Multimodal evaluation of blood-brain barrier opening in mice in response to low-intensity ultrasound and a claudin-5 binder.

Background: The blood-brain barrier (BBB) is a major bottleneck in delivering therapeutics to the brain. Treatment strategies to transiently open this barrier include focused ultrasound combined with intravenously injected microbubbles (FUS+MB) and targeting of molecules that regulate BBB permeability. Methods: Here, we investigated BBB opening mediated by the claudin-5 binder cCPEm (a microorganismal toxin in a truncated form) and FUS+MB at a centre frequency of 1 MHz, assessing dextran uptake, broadband emission, and endogenous immunoglobulin G (IgG) extravasation. Results: FUS+MB-induced BBB opening was detectable at a pressure ≥0.35 MPa when assessed for leakage of 10 and 70 kDa dextran, and at ≥0.2 MPa for uptake of endogenous IgG. Treating mice with 20 mg/kg cCPEm failed to open the BBB, and pre-treatment with cCPEm followed by FUS+MB at 0.2 and 0.3 MPa did not overtly increase BBB opening compared to FUS+MB alone. Using passive cavitation detection (PCD), we found that broadband emission correlated with the peak negative pressure (PNP) and dextran leakage, indicating the possibility of using broadband emission for developing a feedback controller to monitor BBB opening. Conclusions: Together, our study highlights the challenges in developing combinatorial approaches to open the BBB and presents an additional IgG-based histological detection method for BBB opening.

Progress and prospects of biomarker-based targeted therapy and immune checkpoint inhibitors in advanced gastric cancer.

Gastric cancer and gastroesophageal junction cancer represent the leading cause of tumor-related death worldwide. Although advances in immunotherapy and molecular targeted therapy have expanded treatment options, they have not significantly altered the prognosis for patients with unresectable or metastatic gastric cancer. A minority of patients, particularly those with PD-L1-positive, HER-2-positive, or MSI-high tumors, may benefit more from immune checkpoint inhibitors and/or HER-2-directed therapies in advanced stages. However, for those lacking specific targets and unique molecular features, conventional chemotherapy remains the only recommended effective and durable regimen. In this review, we summarize the roles of various signaling pathways and further investigate the available targets. Then, the current results of phase II/III clinical trials in advanced gastric cancer, along with the superiorities and limitations of the existing biomarkers, are specifically discussed. Finally, we will offer our insights in precision treatment pattern when encountering the substantial challenges.

Omega-3 Attenuates Disrupted Neurotransmission and Partially Protects Metabolic Dysfunction Caused by Obesity in Wistar Rats.

Omega-3 (n3) is a polyunsaturated fatty acid well known for its anti-inflammatory and neuroprotective properties. Obesity is linked to chronic inflammation that disrupts metabolism, the intestine physiology and the central nervous system functioning. This study aims to determine if n3 supplementation can interfere with the effects of obesity on the mitochondrial activity, intestinal barrier, and neurotransmitter levels in the brain of Wistar rats that received cafeteria diet (CAF). We examined adipose tissue, skeletal muscle, plasma, intestine, and the cerebral cortex of four groups: CT (control diet), CTn3 (control diet with n3 supplementation), CAF, and CAFn3 (CAF and n3). Diets were offered for 13 weeks, with n3 supplementation in the final 5 weeks. Adipose tissue Electron Transport Chain complexes I, II, and III showed higher activity in CAF groups, as did complexes III and IV in skeletal muscle. Acetate levels in plasma were reduced in CAF groups, and Lipopolysaccharide (LPS) was higher in the CAF group but reduced in CAFn3 group. Claudin-5 in the intestine was lower in CAF groups, with no n3 supplementation effect. In the cerebral cortex, dopamine levels were decreased with CAF, which was reversed by n3. DOPAC, a dopamine metabolite, also showed a supplementation effect, and HVA, a diet effect. Serotonin levels increased in the CAF group that received supplementation. Therefore, we demonstrate disturbances in mitochondria, plasma, intestine and brain of rats submitted to CAF and the potential benefit of n3 supplementation in endotoxemia and neurotransmitter levels.

Global prevalence of claudin 18 isoform 2 in tumors of patients with locally advanced unresectable or metastatic gastric or gastroesophageal junction adenocarcinoma.

BACKGROUND: Limited data exist for global prevalence of claudin 18 isoform 2 (CLDN18.2) positivity and association of CLDN18.2 status with clinical and tumor characteristics in patients with locally advanced (LA) unresectable or metastatic gastric or gastroesophageal junction (mG/GEJ) adenocarcinoma. We report prevalence of CLDN18.2 positivity (phase 3; SPOTLIGHT, NCT03504397; GLOW, NCT03653507) and concordance of CLDN18.2 status between a subset of pair-matched tumor samples (phase 2, ILUSTRO, NCT03505320; phase 1, NCT03528629) from clinical studies of zolbetuximab. METHODS: Tumor samples from patients with LA unresectable or mG/GEJ adenocarcinoma were tested for CLDN18.2 status by immunohistochemistry. Human epidermal growth factor receptor 2 (HER2) expression was tested per central or local assessment. RESULTS: Across SPOTLIGHT and GLOW, the prevalence of CLDN18.2 positivity (≥ 75% of tumor cells demonstrating moderate-to-strong membranous CLDN18 staining) was 38.4%. Prevalence was similar in gastric versus GEJ adenocarcinoma samples and regardless of collection method (biopsy versus resection) or collection site (primary versus metastatic). CLDN18.2 positivity was most prevalent in patients with diffuse-type tumors. In ILUSTRO and the phase 1 study, concordance of CLDN18.2 positivity was 61.1% between archival (i.e., any time before treatment) and baseline (i.e., ≤ 3 months before first treatment) samples, and concordance of any CLDN18 staining (≥ 1% of tumor cells demonstrating moderate-to-strong membranous CLDN18 staining) was 88.9%. CONCLUSIONS: CLDN18.2 was a highly prevalent biomarker in patients with HER2-negative, LA unresectable or mG/GEJ adenocarcinoma. CLDN18.2 positivity remained relatively stable over time in many patients. Biomarker testing for CLDN18.2 should be considered in standard clinical practice in these patients.

[Corrigendum] All­trans retinoic acid alters the expression of the tight junction proteins Claudin­1 and ­4 and epidermal barrier function­associated genes in the epidermis.

Following the publication of the above article, the authors contacted the Editorial Office to explain that they had identified a pair of duplicate images in the control (Vehicle) group of mouse images in Fig. 1A on p. 1792. Specifically, the same image (corresponding correctly to the 'Day 5' experiment) was inadvertently chosen to represent the cutaneous manifestations of mice in the Vehicle group on 'Day 3' and 'Day 5' in Fig. 1A. This error arose as a consequence of repetitive application and duplication procedures within the image set, resulting in the inadvertent reuse of the same photo. Additionally, due to minimal alterations observed in the skin condition of mice from the control group following treatment, each mouse exhibited a similar appearance; this similarity further contributed to the delayed identification of this error during the paper revision stage. Consequently, this duplication of the same image was made as a result of insufficient scrutiny. The revised version of Fig. 1, showing the correct image for the 'Day 3' experiment in Fig. 1A, is shown on the next page. The authors can confirm that the error associated with the assembly of this figure did not have any significant impact on either the results or the conclusions reported in this study, and all the authors agree with the publication of this Corrigendum. The authors are grateful to the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish this; furthermore, they apologize to the readership of the Journal for any inconvenience caused. [International Journal of Molecular Medicine 43: 1789­1805, 2019; DOI: 10.3892/ijmm.2019.4098].

Prinsepia utilis Royle polysaccharides promote skin barrier repair through the Claudin family.

BACKGROUND: Plant polysaccharides have various biological activities. However, few studies have been conducted on the skin barrier of Prinsepia utilis Royle polysaccharide extract (PURP). MATERIALS AND METHODS: The proportions of polysaccharides, monosaccharides and proteins were determined by extracting polysaccharides from fruit meal using water. The healing rate was measured by cell scratch assays. SDS-damaged reconstructed human epidermal models, an acetone-ether-induced mouse model and an IL-4-induced cellular inflammation model were used to detect the effects of polysaccharides on the phenotype, HA, TEWL, and TEER, with further characterizations performed using QRT-PCR, Western blotting, immunofluorescence (IF) assays. RESULTS: PURP contained 35.73% polysaccharides and 11.1% proteins. PURP promoted cell migration and increased skin thickness in a reconstructed human epidermis model. The TEWL significantly decreased, and the HA content significantly increased. PURP significantly increased the TEER and decreased the permeability of the SDS-damaged reconstructed human epidermis model. Claudin-3, Claudin-4, and Claudin-5 were significantly upregulated. IF and Western blot analysis revealed that the Claudin-4 level significantly increased after treatment with PURP. Claudin-1, Claudin-3, Claudin-4, and Claudin-5 gene expression and IF and immunohistochemical staining were significantly increased in mice treated with acetone-ether. PURP promoted the expression of Claudin-1, Claudin-3, Claudin-4, and Claudin-5 after treatment with 100 ng/mL IL-4. PURP also downregulated the expression of NO, IL6, TNFα and NFκB in Raw 264.7 cells and in a mouse model. CONCLUSION: We hypothesize that PURP may repair the skin barrier by promoting the expression of the claudin family and can assist in skin therapy.

Molecular landscape of borderline ovarian tumours: A systematic review.

Borderline ovarian tumours (BOTs) show intriguing characteristics distinguishing them from other ovarian tumours. The aim of the systematic review was to analyse the spectrum of molecular changes found in BOTs and discuss their significance in the context of the overall therapeutic approach. The systematic review included articles published between 2000 and 2023 in the databases: PubMed, EMBASE, and Cochrane. After a detailed analysis of the available publications, we qualified for the systematic review: 28 publications on proto-oncogenes: BRAF, KRAS, NRAS, ERBB2, and PIK3CA, 20 publications on tumour suppressor genes: BRCA1/2, ARID1A, CHEK2, PTEN, 4 on adhesion molecules: CADM1, 8 on proteins: B-catenin, claudin-1, and 5 on glycoproteins: E-Cadherin. In addition, in the further part of the systematic review, we included eight publications on microsatellite instability and three describing loss of heterozygosity in BOT. Molecular changes found in BOTs can vary on a case-by-case basis, identifying carcinogenic mutations through molecular analysis and developing targeted therapies represent significant advancements in the diagnosis and treatment of ovarian malignancies. Molecular studies have contributed significantly to our understanding of BOT pathogenesis, but substantial research is still required to elucidate the relationship between ovarian neoplasms and extraneous disease, identify accurate prognostic indicators, and develop targeted therapeutic approaches.

Resistance Exercise Reduces Sarcopenia by Repairing Leaky Gut in Patients With Alzheimer's Disease.

PURPOSE: Sarcopenia or age-associated muscle loss is common in patients with Alzheimer's disease (AD). We have previously demonstrated the contribution of a leaky gut to sarcopenia in AD. Here, we asked whether resistant exercise (RE) reduces the sarcopenia phenotype by repairing intestinal leakage in patients with AD. METHOD: A prospective, single-center study of older adults, including healthy controls and patients with AD (n = 44-51/group), was conducted to measure plasma zonulin and claudin-3 (markers of intestinal leakage), handgrip strength (HGS), and short physical performance battery (SPPB) as a measure of functional capacity. Measurements in patients with AD were performed at baseline and after 12 weeks of RE. RESULTS: At baseline, patients with AD had higher plasma zonulin and claudin-3 and lower HGS, gait speed, and SPPB scores than controls. RE reduced plasma zonulin and claudin-3 levels and improved HGS, SPPB scores, and gait speed. Regression analysis revealed robust relationships between changes in plasma zonulin and claudin-3 with HGS. Plasma zonulin was also positively associated with SPPB scores. In addition, RE downregulated plasma markers of inflammation and oxidative stress. However, the prevalence of sarcopenia based on low HGS and muscle atrophy or low SPPB was not affected by RE. CONCLUSION: Taken together, disruption of the intestinal mucosal barrier may contribute to functional decline and sarcopenia in AD, which is incompletely recovered by RE. Circulating levels of zonulin and claudin-3 may be valuable in predicting sarcopenia and functional capacity in older adults with AD.

Fetal gut cell-like differentiation in esophageal adenocarcinoma defines a rare tumor subtype with therapeutically relevant claudin-6 positivity and SWI/SNF gene alteration.

Esophageal adenocarcinoma (EAC) is one of the deadliest tumor entities worldwide, with a 5-year survival rate of less than 25%. Unlike other tumor entities, personalized therapy options are rare, partly due to the lack of knowledge about specific subgroups. In this publication, we demonstrate a subgroup of patients with EAC in a large screening cohort of 826 patients, characterized by specific morphological and immunohistochemical features. This subgroup represents approximately 0.7% (6/826) of the total cohort. Morphological features of this subgroup show a striking clear cytoplasm of the tumour cells and the parallel existence of rare growth patterns like yolk sac-like differentiation and enteroblastic differentiation. Immunohistochemistry reveals expression of the fetal gut cell-like proteins Sal-like protein 4 (SALL4), claudin-6, and glypican 3. Interestingly, we find a correlation with alterations of SWI/SNF-complex associated genes, which are supposed to serve as tumor suppressor genes in various tumour entities. Our results suggest a possible implication of rare tumour subtypes in the WHO classification for EACs according to the classification for gastric cancer. Furthermore, claudin-6 positive tumors have shown promising efficacy of CAR T cell therapy in the recently published BNT-211-01 trial (NCT04503278). This represents a personalized therapeutic option for this tumor subtype.

Heterogenous Gene Expression of Bicellular and Tricellular Tight Junction-Sealing Components in the Human Intestinal Tract.

A major site for the absorption of orally administered drugs is the intestinal tract, where the mucosal epithelium functions as a barrier separating the inside body from the outer environment. The intercellular spaces between adjacent epithelial cells are sealed by bicellular and tricellular tight junctions (TJs). Although one strategy for enhancing intestinal drug absorption is to modulate these TJs, comprehensive gene (mRNA) expression analysis of the TJs components has never been fully carried out in humans. In this study, we used human biopsy samples of normal-appearing mucosa showing no endoscopically visible inflammation collected from the duodenum, jejunum, ileum, colon, and rectum to examine the mRNA expression profiles of TJ components, including occludin and tricellulin and members of the claudin family, zonula occludens family, junctional adhesion molecule (JAM) family, and angulin family. Levels of claudin-3, -4, -7, -8, and -23 expression became more elevated in each segment along the intestinal tract from the upper segments to the lower segments, as did levels of angulin-1 and -2 expression. In contrast, expression of claudin-2 and -15 was decreased in the large intestine compared to the small intestine. Levels of occludin, tricellulin, and JAM-B and -C expression were unchanged throughout the intestine. Considering their segment specificity, claudin-8, claudin-15, and angulin-2 appear to be targets for the development of permeation enhancers in the rectum, small intestine, and large intestine, respectively. These data on heterogenous expression profiles of intestinal TJ components will be useful for the development of safe and efficient intestinal permeation enhancers.

Synthesis, preclinical evaluation and pilot clinical translation of [68Ga]Ga-PMD22, a novel nanobody PET probe targeting CLDN18.2 of gastrointestinal cancer.

PURPOSE: Claudin18.2 (CLDN18.2) is a novel target for diagnosis and therapy of gastrointestinal cancer. This study aimed to evaluate the safety and feasibility of a novel CLDN18.2-targeted nanobody, PMD22, labeled with gallium-68 ([68Ga]Ga), for detecting CLDN18.2 expression in patients with gastrointestinal cancer using PET/CT imaging. METHODS: [68Ga]Ga-PMD22 was synthesized based on the nanobody, and its cell binding properties were assayed. Preclinical pharmacokinetics were determined in CLDN18.2-positive xenografts using microPET/CT. Effective dosimetry of [68Ga]Ga-PMD22 was evaluated in 5 gastrointestinal cancer patients, and PET/CT imaging of [68Ga]Ga-PMD22 and [18F]FDG were performed head-to-head in 16 gastrointestinal cancer patients. Pathological tissues were obtained for CLDN18.2 immunohistochemical (IHC) staining and comparative analysis with PET/CT findings. RESULTS: Cell binding assay showed that [68Ga]Ga-PMD22 had a higher binding ability to AGSCLDN18.2 and BGC823CLDN18.2 cells than to AGS and BGC823 cells (p < 0.001). MicroPET/CT images showed that [68Ga]Ga-PMD22 rapidly accumulated in AGSCLDN18.2 and BGC823CLDN18.2 tumors, and high contrast tumor to background imaging was clearly observed. In the pilot study, the effective dose of [68Ga]Ga-PMD22 was 1.68E-02 ± 1.45E-02 mSv/MBq, and the CLDN18.2 IHC staining result was highly correlated with the SUVmax/BKGstomach of [68Ga]Ga-PMD22 (rs = 0.848, p < 0.01). CONCLUSION: A novel [68Ga]Ga-labeled nanobody probe targeting CLDN18.2, [68Ga]Ga-PMD22, was established and preliminarily proved to be safe and effective in revealing CLDN18.2-positive gastrointestinal cancer, providing a basis for the clinical translation of the agent. CLINICAL TRIAL REGISTRATION: This study was registered on the ClinicalTrials.gov (NCT05937919).

A novel function of claudin-5 in maintaining the structural integrity of the heart and its implications in cardiac pathology.

This study aims to investigate the role of claudin-5 (Cldn5) in cardiac structural integrity. Proteomic analysis was performed to screen the protein profiles in enlarged left atrium from atrial fibrillation (AF) patients. Cldn5 shRNA adeno-associated virus (AAV) or siRNA was injected into the mouse left ventricle or added into HL1 cells respectively to knockdown Cldn5 in cardiomyocytes to observe whether the change of Cldn5 influences cardiac morphology and function, and affects those protein expressions stem from the proteomic analysis. Mitochondrial density and membrane potential were also measured by Mitotracker staining and JC-1 staining under the confocal microscope in HL1 cells. Cldn5 was reduced in cardiomyocytes from the left atrial appendage of AF patients compared to non-AF donors. Proteomic analysis showed 83 proteins were less abundant and 102 proteins were more abundant in AF patients. KEGG pathway analysis showed less abundant CACNA2D2, CACNB2, MYL2 and MAP6 were highly associated with dilated cardiomyopathy. Cldn5 shRNA AAV injection caused severe cardiac atrophy, dilation and myocardial dysfunction in mice. The decreases in mitochondrial numbers and mitochondrial membrane potentials in HL1 cells were observed after Cldn5 knockdown. We demonstrated for the first time the mechanism of Cldn5 downregulation-induced myocyte atrophy and myocardial dysfunction might be associated with the downregulation of CACNA2D2, CACNB2, MYL2 and MAP6, and mitochondrial dysfunction in cardiomyocytes.

Extent and clinical significance of the therapy-relevant tight junction protein Claudin 18.2 in pancreatic ductal adenocarcinoma - real-world evidence.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis, wherefore targeted therapies have experienced increasing interest. Zolbetuximab is a novel targeted therapy under investigation in patients with PDAC and targets Claudin 18.2 (CLDN18.2), which is a component of tight junctions and is of significance in various solid tumors. As its role in PDAC is not definitively elucidated, this study aims to clarify the significance of CLDN18.2 expression in PDAC in a real-world setting. METHODS: All patients (n = 309) were recruited at one of the PANCALYZE study centers and received pancreatic resection with curative intention. Paraffin samples were analyzed using an antibody against CLDN18.2, which is known to be comparable to the antibody used by the SPOTLIGHT and GLOW studies. RESULTS: 94 PDACs are positive for CLDN18.2 (30.4 %). Positive CLDN 18.2 expression was associated with significantly better cancer differentiation (p < 0.001). Patients with positive CLDN18.2 expression showed significantly better overall survival when compared to patients with negative expression (median OS: 30 versus 18 months, p = 0.003). Additionally, in multivariable analyses, CLDN18.2 expression was identified as an independent factor for better survival in patients with PDAC (HR = 0.686, 95 %CI = 0.492-0.956, p = 0.026). CONCLUSION: Significant improvement in survival could be demonstrated by adding Zolbetuximab to known chemotherapy regimes in patients with gastro-esophageal junction adenocarcinoma with at least 75 % CLDN18.2 positive cancer cells. Our findings demonstrate, that 30.4 % of the included patients with PDAC would potentially be eligible for therapy with Zolbetuximab in a real-world patient cohort. Results of trials targeting Claudin 18.2 are pending in patients with PDAC.

Pumilio-1 mediated translational control of claudin-5 at the blood-brain barrier.

Claudin-5 is one of the most essential tight junction proteins at the blood-brain barrier. A single nucleotide polymorphism rs10314 is located in the 3'-untranslated region of claudin-5 and has been shown to be a risk factor for schizophrenia. Here, we show that the pumilio RNA-binding protein, pumilio-1, is responsible for rs10314-mediated claudin-5 regulation. The RNA sequence surrounding rs10314 is highly homologous to the canonical pumilio-binding sequence and claudin-5 mRNA with rs10314 produces 25% less protein due to its inability to bind to pumilio-1. Pumilio-1 formed cytosolic granules under stress conditions and claudin-5 mRNA appeared to preferentially accumulate in these granules. Added to this, we observed granular pumilio-1 in endothelial cells in human brain tissues from patients with psychiatric disorders or epilepsy with increased/accumulated claudin-5 mRNA levels, suggesting translational claudin-5 suppression may occur in a brain-region specific manner. These findings identify a key regulator of claudin-5 translational processing and how its dysregulation may be associated with neurological and neuropsychiatric disorders.

Blood-brain barrier dysfunction and decreased transcription of tight junction proteins in epileptic dogs.

BACKGROUND: Epilepsy in dogs and humans is associated with blood-brain barrier (BBB) dysfunction (BBBD), which may involve dysfunction of tight junction (TJ) proteins, matrix metalloproteases, and astrocytes. Imaging techniques to assess BBB integrity, to identify potential treatment strategies, have not yet been evaluated in veterinary medicine. HYPOTHESIS: Some dogs with idiopathic epilepsy (IE) will exhibit BBBD. Identifying BBBD may improve antiepileptic treatment in the future. ANIMALS: Twenty-seven dogs with IE and 10 healthy controls. METHODS: Retrospective, prospective cohort study. Blood-brain barrier permeability (BBBP) scores were calculated for the whole brain and piriform lobe of all dogs by using dynamic contrast enhancement (DCE) magnetic resonance imaging (MRI) and subtraction enhancement analysis (SEA). Matrix metalloproteinase-9 (MMP9) activity in serum and cerebrospinal fluid (CSF) was measured and its expression in the piriform lobe was examined using immunofluorescent staining. Gene expression of TJ proteins and astrocytic transporters was analyzed in the piriform lobe. RESULTS: The DCE-MRI analysis of the piriform lobe identified higher BBBP score in the IE group when compared with controls (34.5% vs 26.5%; P = .02). Activity and expression of MMP9 were increased in the serum, CSF, and piriform lobe of IE dogs as compared with controls. Gene expression of Kir4.1 and claudin-5 in the piriform lobe of IE dogs was significantly lower than in control dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Our findings demonstrate BBBD in dogs with IE and were supported by increased MMP9 activity and downregulation of astrocytic potassium channels and some TJ proteins. Blood brain barrier dysfunction may be a novel antiepileptic therapy target.

Downregulation of chemoresistance by claudin-14 silencing in human colorectal cancer cells.

An exceptional expression of claudins (CLDNs), tight junction (TJ) proteins, is observed in various solid cancer tissues. However, the pathophysiological roles of CLDNs have not been clarified in detail. CLDN14 is highly expressed in human colorectal cancer (CRC) tissues and cultured cancer epithelial cells. We found CLDN14 silencing decreased cell viability without affecting spheroid size in the three-dimensional (3D) spheroid model of DLD-1 cells derived from human CRC. Mitochondria activity and oxidative stress level were reduced by CLDN14 silencing. Furthermore, CLDN14 silencing decreased the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and its target antioxidative genes. CLDN14 was colocalized with ZO-1, a scaffolding protein in the TJ. CLDN14 silencing induced the disruption of TJ barrier such as the reduction of transepithelial electrical resistance and elevation of fluxes of small molecules including glucose in two-dimensional (2D) cultured model,. The depletion of glucose induced the elevation of ROS generation, mitochondria activity, and Nrf2 expression. These results suggest that CLDN14 increases Nrf2 expression in spheroids mediated via the formation of paracellular barrier to glucose. The cytotoxicities of doxorubicin, an anthracycline anticancer drug, and oxaliplatin, a platinum-based agent, were augmented by an Nrf2 activator in 2D cultured cells. The anticancer drug-induced toxicity was enhanced by CLDN14 silencing in 3D spheroids. We suggest that CLDN14 may potentiate chemoresistance mediated by the suppression of paracellular glucose permeability and activation of the Nrf2 signaling pathway in CRC cells.

Probiotics and microbial metabolites maintain barrier and neuromuscular functions and clean protein aggregation to delay disease progression in TDP43 mutation mice.

Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease. The ALS mice expressing human mutant of transactive response DNA binding protein of 43 kDa (hmTDP43) showed intestinal dysfunction before neuromuscular symptoms. We hypothesize that restoring the intestinal and microbial homeostasis with a bacterial metabolite or probiotics delays the ALS disease onset. We investigate the pathophysiological changes in the intestine and neurons, intestinal and blood-brain barriers, and inflammation during the ALS progression. We then cultured enteric glial cells (EGCs) isolated from TDP43 mice for mechanistic studies. TDP43 mice had significantly decreased intestinal mobility, increased permeability, and weakened muscle, compared with the age-matched wild-type mice. We observed increased hmTDP43 and Glial fibrillary acidic protein (GFAP), and decreased expression of α-smooth muscle actin (α-SMA), tight junction proteins (ZO-1 and Claudin-5) in the colon, spinal cord, and brain in TDP43 mice. TDP43 mice had reduced Butyryl-coenzyme A CoA transferase, decreased butyrate-producing bacteria Butyrivibrio fibrisolvens, and increased Bacteroides fragilis, compared to the WT mice. Serum inflammation cytokines (IL-6, IL-17, and IFN-γ) and LPS were elevated in TDP43 mice. EGCs from TDP43 mice showed aggregation of hmTDP43 associated with increased GFAP and ionized calcium-binding adaptor molecule (IBA1, a microglia marker). TDP43 mice treated with butyrate or probiotic VSL#3 had significantly increased rotarod time, increased intestinal mobility and decreased permeability, compared to the untreated group. Butyrate or probiotics treatment decreased the expression of GFAP, TDP43, and increased α-SMA, ZO-1, and Claudin-5 in the colon, spinal cord, and brain. Also, butyrate or probiotics treatment enhanced the Butyryl-coenzyme A CoA transferase, Butyrivibrio fibrisolvens, and reduced inflammatory cytokines in TDP43 mice. The TDP43 EGCs treated with butyrate or probiotics showed reduced GFAP, IBA1, and TDP43 aggregation. Restoring the intestinal and microbial homeostasis by beneficial bacteria and metabolites provide a potential therapeutic strategy to treat ALS.

IFN-γ-responsiveness of lymphatic endothelial cells inhibits melanoma lymphatic dissemination via AMPK-mediated metabolic control.

The integrity of the lymphatic system is critical for preventing the dissemination of tumor cells, such as melanoma, to distant parts of the body. IFN-γ is well studied as a negative regulator for lymphangiogenesis, which is strongly associated with cancer metastasis. However, the exact mechanisms underlying this process remain unclear. In the present study, we investigated whether IFN-γ signaling in lymphatic endothelial cells (LECs) affects tumor cell dissemination by regulating the barrier function of tumor-associated lymphatic vessels. Using LEC-specific IFN-γ receptor (IFN-γR) knockout mice, we found that the loss of IFN-γR in LECs increased the dissemination of melanoma cells into the draining lymph nodes. Notably, IFN-γ signaling in LECs inhibited trans-lymphatic endothelial cell migration of melanoma cells, indicating its regulation of lymphatic barrier function. Further investigations revealed that IFN-γ upregulated the expression of the tight junction protein Claudin-3 in LECs, while knockdown of Claudin-3 in LECs abolished IFN-γ-induced inhibition of trans-lymphatic endothelial migration activity. Mechanistically, IFN-γ inhibits AMPK signaling activation, which is involved in the regulation of fatty acid metabolism. Modulating fatty acid metabolism and AMPK activation in LECs also affected the lymphatic dissemination of melanoma cells, further confirming that this process is involved in IFN-γ-induced regulation of lymphatic barrier function. These results provide novel insights into how IFN-γ modulates tight junctions in LECs, inhibiting the dissemination of melanoma cells via the lymphatic vessels.

Molecular SPECT/CT Profiling of Claudin18.2 Expression In Vivo: Implication for Patients with Gastric Cancer.

Zolbetuximab (IMAB362), a monoclonal antibody targeting Claudin18.2 (CLDN 18.2), demonstrates a significant clinical benefit in patients with advanced gastroesophageal cancers. The noninvasive assessment of CLDN18.2 expression through molecular imaging offers a potential avenue for expedited monitoring and the stratification of patients into risk groups. This study elucidates that CLDN18.2 is expressed at a noteworthy frequency in primary gastric cancers and their metastases. The iodogen method was employed to label IMAB362 with 123I/131I. The results demonstrated the efficient and reproducible synthesis of 123I-IMAB362, with a specific binding affinity to CLDN18.2. Immuno-single-photon emission computed tomography (SPECT) imaging revealed the rapid accumulation of 123I-IMAB362 in gastric cancer xenografts at 12 h, remaining stable for 3 days in patient-derived tumor xenograft models. Additionally, tracer uptake of 123I-IMAB362 in MKN45 cells surpassed that in MKN28 cells at each time point, with tumor uptake correlating significantly with CLDN18.2 expression levels. Positron emission tomography/computed tomography imaging indicated that tumor uptake of 18F-FDG and the functional/viable tumor volume in the 131I-IMAB362 group were significantly lower than those in the 123I-IMAB362 group on day 7. In conclusion, 123I-IMAB362 immuno-SPECT imaging offers an effective method for direct, noninvasive, and whole-body quantitative assessment of tumor CLDN18.2 expression in vivo. This approach holds promise for accelerating the monitoring and stratification of patients with gastric cancer.