Dose-dependent multi-organ injury following lipopolysaccharide gas inhalation.

Lipopolysaccharide (LPS) is widely used to establish various animal models, including models of acute lung injury, cardiomyocyte damage, and acute kidney injury. Currently, there is no consensus on the diagnosis and treatment of LPS-induced disease. We herein present a case series of four patients who developed dose-dependent multi-organ injury, including acute lung injury and acute kidney injury, after inhaling LPS gas in a sealed room. These patients exhibited varying degrees of multi-organ injury characterized by inflammatory cell infiltration and secretion of proinflammatory cytokines. One patient showed progressive symptoms even with active treatment, leading to mild pulmonary fibrosis. This study emphasizes the importance of early diagnosis and treatment of significant LPS exposure and suggests personalized treatment approaches for managing LPS poisoning.

Ultra-efficient radio-immunotherapy for reprogramming the hypoxic and immunosuppressive tumor microenvironment with durable innate immune memory.

Radiosensitization efficacy of conventional tumor radiosensitizers has been frequently limited by insufficient competence for tumor microenvironment (TME) regulation and unfavorable cellular uptake at biological barriers. Here, we reported an ultra-efficient radiotherapy (RT) strategy by synthesizing an extracellular vesicles (EVs)-encapsulated hollow MnO2 to load metformin (Met@HMnER). It demonstrated significant RT enhancement by morphological control of catalyst and cellular respiratory depression against conventional solid MnO2. Furthermore, the target-modified EVs clothing retains outstanding metformin loading capacity while endowing enhanced biological barrier penetration. A noticeably durable innate immune activation of NK cells was triggered with this nanoplatform via the cGAS-STING pathway. The enhanced immunocompetence was verified on distal metastasis and in-situ recurrence model in vivo, This work paved a new path for synergistic and robust innate immunity in clinical cancer treatment.

Efficacy of treatment with N-acetylcysteine inhalation for AECOPD: A propensity-score-matched cohort study.

INTRODUCTION: N-acetylcysteine (NAC) prevents acute exacerbations of chronic obstructive pulmonary disease (AECOPD). However, the value of NAC inhalation in the treatment of patients with AECOPD is still poorly understood. The study was conducted to evaluate the efficacy of NAC inhalation in AECOPD patients requiring hospitalization. METHODS: In this single institutional, retrospective cohort study, all patients with AECOPD requiring hospitalization between January 2021 and January 2022 were included. Patients were divided into NAC group and Non-NAC group according to whether being treated with NAC inhalation and were matched using the propensity score. The primary outcome was a composite of progression to ventilation requirement, in-hospital mortality and readmission for AECOPD within 30 days. The effect on the mean hospitalized days, blood gas indexes and the incidence rate of adverse drug events were compared between the two groups. RESULTS: Ninety-six patients in the NAC group were matched with 96 patients in the Non-NAC group. The differences in the primary composite end point (NAC group vs Non-NAC group, 5.2% vs 16.7%; P = 0.011) were significant. The median time to discharge was shorter in the NAC group (8.3 vs. 9.1 days, P = 0.030). The NAC group presented a larger increase in partial pressure of arterial oxygen (Pa O2 ) and a higher ratio of self-reported symptomatic improvement from admission to day 5. There was no definite difference between the two groups in the frequency of adverse event. CONCLUSION: NAC inhalation is associated with an improved clinical outcome. A further study should be conducted to confirm the clinical usefulness of NAC inhalation in AECOPD patients.

Development of an Effective Neutralizing Antibody Assay for SARS-CoV-2 Diagnosis.

Introduction: Neutralizing antibodies (NAbs) are essential for preventing reinfection with SARS-CoV-2 and the recurrence of COVID-19; nonetheless, the formation of NAbs following vaccination and infection remains enigmatic due to the lack of a practical and effective NAb assay in routine laboratory settings. In this study, we developed a convenient lateral flow assay for the rapid and precise measurement of serum NAb levels within 20 minutes. Methods: Receptor-binding domain-fragment crystallizable (RBD-Fc) and angiotensin-converting enzyme 2-histidine tag (ACE2-His) were expressed by the eukaryotic expression systems of Spodoptera frugiperda clone 9 and human embryonic kidney 293T, respectively. Then, colloidal gold was synthesized and conjugated with ACE2. After optimizing various operating parameters, an NAb lateral flow assay was constructed. Subsequently, its detection limit, specificity, and stability were systematically evaluated, and clinical samples were analyzed to validate its clinical feasibility. Results: RBD-Fc and ACE2-His were obtained with 94.01% and 90.05% purity, respectively. The synthesized colloidal gold had a uniform distribution with an average diameter of 24.15 ± 2.56 nm. With a detection limit of 2 µg/mL, the proposed assay demonstrated a sensitivity of 97.80% and a specificity of 100% in 684 uninfected clinical samples. By evaluating 356 specimens from infected individuals, we observed that the overall concordance rate between the proposed assay and conventional enzyme-linked immunosorbent assay was 95.22%, and we noticed that 16.57% (59/356) of individuals still did not produce NAbs after infection (both by ELISA and the proposed assay). All the above tests by this assay can obtain results within 20 minutes by the naked eye without any additional instruments or equipment. Conclusion: The proposed assay can expediently and reliably detect anti-SARS-CoV-2 NAbs after infection, and the results provide valuable data to facilitate effective prevention and control of SARS-CoV-2. Clinical trial registration: Serum and blood samples were used under approval from the Biomedical Research Ethics Subcommittee of Henan University, and the clinical trial registration number was HUSOM-2022-052. We confirm that this study complies with the Declaration of Helsinki.

Biomimetic Scaffolds for Tendon Tissue Regeneration.

Tendon tissue connects muscle to bone and plays crucial roles in stress transfer. Tendon injury remains a significant clinical challenge due to its complicated biological structure and poor self-healing capacity. The treatments for tendon injury have advanced significantly with the development of technology, including the use of sophisticated biomaterials, bioactive growth factors, and numerous stem cells. Among these, biomaterials that the mimic extracellular matrix (ECM) of tendon tissue would provide a resembling microenvironment to improve efficacy in tendon repair and regeneration. In this review, we will begin with a description of the constituents and structural features of tendon tissue, followed by a focus on the available biomimetic scaffolds of natural or synthetic origin for tendon tissue engineering. Finally, we will discuss novel strategies and present challenges in tendon regeneration and repair.

Liposome fusogenic enzyme-free circuit enables high-fidelity determination of single exosomal RNA.

Accurate determination of single exosomal inclusions in situ presents a significant challenge due to their extremely low abundance as well sub-100 nm vesicle dimensions. Here, we created a Liposome Fusogenic Enzyme-free circuit (LIFE) approach for the high-fidelity identification of exosome-encapsulated cargoes without destroying the vesicle integrity. The probe-loaded cationic fusogenic liposome could capture and fuse with a single target exosome, enabling probes delivery and target biomolecule-initiated cascaded signal amplification in situ. Then the DNAzyme probe encountered conformal change upon exosomal microRNA activation, and generated a convex DNAzyme structure to cleave the RNA site of substrate probe. After that, the target microRNA could be released to introduce a cleavage cycle to yield amplified fluorescence readout. Therefore, trace cargoes in a single exosome could be accurately determined by elaborately controlling the ratio of introduced LIFE probe, paving the way toward the exploration of a universal sensing platform for the assessment of exosomal cargoes to facilitate early disease diagnosis and personalized treatment.

High-fidelity imaging of intracellular microRNA via a bioorthogonal nanoprobe.

The spatiotemporal visualization of intracellular microRNA (miRNA) plays a critical role in the diagnosis and treatment of malignant disease. Although DNAzyme-based biosensing has been regarded as the most promising candidate, inefficient analytical resolution is frequently encountered. Here, we propose a bioorthogonal approach toward high-fidelity imaging of intracellular miRNA by designing a multifunctional nanoprobe that integrates MnO2 nanosheet-mediated intracellular delivery and activation by a fat mass and obesity-associated protein (FTO)-switched positive feedback. MnO2 nanosheets facilitate nanoprobe delivery and intracellular DNAzyme cofactors are released upon glutathione-triggered reduction. Meanwhile, an m6A-caged DNAzyme probe could be bioorthogonally activated by intracellular FTO to eliminate potential off-target activation. Therefore, the activated DNAzyme probe and substrate probe could recognize miRNA to perform cascade signal amplification in the initiation of the release of Mn2+ from MnO2 nanosheets. This strategy realized high-fidelity imaging of intracellular aberrant miRNA within tumor cells with a satisfactory detection limit of 9.7 pM, paving the way to facilitate clinical tumor diagnosis and prognosis monitoring.

Epigenetic remodeling by vitamin C potentiates plasma cell differentiation.

Ascorbate (vitamin C) is an essential micronutrient in humans. The severe chronic deficiency of ascorbate, termed scurvy, has long been associated with increased susceptibility to infections. How ascorbate affects the immune system at the cellular and molecular levels remained unclear. From a micronutrient analysis, we identified ascorbate as a potent enhancer for antibody response by facilitating the IL-21/STAT3-dependent plasma cell differentiation in mouse and human B cells. The effect of ascorbate is unique as other antioxidants failed to promote plasma cell differentiation. Ascorbate is especially critical during early B cell activation by poising the cells to plasma cell lineage without affecting the proximal IL-21/STAT3 signaling and the overall transcriptome. As a cofactor for epigenetic enzymes, ascorbate facilitates TET2/3-mediated DNA modification and demethylation of multiple elements at the Prdm1 locus. DNA demethylation augments STAT3 association at the Prdm1 promoter and a downstream enhancer, thus ensuring efficient gene expression and plasma cell differentiation. The results suggest that an adequate level of ascorbate is required for antibody response and highlight how micronutrients may regulate the activity of epigenetic enzymes to regulate gene expression. Our findings imply that epigenetic enzymes can function as sensors to gauge the availability of metabolites and influence cell fate decisions.

PERK is a critical metabolic hub for immunosuppressive function in macrophages.

Chronic inflammation triggers compensatory immunosuppression to stop inflammation and minimize tissue damage. Studies have demonstrated that endoplasmic reticulum (ER) stress augments the suppressive phenotypes of immune cells; however, the molecular mechanisms underpinning this process and how it links to the metabolic reprogramming of immunosuppressive macrophages remain elusive. In the present study, we report that the helper T cell 2 cytokine interleukin-4 and the tumor microenvironment increase the activity of a protein kinase RNA-like ER kinase (PERK)-signaling cascade in macrophages and promote immunosuppressive M2 activation and proliferation. Loss of PERK signaling impeded mitochondrial respiration and lipid oxidation critical for M2 macrophages. PERK activation mediated the upregulation of phosphoserine aminotransferase 1 (PSAT1) and serine biosynthesis via the downstream transcription factor ATF-4. Increased serine biosynthesis resulted in enhanced mitochondrial function and α-ketoglutarate production required for JMJD3-dependent epigenetic modification. Inhibition of PERK suppressed macrophage immunosuppressive activity and could enhance the efficacy of immune checkpoint programmed cell death protein 1 inhibition in melanoma. Our findings delineate a previously undescribed connection between PERK signaling and PSAT1-mediated serine metabolism critical for promoting immunosuppressive function in M2 macrophages.

Micro but mighty-Micronutrients in the epigenetic regulation of adaptive immune responses.

Micronutrients are essential small molecules required by organisms in minute quantity for survival. For instance, vitamins and minerals, the two major categories of micronutrients, are central for biological processes such as metabolism, cell replication, differentiation, and immune response. Studies estimated that around two billion humans worldwide suffer from micronutrient deficiencies, also known as "hidden hunger," linked to weakened immune responses. While micronutrients affect the immune system at multiple levels, recent studies showed that micronutrients potentially impact the differentiation and function of immune cells as cofactors for epigenetic enzymes, including the 2-oxoglutarate-dependent dioxygenase (2OGDD) family involved in histone and DNA demethylation. Here, we will first provide an overview of the role of DNA methylation in T cells and B cells, followed by the micronutrients ascorbate (vitamin C) and iron, two critical cofactors for 2OGDD. We will discuss the emerging evidence of these micronutrients could regulate adaptive immune response by influencing epigenetic remodeling.

Hexokinases II-mediated glycolysis governs susceptibility to crizotinib in ALK-positive non-small cell lung cancer.

BACKGROUND: Activation of ALK leads to a high level of aerobic glycolysis related to crizotinib insensitivity in anaplastic lymphoma kinase-positive non-small cell lung cancer (ALK+ NSCLC). The strategy and mechanism of glycolysis inhibition in sensitizing ALK+ NSCLC cells to crizotinib requires further investigation. METHODS: The levels of glycolysis in H3122 and H2228 cells were evaluated through detection of glucose consumption and lactate production. MTT assay was used to explore the effects of glycolytic inhibitors on crizotinib sensitivity, and the potential mechanism of action were detected by colony formation, Ki67 incorporation assay, transwell assay, small interfering RNA technology and western blot analysis. RESULTS: ALK+ NSCLC cells exhibited significantly higher levels of glycolysis compared to ALK- NSCLC cells. Long-term exposure to crizotinib could decrease the sensitivity of ALK+ NSCLC cells to crizotinib via increasing the levels of glycolysis related to hexokinases II (HK2). Crizotinib in combination with glycolysis inhibitor 2-deoxy-D-glucose (2DG) synergistically inhibited proliferation, glycolysis, colony formation and invasion ability of ALK+ NSCLC cells. 2DG sensitization crizotinib might be associated with the inhibition of HK2-mediated glycolysis and P-ALK/AKT/mTOR signaling pathway in H3122 and H2228 cells. CONCLUSIONS: These results indicate that HK2-mediated glycolysis plays a crucial role in the increased tolerance of ALK+ NSCLC cells to crizotinib. 2DG may sensitize ALK+ NSCLC to crizotinib via suppression of HK2-mediated glycolysis and the AKT/mTOR signaling pathway.

Molecular Expression Profile of Changes in Rat Acute Spinal Cord Injury.

Background: Spinal cord injury (SCI) is a highly lethal and debilitating disease with a variety of etiologies. To date, there is no effective therapeutic modality for a complete cure. The pathological mechanisms of spinal cord injury at the molecular gene and protein expression levels remain unclear. Methods: This study used single-cell transcriptomic analysis and protein microarray analysis to analyzes changes in the gene expression profiles of cells and secretion of inflammatory factors respectively, around the lesion site in a rat SCI model. Results: Single-cell transcriptomic analysis found that three types of glial cells (microglia, astrocyte, and oligodendrocyte) becomes activated after acute injury, with GO exhibiting a variety of inflammatory-related terms after injury, such as metabolic processes, immune regulation, and antigen presentation. Protein microarray results showed that the levels of four inflammatory cytokines favoring SCI repair decreased while the levels of nine inflammatory cytokines hindering SCI repair increased after injury. Conclusion: These findings thus reveal the changes in cellular state from homeostatic to reactive cell type after SCI, which contribute to understand the pathology process of SCI, and the potential relationship between glial cells and inflammatory factors after SCI, and provides new theoretical foundation for further elucidating the molecular mechanisms of secondary SCI.

Diagnostic possibility of the combination of exhaled nitric oxide and blood eosinophil count for eosinophilic asthma.

BACKGROUND: Tests to identify reversible airflow limitation are important in asthma diagnosis, but they are time-consuming and it may be difficult for patients to cooperate. We aimed to evaluate whether the combination of fractional exhaled nitric oxide (FeNO) and blood eosinophil (B-Eos) can be used to distinguish some asthma patients who could avoid objective tests. METHODS: We conducted a retrospective cohort study on 7463 suspected asthma cases between January 2014 and December 2019 in Chongqing, China, and identified 2349 patients with complete FeNO, B-Eos count, and spirometry data. Asthma was diagnosed by clinicians by the criteria of recurrent respiratory symptoms and a positive bronchial-provocation or bronchodilation test (BPT, BPD). We evaluated the diagnostic accuracy of FeNO or B-Eos alone or both in combination for asthma using receiver operating characteristic (ROC) curve analysis. RESULTS: In this study, 824 patients were diagnosed with asthma. When FeNO and B-Eos counts were used in combination, the area under the ROC curve (AUC) for diagnosing asthma increased slightly (0.768 vs. 0.745 [FeNO] or 0.728 [B-Eos]; both P < 0.001). The odds ratio for having asthma increased progressively with a gradual increase in FeNO or B-Eos count (both P < 0.001; assessed using the Cochran-Armitage trend test). Further analysis of in-series combinations of different threshold values for these biomarkers indicated that moderately elevated biomarker levels (FeNO > 40 ppb and B-Eos > 300 cells/µl) support a diagnosis of asthma because diagnostic specificity was > 95% and the positive likelihood ratio (PLR) was > 10. This conclusion was verified when selecting the 2017-2019 data as the internal validation dataset. CONCLUSION: FeNO or B-Eos count alone is insufficient to accurately diagnose asthma. Patients with moderately elevated biomarkers (FeNO > 40 ppb and B-Eos > 300 cells/µl) could be diagnosed with asthma and avoid objective tests when such tests are not feasible.

Flipped Quick-Response Code Enables Reliable Blood Grouping.

Accurate and rapid blood typing plays a vital role in a variety of biomedical and forensic scenarios, but recognizing weak agglutination remains challenging. Herein, we demonstrated a flipping identification with a prompt error-discrimination (FLIPPED) platform for automatic blood group readouts. Bromocresol green dye was exploited as a characteristic chromatography indicator for the differentiation of plasma from whole blood by presenting a teal color against a brown color. After integrating these color changes into a quick-response (QR) code, prompt typing of ABO and Rhesus groups was automatically achieved and data could be uploaded wirelessly within 30 s using a commercially available smartphone to facilitate blood cross-matching. We further designed a color correction model and algorithm to remove potential errors from scanning angles and ambient light intensities, by which weak agglutination could be accurately recognized. With comparable accuracy and repeatability to classical column assay in grouping 450 blood samples, the proposed approach further demonstrates to be a versatile sample-to-result platform for clinical diagnostics, food safety, and environmental monitoring.

VPS34 suppression reverses osimertinib resistance via simultaneously inhibiting glycolysis and autophagy.

Autophagy and glycolysis are associated with osimertinib resistance. The energy complement and dynamic balance between these two processes make it difficult to block the process of drug resistance; breaking the complementary relationship between them may effectively overcome drug resistance. However, the exact mechanisms and the key players for regulating autophagy and glycolysis remain unclear. In this study, we demonstrate that autophagy and glycolysis levels in osimertinib-resistant cells were markedly higher than parental cells, and a dynamic balance existed between them. Inhibition of the class III phosphoinositide 3-kinase vacuolar protein sorting 34 (VPS34) with 3-methyladenine or small interfering RNA can not only inhibit abnormally enhanced autophagy but also inhibit glycolysis by inhibiting the location of epidermal growth factor receptor (EGFR) and the expression of hexokinase II. By demonstrating that VPS34 is the key player controlling autophagy and glycolysis simultaneously, our study may provide a new strategy for overcoming osimertinib resistance for treatment of EGFR-mutant non-small cell lung cancer patients.

Biological Significance of 18F-FDG PET/CT Maximum Standard Uptake Value for Predicting EGFR Mutation Status in Non-Small Cell Lung Cancer Patients.

PURPOSE: To investigate the potential of maximum standardized uptake value (SUVmax) in predicting epidermal growth factor receptor (EGFR) mutation status in non-small cell lung cancer (NSCLC) patients. METHODS: Clinical data of 311 NSCLC patients who had undergone both EGFR mutation test and 18F-FDG PET/CT scans between January 2013 and December 2017 at our hospital were retrospectively analyzed. Patients were sub-grouped by their origin of SUVmax. Univariate and multivariate analyses were performed to investigate the association between clinical factors and EGFR mutations. Receiver operating characteristic curve (ROC) analysis was performed to confirm the predictive value of clinical factors. In vitro experiments were performed to confirm the correlation between EGFR mutations and glycolysis. RESULTS: EGFR-mutant patients had higher SUVmax than the wild-type patients in both primary tumors and metastases. In the multivariate analysis, SUVmax, gender and histopathologic type were determined as independent predictors of EGFR mutation status for patients whose SUVmax were obtained from the primary tumors; while for patients whose SUVmax were obtained from the metastases, SUVmax, smoking status and histopathologic type were regarded as independent predictors. ROC analysis showed that SUVmax of the primary tumors (cut off >10.92), not of the metastases, has better predictive value than other clinical factors in predicting EGFR mutation status. The predict performance was improved after combined SUVmax with other independent predictors. In addition, our in vitro experiments demonstrated that lung cancer cells with EGFR mutations have higher aerobic glycolysis level than wild-type cells. CONCLUSION: SUVmax of the primary tumors has the potential to serve as a biomarker to predict EGFR mutation status in NSCLC patients.

Metformin attenuates TGF-ß1-induced pulmonary fibrosis through inhibition of transglutaminase 2 and subsequent TGF-ß pathways.

The purpose of this study was to confirm whether metformin can attenuate TGF-ß1-induced pulmonary fibrosis through inhibition of transglutaminase 2 (TG2) and subsequent TGF-ß pathways. In vitro, MTT assay and Annexin V-FITC/PI staining assay were performed to determine the effect of metformin on the proliferation and apoptosis of human fetal lung fibroblasts (HFL-1 cell). Protein expression of TG2, Collagen I (Col I) and α-smooth muscle actin (α-SMA) were determined by western blot. To further confirm the relationship between TG2 and the anti-fibrotic effect of metformin, TG2 siRNA and TG2 overexpression plasmid were used to interfere the expression of TG2. A bleomycin-induced pulmonary fibrosis model was employed to determine the in vivo inhibitory effect of metformin. The concentrations of TG2, both in supernatants of cells and serum of rats, were determined by ELISA assay. Our results showed that metformin concentration-dependently inhibited the proliferation and promoted the apoptosis of TGF-ß1-stimulated HFL-1 cells. The protein expressions of TG2, Col I and α-SMA stimulated by TGF-ß1 were decreased after metformin intervention, which was confirmed in both siRNAs and plasmids treatment conditions. In vivo, metformin attenuated bleomycin-induced pulmonary fibrosis as demonstrated by H&E and Masson staining, as well as the protein expressions of Col I and α-SMA. Besides, phosphorylated SMAD2, phosphorylated SMAD3, phosphorylated Akt and phosphorylated ERK1/2 were all significantly increased after bleomycin treatment and decreased to normal levels after metformin intervention. Taken together, our results demonstrated that metformin can attenuate TGF-ß1-induced pulmonary fibrosis, at least partly, through inhibition of TG2 and subsequent TGF-ß pathways.

Effective Treatment of Lung Adenocarcinoma Harboring EGFR-Activating Mutation, T790M, and cis-C797S Triple Mutations by Brigatinib and Cetuximab Combination Therapy.

INTRODUCTION: Acquired resistance to osimertinib mediated by EGFR cis-C797S is now a growing challenge. No effective treatment strategy is currently available to overcome cis-C797S-mediated resistance. METHODS: In this retrospective cohort study, 15 patients with advanced lung adenocarcinoma and EGFR-activating mutation, T790M, and cis-C797S after osimertinib progression were identified by targeted next-generation sequencing. Five of these patients received a combined therapy of brigatinib and cetuximab, and 10 patients received cisplatin-based doublet chemotherapy. RESULTS: Among the five patients who were positive for EGFR 19del-T790M-cis-C797S mutations, and who received brigatinib and cetuximab combination therapy, three patients achieved partial response, and two had stable disease, resulting in an overall objective response rate of 60% and disease control rate of 100%. Among the 10 patients who were positive for EGFR 19del or L858R-T790M-cis-C797S mutations and received chemotherapy, only one patient achieved partial response, five had stable disease, and the other four did not benefit from chemotherapy, resulting in an overall objective response rate and disease control rate of 10% and 60%, respectively. The median progression-free survival of patients who received combined targeted therapy was 14 months, and 3 months for those treated with chemotherapy. No grade III to IV adverse events were observed in any patient. CONCLUSIONS: Our retrospective study provides clinical evidence that a combined targeted therapy of brigatinib and cetuximab could be of benefit and may potentially be an effective treatment strategy to improve survival outcomes in patients who acquire EGFR T790M-cis-C797S-mediated resistance to osimertinib.

Metformin reduces HGF-induced resistance to alectinib via the inhibition of Gab1.

Alectinib is a second-generation anaplastic lymphoma kinase (ALK) inhibitor that has sufficient clinical efficacy and satisfactory safety in ALK-positive non-small cell lung cancer (NSCLC) patients with or without brain metastasis. Alectinib has now become an important drug in the first-line treatment of advanced ALK-positive NSCLC; however, resistance is almost inevitable. The increased expression of hepatocyte growth factor (HGF) and its physiological receptor tyrosine kinase MET have been shown to be linked to acquired resistance to various tyrosine kinase inhibitors (TKIs), and this phenomenon has been observed in some ALK-positive NSCLC tumour tissues. In this study, we found that HGF levels in the culture supernatant of an ALK-positive cell line tended to increase with time and could be further increased by alectinib in a time-dependent manner. Exogenous or endogenous HGF did not cause resistance to the ALK/MET double-targeted small molecule inhibitor crizotinib, but it was an important cause of alectinib resistance. Furthermore, Gab1 was a key effector in the HGF/MET signal transduction pathway that mediated alectinib resistance. The antidiabetic drug metformin combined with alectinib overcame alectinib resistance triggered by HGF/MET through disrupting the complex between MET and Gab1, thereby inhibiting Gab1 phosphorylation and the activation of downstream signal transduction pathways. These results suggest that metformin combined with alectinib may be useful for overcoming alectinib resistance induced by the activation of the HGF/MET signalling pathway and improving the efficacy of alectinib.

Intracellular Galectin-9 Enhances Proximal TCR Signaling and Potentiates Autoimmune Diseases.

Galectin-9 is a risk gene in inflammatory bowel disease. By transcriptomic analyses of ileal biopsies and PBMCs from inflammatory bowel disease patients, we identified a positive correlation between galectin-9 expression and colitis severity. We observed that galectin-9-deficient T cells were less able to induce T cell-mediated colitis. However, several mouse-based studies reported that galectin-9 treatment induces T cell apoptosis and ameliorates autoimmune diseases in an exogenously modulated manner, indicating a complicated regulation of galectin-9 in T cells. We found that galectin-9 is expressed mainly inside T cells, and its secreted form is barely detected under physiological conditions. Endogenous galectin-9 was recruited to immune synapses upon T cell activation. Moreover, proximal TCR signaling was impaired in galectin-9-deficient T cells, and proliferation of these cells was decreased through an intracellularly modulated manner. Th17 cell differentiation was downregulated in galectin-9-deficient T cells, and this impairment can be rescued by strong TCR signaling. Taken together, these findings suggest that intracellular galectin-9 is a positive regulator of T cell activation and modulates the pathogenesis of autoimmune diseases.