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关键词： MHC   Human leukocyte antigen   Mouse model   Transgenic mice   Bacterial artificial chromosome   Chromosome engineering  

摘要： Humanized transgenic mice carrying human genes are useful for research on gene function and disease. Bacterial artificial chromosomes (BACs) that carry human genomic sequences with regulatory elements enable the expression of transgenes at physiological levels in vivo. To study complex biological phenomena involving multiple genes, techniques for co-introducing transgenes into mice have been developed;however, the introduction of multiple BACs remains laborious. The simultaneous integration of multiple gene loading vectors (SIM) system was developed to incorporate three or more gene-loading vectors (GLVs) using a mouse artificial chromosome (MAC) vector. This system allows for simultaneous site-specific incorporation of three GLVs into a single MAC with only one screening. However, the capacity for large constructs, such as BACs, has yet to be evaluated. This study is the first to demonstrate the development of multi-BAC transchromosomic (Tc) mice targeting the human leukocyte antigen (HLA) class I gene cluster (HLA-A, HLA-B, HLA-C) and beta-2-microglobulin (B2M) using the SIM system. By constructing a MAC using three BACs containing these genomic regions, we successfully generated HLA class I Tc mice. The technology to generate multi-BAC Tc mice will accelerate the analysis of complex life mechanisms involving multiple factors.

关键词： microtia   IL-6   intercellular communication   cartilage microspheres   development and regeneration of auricular cartilage  

摘要： Introduction Congenital microtia is a birth defect characterized by auricular underdevelopment, with unclear pathogenesis and unidentified pathogenic *** Differential expression analysis, weighted co-expression network analysis (WGCNA), protein-protein interaction (PPI) networks and support vector machine recursive feature elimination (SVM-RFE) identified the key biomolecules in microtia. Single-cell and intercellular communication analysis were used to decipher the key intercellular signaling pathway. We extracted primary cells and conducted Immuno precipitation mass spectrometry (IP-MS), co-Immuno precipitation (Co-IP) and RNA-sequencing (RNA-seq) to confirmed the mechanism. The intercellular communication network was confirmed through the cell co-culture system. Organoid and animal models further validated the role of key *** We found that IL-6 may be the key biomolecule in microtia. Normal ear cartilage tissue is mainly composed of chondrocytes, but microtia auricular ear tissue contained chondrocytes and stem cells. IL-6 signaling pathway is the main intercellular communication pathways in microtia. We extracted primary chondrocytes and stem cells, and proved that IL-6 promotes the growth and migration of primary cells. The binding of IL-6 and IL-6R and Glycoprotein 130 (GP130) and the activation of their downstream were confirmed. Furthermore, IL-6 signaling pathway was proved to involve in the intercellular communication of microtia. Cartilage microspheres demonstrated the role of IL-6 in regeneration of ear cartilage. The preventive intervention of adeno-associated virus (AAV) on pregnant mice confirmed the role of IL-6 in *** IL-6 signaling is the key biomolecule in the development and regeneration of auricular cartilage in microtia. IL-6 is a potential biomarker and preventive and therapeutic target for microtia patients.

关键词： epithelial-mesenchymal transition (EMT)   fibrinogen-like protein 1 (FGL1)   IL-6/STAT3   metastasis   Non-small cell lung cancer (NSCLC)  

摘要： Background: It has been reported that IL-6 induces the synthesis and secretion of fibrinogen-like protein 1 (FGL1) in liver cells as well as promotes the regeneration of liver cells. FGL1 is upregulated in human cancers, especially in non-small cell lung cancer (NSCLC). FGL1 is involved in the regulation of epithelial-mesenchymal transition (EMT) in gastric cancer (GC) cells. However, the role of IL-6/FGL1 signaling axis in the EMT process of NSCLC cells and its mechanism remain unclear. In this study, we investigated the role of FGL1 in mediating the metastasis and EMT processes of NSCLC cells, as well as the underlying signaling mechanisms. Methods: The Cancer Genome Atlas (TCGA) database and STARBASE database were used to analyze the biological information of FGL1 gene expression in NSCLC patients, and the database information was verified by clinical data. Transwell, Western blotting and microscopy were used to observe the effects of FGL1 on the metastasis and proliferation of NSCLC cells and the occurrence of EMT and its potential signaling pathway proteins. Results: Database analysis and clinical data showed that the prognosis of NSCLC patients with high FGL1 expression was poor. Cell phenotypic experiments showed that FGL1 silencing significantly reduced proliferation, migration and EMT of NSCLC cells, suggesting that the expression level of FGL1 may be significantly correlated with migration ability and EMT in lung cancer cells. In addition, the FGL1-neutralizing antibody inhibited EMT and metastasis of NSCLC cells in vivo. Further studies showed that IL-6 may mediate EMT by inducing FGL1 expression through the STAT3 pathway in lung cancer cells. Furthermore, treatment with a STAT3 inhibitor significantly inhibited the IL-6-induced FGL1 expression and EMT in NSCLC cells. Conclusions: IL-6 regulates FGL1 expression to mediate metastasis and EMT of NSCLC cells through the STAT3 signaling pathway. © AME Publishing Company.

关键词： REG gamma   PPP2CB   TBK1   IFN beta   Viral infection  

摘要： Although significant progressions in antiviral studies of IFN beta have been demonstrated, the role of the proteasome in modulating cross-talk between TBK1-IFN beta signaling and viral replication during viral infection is not fully elucidated. Here, we discover that deficiency of REG gamma, a proteasome activator, significantly reduces IFN beta production and increases viral replications in mice, leading to increased mortality in virus infection models. Our mechanistic study indicates that REG gamma interacts with and degrades the protein phosphatase subunit Protein Phosphatase 2 Catalytic Subunit Beta (PPP2CB). This degradation disrupts the dephosphorylation of TBK1 and its interaction with IRF3, resulting in the activation of IFN beta-mediated antiviral signaling. In response to viral infection, up-regulation of REG gamma in macrophages accelerates the degradation of PPP2CB, which increases the activation of TBK1-IRF3-IFN beta axis and thereby restricts viral replications and pathology. Interestingly, IFN beta enhances REG gamma expression in viral infection, forming a positive feedback regulatory loop. In conclusion, our work demonstrates that REG gamma is a positive modulator of IFN beta signaling during antiviral response, highlighting that this procedure is regulated via REG gamma degradation of PPP2CB and provides a new insight into the coordination between antiviral response and proteasome activity. Thus, REG gamma-proteasome activity and phosphatase PPP2CB may be potential targets in host defense against viruses.

摘要： Objectives Idiopathic inflammatory myopathies (IIMs) are rare autoimmune disorders. Genetic association studies have highlighted the role of human leukocyte antigen (HLA) polymorphisms in IIM. We aimed to characterise the nonadditive effects (dominance and interaction) of HLA alleles on IIM risk. Methods This study included a total of 3206 IIM cases and 11,697 controls of European ancestry. HLA alleles were imputed using a multiancestry HLA reference panel. Logistic regressions were conducted to estimate the nonadditive effects of HLA alleles. Clinical subgroup analysis, calculation of phenotypic variance explained, and stepwise conditional analyses were conducted to further characterise these effects. Results We identified significant nonadditive effects in 5 HLA genes, particularly in the core alleles of ancestral haplotype 8.1 (8.1 AH), including HLA-B*08:01 ( P = 3.93 × 10 −13 ), HLA-C*07:01 ( P = 3.14 × 10 −8 ), HLA-DQA1*05:01 ( P = 3.03 × 10 −9 ), HLA-DQB1*02:01 ( P = 3.53 × 10 −23 ), and HLA-DRB1*03:01 ( P = 8.47 × 10 −21 ). Notable risk difference between heterozygotes and homozygotes was observed in IIM, such as HLA-DRB1*03:01 (homozygote odds ratio [OR], 2.17; heterozygote OR, 3.13). In the interaction model, HLA-DQA1 and HLA-DRB1 showed specific significant allelic interactions. The nonadditive effect model explained a larger proportion of phenotypic variance than the model with additive effects alone. Conditional analysis indicated the independent nonadditive effect of HLA-DRB1*03: 01 in 8.1 AH and amino acid residue Arg-74 in HLA-DRB1. Conclusions This study identified significant nonadditive effects within the HLA region of IIM. A genetic risk model including nonadditive effects could provide more accurate individual risk estimates. These findings highlight a complex role of HLA heterozygosity in the development of IIM and support further research into HLA nonadditive effects with clinical relevance.

关键词： SARS-CoV-2疫苗   腺病毒载体   细胞免疫   体液免疫  

摘要： 目的 研究融合表达新型冠状病毒（Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2）Omicron变异株受体结合域蛋白（RBD）与衣壳蛋白（N）的非复制型5型重组腺病毒的免疫原性，旨在为开发针对SARS-CoV-2 Omicron变异株的高效疫苗提供理论依据和实验基础。方法 利用重叠PCR方法获得RBDgs3N片段，同源重组得到质粒pKAd5ESRBDgs3N，重组质粒转染HEK293细胞后收获和扩增重组病毒，氯化铯密度梯度离心法纯化病毒，有限稀释分析法测定病毒滴度，Western Blot方法鉴定融合蛋白表达情况。将重组腺病毒肌注免疫BALB/c小鼠，利用间接ELISA方法和酶联免疫斑点技术检测小鼠特异性免疫应答。结果 体外纯化获得滴度为1.168×1010IU/mL的重组腺病毒AdV5-RBDgs3N。重组腺病毒体外感染HEK293细胞可成功表达RBDgs3N融合蛋白。单剂肌注免疫能显著诱导小鼠产生针对新冠野毒株（WT）和变异株（Delta,Omicron）RBD的IgG抗体，抗体滴度分别为103.677 8,103.878 5,104.454 9，针对保守的N蛋白产生的特异性抗体滴度为104.942 2。RBD特异性细胞免疫的IFN-γ水平为1 452 SFC/10～6个细胞。结论 本研究构建的RBD和N基因融合的非复制型5型重组腺病毒能在BALB/c小鼠体内分别诱导高水平的针对RBD和N蛋白的特异性抗体及RBD特异的细胞免疫，为进一步评价该重组腺病毒作为潜在新冠疫苗提供了研究基础。

关键词： Fibrosis   Immunology   Nephrology   Therapeutics  

摘要： Low-intensity pulsed ultrasound stimulation (LIPUS) has become increasingly appreciated for its therapeutic effect on kidney diseases. However, its role and biological mechanism in treating chronic kidney disease (CKD) remain poorly defined. Here, we revealed that LIPUS was applied in a safe range with an intensity of 25-315 mW/cm2. Daily LIPUS at an intensity of 315 mW/cm2 ameliorated ischemia-reperfusion (IR)-induced tubular injury and renal fibrosis, accompanied by the remarkable downregulation of IL-1R. Transcriptome sequencing showed that LIPUS significantly down-regulated IL-1R and its downstream genes in IL-1β-stimulated IR-injured mice. LIPUS effectively reversed IL-1β-induced tubular injury and reduced the production of profibrotic cytokines by down-regulating IL-1R in vivo and in vitro. Renal proximal tubule-specific Il1r1 knockout mice exhibited milder renal tubular injury and fibrosis after IR injury. However, LIPUS did not ameliorate IR injury in proximal tubule-specific Il1r1 knockout mice. Collectively, daily LIPUS at an intensity of 315 mW/cm2 relieves IR-induced tubular injury and fibrosis, potentially through down-regulating tubular IL-1R.

摘要： The immune response induced by gluten is the result of molecular mechanisms involving gliadin peptides, DQ2 or DQ8 glycoproteins, and the interaction with T lymphocyte receptors. DQ8-glia-alpha 1 is an immunodominant peptide present in gliadin from wheat Triticum spelta that interacts with the DQ8 protein, as proven through transgenic mouse models. The research was carried out by performing a computational analysis aimed at finding antagonistic peptides of the DQ8-glia-alpha 1 peptide, i.e. peptides obtained by varying its amino acids to maintain or even enhance the binding towards DQ8 and at the same time to prevent an immune response by a reduced interaction with the T lymphocyte receptors. Crystallographic structures of DQ8 and three different T-cell receptors were taken as experimental starting systems, the peptide-protein interaction was modelled by molecular dynamics simulations and molecular interaction field calculations, and the optimal mutations of the peptide sequence were identified by using multivariate analysis. The method provided a list of nine immunodominant peptide candidates, which were produced by chemical synthesis and validated by tests on transgenic mice. The results showed that immunization with the peptide (DQ8-glia-alpha 1, designated M1) induced in vitro antigen-specific secretion of IFN-gamma restricted to the M1 peptide alone. M1 also stimulated antigen-specific secretion of the regulatory cytokine IL-10. A peptide (i.e., M10) was identified as a potential therapeutic molecule for down-regulating the inflammatory condition triggered by the DQ8-glia-alpha 1 immunodominant peptide in CD.

关键词： 疗效   上呼吸道感染   磷酸奥司他韦   炎性反应   喜炎平注射液  

摘要： 探讨磷酸奥司他韦配合喜炎平注射液对儿童病毒性上呼吸道感染INF-γ、Th1/Th2平衡的影响。方法 取2023年8月~2024年8月本院收治的病毒性上呼吸道感染患儿为样本开展研究,共有94例患儿参与本次研究,基于电脑生成随机数法将上述患儿分为两组,在对症处理基础上,分别给予磷酸奥司他韦、喜炎平注射液+磷奥司他韦治疗,依次记作对照组(n=47)、试验组(n=47),观察两组治疗前后症状、炎性因子指标、INF-γ水平、Th1/Th2差异,分析不同治疗方案对患儿Th1/Th2平衡、预后的影响。结果 试验组治疗3天后的体温比对照组低(P<0.05)。两组治疗1周后的症状评分、炎性因子(PCT、CRP、SAA、IL-6)水平均较治疗前下降,INFγ、NO、Th1/Th2较治疗前升高,而且试验组变化幅度更大(P<0.05)。试验组发热、咽喉肿痛、鼻塞流涕和咳嗽症状消退时间均短于对照组(P<0.05)。结论 磷酸奥司他韦配以喜炎平注射液疗法可进一步改善病毒性上呼吸道感染患儿的病情、炎性反应、免疫应答情况,有助于患儿身体早期恢复。