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关键词： 祛风壮骨合剂   类风湿性关节炎   体内成分分析   细胞因子   UPLC-Q-exactive orbitrap-MS  

摘要： 目的：采用类风湿性关节炎（RA）动物模型评价祛风壮骨合剂活性，并使用超高压色谱串联高分辨质谱（UHPLC-HRMS）解析大鼠血清、尿液和粪便中的化学成分。方法：48只大鼠随机分为空白组、模型组、甲氨蝶呤组、雷公藤组和祛风壮骨合剂高、低剂量组，每组8只。除空白组外，其余大鼠采用完全弗式佐剂（FCA）法建立RA动物模型。造模成功后，甲氨蝶呤组灌胃给予甲氨蝶呤1.05 mg/（kg·w），雷公藤组灌胃给予雷公藤多苷3.15 mg/（kg·d），祛风壮骨合剂高、低剂量组分别灌胃给予祛风壮骨合剂2.0、0.5 mL/（kg·d），连续14 d。观察各组大鼠体质量、足趾肿胀度、肝脏和踝关节组织受损程度及血清内细胞因子含量水平；同时，收集祛风壮骨合剂低剂量组大鼠血清、尿液、粪便三种生物样本，运用UPLC-QE-MS技术对祛风壮骨合剂入血、入尿、入粪成分进行定性分析与系统解析。结果：与模型组比较，祛风壮骨合剂能恢复模型组大鼠降低的体质量，并能有效抑制RA大鼠足肿胀，下调RA大鼠血清中TNF-α的含量水平，上调SOD和IL-10含量水平（P <0.05）。在祛风壮骨合剂中共发现化合物45个，其中酚类13个、蒽醌类6个、脂肪酸类6个、黄酮类4个、环烯醚萜苷类4个和香豆素类化合物2个；在祛风壮骨合剂低剂量组大鼠血清、尿液、粪便三种生物样品中共发现成分29个；其中，仅在血清中检测到的成分有9个，仅在尿液中检测到的成分有4个，仅在粪便中检测到的成分有3个；仅在血清与尿液中检测到的共有成分0个，仅在血清与粪便中检测到的共有成分1个，仅在尿液与粪便中检测到的共有成分6个；血清、尿液、粪便三种生物样本中均检测到的有成分有6个。结论：祛风壮骨合剂的抗类风湿关节炎活性良好，UHPLC-QE-MS技术能快捷、准确、较全面地鉴定RA模型大鼠体内的化学成分，为祛风壮骨合剂的药效物质基础研究奠定基础。

关键词： benralizumab   eosinophil   eosinophilic granulomatosis with polyangiitis   interleukin-5   mepolizumab  

摘要： Eosinophilic granulomatosis with polyangiitis (EGPA) is a necrotizing vasculitis characterized by extravascular granulomas and eosinophilia in both blood and tissues. Eosinophils, which play a critical role in the pathophysiology of EGPA, require interleukin (IL)-5 for maturation in the bone marrow and migration to tissues. Glucocorticoids and immunosuppressants have been the cornerstone of treatment;however, their side effects have imposed a significant burden on many patients. Mepolizumab, an antibody that binds to and neutralizes IL-5, demonstrated efficacy in controlling disease activity in EGPA in the MIRRA trial conducted in 2017. In 2024, benralizumab, an IL-5 receptor alpha antagonist, was shown to be non-inferior to mepolizumab in efficacy against EGPA in the MANDARA trial. Both drugs were originally used for severe asthma and have benefited EGPA by reducing eosinophil counts. Due to differences in pharmacological structure and pharmacokinetics, the degree of eosinophil suppression varies between the two agents, and recent studies suggest that they may also affect inflammatory and homeostatic eosinophils differently. This review summarizes the latest insights into the pathophysiology of EGPA, highlights the similarities and differences between the two drugs, and discusses future treatment strategies for EGPA based on current clinical unmet needs, including drug selection.

摘要： Long-lived memory B cells developing from germinal centers (GCs) are important contributors to protective immunity. In this issue of Immunity, Luo et al. report that during primary responses, T cell-derived interleukin-9 potentiates differentiation of GC memory precursors into functional memory B cells in a manner dependent on the transcriptional regulator ZBTB18.

关键词： eosinophils   Th2 cytokines   tartaric acid   colitis   mouse model  

摘要： Eosinophils are granulocytes involved in the effector phase of type 2 T cell immune responses, which are elevated in inflammatory conditions like ulcerative colitis (UC) and other allergic diseases. UC is a chronic inflammatory colon disease, marked by excessive eosinophil infiltration and elevated Th2 cytokines, which contribute to mucosal inflammation and tissue damage. Dietary factors, including certain organic acids, can influence UC progression by modulating gut immune responses. This research is the first to explore the dose-dependent effects of tartaric acid (TA), a naturally occurring organic acid widely used in the food industry, on eosinophil activation and Th2 cytokine response in both normal mice and a dextran sulfate sodium (DSS)-induced colitis model. Normal mice were treated with TA at varying doses (5 mu g, 25 mu g, and 50 mu g/mouse/day), while colitis mice received 50 mu g TA. Eosinophil activation markers (CD11b+, SiglecF+, and CCR3+), Th2 cytokines (IL-4, IL-13, and IL-31), and IL-17 were assessed in peripheral blood leukocytes, lymph nodes, and splenocytes using flow cytometry. Additionally, mRNA expression levels of eosinophil-associated chemokines and cytokines in the splenocytes were quantified with real-time qPCR. Our results demonstrate a dose-dependent effect of TA, with the highest dose (50 mu g) significantly increasing eosinophil activation markers, Th2 cytokines, IL-17, and mRNA expression of SiglecF, CCL11, and toll-like receptor 4 in normal mice. In colitis mice, treatment with 50 mu g TA showed marked increases in IL-13 levels compared to those of untreated colitis mice, reflecting increased eosinophil recruitment to inflamed tissues. Moreover, mRNA expression of IL-5R alpha was elevated in normal mice and colitis mice administered with TA. These results suggest that TA enhances eosinophil proliferation, the upregulation of their regulatory molecules, and Th2 immune profiles, potentially worsening the severity of colitis.

关键词： 布鲁氏菌   BP26蛋白   单克隆抗体   单链抗体  

摘要： 犬布鲁氏菌病是一种主要由犬种布鲁氏菌感染引起的重要人畜共患病，其精准诊断是控制该病的重要策略之一。BP26蛋白是布鲁氏菌的一种主要免疫原性蛋白，其作为靶标在开发布鲁氏菌诊断试剂中具有重要价值。本研究首先通过PCR方法将bp26基因克隆至表达载体pCold I中，转化至大肠杆菌BL21（DE3）中进行表达并纯化BP26蛋白。随后，将该蛋白免疫接种BALB /c雌性小鼠，采用传统杂交瘤技术制备抗BP26蛋白单克隆抗体，并通过免疫印迹实验验证其反应原性。之后，提取杂交瘤细胞株总RNA，反转录为cDNA，通过PCR方法获得抗体重链可变区(VH)和轻链可变区(VL)基因片段，进而通过重叠PCR构建成VL-linker-VH结构的单链抗体基因（scFv），将其克隆至表达载体pCold TF，转化至大肠杆菌BL21（DE3）中进行表达纯化，获得TF-scFv单链抗体。最后，通过免疫印迹实验和ELISA实验分析TF-scFv与单克隆抗体竞争结合BP26蛋白的反应。本研究制备的BP26单克隆抗体和scFv抗体为开发犬布鲁氏菌病诊断试剂奠定基础。

关键词： Canine   Intestinal epithelial cells   Intestinal organoids   MHC class II   IFN-gamma   Translational medicine   Comparative medicine  

摘要： BackgroundsThe interplay between intestinal epithelial cells (IECs), the immune system, and the gut microbiome is pivotal for maintaining gastrointestinal homeostasis and mediating responses to ingested antigens. IECs, capable of expressing Major Histocompatibility Complex (MHC) class II molecules, are essential in modulating immune responses, especially CD4 + T cells, in both physiological and pathological contexts. The expression of MHC class II on IECs, regulated by the class II transactivator (CIITA) and inducible by cytokine IFN-gamma, has been traditionally associated with professional antigen-presenting cells but is now recognized in the context of inflammatory conditions such as inflammatory bowel disease (IBD). In veterinary medicine, particularly among canine populations, MHC (or Dog Leukocyte Antigen, DLA) expression on IECs underlines its significance in intestinal immune pathologies, yet remains underexplored. This study aims to leverage canine intestinal organoids as a novel in vitro model to elucidate MHC class II expression dynamics and their implications in immune-mediated gastrointestinal diseases, bridging the gap between basic research and clinical application in canine *** colonoids derived from healthy dogs showed significant expression of MHC class II and its promoter gene, CIITA, after IFN-gamma treatment. This MHC class II induction was even more pronounced in differentiated colonoids cultured in Wnt-3a-depleted *** study provides insights into the role of IECs as antigen-presenting cells and demonstrates the use of intestinal organoids for investigating epithelial immune responses in inflammatory conditions.

关键词： Epitopes  

摘要： Therapeutic drug monitoring (TDM) is increasingly valuable for tailoring personalized therapy, particularly in managing chronic inflammatory diseases where overtreatment can cause significant side effects. Monoclonal antibodies (mAbs), a primary therapeutic approach for these conditions, face challenges from antidrug antibodies (ADAs), which can reduce mAb bioavailability and efficacy. To address these issues, we utilized Tumor Necrosis Factor alpha (TNF-alpha) as a binding moiety in a fluorescence-based programmable nanosensor within the NanoHYBRID (NH) platform developed by Ulisse Biomed S.p.A. By directly conjugating TNF-alpha to DNA probes, we developed a rapid, homogeneous, affinity-based assay capable of detecting multiple mAbs targeting distinct epitopes on the same protein. This NH platform effectively detected therapeutic concentrations of clinically relevant mAbs, such as Infliximab, Adalimumab, and Golimumab, in blood serum samples in a one-step process, bypassing the need for time-intensive washing steps. Moreover, the NH sensor exhibited heightened sensitivity to the presence of ADA, which impacted drug quantification, indicating its utility for monitoring bioavailable mAb levels. Compared to ELISA, the NH platform demonstrated superior sensitivity to ADAs, suggesting its potential as a highly specific, modular solution for TDM. This modular design allows the NH platform to create multiepitope nanosensors capable of measuring bioavailable mAbs in a single step.

关键词： Cyclic voltammetry  

摘要： Methyl parathion (MP) is a widely used pesticide;it is recognized as being toxic to both target and non-target species, posing serious risks to environmental and human health. Monitoring and controlling MP residues is thus essential, necessitating the development of innovative sensors that are highly sensitive, selective, and reproducible. In the present study, an efficient electrochemical MP sensor is proposed based on silver nanoparticles (AgNPs) in conjunction with graphene oxide/ionic liquid (GO/IL) on screen printed electrodes (AgNPs@GO/IL@SPCE). The AgNPs were synthesized via a cost-effective wet-chemical process and characterized using UV-Vis spectroscopy and transmission electron microscopy (TEM). The modified electrodes were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). The active surface area and charge transfer were examined by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), respectively. The modified electrodes' electrocatalytic performance towards the reduction of MP was investigated by CV, complemented by semiempirical quantum chemistry calculations to elucidate the interaction and the electrochemical reduction mechanism of MP. The sensor demonstrates a remarkable limit of detection of 0.009 mu mol L-1 within a linear range of 0.025 to 200 mu mol L-1. It has an excellent analytical performance in terms of selectivity, reproducibility, and long-term stability over 60 days. The designed sensor was effectively used to inspect MP in groundwater and surface water samples, with recovery values ranging from 95.60% to 99.68%.

关键词： nail psoriasis   dermoscopy   IL-17A inhibitors   IL-23 inhibitor   real-world cohort   NAPSI  

摘要： Objective To compare the real-world clinical efficacy and safety of interleukin (IL)-17A inhibitors (secukinumab [SEC] and ixekizumab [IXE]) versus the IL-23 inhibitor guselkumab (GUS) in patients with nail psoriasis, with a focus on site-specific biologic therapeutic responses (nail matrix vs. nail bed) in a 24-week prospective observational cohort. Methods This cohort enrolled 65 adult patients with plaque psoriasis and dermoscopy-confirmed nail involvement, stratified into three treatment groups: SEC (n=25), IXE (n=20), and GUS (n=20). Outcome assessments at baseline and week 24 included: Nail Psoriasis Severity Index (NAPSI) with domain-specific scoring (matrix/bed) by dermoscopic evaluation using a 10x polarized handheld device;Psoriasis Area and Severity Index (PASI), Body Surface Area (BSA);Dermatology Life Quality Index (DLQI). Safety was monitored through treatment-emergent adverse events (TEAEs). Results (1) By week 24, PASI, BSA, DLQI and NAPSI scores had significantly decreased from baseline in all groups (P<0.001). (2) By week 24: SEC, IXE, and GUS groups saw nail matrix NAPSI score improvements of 65.9%, 60.5%, and 51.5%, with 68%, 55%, and 30% achieving NAPSI 60;Nail bed NAPSI score improvements were 58.8%, 68.6%, and 65.8%, with 28%, 65%, and 40% achieving NAPSI 60;Total NAPSI score improvements were 62.7%, 64.6%, and 53.7%, with 44%, 70%, and 30% achieving NAPSI 60. (3) All patients in the SEC and IXE groups achieved PASI 75, compared to 85% in the GUS group. SEC showed PASI 90 and PASI 100 response rates of 80% and 36%, while IXE of 60% and 30%. (4) TEAEs were mild, including: injection site reactions: 15% (IXE group);eczematous rashes: 8% (SEC group). No TEAEs were reported in the GUS group, and no serious adverse events occurred in any group. Conclusion IL-17A inhibitors and the IL-23 inhibitor demonstrated significant efficacy in improving both nail and skin lesions in psoriasis. Notably, IL-17A inhibitors exhibited superior overall efficacy com

关键词： brain drug delivery   endothelial inflammation   hemorrhagic stroke   intracerebral hemorrhage   targeted nanoparticles  

摘要： Intracerebral hemorrhage (ICH) is a lethal and highly morbid form of stroke for which there is no disease-specific therapy. Inflammation after ICH is an important mechanism of secondary damage, and the inflamed endothelium in ICH is a promising therapeutic target as it is the gateway for inflammation in the brain. Systemic therapies that target inflammation have been unsuccessful in stroke, in part due to side effects or poor brain delivery. We hypothesized that targeting mRNA encoding IL-10, a potent anti-inflammatory cytokine, to the brain vasculature would improve outcomes in an experimental mouse model of ICH. We manufactured lipid nanoparticles (LNPs) using microfluidics, packaged them with IL-10 mRNA, and decorated them with antibodies against vascular cellular adhesion molecule (VCAM), which can bind the inflamed brain endothelium after ICH. VCAM LNPs distributed to the brain ~4x more than nonspecific LNPs and expressed their cargo in the brain at 10x higher levels. Treatment with VCAM-LNPs containing IL-10 mRNA led to ~69% reduction in hematoma size at 72 hours after ICH and ~65% improvement in motor behavior in our model, with no improvement in vascular leakage. Finally, we observed that VCAM-LNPs primarily target infiltrated macrophages and neutrophils. VCAM-IL10-LNPs significantly increased plasma and brain levels of IL10. Our data provide preliminary data for a promising therapeutic and delivery strategy for ICH, and shed light on the relationship between inflammation and vascular leakage. Future experiments will seek to understand how serial dosing affects LNP expression in our model and whether treatment at later time points after ICH can still confer therapeutic effects.