Expression Of Claudin-1 Research Articles

Aqueous extract of Chuan Xiong Rhizoma enhances inhibitory effect of temozolomide against brain metastasis of melanoma in mice

To investigate the effect of the aqueous extract of Chuan Xiong Rhizoma (CR) on brain metastasis of melanoma B16F10 cells in mice. C57BL/6J mouse models of brain metastasis of melanoma were established by ultrasound-guided intraventricular injection of Luc-labeled B16F10 cells, and brain tumor growth was monitored by in vivo imaging. The mouse models were then randomized for daily gavage of saline or aqueous extract of CR (equivalent crude drug concentration of 1 mg/g). Behavioral tests were used to evaluate the neuroprotective effects of CR in the tumor-bearing mice, and the changes in proteins associated with blood-brain barrier integrity, neuronal cell proliferation and apoptosis, and microglial cell apoptosis and activation were observed using immunofluorescence assay. The efficacy of CR combined with temozolomide (25 mg/kg) against brain metastases of B16F10 cells was observed by in vivo imaging. CR-treated mouse models did not show obvious progression of brain metastases and had a reduced rate of body weight loss and lowered protein expressions of ZO-1, claudin-5, occludin, P-gp, TNF-α, AQP4 and PDGFRβ. In the behavioral tests, the CR-treated mice showed prolonged stay on the wooden stick with a shortened time of sticky stick removal. Immunofluorescence assay showed increased proliferation and decreased apoptosis of neuronal cells and microglia in CR-treated mice. CR treatment significantly increased the levels of CD86, CD206, IL-4 and IL-10 and decreased the levels of CD163 and IL-1β in the microenvironment of brain metastases. The mice receiving combined treatments with CR and temozolomide showed significantly lower intensity of fluorescent signals in the brain than those treated with temozolomide alone. CR does not promote brain metastasis of melanoma while inducing opening of the blood-brain barrier, and its combined use with TMZ results in enhanced inhibition against brain metastasis of melanoma B16F10 cells in mice.

Improved Preservation of Rat Small Intestine Transplantation Graft by Introduction of Mesenchymal Stem Cell-Secreted Fractions.

Segmental grafts from living donors have advantages over grafts from deceased donors when used for small intestine transplantation. However, storage time for small intestine grafts can be extremely short and optimal graft preservation conditions for short-term storage remain undetermined. Secreted factors from mesenchymal stem cells (MSCs) that allow direct activation of preserved small intestine grafts. Freshly excised Luc-Tg LEW rat tissues were incubated in preservation solutions containing MSC-conditioned medium (MSC-CM). Preserved Luc-Tg rat-derived grafts were then transplanted to wild-type recipients, after which survival, injury score, and tight junction protein expression were examined. Luminance for each graft was determined using in vivo imaging. The findings indicated that 30-100 and 3-10kDa fractions of MSC-CM have superior activating effects for small intestine preservation. Expression of the tight-junction proteins claudin-3, and zonula occludens-1 preserved for 24h in University of Wisconsin (UW) solution containing MSC-CM with 50-100kDa, as shown by immunostaining, also indicated effectiveness. Reflecting the improved graft preservation, MSC-CM preloading of grafts increased survival rate from 0% to 87%. This is the first report of successful transplantation of small intestine grafts preserved for more than 24h using a rodent model to evaluate graft preservation conditions that mimic clinical conditions.

EPEN-18. MULTI-OMICS APPROACH REVEALED A TIGHT BLOOD-BRAIN BARRIER IN EPENDYMOMA

Abstract BACKGROUND A major challenge in the treatment of brain tumors is the limited penetration of many drugs through the blood-brain barrier (BBB). BBB characteristics include endothelial cells connected by tight junction proteins (e.g. Claudin5, ZO1, occludin) and the expression of efflux transporters (e.g. P-gp) with the physiological role to limit the accumulation of potentially toxic substances in brain tissue. Our study aims to enhance our understanding of the BBB composition across different molecular groups of ependymoma (EPN) with the goal of leveraging this knowledge to improve therapeutic strategies against these tumors. METHODS We applied a multi-omics approach integrating single-nuclear RNA sequencing and ultra-high content imaging to unravel BBB composition at both transcriptomic and proteomic levels. Patient-derived xenograft (PDX) models were utilized to explore differences in BBB penetration between tumor and healthy brain tissue following drug treatments. RESULTS Expression of tight junction and transporter proteins revealed strong dependencies specific to molecular groups, but were independent of the corresponding brain regions. Human tissue of ST-EPN-ZFTA exhibited highest expression of claudin5, while both ST-EPN-ZFTA and PF-EPN-A represented upregulation of occludin and ZO1 in comparison to healthy tissue. These findings on claudin5 and ZO1 were further confirmed in PDX models. Single-cell data analysis from patients localized the expression of relevant BBB factors to tumor-associated endothelial cells. Treatment of PDX models with drugs revealed disparities in drug penetration between EPN tumors and healthy brain regions. Notably, for most drugs BBB penetration was lower in ST-EPN-ZFTA tumors than healthy brain region. CONCLUSIONS Molecular BBB specifics may contribute to drug resistance of aggressive EPN particularly in ST-EPN-ZFTA tumors, which presented high tight junction expressions and lower drug penetration. This resource aims to improve BBB penetration prediction in EPN, to identify combination therapies targeting BBB components and to select innovative drug delivery approaches.

DIPG-35. OVERCOMING THE BLOOD-BRAIN BARRIER CHALLENGE IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas (DMGs) are aggressive paediatric brainstem tumours with no known cure. The development of effective treatments is greatly impeded by the failure of most anti-tumour agents to penetrate the blood-brain barrier (BBB). Our research, along with that of others, has revealed that the BBB remains intact in DMG. We employed DMG orthotopic models to explore potential region-specific variations in response to the therapeutic agent temsirolimus as well as confocal microscopy and single-cell RNA sequencing (scRNAseq) to elucidate the impact of DMGs on brain endothelial morphology and pathways governing BBB integrity. We first assessed the effectiveness of the mTOR inhibitor temsirolimus when administered in cortex-injected DMG models compared to brainstem-injected DMG. We found significantly increased survival rates, elevated temsirolimus levels, and reduced tumour cell proliferation in the cortex versus the brainstem. Confocal imaging analysis indicated no structural differences between DMG and Matrigel-injected animals. However, scRNAseq unveiled distinct transcriptomic changes: brainstem-injected DMG exhibited downregulation of genes related to inflammatory and apoptotic pathways, whereas cortex-injected DMG showed downregulation of interferon pathways. To overcome the tightened barrier in the brainstem, we explored three MCL1 inhibitors and SNGR-TNFa (targeting CD13) as potential blood-brain barrier modulators. These inhibitors significantly reduced transendothelial electrical resistance, increased tracer dye leakage in an in vitro DMG BBB model, and decreased the protein expression of claudin-5. Moreover, in vivo, we found that administration of a single dose of either SNGR-TNFa or the MCL1 inhibitor S63845 improved penetration of Texas-Red (3KDa) in DMG-engrafted animals, indicating the effective opening of the BBB. In summary, our study revealed that DMG directly affects BBB pathways in endothelial cells, tightening the barrier and reducing treatment efficacy. MCL1 inhibitors and SNGR-TNFa show promise in modulating the BBB and have the potential to enhance therapeutic activity.

Flagellin conjugated Per a 10 and its T cell peptides attenuate airway inflammation and restore cellular function.

Adjuvants were used to modulate response towards relevant immune cells. The present study aims to investigate FlaA-conjugated Per a 10 and T cell peptides in amelioration of allergic airway disease in mice. Mice given Per a 10 showed allergic features with higher cellular infiltration, IgE, Th-2 cytokines and alarmins. Fusion protein treatment reduced lung inflammation (p < 0.0001) and cellular infiltrates (p < 0.001) with higher IgG2a/IgE indicating resolution of disease. Immunotherapy with FPT1 and FPT3 reduces IL-4, IL-5 and IL-13 levels (p < 0.0001) with a fourfold increase in IFN-γ secretion in BALF. FPT1- and FPT3-treated mice have increased IL-10 and TGF-β levels (p < 0.001) with CD4+Foxp3+ T cells (p < 0.01) indicating Treg response. There was enhanced expression of claudin-1 (1.7-fold) and occludin (fourfold) in lungs of FPT1- and FPT3-treated mice with reduced TSLP (p < 0.01) and IL-33 (p < 0.0001) secretion in BALF indicating recovery of epithelial function. Peptide-conjugated FlaA proteins showed protective immunity in mice and have potential for immunotherapy with restoration of cellular function.

Glycerol monolaurate extracts protective effects in Caco-2 cells and dextran sulfate sodium-induced colitis in C57BL/6 mice

Glycerol monolaurate (GML) is a common food additive known for its antimicrobial and anti-inflammatory properties. This study aims to assess the impact of GML on experimental colitis, both in vivo and in vitro. The results indicated that GML significantly inhibited the expression of MIP-3α, TNF-α, IL-1β, and IL-6, while upregulating the expression of claudin-3 and claudin-4 in DSS-treated Caco-2 cell monolayers. Oral administration of GML effectively alleviated colitis in mice, as demonstrated by mitigating body weight loss, lowering the DAI score, and improving colon shrinkage. GML significantly suppressed the expression of NF-κB p65, IL-1β, IL-6, TNF-α, and IL-18 in DSS-treated mice. Moreover, GML increased the cecal microbial alpha-diversity index and restored the dysbiosis. GML significantly increased the relative abundance of Lactobacillus, Alistipes, and Bacteroides, which were found to be negatively correlated with the DAI score and pro-inflammatory cytokine expression. In conclusion, the results highlight the potential therapeutic benefits of GML in preventing colitis and emphasize the role of gut microbiota in this process.

Characteristics of Retinal Vascular Degeneration and the Expression of Vessel-Related Claudin Proteins in Retinal Degeneration Mouse.

This study aimed to investigate the characteristics of retinal vascular degeneration and the expression of vessel-related claudin (CLD) proteins in retinal degeneration mouse (Pde6βrd1/rd1 rd1 mouse). Retinas from wild-type (WT) mice and rd1 mice at postnatal day 3 (P3), P5, P8, P11, P13, P15, P18, and P21 were collected. Immunofluorescence staining was used to assess the retinal vascular plexus, cell proliferation, CLD expression, and retinal ganglion cells (RGCs). The distribution of retinal superficial and deep vessels was determined by isolectin B4 fluorescence staining of retinal flat mounts and frozen sections. Hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dNTP nick-end labeling were used to investigate retinal histological degeneration and apoptosis in rd1 mice, respectively. Quantitative real-time PCR and Western blot were used to measure the expression of vessel-related CLD-1, -2, -3, and -5, vascular endothelial growth factor A (VEGFA), and vascular endothelial growth factor receptor 2 (VEGFR2) in the retinas. Compared to the WT mice, the rd1 mice displayed delayed but completed progressive development in the retinal superficial vascular plexuses (SVPs) and deep vascular plexuses (DVPs). In the rd1 mice, the thickness of retinal layers gradually decreased and the retinas underwent progressive atrophy and degeneration. The deterioration got worse at the late developmental stage. The declined vessel density of SVP and DVP correlated with the decreased thickness of the full and inner parts of the retina and the reduced number of RGCs. DVP degeneration and the thinning of the outer nuclear layer exhibited an obvious reduction at P15. The expression levels of CLD-1, CLD-2, CLD-3, CLD-5, VEGFA, and VEGFR2 decreased and were consistently lower in the rd1 mice than in WT mice since P15. Rd1 mice exhibited progressive vascular degeneration of retinal SVP and DVP, the thinning and atrophy of retinal ONL and RGC, and the downregulation of vessel-related CLD proteins during the late developmental period. Thus, the rd1 mouse is a useful model of not only retinal neuro-degeneration but also retinal vascular degeneration.

Tomato juice regulates bile acid-TGR5 signaling and reverses cognitive deficit and neuroinflammation in high-fat high-cholesterol diet-induced mice

Cognitive deficits and neuroinflammation are significant complications associated with metabolic diseases induced by a high-fat, high-cholesterol (HFHC) diet. Although tomato juice (TJ) has shown potential benefits for brain health, its specific interventional role remains unclear. This research examined the capability of TJ to mitigate HFHC-induced cognitive deficits and delineated TJ's specific mechanisms against neuroinflammation. The results demonstrated that TJ effectively ameliorated cognitive impairments triggered by HFHC diets, improving spatial working memory, novel object recognition, and reducing anxiety-like behaviors in open space and elevated maze tests. Moreover, TJ mitigated neuronal damage, decreased the activity of immune cells, and diminished the level of COX-2 and iNOS in the hippocampus. Additionally, TJ decreased serum TNF-α and lipopolysaccharide levels, reduced liver inflammation, and improved intestinal barrier markers Claudin-1 and Mucin-2 expression. Fecal metabolomics demonstrated that TJ significantly upregulated bile acid metabolism-related pathways. TJ also altered the profile of serum bile acids, elevating the levels of Takeda G protein-coupled receptor 5 (TGR5)-activating bile acids, including deoxycholic acid, ursodeoxycholic acid, and tauroursodeoxycholic acid. TJ upregulated TGR5 protein expression and inhibited the phosphorylation of protein kinase B and nuclear factor kappa B proteins in the hippocampus. Notably, the positive effects of TJ on cognition and TGR5 activation were nullified in an antibiotic-treated mouse model, underscoring the essential role of the gut-brain axis in mediating TJ's neuroprotective effects. These findings propose that bile acid-TGR5 signaling may be associated with the anti-neuroinflammatory effects of TJ, which may represent a novel approach for neurogenic disease prevention.

In vitro study of momordin Ic in improving atopic dermatitis by regulating claudin-1/MyD88 and targeting NLRP3

Atopic dermatitis (AD) is a non-infectious inflammatory skin disease characterized by persistent itching of the skin, and it has become a global health problem with increasing incidence and complex pathogenesis. Momordin Ic (MMI) is a triterpenoid saponin that has anti-inflammatory effect and may play an effective role in the treatment of AD. To explore the molecular mechanism of MMI in relieving AD, and to provide some experimental data for the clinical application of MMI in AD. The mRNA levels of IL-33, IL-4 and NF-κB were detected by RT-qPCR. The protein expressions of Filaggrin, Claudin-1, MyD88 and NLRP3 were detected by western blotting. Transepithelial/transendothelial electrical resistance (TEER) method was used to detect the tight-junction function of HaCaT cells. Molecular docking experiment was performed on CB-DOCK2 to predict the binding of MMI and NLRP3. MMI reversed the decrease of filaggrin protein expression and intercellular resistance in HaCaT cells induced by TNF-α and IFN-γ. In addition, MMI reversed the increase in IL-33 and IL-4 mRNA levels in HaCaT cells treated with TNF-α and IFN-γ. In addition, MMI promoted the expression of Claudin-1 and inhibited the expression of MyD88 and NLRP3. In addition, the molecular docking results showed that MMI and NLRP3 had interaction sites. MMI improved the inflammatory response of HaCaT cells by regulating the expression of Claudin-1/MyD88 and targeting NLRP3, which may act a positive role in relieving AD.

Protective Effect of Red Light-Emitting Diode against UV-B Radiation-Induced Skin Damage in SKH:HR-2 Hairless Mice.

In this in vivo study on hairless mice, we examined the effects of light-emitting diode (LED) treatment applied prior to ultraviolet B (UVB) irradiation. We found that pre-treating with LED improved skin morphological and histopathological conditions compared to those only exposed to UVB irradiation. In our study, histological evaluation of collagen and elastic fibers after LED treatment prior to UVB irradiation showed that this pretreatment significantly enhanced the quality of fibers, which were otherwise poor in density and irregularly arranged due to UV exposure alone. This suggests that LED treatment promotes collagen and elastin production, leading to improved skin properties. Additionally, we observed an increase in Claudin-1 expression and a reduction in nuclear factor-erythroid 2-related factor 2 (Nrf-2) and heme-oxygenase 1 (HO-1) expression within the LED-treated skin tissues, suggesting that LED therapy may modulate key skin barrier proteins and oxidative stress markers. These results demonstrate that pretreatment with LED light can enhance the skin's resistance to UVB-induced damage by modulating gene regulation associated with skin protection. Further investigations are needed to explore the broader biological effects of LED therapy on other tissues such as blood vessels. This study underscores the potential of LED therapy as a non-invasive approach to enhance skin repair and counteract the effects of photoaging caused by UV exposure.

Protective effects of chlorogenic acid on the intestinal barrier of broiler chickens: an immunological stress model study

This study was conducted to investigate the protective effects of chlorogenic acid (CGA) on inflammatory responses and intestinal health of lipopolysaccharide (LPS)-challenged broilers. One hundred and forty-four 1-day-old male broiler chicks were divided into 3 groups with 6 replicates of 8 birds each. The groups were as follows: 1) Control group: birds fed a basal diet; 2) LPS group: LPS-challenged birds fed a basal diet; 3) CGA group: LPS-challenged birds fed a CGA-supplemented diet. The LPS was intraperitoneally administered at a dose of 1 mg/kg of body weight. CGA increased the weight gain and feed intake of LPS-challenged birds by 37.05% and 24.29%, respectively (P < 0.05). CGA also alleviated LPS-induced inflammation, as evidenced by lower levels of pro-inflammatory cytokines in the serum and jejunum (tumor necrosis factor-α, interferon-γ, interleukin-1β, and interleukin-6), and the decreased myeloperoxidase activity in the jejunum (P < 0.05). These effects were accompanied by a decrease in the mRNA abundance of toll-like receptor 4 and myeloid differentiation factor 88 and an inhibition of nuclear factor kappa-B translocation in the jejunum (P < 0.05). CGA reduced circulating diamine oxidase activity and levels of D-lactate and endotoxin, and positively regulated the expression of jejunal claudin-3 and zonula occludens-1 in LPS-challenged broilers (P < 0.05). Compared to the LPS group, CGA reduced the apoptotic rate of epithelial cells and cytochrome c concentration in the jejunum, and normalized the expression of genes responsible for proliferation and apoptosis in jejunal epithelial cells, including cysteine aspartate-specific protease-9, B cell lymphoma-2, and proliferating cell nuclear antigen (P < 0.05). Furthermore, CGA normalized the altered phosphorylation of protein kinase B and glycogen synthase kinase-3β, as well as the translocation of nuclear β-catenin in the jejunum of LPS-challenged broilers (P < 0.05). These results suggested that CGA supplementation improved growth performance, alleviated inflammation, and helped maintain intestinal integrity and barrier function in LPS-challenged broilers, possibly through the regulation of the toll-like receptor 4/nuclear factor kappa-B and protein kinase B/Wnt/β-catenin pathways.

Safety and efficacy of IBI343 (anti-claudin18.2 antibody-drug conjugate) in patients with advanced pancreatic ductal adenocarcinoma or biliary tract cancer: Preliminary results from a phase 1 study.

3037 Background: Prognosis for advanced pancreatic ductal adenocarcinoma (PDAC) and biliary tract cancer (BTC) remains poor, with limited treatment options available. Expression of claudin18.2 (CLDN18.2) has emerged as a potential target for anti-cancer treatment. Herein, we report preliminary safety and efficacy results of IBI343, an antibody-drug conjugate (ADC) consisting of anti-CLDN18.2 monoclonal antibody conjugated to exatecan (topoisomerase I inhibitor), in patients (pts) with advanced PDAC or BTC in a phase 1 study. Methods: Eligible pts who failed or were intolerant to standard treatment were enrolled. IBI343 were intravenously administered at 6 mg/kg or 8 mg/kg Q3W. In dose escalation, pts were enrolled regardless of CLDN18.2 expression. In dose expansion, pts were required to have CLDN18.2 expression ≥40% (1+/2+/3+ staining intensity by immunohistochemistry). Primary endpoint was safety. Secondary endpoints included objective response rate (ORR), disease control rate (DCR), duration of response (DoR) and progression free survival (PFS) assessed by investigator per RECIST v1.1. Results: As of December 19, 2023, 35 pts (1 pt in dose escalation and 34 pts in dose expansion) were enrolled from China and Australia (males: 57.1%, median age: 58.0 years, ECOG PS 1: 71.4%, stage IV: 91.4%, median lines of prior treatment: 2) including 28 PDAC pts and 7 BTC pts. Pts received IBI343 at 6 mg/kg (n=17) or 8 mg/kg (n=18). Median duration of treatment was 7.0 weeks (range: 3.0-23.6) with 23 (65.7%) pts still on treatment. In all pts, treatment-related adverse events (TRAEs) occurred in 28 (80.0%) pts including grade ≥3 TRAEs in 9 (25.7%) pts. Common TRAEs (≥20%) were anemia (42.9%), neutrophil count decreased (28.6%), nausea (25.7%), vomiting (25.7%) and white blood cell count decreased (22.9%). Serious TRAEs occurred in 4 (11.4%) pts. TRAEs leading to dose interruption and treatment discontinuation occurred in 7 (20.0%) pts and 1 (2.9%) pt respectively. No TRAE led to death. Safety profiles of IBI343 in PDAC and BTC were comparable with the whole study cohort and no new safety signal was observed. As of January 15, 2024, 25 pts at 6 mg/kg and 8 mg/kg were efficacy evaluable. Partial response (PR) was observed in 7 pts (5 PDAC and 2 BTC). The ORR was 28.0% (95%CI: 12.1-49.4) and DCR was 80.0% (95%CI: 59.3-93.2). In evaluable pts at 6 mg/kg with CLDN18.2 expression ≥60% (1+/2+/3+, n=13), 5 pts had PR with ORR of 38.5% (95%CI: 13.9-68.4) and DCR of 84.6% (95%CI: 54.6-98.1). Among 10 PDAC pts in this subgroup, ORR was 40% (95%CI: 12.2-73.8). DoR and PFS data were immature. More updated data on safety and efficacy will be presented at the meeting. Conclusions: IBI343 was well tolerated with favorable safety profiles and encouraging efficacy in CLDN18.2-positive PDAC and BTC. Clinical trial information: NCT05458219 .

Protein expression of claudin 18.2, Nectin-4, HER-3, and PD-L1 in small bowel adenocarcinoma: Exploring potential targets of antibody drugs.

4176 Background: Small bowel adenocarcinoma (SBA) is a rare cancer and improved treatment strategies are needed. To explore potential targets of existing antibody drugs for SBA, we examined the protein expression of Claudin 18.2, Nectin-4, HER-3, and PD-L1 in SBA and analyzed the associated clinicopathological features and prognosis. Methods: We retrospectively reviewed patients diagnosed with SBA who underwent adjuvant or palliative chemotherapy between July 2010 and July 2023 at our hospital. Immunohistochemistry staining of Claudin 18.2, Nectin-4, HER-3, and PD-L1 was performed for pathological samples. We also assessed overall survival (OS) in patients receiving palliative chemotherapy to examine the association between prognosis and expression of each protein, excluding patients with microsatellite instability-high (MSI-H) treated with immunotherapy. Results: Pathological samples and clinical data were available for 51 patients aged 23–82 years. The primary lesion was in the duodenum in 49% of these patients and in the jejunum or ileum in 51%. There was no recurrence after surgery and adjuvant chemotherapy in 12% of the patients, while 88% received oxaliplatin-based palliative chemotherapy for unresectable SBA. Among those with unresectable SBA, 4 (8%) had MSI-H and were treated with immunotherapy. Claudin 18.2, Nectin-4, and HER-3 positivity was 35%, 82%, and 88%, respectively. All cases had expression of at least one of these molecules, and 25% had expression of all three. Two molecules were co-expressed in 55% of the patients, most frequently Nectin-4 and HER-3. Nectin-4 expression was observed in all primary lesions in the jejunum or ileum (100%), compared with 60% in the duodenum ( p &lt; 0.001). The distribution of combined positive score (CPS) for PD-L1 was &lt; 1, 1-5, and ≥ 5 for 33%, 32%, and 35%, respectively. CPS was higher in Nectin-4 positive samples compared with negative samples (median CPS 2.5% vs 0%, p = 0.077). Among patients receiving palliative chemotherapy, OS did not differ according to the Claudin 18.2 expression (17.0 vs. 13.7 months, HR 0.91, p = 0.800) or HER-3 expression (17.0 vs. 9.7 months, HR 0.40, p = 0.166). However, patients with Nectin-4 expression had significantly shorter OS than those without Nectin-4 expression (12.6 vs. 43.2 months, HR 5.12, p = 0.006). Similarly, OS was shorter in patients with PD-L1 CPS ≥ 5 compared with CPS &lt; 5 (9.7 vs. 18.0 months, HR 2.60, p = 0.028). Multivariate analysis revealed that Nectin-4 expression (HR 4.55, p = 0.020) was an independent adverse prognostic factor for OS. Conclusions: Claudin 18.2, Nectin-4, and HER-3 are highly expressed in SBA, indicating they are potential targets for existing antibody drugs. In particular, Nectin-4-positive SBA exhibited poor prognosis and high PD-L1 expression, suggesting that a combination of immunotherapy and Nectin-4-targeting therapy may be effective.

Systematic proteomics analysis revealed different expression of laminin interaction proteins in breast cancer: lower in luminal subtype and higher in claudin-low subtype.

Breast cancer is a major public health concern. Proteomics enables identification of proteins with aberrant properties. Here, we identified proteins with abnormal expression levels in breast cancer tissues and systematically analyzed and validated the data to locate potential diagnostic and therapeutic targets. Protein expression level in breast cancer tissues and para-carcinoma tissues were detected by Isobaric Tags for Relative and Absolute Quantification (iTRAQ) technology and further screened through Gene Expression Profiling Interactive Analysis (GEPIA) database. Cellular components, protein domain and Reactome pathway analysis were performed to screen functional targets. Abnormal expression levels of functional targets were validated by Oncomine database, quantitative real time polymerase chain reaction (qRT-PCR) and proteomics detection. Protein correlation analysis was performed to explain the abnormal expression levels of potential targets in breast cancer. Overall, 207 and 207 proteins were up- and down-regulated, respectively, in breast cancer tissues, and approximately 50% were also detected in the GEPIA database. The overlapping proteins were mainly extracellular proteins containing epidermal growth factor-like domain in leukocyte adhesion molecule (EGF-Lam) domain and enriched in laminin interaction pathway. Moreover, the downregulated laminin interaction proteins could be functional targets, which were also validated through Oncomine-Richardson and Oncomine-Curtis database. However, the lower expression level of laminin interaction proteins only fit for luminal breast cancer cells with no or low metastasis ability because the proteins achieved higher expression level in more invasive claudin-low breast cancer cells. In addition, when compared with corresponding in situ carcinoma tissues, above-mentioned proteins also showed higher expression levels in invasive carcinoma tissues. Finally, we have revealed the negative correlation between the laminin interaction proteins and the claudins. The laminin interaction protein, especially for laminins with β1 and γ1 subunits and their integrin receptors with α1 and α6 subunits, showed lower expression levels in luminal breast cancer with no or lower metastatic ability, but showed higher expression levels in claudin-low breast cancer with higher metastatic ability; and their higher expression could be related to the low claudin expression.

Disruption of pulmonary microvascular endothelial barrier by dysregulated claudin-8 and claudin-4: uncovered mechanisms in porcine reproductive and respiratory syndrome virus infection

The pulmonary endothelium is a dynamic and metabolically active monolayer of endothelial cells. Dysfunction of the pulmonary endothelial barrier plays a crucial role in the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), frequently observed in the context of viral pneumonia. Dysregulation of tight junction proteins can lead to the disruption of the endothelial barrier and subsequent leakage. Here, the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) served as an ideal model for studying ALI and ARDS. The alveolar lavage fluid of pigs infected with HP-PRRSV, and the supernatant of HP-PRRSV infected pulmonary alveolar macrophages were respectively collected to treat the pulmonary microvascular endothelial cells (PMVECs) in Transwell culture system to explore the mechanism of pulmonary microvascular endothelial barrier leakage caused by viral infection. Cytokine screening, addition and blocking experiments revealed that proinflammatory cytokines IL-1β and TNF-α, secreted by HP-PRRSV-infected macrophages, disrupt the pulmonary microvascular endothelial barrier by downregulating claudin-8 and upregulating claudin-4 synergistically. Additionally, three transcription factors interleukin enhancer binding factor 2 (ILF2), general transcription factor III C subunit 2 (GTF3C2), and thyroid hormone receptor-associated protein 3 (THRAP3), were identified to accumulate in the nucleus of PMVECs, regulating the transcription of claudin-8 and claudin-4. Meanwhile, the upregulation of ssc-miR-185 was found to suppress claudin-8 expression via post-transcriptional inhibition. This study not only reveals the molecular mechanisms by which HP-PRRSV infection causes endothelial barrier leakage in acute lung injury, but also provides novel insights into the function and regulation of tight junctions in vascular homeostasis.

Mechanisms of Peitu Yimu acupuncture in repairing intestinal mucosal barrier by regulating CRF/CRFR1 pathway in diarrhea-predominant irritable bowel syndrome rats.

To investigate the effect of Peitu Yimu(strengthening spleen and soothing liver) acupuncture on intestinal mucosal barrier function and corticotropin-releasing factor (CRF)/CRF receptor 1 (CRFR1) pathway in rats with diarrhea-predominant irritable bowel syndrome (IBS-D), so as to explore its underlying mechanism in alleviating IBS-D. Forty female SD rats were randomly divided into blank, model, electroacupuncture (EA), and agonist groups, with 10 rats in each group. Except for the blank group, rats in the other groups were given folium sennae infusion by gavage combined with chronic unpredictable mild stress to establish IBS-D model. Rats in the EA group received acupuncture at "Tianshu"(ST25) and EA at "Zusanli"(ST36) and "Taichong"(LR3) (2 Hz/15 Hz) on one side for 20 min, with the side chosen alternately every other day, for 14 days after modeling. Rats in the agonist group received acupuncture 30 min after intravenous injection of CRFR1 agonist urocortin, with the same manipulation method and time as the EA group. Before and after intervention, visceral pain threshold and stool Bristol scores were measured. Elevated plus maze test and open field test were used to detect anxiety and depression like behavior of rats. ELISA was used to detect the contents of CRF and CRFR1 in rats serum. Immunohistochemistry was used to detect the positive expressions of CRF, CRFR1, zonula occludens protein 1(ZO-1), occlusal protein(Occludin), and closure protein 1 (Claudin-1) in colon tissue. Compared with the blank group, the visceral pain threshold, open arm time percentage (OT%), total distance of movement in the open field test, and positive expression of ZO-1, Occludin, and Claudin-1 in colon were decreased (P<0.01, P<0.05), while Bristol stool scores, serum CRF and CRFR1 contents, and positive expressions of CRF and CRFR1 in colon were increased (P<0.01) in the model group. After intervention and compared with the model group, the visceral pain threshold, OT%, total distance of movement in the open field test, and positive expressions of ZO-1, Occludin, and Claudin-1 in colon were increased (P<0.05, P<0.01), while Bristol stool scores, serum CRF and CRFR1 contents, and positive expressions of CRF and CRFR1 in colon were decreased (P<0.01) in the EA group；the Bristol stool scores, serum CRF content, and CRF positive expression in colon were significantly decreased in the agonist group (P<0.01). Peitu Yimu acupuncture can significantly improve visceral hypersensitivity and anxiety-depression state in IBS-D rats. Its mechanism may be related to the inhibition of CRF/CRFR1 pathway and restoration of intestinal tight junction protein expressions.

Outer Membrane Vesicles Derived from Adherent-Invasive Escherichia coli Induce Inflammatory Response and Alter the Gene Expression of Junction-Associated Proteins in Human Intestinal Epithelial Cells.

Adherent-invasive Escherichia coli (AIEC) pathobionts, which are characterized by their ability to adhere to and invade intestinal epithelial cells, are associated with the etiopathogenesis of inflammatory bowel diseases (IBDs). Outer membrane vesicles (OMVs) released by AIEC strains can facilitate the interaction of these bacteria with host cells through delivering bacterial effectors. The aim of this study was to determine the ability of OMVs derived from AIEC strain LF82 to induce the host immune response, leading to production of proinflammatory cytokines and also altering the gene expression of junction-associated proteins in the human epithelial colorectal adenocarcinoma Caco-2 cell line. OMVs were extracted from AIEC strain LF82, and the cell viability of Caco-2 cells treated with these vesicles was assessed by MTT assay. The morphology and size distribution of vesicles were analyzed using transmission electron microscopy and dynamic light scattering, respectively. Gene expression of occludin, ZO-1, claudin-2, E-cadherin, TLR-2, and TLR-4 in response to OMVs was assessed in Caco-2 cells by RT-qPCR. Moreover, the secretion of IL-8 and TNF-α into the supernatant of Caco-2 cells upon treatment with OMVs was measured using ELISA. Our results demonstrated that OMVs upregulated the gene expression level of TLRs and also altered the gene expression level of junction-associated proteins. OMVs derived from AIEC may play a major role in the promotion of intestinal inflammation and epithelial barrier dysfunction. However, further investigations are needed to elucidate the putative role of OMVs in the pathogenesis of AIEC and IBD.

#2837 Novel therapeutic for crescentic glomerulonephritis through targeting CLDN1 in parietal epithelial cells

Abstract Background and Aims Crescentic glomerulonephritis (CrGN) is the final mode of kidney injury common to several immune-mediated kidney diseases. CrGN is characterized by extensive glomerular parietal epithelial cells (PECs) proliferation forming crescents. The destructive crescent is then progressively replaced by fibrosis. Currents therapies for CrGN do not directly target the deleterious response of resident kidney cells. In a pathological context, claudin-1 (CLDN1), a transmembrane protein involved in epithelial tight junctions, can be exposed outside the tight junctions and mediate pro-fibrotic pathways and extracellular matrix (ECM) remodelling. Lixudebart (formerly ALE.F02) is a first-in-class monoclonal antibody that specifically targets and blocks exposed CLDN1 in injured epithelial cells. A phase II clinical trial is under way for patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV) with rapidly progressive glomerulonephritis which induces kidney fibrosis (RENAL-F02, NCT06047171). CLDN1 is highly expressed by glomerular PECs. This study investigated the functional role of CLDN1 in CrGN and the potential benefit to target CLDN1 in CrGN. Method CLDN1 expression in renal tissues of CrGN patients was analyzed using kidney multicolor immunofluorescence staining and spatial transcriptomics. Correlation between CLDN1 expression and clinical endpoints (eGFR, proteinuria), disease biomarkers and crescent evolution were studied. A spatially resolved molecular roadmap from CLDN1+ crescentic glomeruli was conducted. Proof-of-concept studies using anti-CLDN1 mAb were performed in preclinical models of CrGN. Results In tissues (n = 150) of patients with AAV and IgAN, multicolor immunofluorescence revealed up-regulated CLDN1 expression by cellular and fibrocellular crescents. At the time of diagnostic kidney biopsy, glomerular CLDN-1 expression was correlated with podocyte loss (as measured with P57 staining) and fibronectin area. The extent of dual expression of CLDN1 and CD44 at the surface of active PECs was associated with poor renal outcome (eGFR &amp;lt; 30 ml/min) in AAV and IgAN patients with a median follow-up of 2.5 and 3.7 years respectively. Spatial transcriptomics analysis highlighted the association between CLDN1+ crescentic glomeruli and ECM genes. Treatment with anti-CLDN1 mAb reduced albuminuria, improved kidney function and decreased fibrosis biomarkers in nephrotoxic serum-induced CrGN in mice. Conclusion Our results suggest a functional role of CLDN1 in the pathogenesis of CrGN, providing preclinical proof-of-concept for anti-CLDN1 antibodies as a novel therapeutic approach in patients with CrGN.

#794 LncRNA NEAT1 protects uremic toxin-induced intestinal epithelial barrier injury by regulating miR-122-5p/Occludin axis

Abstract Background and Aims Long non-coding RNA(LncRNA) has been reported to be associated with intestinal barrier damage. The aim of this study was to explore the mechanism of lncRNA Nuclear enriched abundant transcript 1 (NEAT1) in uremic toxin-induced intestinal epithelial barrier injury. Method Human colon cancer cells (Caco-2) were used to establish intestinal epithelial injury models with the urea treatment in different conditions. Cell Counting Kit-8 (CCK-8) and Western blot screening the best concentration and time. The expressions of lncRNA NEAT1 and miR-122-5p were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot and immunofluorescence were used to detect the expression of tight junction proteins Occludin, ZO-1 and Claudin-1. Sodium fluorescein was used to detect the paracellular permeability of intestinal epithelial injury models. The binding of miR-122-5p to lncRNA NEAT1 and Occludin was determined by bioinformatics analysis and dual luciferase reporter assay. Results The best condition for the injury model was urea treatment in 144 mg/dl for 48 hours. With the increase of urea intervention time and concentration, the damage degree of intestinal epithelial cells is aggravated. Based on the qRT-PCR results, lncRNA NEAT1 was significantly down-regulated in the model group. Meanwhile, the tight junction proteins Occludin, ZO-1 and Claudin-1 were significantly reduced. The permeability of sodium fluorescein was significantly increased in the model group. Overexpression of lncRNA NEAT1 can alleviate the above performances. As the target gene of lncRNA NEAT1, miR-122-5p is significantly up-regulated in the model group. The dual luciferase reporter assay proved that miR-122-5p was targets to Occludin. The protective effect of overexpression lncRNA NEAT1 on intestinal epithelial barrier function is reversed by miR-122-5p mimics. Conclusion LncRNA NEAT1 protects uremic toxin-induced intestinal epithelial barrier injury by regulating miR-122-5p/Occludin axis.

Quorum-Sensing Signal DSF Inhibits the Proliferation of Intestinal Pathogenic Bacteria and Alleviates Inflammatory Response to Suppress DSS-Induced Colitis in Zebrafish.

The imbalance of gut microbiota is an important factor leading to inflammatory bowel disease (IBD). Diffusible signal factor (DSF) is a novel quorum-sensing signal that regulates bacterial growth, metabolism, pathogenicity, and host immune response. This study aimed to explore the therapeutic effect and underlying mechanisms of DSF in a zebrafish colitis model induced by sodium dextran sulfate (DSS). The results showed that intake of DSF can significantly improve intestinal symptoms in the zebrafish colitis model, including ameliorating the shortening of the intestine, reducing the increase in the goblet cell number, and restoring intestinal pathological damage. DSF inhibited the upregulation of inflammation-related genes and promoted the expression of claudin1 and occludin1 to protect the tightness of intestinal tissue. The gut microbiome analysis demonstrated that DSF treatment helped the gut microbiota of the zebrafish colitis model recover to normal at the phylum and genus levels, especially in terms of pathogenic bacteria; DSF treatment downregulated the relative abundance of Aeromonas hydrophila and Staphylococcus aureus, and it was confirmed in microbiological experiments that DSF could effectively inhibit the colonization and infection of these two pathogens in the intestine. This study suggests that DSF can alleviate colitis by inhibiting the proliferation of intestinal pathogens and inflammatory responses in the intestine. Therefore, DSF has the potential to become a dietary supplement that assists in the antibiotic and nutritional treatment of IBD.