Endosomal toll-like receptors play a key role in activation of primary human monocytes by cowpea mosaic virus

The plant virus, cowpea mosaic virus (CPMV), has demonstrated a remarkable capacity to induce anti-tumour immune responses following direct administration into solid tumours. The molecular pathways that account for these effects and the capacity of CPMV to activate human cells are not well defined. Here, we examine the ability of CPMV particles to activate human monocytes, dendritic cells (DCs) and macrophages. Monocytes in peripheral blood mononuclear cell cultures and purified CD14+ monocytes were readily activated by CPMV in vitro, leading to induction of HLA-DR, CD86, PD-L1, IL-15R and CXCL10 expression. Monocytes released chemokines, CXCL10, MIP-1α and MIP-1β into cell culture supernatants after incubation with CPMV. DC subsets (pDC and mDC) and monocyte-derived macrophages also demonstrated evidence of activation after incubation with CPMV. Inhibitors of spleen tyrosine kinase (SYK), endocytosis or endocytic acidification impaired the capacity of CPMV to activate monocytes. Furthermore, CPMV activation of monocytes was partially blocked by a TLR7/8 antagonist. These data demonstrate that CPMV activates human monocytes in a manner dependent on SYK signalling, endosomal acidification and with an important contribution from TLR7/8 recognition.     

Influence of HIV-1 V2 sequence variability on bacterial translocation in antiretroviral naïve HIV-1 infected patients

HIV-1 V2 domain binds α4β7, which assists lymphocyte homing to gut-associated lymphoid tissue. This triggers bacterial translocation, thus contributing to immune activation. We investigated whether variability of V2 ^179-181binding site could influence plasma levels of lipopolysaccharide (LPS) and soluble cluster of differentiation 14 (sCD14), markers of microbial translocation/immune activation. HIV gp120 sequences from antiretroviral naïve patients were analyzed for V2 tripeptide composition, length, net charge, and potential N-linked-glycosylation sites. LPS and sCD14 plasma levels were quantified. Clinical/immuno-virologic data were retrieved. Overall, 174 subjects were enrolled, 8% with acute infection, 71% harboring a subtype B. LDV^179-181 was detected in 41% and LDI in 27%. No difference was observed between levels of LPS or sCD14 according to different mimotopes or according to other sequence characteristics. By multivariable analysis, only acute infection was significantly associated with higher sCD14 levels. In conclusion, no association was observed between V2 tripeptide composition and extent of bacterial translocation/immune activation.     

TRPM7干扰载体修复低氧/复氧致大鼠心肌细胞系H9C2损伤

目的研究瞬时受体电位通道7(TRPM7)干扰载体对低氧大鼠心肌细胞系(H9C2)修复功能的影响及其机理。方法建立H9C2心肌细胞低氧/复氧模型,构建TRPM7干扰表达载体转染H9C2细胞,用RT-qPCR与Western blot检测H9C2细胞低氧诱导因子(HIF1-α)和TRPM7的表达,流式细胞计量术检测胞内钙离子水平([Ca^2+]i)和细胞凋亡,确定TRPM7对H9C2心肌细胞的修复作用。结果H9C2细胞转染TRPM7干扰载体后,细胞的HIF1-α和TRPM7表达显著下降(P<0.05),[Ca^2+]i降低及凋亡率减少(P<0.05)。结论TRPM7干扰载体可能通过PI3K/Akt通路对低氧H9C2心肌细胞有显著的修复作用。     

一个Usher综合征Ⅰ型家系的临床表型分析及基因诊断

目的筛查一个Usher综合征Ⅰ型家系的致病变异位点,分析其基因型-表型对应关系。方法详细采集先证者的临床表型及家族史,应用高通量测序技术对先证者进行全外显子组检测。应用Sanger测序对疑似致病变异以及家系其他成员的携带情况进行验证。结果先证者10岁时出现夜盲、白内障等症状,之后发生视网膜变性,并随年龄增加出现耳聋症状。高通量测序及Sanger测序提示其携带MYO7A基因c.2694+2T>G及c.6028G>A复合杂合变异。其姐携带相同的变异位点,且表型与先证者相似。先证者女儿携带MYO7A基因c.6028G>A杂合变异,表型无异常。结论明确了一个Usher综合征Ⅰ型家系的遗传学病因,并丰富了该病的表型与基因型数据库,为遗传咨询提供了依据。     

COL7A1基因复合杂合变异所致的隐性营养不良型大疱性表皮松解症家系的遗传学分析

目的对1个隐性营养不良型大疱性表皮松解症家系进行基因检测及产前诊断。方法利用PCR-Sanger测序技术检测患者COL7A1基因的全部外显子及其侧翼区的潜在变异,之后进行家系验证及产前基因诊断。结果Sanger测序显示患者COL7A1基因存在c.7289delC(p.Pro2430Glnfs*36)及c.7474C>T(p.Arg2492*)复合杂合变异,分别遗传自其母亲和父亲,在100名健康对照中未检测到上述变异。产前诊断胎儿COL7A1基因c.7289位置未见变异,c.7474位置存在C>T杂合变异,判断为携带者。结论明确了1例隐性营养不良型大疱性表皮松解症家系COL7A1基因的致病性变异,并成功进行了产前诊断。     

儿童7型腺病毒肺炎特点分析

目的 分析儿童7型腺病毒肺炎的临床特征,以提高诊疗水平、改善预后。方法 回顾性分析2019年4月~7月我院收治的42例7型腺病毒肺炎患儿的临床资料,包括临床表现、实验室检查指标及影像学结果、恢复期肺功能检查、脏器功能受累情况、合并其他病原感染情况、治疗结局。结果 共42例患儿中,2岁以下29例（69.05%）,均有发热,热峰＞40℃ 33例（78.57%）,热程≥2周19例（45.24%）。42例患儿LDH、AST、ALT均升高,ALB降低;随着热程时间延长,LDH、AST水平升高,ALB下降,其余指标均无明显差异。影像学表现节段性实变,9例患儿初期胸片或CT表现以单侧节段性实变为主,23例患儿以双侧散发与节段性实变为主,入院1~3 d内复查胸片或CT,双侧实变迅速发展至26例（61.90%）。肺外合并症主要为血液系统损害（30.95%）为主。病原感染主要以合并肺炎支原体IgM阳性13例（30.95%）为主。呼吸支持包括鼻导管给氧9例（21.42%）,无创通气24例（57.14%）,机械通气9例（21.42%）。住院期间死亡3例,3例患儿因病情危重转至上级医院,其中2例死亡,1例遗留严重后遗症;其余36例（85.71%）治愈、好转出院。结论 儿童7型腺病毒肺炎热峰高,热程长,LDH、AST明显升高,影像学进展快,常伴肺内外各种损害。临床疑诊7型腺病毒肺炎时应尽快完善病原学,动态监测LDH、AST水平及影像学变化,及早治疗。     

Characterization of antibody and memory T-cell response in H7N9 survivors: a cross-sectional analysis

ObjectivesDespite the importance of immunological memory for protective immunity against viral infection, whether H7N9-specific antibodies and memory T-cell responses remain detectable years after the original infection is unknown.MethodsA cross-sectional study was conducted to investigate the immune memory responses of H7N9 patients who contracted the disease and survived during the 2013–2016 epidemics in China. Sustainability of antibodies and T-cell memory to H7N9 virus were examined. Healthy individuals receiving routine medical examinations in a physical examination centre were recruited as control.ResultsA total of 75 survivors were enrolled and classified into four groups based on the time elapsed from illness onset to specimen collection: 3 months (n = 14), 14 months (n = 14), 26 months (n = 28) and 36 months (n = 19). Approximately 36 months after infection, the geometric mean titres of virus-specific antibodies were significantly lower than titres in patients 3 months after infection, but 16 of 19 (84.2%) survivors in the 36-month interval had microneutralization (MN) titres ≥40. Despite the overall declining trend, the percentages of virus-specific cytokine-secreting memory CD4⁺ and CD8⁺ T cells remained higher in survivors at nearly all time-points in comparison with control individuals. Linear regression analysis showed that severe disease (mean titre ratio 2.77, 95% CI 1.17–6.49) was associated with higher haemagglutination inhibition (HI) titre and female sex for both HI (1.92, 1.02–3.57) and MN (3.33, 1.26–9.09) antibody, whereas female sex (mean percentage ratio 1.69, 95% CI 1.08–2.63), underlying medical conditions (1.94, 95% CI 1.09–3.46) and lack of antiviral therapy (2.08, 95% CI 1.04–4.17) were predictors for higher T-cell responses.ConclusionsSurvivors of H7N9 virus infection produced long-term antibodies and memory T-cell responses. Our findings warrant further serological investigation in general and high-risk populations and have important implications for vaccine design and development.     

A familial case of overgrowth syndrome caused by a 9q22.3 microdeletion in a mother and daughter

Microdeletions in the 9q22.3 chromosomal region can cause macrosomia with characteristic features, including prenatal-onset overgrowth, metopic craniosynostosis, hydrocephalus, developmental delay, and intellectual disability, in addition to manifestations of nevoid basal cell carcinoma syndrome (NBCCS). Haploinsufficiency of PTCH1 may be responsible for accelerated overgrowth, but the mechanism of macrosomia remains to be elucidated. We report a familial case with a 9q22.3 microdeletion, manifesting with prenatal-onset overgrowth in a mother and post-natal overgrowth in her daughter. Although both were clinically diagnosed with NBCCS, they had characteristic features of 9q22.3 microdeletion, especially the daughter. Microarray comparative genomic hybridization analysis revealed a 4.0 Mb deletion of chromosome 9q22.3 in both individuals. Among the 11 reported patients of overgrowth and/or macrosomia, a 550 Kb region encompassing PTCH1, C9orf3, FANCC, and 5 miRNAs is the most commonly deleted region. The let-7 family miRNAs, which are involved in diverse cellular processes including growth and tumor processes, were identified in the deleted regions in 10 of 11 patients. Characteristic features of 9q22.3 microdeletion might be associated with decreased expression of let-7.     

The Generation of Zebrafish Mariner Model Using the CRISPR/Cas9 System

Targeted genome editing mediated by clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) technology has emerged as a powerful tool for gene function studies and has great potential for gene therapy. Although CRISPR/Cas9 has been widely used in many research fields, only a few successful zebrafish models have been established using this technology in hearing research. In this study, we successfully created zebrafish mariner mutants by targeting the motor head domain of Myo7aa using CRISPR/Cas9. The CRISPR/Cas9-generated mutants showed unbalanced swimming behavior and disorganized sterocilia of inner ear hair cells, which resemble the phenotype of the zebrafish mariner mutants. In addition, we found that CRISPR/Cas9-generated mutants have reduced number of stereociliary bundles of inner ear hair cells and have significant hearing loss. Furthermore, phenotypic analysis was performed on F0 larvae within the first week post fertilization, which dramatically shortens data collection period. Therefore, results of this study showed that CRISPR/Cas9 is a quick and effective method to generate zebrafish mutants as a model for studying human genetic deafness. Anat Rec, 303:556–562, 2020. © 2019 American Association for Anatomy     

缺血预适应对大鼠保留肾单位手术中缺血再灌注损伤的保护作用及其机制研究

目的 探讨缺血预适应(IPC)动员肾祖细胞(RPC)归巢在保留肾单位手术(NSS)中对肾脏缺血再灌注损伤(IRI)的保护作用及其发生机制。方法 选取2~3月龄、体质量250~300 g的雄性SD大鼠54只,建立切除右肾的单肾模型后采用数字表法随机分为3组,每组18只:假手术组(Sham组,无血管夹闭),NSS组(肾动脉夹闭45 min后行NSS),IPC组(先进行肾动脉夹闭15 min,再灌注10 min预处理,再行NSS)。分别在术后12、24、72 h每组各取出6只大鼠,收集血液及肾组织标本,之后采用颈椎脱臼法处死大鼠。观察项目:(1)检测血肌酐(SCr)、尿素氮(BUN);(2)组织病理学检查及肾小管损伤评分;(3)在24 h观察IPC对肾组织中RPC数量的影响,以及IPC对基质细胞衍生因子(SDF-1)、受体CXCR7表达水平的影响。结果 (1)术后12、24、72 h 时3组大鼠SCr和BUN值比较, IPC组分别为(65.0±10.78)、(91.5±15.12)、(52.6±11.68)μmol/L和(14.78±2.77)、(18.31±4.99)、(9.41±2.73)mmol/L,NSS组分别为(80.5±12.63)、(116.9±14.32)、(83.7±11.43)μmol/L和(18.58±4.18)、(28.86±5.64)、(19.49±3.83)mmol/L, Sham组分别为(41.5±7.36)、(39.7±7.55)、(42.7±7.15)μmol/L和(7.72±1.75)、(7.40±1.98)、(6.83±2.09)mmol/L;除72 h时IPC组与Sham组SCr和BUN值比较差异无统计学意义(P＞0.05)外,在12 h和24 h,IPC组均高于Sham组、低于NSS组,差异均有统计学意义(P值均＜0.05)。(2)术后12、24、72 h肾小管损伤评分IPC组和NSS组均高于Sham组,术后12、24 h IPC组较NSS组肾小管损伤评分低,差异均有统计学意义(P值均＜0.05)。(3)术后24 h,IPC组和NSS组大鼠肾组织中RPC数量明显增加、SDF-1和CXCR7表达显著升高,差异均有统计学意义(P值均＜0.05)。结论 IPC可促进RPC归巢,缓解NSS中IRI损伤程度,保护肾功能,SDF-1/CXCR7轴可能在这一动员过程中发挥了重要作用。