and a reduction in average daily feed intake (ADFI) and feed-to-gain ratio (F:G) during days 1-35 and days 15-35 in 0.2% S compared with CON (P > 0.05). Furthermore, villus height in the ileum and duodenum was increased, and crypt depths in the colon and jejunum were reduced in both groups (P < 0.05). Moreover, the ratio of villus height and crypt depth (V/C) in 0.2% S both in the ileum and jejunum was significantly increased (P < 0.05) compared with CON. The relative mRNA expression of PKC, MUC1, CLDN1, and ITGB1 was upregulated in the ileum of 0.2% S compared with CON (P < 0.05). The digesta samples of 0.2% S, both in the ileum (P < 0.05) and colon, contained greater intestinal bacterial abundance and diversity of probiotics, including Lactobacillus, Streptococcus, Megasphaera, and Blautia, which promoted amino acid metabolism and energy production and conversion in the colon and the synthesis of carbon-containing biomolecules in the ileum. In summary, dietary supplementation with 0.2% sodium butyrate was shown to have a tendency to improve the growth performance of weaned piglets and enhance intestinal mucosal barrier function via altering the gut microbiota.

Intestinal Epithelial Digestive, Transport, and Barrier Protein Expression Is Increased in Environmental Enteric Dysfunction

Environmental enteric dysfunction (EED) is characterized by malabsorption and diarrhea that result in irreversible deficits in physical and intellectual growth. We sought to define the expression of transport and tight junction proteins by quantitative analysis of duodenal biopsies from patients with EED. Biopsies from Pakistani children with confirmed EED diagnoses were compared to those from age-matched North American healthy controls, patients with celiac disease, and patients with nonceliac disease with villous atrophy or intraepithelial lymphocytosis. Expression of brush border digestive and transport proteins and paracellular (tight junction) proteins was assessed by quantitative multiplex immunofluorescence microscopy. EED was characterized by partial villous atrophy and marked intraepithelial lymphocytosis. Epithelial proliferation and enteroendocrine, tuft, and Paneth cell numbers were unchanged, but there was significant goblet cell expansion in EED biopsies. Expression of proteins involved in nutrient and water absorption and that of the basolateral Cl− transport protein NKCC1 were also increased in EED. Finally, the barrier–forming tight junction protein claudin-4 (CLDN4) was significantly upregulated in EED, particularly within villous enterocytes. In contrast, expression of CFTR, CLDN2, CLDN15, JAM-A, occludin, ZO-1, and E-cadherin was unchanged. Upregulation of a barrier–forming tight junction protein and brush border and basolateral membrane proteins that support nutrient and water transport in EED is paradoxical, as their increased expression would be expected to be correlated with increased intestinal barrier function and enhanced absorption, respectively. These data suggest that EED activates adaptive intestinal epithelial responses to enhance nutrient absorption but that these changes are insufficient to restore health.

Effect of circadian clock and claudin regulations on inulin-induced calcium absorption in the mouse intestinal tract.

Dietary calcium supplementation has been shown to be an effective adjunct therapy in an inflammatory bowel disease model. Soluble dietary fiber reduces intestinal pH and is known to enhance calcium absorption. Although many circadian clock regulations of nutrient absorption in the intestinal tract have been reported, the effects of clock regulation on calcium absorption have yet to be understood. In this study, we investigated the timing of efficient calcium intake by measuring urinary calcium excretion in mice. The diurnal variations in channel-forming tight junctions (claudins) were detected in both the jejunum and ileum. Following 2 days of feeding with a Ca2+-free diet, Ca2+-containing diets with or without soluble fiber (inulin) were fed at specific timings, and urine was subsequently examined every 4 hr. There was an evident increase in urinary calcium concentration when the inulin diet was fed at the beginning of the resting period. The Claudin 2 (Cldn2) expression level also showed a significant day-night change, which seemed to be a mechanism for the increased calcium excretion after inulin intake. This diurnal rhythm and enhanced Cldn2 expression were abolished by disruption of the suprachiasmatic nucleus, the central clock in the hypothalamus. This study suggests that intestinal calcium absorption might be modulated by the circadian clock and that the intake of inulin is more effective at the beginning of the resting period in mice.

Abstract B021: Claudin-1 expression promotes chemoresistance and cancer stem cell characteristics in colorectal cancer

Abstract Background: Claudins are integral components of tight junction complexes. Beyond just passive cell adhesion molecules, they influence structural dynamics, molecular trafficking, and important cell signaling pathways. Claudins are often dysregulated in cancer, leading to changes in adhesion and metastatic potential. Claudin-1 (Cldn1) is expressed at low levels in normal colon but is highly expressed in colorectal cancer (CRC) and promotes epithelial-mesenchymal transition and metastasis. Cancer stem cells (CSCs) are a small subset of cancer cells that display increased capacity for self-renewal and can undergo symmetric and asymmetric division to reform tumors after treatment. CSCs are resistant to cytotoxic drugs and are thought to be responsible for treatment failure, metastasis, and relapse. Significance of problem: CRC is the second leading cause of cancer-related deaths in the United States with an estimated 151,030 new cases in 2022. Although CRC has an overall relative 5-year survival rate of 64%, prognosis varies dramatically with the stage at diagnosis. Localized CRC can be treated surgically and has a 5-year survival rate of 91%, while the survival rate of metastatic CRC falls to 15%. The primary problem in treating metastatic CRC is a combination of intrinsic and acquired chemoresistance. Therefore, understanding the mechanisms of chemoresistance is vital to developing effective therapies. Hypothesis: Claudin-1 expression in colorectal cancer increases resistance to chemotherapeutic drugs and promotes cancer stem cell characteristics. Experimental design: Bioinformatic analysis was performed on a database of responders and non-responders to standard-of-care CRC chemotherapy. Cldn1 expression was manipulated in CRC cell lines, and assays were performed to evaluate chemoresistance and CSC characteristics. Results: Bioinformatic analysis of a patient dataset of responders and non-responders to chemotherapy revealed that Cldn1 expression was positively correlated with resistance to FOLFOX treatment. It was also significantly correlated with colorectal CSC marker expression including CD44, CD133, and Lgr5. Overexpression of Cldn1 in SW480 cells led to a significant increase in resistance to 5-FU, side population and ALDH-high percentage, colorectal CSC markers, and tumorigenic potential, while knockout of Cldn1 in SW620 and DLD-1 cells led to a decreased response in these assays. Follow-up experiments identified the PDZ-binding domain of Cldn1 as essential for the chemoresistant phenotype. Specifically, both CD44 and its downstream target phospho-AKT are increased with the expression of full-length but not mutated Cldn1. Conclusion: Cldn1 expression induces chemoresistance and cancer stem cell properties in colorectal cancer. Because of this, Cldn1 may serve as a biomarker in CRC for prescribing more aggressive adjuvant therapy or as a molecule for targeted treatment aimed at the resistant tumor cells responsible for treatment failure and recurrence. Citation Format: Mark Primeaux, Saiprasad Gowrikumar, Dhundy Bastola, Punita Dhawan, Amar Singh. Claudin-1 expression promotes chemoresistance and cancer stem cell characteristics in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B021.

Development and comparison of 68Ga/18F/64Cu-labeled nanobody tracers probing Claudin18.2

Claudin 18.2 (CLDN18.2) is an emerging target for the treatment of gastric cancers. We aim to develop tracers to image the expression of CLDN18.2. A humanized nanobody targeting CLDN18.2 (clone hu19V3) was produced and labeled with 68Ga, 64Cu, and 18F. The tracers were investigated in subcutaneous and metastatic models established using two different mouse types (nude and Balb/c mice) and two different cell lines (CHO-CLDN18.2 and CT26-CLDN18.2). Gastric cancer patient-derived xenograft (PDX) models were further established for validation experiments. Three novel CLDN18.2-targeted tracers (i.e., [68Ga]Ga-NOTA-hu19V3, [64Cu]Cu-NOTA-hu19V3, and [18F]F-hu19V3) were developed with good radiochemical yields and excellent radiochemical purities. [68Ga]Ga-NOTA-hu19V3 immuno-positron emission tomography (immunoPET) rapidly delineated subcutaneous CHO-CLDN18.2 lesions and CT26-CLDN18.2 tumors, as well as showing excellent diagnostic value in PDX models naturally expressing CLDN18.2. While [68Ga]Ga-NOTA-hu19V3 had high kidney accumulation, [64Cu]Cu-NOTA-hu19V3 showed reduced kidney accumulation and improved image contrast at late time points. Moreover, [18F]F-hu19V3 was developed via click chemistry reaction under mild conditions and precisely disseminated CHO-CLDN18.2 lesions in the lungs. Furthermore, region of interest analysis, biodistribution study, and histopathological staining results correlated well with the invivo imaging results. Taken together, immunoPET imaging with the three tracers can reliably visualize CLDN18.2 expression.