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关键词： atopic dermatitis   dupilumab (anti-IL4R alpha antibody)   electrical impedance spectroscopy   ex vivo human skin   inflammation   interleukin-13   interleukin-22   interleukin-4   skin barrier  

摘要： Background Atopic dermatitis (AD) is a chronic type-2 inflammatory skin disease characterized by eczema and epithelial barrier dysfunction. Along with the type-2 cytokines IL-4 and IL-13, IL-22 contributes to AD pathogenesis. To date, most skin studies rely on reconstructed keratinocytes, which do not represent the real skin *** Here, we report the distinct effects of IL-4, IL-13, and IL-22 on bio-stabilized human skin with intact barriers and immune *** Spatial transcriptomics on AD-lesions and non-lesional skin was performed. Ex vivo skin barrier integrity was evaluated using electrical impedance spectroscopy (EIS), RNA-sequencing, and untargeted proteomics, complemented by analyses of skin biopsies from dupilumab-treated AD *** Spatial transcriptomics demonstrated that AD lesions showed reduced expression of key barrier genes, including CLDN1, FLG, and FLG2. IL-4, IL-13, and IL-22 disrupted the skin barrier in the ex vivo human skin. Combining type-2 cytokines and IL-22 alone downregulated genes critical for barrier function and keratinization. In addition, IL-4 and IL-13 downregulated antimicrobial peptides, while IL-22 upregulated them. Interestingly, IL-4 and IL-13 reduced IL-22R alpha 1, and IL-22 upregulated IL-4R alpha, suggesting immune cross-regulation. Proteomic analysis confirmed that all three cytokines (IL-4, IL-13, and IL-22) reduced the expression of key skin barrier proteins, particularly filaggrin and claudin-1. Dupilumab treatment of AD patients for 3 months restored IL-4/IL-13-dysregulated genes, whereas it had limited effect on IL22-associated *** This comprehensive study provides insights into the distinct immune profiles following IL-4, IL-13, and IL-22 stimulation on human skin, highlighting their complex interplay in disrupting skin barrier function and modulating innate immune responses.

关键词： HIV reservoir   spatial transcriptomics   lymph node immune regulation   CD8 T cell regulation   HLA-E  

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摘要： Bone metastasis, which is associated with adverse outcomes, is a serious health concern for renal cell carcinoma (RCC) patients, especially considering the limited therapeutic options. In this study, we investigated the expression profiling of circRNAs in five primary RCC samples and RCC-bone metastases (Bone Met) using high-throughput screening and identified an upregulated circRNA in Bone Met (hsa_circ_0016459, circKCNK2). Notably, overexpression of circKCNK2 could promote osteoclast differentiation and accelerate the destruction of osteolytic bone metastasis by stimulating IL-11 secretion. Additionally, we observed that RCC with a high circKCNK2 expression could benefit from an anti-IL-11 strategy rather than a denosumab-based therapeutic regimen. At the molecular level, circKCNK2 is competitively bound to EDC4 (a scaffold protein of P-bodies). The interaction between circKCNK2 and EDC4 alpha-helical disrupted the combination of DCP1 and DCP2, which weakened the function of P-bodies and resulted in an increased level of IL-11 mRNA and finally activated STAT-3 signaling in osteoclast precursors (OPs). This axis could be blocked with a mutation of EDC4 alpha-helical. Further experiments revealed that increased circKCNK2 production in bone metastases was attributed to decreasing expression of heterogeneous nuclear ribonucleoprotein U (hnRNPU) under an acidic microenvironment. Our findings suggest that circKCNK2 could have a critical role in linking P-bodies to IL-11/STAT-3 signaling. Developing a secure and effective gene delivery system targeted at circKCNK2 is promising for RCC therapy.

摘要： Adaptive immunity is critical for combating pathogens and generating immunological memory. Central to adaptive immunity are myeloid cells, which activate upon pathogen detection. Activation is essential for inflammatory cytokine release and requires a complex series of molecular events to facilitate cytokine expression. However, although the transcriptional machinery regulating cytokine expression is well defined, it is apparent that trafficking machinery also has to be re-programmed to facilitate cytokine secretion. We demonstrate through quantitative total internal-resonance fluorescence (TIRF) microscopy that short-term inflammatory stimulation with lipopolysaccharide (LPS) is sufficient to up-regulate interleukin-6 (IL-6) secretion in dendritic cells. Through bioinformatic analysis of phosphoproteomic data, we demonstrate that the activation of dendritic cells rapidly reprograms SNARE-associated trafficking machinery. We link the enhanced rate of IL-6 secretion to the phosphorylation of the SNARE protein VAMP3. This releases VAMP3 from the chaperone WDFY2, enabling the trafficking of IL-6/VAMP3+ vesicles to the plasma membrane. VAMP3 then complexes with STX4, facilitating IL-6 secretion. Finally, we found that VAMP3-dependent IL-6 secretion is polarised, reconciling findings that, in dendritic cells, IL-6-positive vesicles are non-polarised, yet VAMP3 vesicles are largely polarised.

关键词： Autoimmune disease   Humanized mouse model   Inflammatory markers   NOD-scid IL2Rynull (NSG) mice   Rheumatoid arthritis   Therapeutic evaluation  

摘要： Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation and joint destruction. Replicating human manifestations of RA in animal models remains challenging, however, due to heterogeneity of the disease. In this study, a humanized mouse model for RA was developed and validated using NOD-scid IL2Rnull (NSG) mice engrafted with peripheral blood mononuclear cells (PBMCs) from RA patients (NSG-RA). RA symptoms were induced using lipopolysaccharide and a cocktail of antibodies against type II collagen. Pathological manifestations were assessed through clinical scoring of hind paw swelling, histological analysis, and evaluation of RA-specific markers in plasma and joints using Luminex, RT-PCR, and RNA-seq. NSG-RA mice exhibited increased levels of RA-specific markers, an influx of inflammatory cells into the synovium, bone erosion, and elevated levels of human autoantibodies. Enriched RNA-seq pathway analysis revealed activation of the RA disease pathway, along with the TNF and IL-17 signaling pathways. Treatment with prednisolone or infliximab ameliorated disease symptoms and decreased levels of inflammatory markers. These findings indicate that the NSG-RA model offers a translational tool for studying RA pathogenesis and testing novel therapeutic approaches.

关键词： 牛病毒性腹泻病毒   核心蛋白C   原核表达   多克隆抗体   间接ELISA  

摘要： 为建立基于牛病毒性腹泻病毒(bovine viral diarrhea virus，BVDV)核心蛋白C的间接ELISA检测方法，本研究通过PCR扩增出BVDV C基因，克隆至原核表达载体pET-32a，构建重组质粒pET-32a-C，IPTG诱导表达纯化后免疫BABL/c小鼠制备多克隆抗体，以C蛋白为包被抗原，建立间接ELISA检测方法。结果显示，pET-32a-C重组质粒构建成功，重组C蛋白大小约为30 kDa，能够以可溶形式表达，30 ℃、0.8 mmol/L IPTG诱导6 h可溶性表达最佳；免疫BABL/c小鼠后制备血清抗体效价为1∶128 000，能够特异性识别C蛋白。该ELISA检测方法的最适条件为：0.5 μg/mL抗原37 ℃包被2 h，1% BSA 37 ℃封闭3 h，1∶16 000稀释血清孵育2 h，二抗孵育40 min，避光显色10 min，阴阳性临界值判定标准为0.224，具有良好的特异性和重复性。利用本方法对未免疫接种的疑似BVDV感染的牛血清进行检测，与RT-qPCR核酸检测方法相比，二者阳性符合率达94.73%。上述结果表明，基于C蛋白建立的间接ELISA抗体检测方法特异性强、灵敏度高、重复性好，为BVDV临床诊断及开发基于C蛋白的间接ELISA检测试剂盒奠定基础。

关键词： bystander activation   IL-2   T cells  

摘要： In response to an infection, the host T cell compartment develops immunological memory to ensure rapid responses upon re-infection with the same pathogen. However, these memory responses can also modulate immune reactions to unrelated pathogens through bystander activation. Herein, T cells are activated in an antigen-independent manner, often triggered by innate cytokines or Toll-like receptor ligands. To uncover new strategies for modulating immune responses, it is essential to deepen our understanding of this alternative mechanism of T cell activation, particularly in humans. Therefore, we studied the response of human CD8+ T cells to innate bystander stimuli in vitro. Thereby, we measured the induction of activation markers and proliferation using flow cytometry, and depleted or blocked several potential modulatory components to identify mediators of bystander CD8+ T-cell responses. Our study demonstrates that CD8+ T cells can be activated as bystander cells in response to innate stimuli present during a viral infection, including IL-15, IFN-alpha, and TLR7/8 and 9 agonists (R848 and CpG). Depletion experiments demonstrated that these bystander responses are dependent on monocytes and CD4+ T cells. In addition, we revealed that the bystander responses to the innate stimuli are highly reliant on IL-2 signaling. Altogether, our study underscores the pivotal role of IL-2 in mediating bystander responses of CD8+ T cells to innate stimuli, revealing a novel mechanism of immune response modulation during viral infections.