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关键词： 猴痘病毒   E8L抗原   单克隆抗体   mRNA免疫   单细胞测序  

摘要： 目的 基于mRNA免疫和单细胞测序技术筛选鉴定特异性靶向猴痘病毒细胞内成熟粒子表面包膜蛋白E8L的单克隆抗体。方法 体外合成E8L-mRNA，制备E8L-mRNA脂质纳米粒，免疫小鼠，引发免疫反应。根据血清抗体滴度，判断小鼠体内产生足够抗体后，对小鼠脾B细胞进行流式分选、建库测序，基于测序数据分析抗体轻重链序列，根据丰度排序筛选候选单克隆抗体并进行表达。采用酶联免疫吸附实验（ELISA）鉴定单克隆抗体的亲和力，将获得的高亲和力抗体结合免疫层析技术，应用于猴痘病毒蛋白的检测。结果 E8L-mRNA脂质纳米粒免疫小鼠，经流式分选出约150万个B细胞，单细胞测序后，筛选并表达纯化获得17个E8L候选单克隆抗体；经ELISA测定，其中2个单克隆抗体对猴痘病毒蛋白E8L具有高亲和力；将这2个抗体配对，结合免疫层析技术对猴痘病毒蛋白E8L进行检测，灵敏度可达0.5 ng/mL。结论 基于mRNA免疫和单细胞测序技术筛选鉴定获得了2个具有较高亲和力的抗猴痘病毒E8L单克隆抗体，具有检测猴痘病毒E8L蛋白的潜力。

关键词： 口蹄疫病毒   非复制型mRNA   非编码区   蛋白表达  

摘要： 口蹄疫是目前全球范围内最重要的动物传染病之一，所有偶蹄动物均易感。疫苗免疫是口蹄疫防控的首要选择。mRNA疫苗可以在体外快速设计合成和大规模生产，且能够诱导高效的保护性免疫应答，具有开发速度快、易制备、生物安全风险低等优点。其中，非编码区的设计是增强mRNA疫苗表达和效力的关键。为了获取获得高效的FMD mRNA疫苗，本研究体外合成了3种具有不同非编码区的口蹄疫P12A3C DNA与对应的mRNA，通过比较其在不同时间点和细胞系中的表达量差异，发现mRNA P12A3C-UTR3表达量优异且普适性最好。本研究为口蹄疫mRNA疫苗的研制奠定了基础，也为高效mRNA的序列设计提供了理论依据。

关键词： Cytokines   Immunology   Infectious disease   Malaria   NK cells  

摘要： *** infection can trigger high levels of inflammation that lead to fever and sometimes severe disease. People living in malaria-endemic areas gradually develop resistance to symptomatic malaria and control both parasite numbers and the inflammatory response. We previously found that adaptive NK cells correlated with reduced parasite load and protection from symptoms. We also found that murine NK cell production of IL-10 protected mice from experimental cerebral malaria. Human NK cells can also secrete IL-10, but it is unknown what NK cell subsets produce IL-10 or if this is affected by malaria experience. We hypothesized that NK cell immunoregulation may lower inflammation and reduce fever induction. Here, we showed that NK cells from participants with malaria experience make significantly more IL-10 than participants with no malaria experience. We then determined the proportions of NK cells that are cytotoxic and produce IFN-gamma and/or IL-10 and identified a signature of adaptive and checkpoint molecules on IL-10-producing NK cells. Lastly, we found that coculture with primary monocytes, Plasmodium-infected RBCs, and antibody induced IL-10 production by NK cells. These data suggest that NK cells may contribute to protection from malaria symptoms via IL-10 production.

关键词： Cell biology   Collagens   Extracellular matrix   Fibrosis   Therapeutics  

摘要： Dupuytren's disease is a common fibroproliferative disease of the palmar fascia of the hand, with advanced cases treated surgically. Anti-TNF injection has undergone phase 2 trials and may be effective in slowing early-stage disease progression. Here we sought to determine how new synthesis of type I collagen in Dupuytren's differs from normal palmar fascia samples and to analyze the role of TNF in aberrant collagen synthesis. Model nonfibrotic but fibrous connective tissues were used to analyze active type I collagen protein synthesis in development, aging, and degenerative disease, where it was restricted to early development and ruptured tissue. Dupuytren's tissue was shown to actively synthesize type I collagen, including abnormal type I collagen homotrimer. TNF-alpha reduced COL1A2 gene expression only in the presence of serum in 2D cell culture and had opposing effects on collagen protein production in the presence or absence of serum. TNF-alpha had only limited effects in 3D tendon-like constructs. Anti-TNF did not reduce type I collagen synthesis in 3D tendon-like constructs or prevent type I collagen homotrimer synthesis in Dupuytren's tissue. Hence, modulation of the TNF-alpha pathway in Dupuytren's disease is unlikely to prevent the pathological collagen accumulation that is characteristic of fibrosis.

关键词： 肾移植   IgA肾病   移植后排斥反应  

摘要： 目的：总结1例肾移植术后排斥反应合并免疫球蛋白A(IgA)肾病复发患者的临床资料，为肾移植术后相关并发症的诊治提供参考。方法：回顾性分析1例肾移植术后排斥反应合并IgA肾病复发患者的临床资料。结合文献复习，对该类患者的临床诊疗进行总结。结果：一位43岁男性终末期肾病患者，移植前患有IgA肾病且供体特异性抗体检测呈阴性，既往无致敏事件，移植后7个月出现混合性排斥反应且IgA肾病复发。给予强化免疫抑制和静脉注射免疫球蛋白治疗后症状好转，继续随访观察。结论：该病例主要特点包括诊断具有复杂病理变化，但临床表现不明显病人的挑战，这增加了误诊或延误诊断的风险，长期随访对于有效管理和监测这类病人至关重要。

关键词： germinal center   resident memory T   Tfh   T follicular helper   T regulatory type 1  

摘要： Germinal center (GC) reactions are tightly regulated to generate high-affinity antibodies. Although IL10+ Foxp3- follicular T cells have recently been described as contributing to the suppression of GC reactions, their differentiation, localization, and heterogeneity remain incompletely understood. Additionally, it remains unclear whether IL10+ Foxp3- follicular T cells represent a transient status or an independent subset. To address these gaps, we performed integrative single-cell analysis of transcriptomes, epigenomes, surface proteomes, and TCR repertoires in human tonsillar CD4+ T cells. Unbiased clustering revealed IL10+ Foxp3- follicular T cells as a transcriptionally and epigenetically unique subset. This subset exhibited features of both T follicular helper (Tfh) and T regulatory type 1 (Tr1) cells, and accordingly, hereafter, we call them T follicular regulatory type 1 (Tfr1) cells. Analysis using imaging mass cytometry and spatial RNA-TCR sequencing demonstrated their presence within GCs in humans. Bioinformatic analysis suggested that Tfr1 cells differentiate from GC-Tfh cells upon strong TCR stimulation, a finding corroborated by mouse in vivo experiments and time-series single-cell RNA-TCR sequencing of human in vivo CD4+ T cells. Of note, our bioinformatic analysis suggested that Tfr1 cells receive strong TCR signals from ICOS-Lhigh GC-B cells, likely representing high-affinity GC-B cells. Finally, we show that Tfr1 cells acquire a resident memory phenotype following an effector phase. Together, our findings suggest that high-affinity ICOS-Lhigh GC-B cells transform follicular T cells from GC-Tfh cells to Tfr1 cells, which likely become memory cells and reside in the lymphoid organ to support effective antibody production.

关键词： CTL   lipopeptide   MHC class I   TAP  

摘要： A novel subset of classical major histocompatibility complex class I molecules has recently been identified in rhesus monkeys that mediates the presentation of N-myristoylated lipopeptides, rather than conventional peptides, to CD8+ cytotoxic T lymphocytes (CTLs). For example, the rhesus Mamu-B*098 allomorph binds an N-terminal 5-mer fragment (C14 fatty acid-Gly-Gly-Ala-Ile-Ser;C14nef5) derived from the N-myristoylated SIV Nef protein and activates C14nef5-specific CTLs. Additionally, a transporter for antigen presentation (TAP)-independent cell-surface expression was observed for Mamu-B*098 in the in vitro transfection experiments, leading us to hypothesize that TAP-independent pathways may exist for CTL activation. To address this directly, we generated transgenic mice expressing Mamu-B*098 and analyzed its function under TAP-deficient conditions. We first confirmed that its expression level was unchanged on the surface of TAP-deficient cells compared with that of TAP-sufficient cells. Second, the CD8+ T cell population, but not the CD4+ T cell population, increased in TAP knockout (KO) mice as a result of the acquisition of Mamu-B*098 expression. Third, C14nef5-specific, Mamu-B*098-restricted CD8+ T cells were readily inducible in Mamu-B*098 transgenic/TAP KO but not in nontransgenic/TAP KO mice. Finally, the CD8+ T cells expressed cytolytic granule contents and functioned as CTLs. These findings provide evidence that in addition to conventional peptide-specific CTL responses that require TAP, an alternative TAP-independent pathway for CTL activation exists in primates. This novel pathway may be valuable when TAP is targeted by pathogenic viruses for immune evasion. We propose that the established concept of major histocompatibility complex class I biology may require modifications to incorporate TAP-independent pathways of lipopeptide-specific CTL responses.

关键词： 水稻矮缩病毒(RDV)   次要外壳蛋白P9   多克隆抗体  

摘要： 由水稻矮缩病毒（Rice dwarf virus,RDV）引起的水稻矮缩病是危害水稻安全生产的主要病毒病害，建立RDV的监测技术体系对于水稻矮缩病的田间早期预警与防控具有重要意义。本研究通过反转录PCR(Reverse transcription-PCR, RT-PCR)从RDV感染的水稻病株中扩增1056 bp的P9基因片段，并将其在大肠杆菌（Escherichia coli）BL21(DE3)中表达。以纯化的P9蛋白为抗原免疫新西兰雄兔，制备P9多克隆抗血清。酶联免疫吸附测定(Enzyme-linked immunosorbent assay, ELISA）结果显示该抗血清效价为1:32 000。蛋白质印迹（Westernblotting）结果表明经抗原亲和纯化获得的多克隆抗体可特异性识别RDV感染的水稻病株及带毒介体黑尾叶蝉中的RDV P9蛋白。进一步将该多克隆抗体应用于间接ELISA和斑点ELISA(dot-ELISA)分析，并结合RT-PCR对采集自福建省莆田市仙游县的田间样品，包括水稻、稗草及3种昆虫介体（黑尾叶蝉、电光叶蝉、褐飞虱）共223份样本进行检测。结果显示：相对于RDV，水稻和稗草的感病率分别为29.03%和19.05%，黑尾叶蝉、电光叶蝉和褐飞虱的带毒率分别为12.28%、2.56%和0。本研究成功制备了高效价、高特异性的RDV P9多克隆抗体，基于此建立的血清学检测方法不仅提升了水稻矮缩病诊断的可靠性，更为后续P9蛋白功能研究及RDV检测提供了重要技术支撑。

关键词： Analgesic Effects   Cancer Induced Bone Pain   Electroacupuncture   IFN-1   STING  

摘要： This study aimed to evaluate the effects of electroacupuncture (EA) on cancer-induced bone pain (CIBP) and investigate its interaction with the STING/IFN-1 pathway. A CIBP model was established in female rats. EA was administered for six consecutive days at bilateral L3-L5 Jia Ji points (EX-B2). EA-induced antinociception was evaluated through mechanical, thermal, and cold sensitivity assessments. EA significantly increased the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) in rats with CIBP (P<0.01). In the spinal cord of CIBP model rats, western blot analysis demonstrated that the application of EA upregulated the expression of STING, IRF3, and IFNAR (P<0.05). The ELISA results indicated that EA significantly increased the expression of IFN-α (P<0.005) and IFN-β (P<0.01) and reduced the expression of TNF-α and IL-1β (P<0.05). Immunofluorescence analysis revealed that STING was predominantly localized in microglia, with a minimal presence in neuronal cells. Furthermore, intrathecal administration of the STING antagonist C-176 attenuated the analgesic effects of EA in CIBP (P<0.05). In particular, EA reduced pro-inflammatory cytokines and increased anti-inflammatory cytokines, whereas the STING agonist, despite its analgesic effects, aggravated the inflammatory response. Both EA and STING agonists were effective in alleviating pain in rats with CIBP, possibly through the activation of the STING/IFN-1 pathway. In addition, EA exhibited an anti-inflammatory effect, underscoring its therapeutic potential in the management of CIBP.