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摘要： Given the undeniable and promising outcomes of chimeric antigen receptor (CAR) technology-based immunotherapy reported in recent years, this study aimed to develop anti-HER2 CAR NK cells as a novel therapeutic strategy for cancer immunotherapy. The NK-92 cell line was transduced with a recombinant lentiviral vector encoding an anti-HER2 construct, either with or without IL-15 co-expression. This yielded two distinct CAR NK cell populations: (1) anti-HER2 CAR NK cells and (2) IL-15-secreting anti-HER2 CAR NK cells. The cytotoxic effects of these engineered cells against the HER2-positive SK-BR-3 target cells were then evaluated using the PE-Annexin V and 7-AAD assays. Flow cytometry analyses were performed to assess CAR NK cell activity by measuring the expression of degranulation marker CD107a and intracellular levels of granzyme B and perforin, following surface and intracellular staining. Our findings demonstrated that anti-HER2 CAR NK cells and IL-15-secreting anti-HER2 CAR NK cells induced significantly higher levels of total apoptosis in HER-positive SK-BR-3 cells compared to mock-transduced (control) and non-transduced NK cells (control). The mean percentage (± SD) of CD107a was significantly higher in CAR NK cells co-cultured with SK-BR-3 cells compared to both control groups. Moreover, the mean expression (based on MFI) of granzyme B and perforin was significantly elevated in both CAR NK cell types following co-culture with HER2-positive SK-BR-3 cells. Notably, IL-15-secreting anti-HER2 CAR NK cells exhibited superior cytotoxic potential compared to their non-secreting counterparts. In summary, our findings demonstrate potent antitumor activity of anti-HER2 CAR NK cells. The promising results suggest that these engineered CAR NK cells, particularly those capable of IL-15 secretion, hold significant potential as a novel immunotherapeutic strategy for HER2-positive malignancies.

关键词： 肢体缺血-再灌注损伤   miR-675   CaMK   TNF-a   桃红四物汤  

摘要： 目的 基于miR-675-CaMKⅡ-炎症因子信号轴探讨桃红四物汤对肢体缺血-再灌注损伤的干预机制。方法 将雄性SD大鼠78只随机分为4组（对照组、模型组、mir-675阻断剂组和桃红四物汤组）；除对照组外，余下各组均复制缺血-再灌注损伤模型；成功后选取各组0、2、4、8h时段取大鼠骨骼肌和血清拿来做指标检测：HE染色法和DAPI法观察大鼠骨骼肌形态及病理性和细胞凋亡情况；RT-qPCR检测miR-675、CaMKⅡmRNA的表达，Western blot检测CaMKⅡ蛋白表达，ELISA法检测血清TNF-a表达情况。结果 HE染色结果显示，模型组骨骼肌损伤最严重，miR-675阻断剂组和桃红四物汤组损伤较模型组减轻。DAPI荧光染色显示，模型组细胞凋亡数量最多，凋亡率高于其他各组，差异有统计学意义（P<0.05）。RT-qPCR和Western blot的结果提示在同时段相比对照组：模型组、mir-675阻断剂组和桃红四物汤组的miR-675、CaMKⅡmRNA表达均高出对照组，CaMKⅡ蛋白的表达也增高（P<0.05）;mir-675阻断剂组和桃红四物汤组的miR-675、CaMKⅡmRNA的表达均低于模型组，CaMKⅡ蛋白的表达也降低（P<0.05），差异有统计学意义。ELISA血清检测提示各组TNF-a含量表达均高于对照组（P<0.05）;mir-675阻断剂组和桃红四物汤组的TNF-a含量表达均低于模型组（P<0.05）差异有统计学意义。结论 桃红四物汤可以减轻大鼠骨骼肌细胞在肢体缺血-再灌注中的损伤，机制可能与桃红四物汤参与调控miR-675并进一步影响CaMKⅡ蛋白、TNF-a等炎症因子的表达有关。

关键词： B淋巴细胞   细胞重编程   巨核细胞   UNC1999  

摘要： 血小板短缺是临床实践中的常见问题，基于细胞重编程的工程化血小板制备技术是解决该难题的一种潜在方案。本研究以B淋巴细胞为起始细胞，探索通过转录因子Gata1和FLI1驱动细胞重编程生成巨核细胞的可能性。研究发现，通过过表达转录因子Gata1，部分小鼠原代B淋巴细胞能够被诱导表达巨核细胞特异性标志物Cd41和Cd42，显示其向巨核细胞样细胞转分化的潜力。进一步研究表明，共表达FLI1和Gata1或使用Ezh2/Ezh1抑制剂UNC1999处理，均可显著提高细胞重编程效率。重编程细胞经过10～14 d培养，部分细胞呈现前血小板形态特征。值得注意的是，过表达BCR-ABL诱导的永生化B淋巴细胞仍保持对Gata1/FLI1诱导的响应性。综上所述，本研究表明B淋巴细胞具有重编程为巨核细胞的潜力，有望成为工程化血小板制备的起始细胞。此外，由于B淋巴细胞具备悬浮培养特性，并且在永生化过程中仍保持对重编程因子的响应性，基于B淋巴细胞的重编程体系可能在实际应用中更易于实现大规模制备。

关键词： OSMR pathway   chronic prurigo   neuroimmune itch pathways   prurigo nodularis   pruritus  

关键词： 慢性阻塞性肺疾病   急性加重   2型糖尿病   T细胞亚群   T细胞因子  

摘要： 目的 探讨合并2型糖尿病（T2DM）的慢性阻塞性肺疾病（慢阻肺）患者外周血T细胞亚群、T细胞因子水平及其预测慢阻肺急性加重的价值。方法 选取我院2022年11月至2024年8月收治的合并T2DM的慢阻肺患者,分为急性加重组和稳定组。收集所有纳入患者的临床资料及外周血T细胞亚群、T细胞因子水平,采用单因素及多因素Logistic回归分析急性加重的危险因素,并分析T细胞亚群与T细胞因子水平对慢阻肺急性加重的预测价值。结果 研究共纳入180例患者,急性加重组95例,稳定组85例。与稳定组相比,急性加重组患者调节性T细胞（Treg）比例、CD4+/CD8+比值及白细胞介素（IL）-10水平显著降低,IL-6、肿瘤坏死因子α（TNF-α）水平显著升高（均P<0.05）;多因素Logistic回归示CD4+/CD8+比值（OR=0.020,95%CI:0.002～0.183,P <0.001）、Treg细胞比例（OR=0.084,95%CI:0.034～0.209,P <0.001）、IL-10（OR=0.779,95%CI:0.661～0.918,P=0.003）是慢阻肺患者急性加重的保护因素,IL-6（OR=1.217,95%CI:1.077～1.375,P=0.002）、TNF-α（OR=1.302,95%CI:1.152～1.472,P <0.001）是慢阻肺患者急性加重的危险因素。基于独立影响因素构建回归预测模型:log（X|急性发作）=2.085-3.903×（CD4+/CD8+比值）-2.475×（Treg细胞亚群比例）+0.196×（IL-6）-0.250×（IL-10）+0.264×（TNF-α）。ROC分析示该模型预测患者急性加重的AUC为0.922（95%CI:0.882～0.963,P <0.001）;DCA曲线示该预测模型预测患者急性加重的净收益大于0。结论Treg细胞亚群、CD4+/CD8+比值、IL-6、IL-10及TNF-α均是影响合并T2DM的慢阻肺患者急性加重风险的重要影响因素,基于上述指标构建的回归模型对于预测合并T2DM的慢阻肺患者急性加重具有重要价值。

关键词： IL‐13   chronic rhinosinusitis with nasal polyps   club cells   dupilumab   nasal epithelial repair  

摘要： BACKGROUND:Club cells, originating from basal cells, can differentiate into ciliated or goblet cells in the lower respiratory tract, but their role in nasal epithelium during chronic rhinosinusitis with nasal polyps (CRSwNP) is not well understood. We aim to investigate the role of club cells in CRSwNP and their biological mechanism in an IL-13-induced inflammation model of human nasal epithelial cells (HNECs). METHODS:CRSwNP was classified into four endotypes (NE, NE, NE, and NE) based on neutrophil and eosinophil infiltration. The expression of basal (TP63), club (SCGB1A1), ciliated (βIV-tubulin), and goblet cells (MUC5AC) was assessed in healthy controls (n = 23) and CRSwNP (n = 73) using RT-qPCR, western blot (WB), and immunofluorescence. The effects of IL-13, Dupilumab, and STAT6 inhibitor (AS1517499) on cell differentiation were investigated in HNECs (n = 9) through RT-qPCR, WB, and flow cytometry (FCM). RESULTS:Club and ciliated cells significantly decreased in four endotypes of CRSwNP (all p < 0.001), while basal and goblet cells increased in neutrophilic and eosinophilic CRSwNP, respectively (all p < 0.05). In vitro, IL-13-induced significant cilia loss and MUC5AC secretion compared to IFN-γ and IL-17A (all p < 0.05), and FCM revealed a shift from SCGB1A1βIV-tubulin to SCGB1A1MUC5AC cells by IL-13-treated (all p < 0.05). Furthermore, Dupilumab mitigated IL-13-induced changes more effectively than AS1517499 (all p < 0.05). CONCLUSIONS:Club cells are crucial in nasal epithelial repair, which is notably compromised in CRSwNP. This impairment is highlighted in an IL-13-induced inflammation model of nasal epithelium and further underscored by the therapeutic effect of Dupilumab in restoring epithelial repair mechanisms.

摘要： Sweet syndrome (SS), also known as acute febrile neutrophilic dermatosis, is characterized by painful erythematous plaques or nodules with diffuse infiltration of mature neutrophils in the dermis. Most patients respond well to systemic corticosteroids, except for a few resistant cases. SS may be idiopathic or triggered by trauma, infection, drugs, and immune disorders. The exact pathogenesis, pathophysiology and precise treatment options beyond corticosteroids remain unclear. We applied anti-IL-36R monoclonal antibody, Spesolimab, to treat two SS patients and achieved favorable outcomes, however, the molecular mechanism is unproved. Here, we applied spatiotemporal transcriptomics at single-cell resolution and multiplex immunofluorescence on the skin lesion and blood samples from SS patients before, during, and after anti-IL-36R treatment. Releasing of neutrophil extracellular traps (NETs) was observed in the distinct NETosis neutrophil lineage infiltrating SS lesions, which secreted neutrophil elastase to splice full-length IL-36 proteins into their active forms, amplifying the inflammatory signal. From spatial view, the co-localized niches of heterogenous maturation neutrophils and IL36RN+ differentiated keratinocytes were found at the interface of the epidermis and dermis. The heterogenous maturation neutrophil activated IL-36 signaling was considered as rapid inflammatory response and was eliminated after treatment. Meanwhile, CD8+ T cells were also recruited and participated in the interferon signaling interacted with keratinocytes and neutrophils in the SS lesion, which was considered as delayed inflammatory response. Overall, a forward loop consisting of keratinocytes, heterogenous maturation neutrophils and CD8+ T cells was constructed to decipher the potential pathogenesis of SS, and IL-36 activated by neutrophil elastase released from heterogenous maturation neutrophils was one of the drivers of the loop. Moreover, IL-36 signaling was confirmed as a novel a

关键词： Alzheimer's disease   brain microvascular endothelial cells   mast cells   neuroinflammation   rapamycin   suramin  

摘要： Chronic blood-brain barrier (BBB) disruption due to impaired function of brain microvascular endothelial cells (BMECs) is commonly observed in neuroinflammatory and neurodegenerative conditions. Current treatment approaches are generally limited in their capacity to reduce this dysfunction, with the Akt/mTOR/GSK pathway modulator rapamycin showing recent promise in ameliorating neuroinflammatory BBB dysfunction. Understanding the role of early, cellular level BMEC dysfunction, particularly in the context of interplay with immune cells involved in neuroinflammation, such as mast cells (MCs), is important for identifying targets for therapeutic intervention related to BBB disruption. In the present work, we investigate primary human BMECs and human MC line HMC-1.2 dysfunction in response to inflammatory insult with TNF-α and paracrine interactions, with an emphasis on the Akt/mTOR/GSK pathway-an upstream regulator of angiogenesis and MC activation-and associated extracellular and intracellular cytokine production and oxidative stress. We further compare alterations in BMEC-MC paracrine inflammatory crosstalk in response to Akt/mTOR/GSK pathway modulator suramin (100 µM) relative to rapamycin (250 nM). In monoculture, TNF-α stimulation significantly increased oxidative stress-assessed through measuring PGE2-extracellularly in BMECs. Similarly, proangiogenic and pro-inflammatory cytokine and chemokine secretion was increased in both TNF-α stimulated BMEC and MC monocultures. Additionally, TNF-α stimulation increased BMEC levels of the Akt/mTOR/GSK pathway intermediates p-p70S6K and p-RPS6 and MC levels of p-GSK3α and p-GSK3β. Coculture of TNF-α stimulated BMECs and MCs resulted in a modest increase in extracellular PGE2, and effects on extracellular cytokine/chemokine levels were primarily limited to increases in pro-inflammatory CCL2, CCL3 and CCL5 relative to TNF-α-stimulated BMEC monoculture. In contrast, the levels of intracellular cytokines in MCs increased 2-100 f

摘要： Multiple sclerosis (MS) is an autoimmune and neuroinflammation disease characterized by axonal damage, inflammatory demyelination, and neurodegeneration. However, the precise mechanisms underlying MS pathogenesis remain largely unclear. Here, we identify ZNRF1, an E3 ubiquitin ligase, as a critical regulator of experimental autoimmune encephalomyelitis (EAE), a murine model that recapitulates the autoimmune demyelination features of MS. Mice lacking ZNRF1 exhibit increased susceptibility to EAE progression. Notably, ZNRF1 depletion in peripheral myeloid cells, but not in microglia, leads to enhanced immune cell infiltration into the central nervous system, resulting in demyelination and exacerbated disease severity. The heightened EAE severity in Znrf1-deficient mice is associated with increased polarization of Th1 and Th17 cells, elevated antigen-specific T helper cell proliferation, and amplified immune responses. Furthermore, following EAE induction, macrophages from Znrf1-deficient mice display elevated surface expression of MHC class II (MHC-II) molecules. Collectively, our findings suggest that ZNRF1 in peripheral myeloid cells plays a suppressive role in neuroinflammation by regulating MHC-II surface expression, thereby controlling antigen-specific T-cell proliferation and activation.

关键词： 登革病毒   NS1抗原   检测试剂   监管要求   技术审评  

摘要： 登革热是因登革病毒感染引起的急性蚊媒传染病，在热带和亚热带地区广泛流行。目前，我国登革热疫情主要是因输入引起的本土传播，疫情累及区域逐渐扩大，且发病率呈上升趋势，对人民群众的健康构成巨大威胁。早发现、早诊断、早治疗是登革热的核心防控原则，早期识别和及时救治是降低病死率的关键。登革病毒NS1抗原检测可作为实验室早期诊断的特异性指标，在登革热的早期诊断中具有重要作用。本文阐述了目前登革病毒NS1抗原检测试剂在我国的总体上市监管要求，同时根据作者的审评经验并结合登革病毒的病原学特点从主要原材料、分析性能研究、阳性判断值等方面探讨了登革病毒NS1抗原检测试剂的注册审评思路和审评要求，希望能够为相关检测试剂的开发和注册提供参考和帮助。