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关键词： AMD   Aqueous humor   Assay qualification   Biomarker   IL-33   Immuno-PCR  

摘要： Immuno-polymerase chain reaction (iPCR) is an analytical technology that combines the specificity of antibody reagents with the sensitivity of polymerase chain reaction (PCR) signal amplification. Here we describe the optimization and qualification of an ultra-sensitive iPCR method designed to detect and quantify active (reduced form) interleukin-33 (IL-33) in human samples. The modified assay incorporates several improvements over a previous version utilized in research studies. The changes aimed at making the assay more suitable to the rigorous requirements promulgated to biomarker assays supporting clinical development of new human therapeutics. The assay can accurately measure sub-picogram/mL levels of reduced IL-33, and has a dynamic range of 977–1000,000 fg/mL. We found this iPCR-based format to be versatile, and we believe it can be adapted to detect and quantify other low-level cytokines in human matrices when limited sample volumes are available.

关键词： Glutathione transferase Omega-1   NLRP3 inflammasome   GSTO1-1 inhibitor   Covalent inhibitor   SAR   Anti-inflammation   Anti-Cancer   ALARM NMR   a La assay to detect reactive molecules by nuclear magnetic resonance   AMD   age-related macular degeneration   ASC   apoptosis-associated speck-like protein containing a caspase-recruitment domain   ATP   adenosine triphosphate   BMDM   bone marrow-derived macrophage   Boc   tert   butyloxycarbonyl   C   max   maximum plasma concentration   DCE   1,2-dichloroethane   DCM   dichloromethane   DHA   dehydroascorbate   DIAD   diisopropyl azodicarboxylate   DIPEA   N   N   diisopropylethylamine   DMAP   4-dimethylaminopyridine   DMEM   Dulbecco's modified eagle media   DMF   dimethylformamide   DMSO   dimethyl sulfoxide   dppf   1,1′-Bis(diphenylphosphino)ferrocene   DTT   dithiothreitol   EDC   1-ethyl-3-(3-dimethylaminopropyl)carbodiimide   EDTA   ethylenediaminetetraacetic acid   FBS   fetal bovine serum   GSH   glutathione   GST   glutathione transferase   GSTO1-1   glutathione transferase Omega-1   GSTO2-2   glutathione transferase Omega-2   HATU   hexafluorophosphate azabenzotriazole tetramethyl uranium   hLa   human Lupus antigen   HMDS   hexamethyldisilazane   HMQC   heteronuclear multiple quantum correlation   HPLC   high-performance liquid chromatography   HRMS   high-resolution mass spectrometry   HWE   Horner-Wadsworth-Emmons   IBD   inflammatory bowel disease   IC   50   half-maximal inhibitory concentration   IL   interleukin   ip   intraperitoneal   K   I   the concentration of inhibitor required for half of the maximum potential rate of covalent bond formation   k   inact   the maximum potential rate of covalent bond formation   LLOQ   lower limit of quantitation   LPS   lipopolysaccharide   MS   multiple sclerosis   NADPH   reduced nicotinamide adenine dinucleotide phosphate   NEK7   NIMA-related kinase 7   NF-κB   nuclear factor κB   NLRP3   nucleotide-binding oligomerization domain-like receptor family protein 3   4-NPG   S   (4-nitrophenacyl)glutathione   PBS   phosphate buffered saline   PK   pharmacokinetics   ROS   reactive oxygen species   SAR   structure-activity relationship   TFA   trifluoroacetic acid   THF   tetrahydrofuran   TLC   thin-layer chromatography   TLR4   Toll-like recep  

摘要： Glutathione transferase Omega-1 (GSTO1-1) plays a key role in the activation of the NLRP3 inflammasome. Consequently, it is involved in the pathology of multiple inflammatory conditions as well as cancer. Small-molecule inhibitors that bind covalently to its active-site cysteine have been developed as potential therapeutics. In this study, the X-ray co-crystal structure of the reported GSTO1-1 inhibitor C5-1 in complex with GSTO1-1 was solved, and used to elaborate a comprehensive SAR analysis of C5-1 . The inhibitory profile of compounds was evaluated in a spectrophotometric assay with purified recombinant GSTO1-1 and in a cell-based assay measuring IL-1β release. The k inact / K I values of selected covalent inhibitors were determined as well as the biochemical selectivity of these compounds for GSTO1-1 over GSTO2-2, GSTA1-1 and GSTP1-1. The C5-1 chemotype was assessed to be a useful biochemical tool for GSTO1-1 inhibitor development with our analysis revealing that compound 10u to be the most potent GSTO1-1 inhibitor identified in this study. Both C5-1 and 10u showed a capacity to attenuate inflammation in mice and to significantly enhance the cytotoxicity of cisplatin, suggesting their future potential application in the treatment of inflammation and cancer.

关键词： Cancer-associated fibroblasts   Chemotherapy   IL-6   Lung squamous cell carcinoma   Tumor microenvironment  

摘要： Chemoresistance remains a major obstacle in the treatment of lung squamous cell carcinoma (LUSC), often leading to suboptimal clinical outcomes. Among the key contributors to this resistance are cancer-associated fibroblasts (CAFs), which are increasingly recognized for their tumor-supportive roles. Despite this, the molecular pathways through which CAFs promote chemoresistance in LUSC are not fully elucidated. This study found that CAFs-derived interleukin-6 (IL-6) upregulated the expression of Specificity Protein 1 (SP1) and the ATP-binding cassette transporter B7 (ABCB7) in LUSC cells exposed to cisplatin and docetaxel. In vitro assays showed a marked decrease in apoptosis in tumor cells co-cultured with CAFs. Consistent with these findings, in vivo xenograft models demonstrated that IL-6-producing CAFs reduced the antitumor efficacy of both chemotherapeutic agents. Elevated serum IL-6 levels also emerged as a potential indicator of poor response to chemotherapy. Our findings suggest that IL-6 secreted by CAFs impairs the cytotoxic effects of cisplatin and docetaxel in LUSC, partly through activation of the PI3K/AKT/NF-κB signaling axis. Targeting this IL-6-mediated pathway may offer a promising strategy to overcome chemoresistance and enhance therapeutic outcomes in patients with LUSC.

关键词： Flexography printing   Graphite   Carbon dots   Interleukin 8   Non-faradaic biosensor   Disability  

摘要： Interleukin-8 (IL-8) is a key biomarker linked to inflammation and disability in neonates. However, current IL-8 detection methods are often costly, labor-intensive, and require highly trained personnel. While electrochemical techniques have been employed for sensitive IL-8 quantification, they typically rely on redox probes and threeelectrode electrochemical cells, leading to issues such as toxicity, prolonged fabrication time, and increased waste generation. Additionally, conventional electrochemical biosensors fabrication techniques are expensive and time-consuming, limiting their scalability for mass disease screening. In this study, we introduce a low-cost, non-faradaic electrochemical nanobiosensor for the direct detection of IL-8. The sensor consists of interdigitated graphite/carbon dot conjugates flexographically printed onto a flexible polyimide substrate. The printed layer's physical properties were systematically characterized using SEM, AFM and surface profilometer, and biofunctionalization was achieved using aminopropyltriethoxysilane (APTES) and glutaraldehyde. Successful surface modification was confirmed through ATR-FTIR and EDS elemental mapping. Electrochemical impedance spectroscopy (EIS) analysis demonstrated the nanobiosensor's response to varying IL-8 concentrations, with capacitance, Zmod, Zreal, and Zimag measurements. Among these, Zimag exhibited the highest sensitivity, with a response of 2.7 k Omega/log(ng/mL) and a detection limit of 50 pg/mL-well below the clinically established threshold of 600 pg/mL. This study demonstrates that, in addition to capacitance, there are multiple parameters that warrant exploration in non-faradaic biosensors to improve their sensing performance. The nanobiosensor fabrication via flexographic printing enables scalable, cost-effective production while maintaining high sensitivity and selectivity through a non-faradaic detection mechanism. This work paves the way for the development of affordable, mass-produ

摘要： Mast cells (MC) serve as pivotal sentinels in the regulation of immune responses and inflammation, yet their function in lung adenocarcinoma (LUAD) remains largely neglected. To decode their heterogeneity, we perform single-cell transcriptomic analysis of LUAD-infiltrating MCs. Our study uncovers the complexity in MC composition and identifies 9 distinct states, including proinflammation, chemotaxis, and antigen presentation. The proinflammatory MC subset, characterized by high IL-18 expression, is associated with improved outcomes for LUAD patients. This pro-inflammatory property is regulated by the activation of NLRP3 inflammasome within MCs, resulting in the formation of GSDMD pores and successive pyroptosis. Moreover, these MCs enhance the innate-like anti-tumor activity of MAIT cells by upregulating NKG2D and IFN-γ through the cytokine-activation mechanism. Our results uncover an unappreciated state of MCs and describe an inflammasome-dependent, MC-mediated regulation of MAIT cells in LUAD. These findings diversify our understanding of the functional repertoire and mechanistic equipment of MCs and MAIT cells, and suggest a potential therapeutic target for cancer treatment. © The Author(s) 2025.

摘要： Glioblastoma remains a challenging indication for immunotherapy: the blood-brain barrier hampers accessibility for systemic treatments and the immunosuppressive microenvironment impedes immune attack. Intratumoral therapy with the proinflammatory cytokine interleukin-12 (IL-12) can revert immunosuppression but leakage into the circulation causes treatment-limiting toxicity. Here we engineer an IL-12Fc fusion cytokine with reduced binding to the neonatal Fc receptor FcRn. FcRn-silenced IL-12Fc avoids FcRn-mediated brain export, thus exhibits prolonged brain retention and reduced blood levels, which prevents toxicity. In murine glioblastoma, FcRn-silenced IL-12Fc induces more durable responses with negligible systemic cytokine exposure and boosts the efficacy of radio- and chemotherapy. It triggers anti-tumor responses independently of peripheral T cell influx or lymphopenia and leads to inflammatory polarization of the tumor microenvironment in patient-derived glioblastoma explants. FcRn-silencing of IL-12Fc may unlock the full potential of IL-12 for brain cancer therapy and could be further applied to containing the activity of other therapeutics targeting neurological diseases. © The Author(s) 2025.

摘要： Although the immune system plays a crucial role in hypertension, its underlying mechanisms remain unclear. While extensive research has explored the gut-immune relationship in various diseases, the role of gut immunity in salt-sensitive (SS) hypertension is not well understood. In this study, we aimed to elucidate the role of gut immunity in the development of SS hypertension by analyzing immune cells in the gut and blood using single-cell RNA sequencing (scRNA-seq). Six-week-old Dahl SS rats were fed either a normal salt diet (NSD;0.4% NaCl) or a high-salt diet (HSD;4% NaCl) for four weeks. Blood pressure was measured weekly using the tail-cuff method. After four weeks, small intestine lamina propria cells (SILPs) and peripheral blood mononuclear cells (PBMCs) were isolated and subjected to scRNA-seq. Our results demonstrated an increase in gut B and T follicular helper (Tfh)-like cells in SS rats. Furthermore, KEGG pathway analysis revealed that HSD upregulated IL-6 receptor (IL-6R)-JAK-STAT signaling in B cells and Tfh-like cells. These findings provide novel insights into the potential of targeting the gut immunity in the treatment of SS hypertension. © The Author(s) 2025.

关键词： 嵌合抗原受体T细胞   质量控制   放行检测  

摘要： 嵌合抗原受体T细胞免疫疗法(CAR-T)作为肿瘤免疫治疗的突破性技术,通过基因修饰患者T细胞靶向杀伤肿瘤,展现出特殊疗效,为癌症患者带来了新希望。CAR-T治疗产品的质量与其临床疗效紧密相关,如何持续生产高质量的CAR-T治疗产品,已成为CAR-T细胞疗法实现广泛应用的关键。本文对自体CAR-T细胞产品生产过程的质量控制要点和产品的质量放行进行了综述,以期为更多CAR-T细胞产品的研究和应用提供参考。