膜辅助蛋白CD46 rs1970530遗传变异对肝癌发病风险的影响

背景与目的：膜辅助蛋白CD46可保护宿主细胞免受补体依赖的细胞毒性作用，研究表明，CD46可能作为肝细胞癌（hepatocellular carcinoma，HCC）患者的潜在生物标志物。探讨CD46基因表达及启动子区遗传变异与HCC发病风险的关系。方法：通过基因表达谱交互分析（gene expression profiling interactive analysis，GEPIA）在线网站分析HCC组织和正常肝组织CD46表达的差异；2011—2015年在华北理工大学附属唐山市工人医院和华北理工大学附属唐山市人民医院经病理学检查确诊且未经放化疗的240例HCC患者作为病例组，对照组为同时期入院体检的500名健康人群。采用聚合酶链反应-限制性片段长度多态性分析（polymerase chain reaction-restriction fragment length polymorphism，PCR-RFLP）法进行基因分型，检测两组基因型频率和等位基因频率，评估CD46 rs1970530遗传变异与HCC发病风险的关系。结果：CD46在HCC组织与正常组织中的表达差异有统计学意义（P<0.05）。经非条件logistic回归分析发现，CD46 rs1970530至少携带一个G等位基因型在病例组及对照组之间差异有统计学意义（OR=0.666，95% CI：0.448~0.990，P<0.05）；两组间等位基因G频率差异有统计学意义（OR=0.689，95% CI：0.478~0.994，P<0.05）。分层分析结果显示，至少携带一个G等位基因者可降低高年龄组（>60岁）（P=0.048）和男性（P=0.023）人群的HCC发病风险，在女性和低年龄组（≤60岁）中差异无统计学意义（P>0.05）。按吸烟状态进行分层分析，rs1970530变异与HCC易感性无明显关联（P>0.05）。结论：HCC中CD46基因高表达及CD46 rs1970530遗传变异影响HCC发病风险。     

Methodological advances in the design of peptide-based vaccines

The tremendous advances in genomics, recombinant DNA technology, bioengineering and nanotechnology, in conjunction with the development of high-end computations, have been instrumental in the process of rational design of peptide-based vaccines. The use of peptide vaccines was limited owing to their inherent instability when systemically administered; however, advanced formulation techniques have been developed for their systemic delivery, thereby overcoming their degradation, clearance, cellular uptake and off-target effects. With the rise of sophisticated immunological predictors and experimental techniques, several methodological advances have occurred in this field. This review examines contemporary methods to identify and optimize epitopes, engineer their immunogenic properties and develop their safe and efficient delivery into the host.     

Tfh-cell-derived interleukin 21 sustains effector CD8+ T cell responses during chronic viral infection

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中药影响实验性自身免疫性脑脊髓炎CD4+T细胞亚群分化的研究进展

实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)是由CD4⁺T细胞介导的中枢神经系统脱髓鞘性疾病,是国际公认研究多发性硬化(multiple sclerosis,MS)的动物模型。CD4⁺T细胞作为EAE模型的主要免疫应答细胞,存在多个亚群,可分泌多种细胞因子,参与病情进展。CD4⁺T细胞增殖分化及功能紊乱与EAE发病机制密切相关。目前临床尚无治疗MS的有效药物,多以对症应用皮质类固醇激素为主,但不良反应严重,且易产生药物依赖。近年来中药因作用温和、药物安全性较高、成本低、耐受性好等优点逐渐走进大众视野,针对中药对EAE中CD4⁺T细胞亚群分化的影响展开论述,为临床用药提供更多理论基础。     

CD4 receptor diversity represents an ancient protection mechanism against primate lentiviruses

Infection with human and simian immunodeficiency viruses (HIV/SIV) requires binding of the viral envelope glycoprotein (Env) to the host protein CD4 on the surface of immune cells. Although invariant in humans, the Env binding domain of the chimpanzee CD4 is highly polymorphic, with nine coding variants circulating in wild populations. Here, we show that within-species CD4 diversity is not unique to chimpanzees but found in many African primate species. Characterizing the outermost (D1) domain of the CD4 protein in over 500 monkeys and apes, we found polymorphic residues in 24 of 29 primate species, with as many as 11 different coding variants identified within a single species. D1 domain amino acid replacements affected SIV Env-mediated cell entry in a single-round infection assay, restricting infection in a strain- and allele-specific fashion. Several identical CD4 polymorphisms, including the addition of N-linked glycosylation sites, were found in primate species from different genera, providing striking examples of parallel evolution. Moreover, seven different guenons (Cercopithecus spp.) shared multiple distinct D1 domain variants, pointing to long-term trans-specific polymorphism. These data indicate that the HIV/SIV Env binding region of the primate CD4 protein is highly variable, both within and between species, and suggest that this diversity has been maintained by balancing selection for millions of years, at least in part to confer protection against primate lentiviruses. Although long-term SIV-infected species have evolved specific mechanisms to avoid disease progression, primate lentiviruses are intrinsically pathogenic and have left their mark on the host genome.

Simian immunodeficiency viruses (SIVs) comprise a large group of lentiviruses that infect over 45 African primate species, including numerous guenons (Cercopithecus spp.), African green monkeys (Chlorocebus spp.), mandrills and drills (Mandrillus spp.), mangabeys (Cercocebus spp.), colobus monkeys (Colobus spp., Piliocolobus spp.), as well as chimpanzees (Pan troglodytes) and western gorillas (Gorilla gorilla) (1). Although the prevalence rates and geographic distribution of these infections vary widely, most SIVs are host-specific (i.e., their genomes form species-specific clusters in phylogenetic trees) (2–5). This has enabled the identification of instances when SIVs have crossed species barriers, including from apes and monkeys to humans (6). Phylogenetic analyses have shown that both pandemic and nonpandemic forms of HIV type 1 (HIV-1) resulted from the cross-species transmission of SIVs infecting central chimpanzees (P. troglodytes troglodytes) and western lowland gorillas (G. gorilla gorilla), while the various groups of HIV type 2 (HIV-2) emerged following the transfer of SIVsmm strains naturally infecting sooty mangabeys (Cercocebus atys) (6–9).SIVs have also jumped between nonhuman primate species, generating new SIV lineages. Cross-species transmission and recombination between ancestors of viruses today infecting greater spot-nosed (Cercopithecus nictitans), mustached (Cercopithecus cephus) and mona (Cercopithecus mona) monkeys (SIVgsn/SIVmus/SIVmon), and SIVrcm infecting red-capped mangabeys (Cercocebus torquatus) gave rise to SIVcpz in chimpanzees (10), and onward transmission of this virus to western lowland gorillas generated SIVgor (11). Additional cross-species transmissions and recombination events have generated mosaic SIV lineages in green monkeys (Chlorocebus sabaeus) and mandrills (Mandrillus sphinx) (12, 13). Finally, repeated introductions of diverse SIVs into the same primate species have resulted in cocirculating lineages, such as SIVkcol1 and SIVkcol2 in Kibale black-and-white colobus (Colobus guereza), and SIVmus-1, SIVmus-2, and SIVmus-3 in mustached monkeys (14, 15). Thus, primate lentiviruses have a high propensity to cross species barriers and have done so on numerous occasions throughout their evolutionary history.Lentiviruses have existed for tens of millions of years as evidenced by the finding of endogenous viruses in the genomes of species from four orders of mammals, including lemurs (16, 17), colugos (18), rabbits (19, 20), and weasels (21, 22). Some SIVs, such as those infecting green monkeys (Chlorocebus spp.) (23, 24) and the lhoesti group of guenons (Allochrocebus spp.) (25, 26) are at least several million years old because they appear to have coevolved with their respective hosts since these diverged from a common ancestor. Although an upper limit of 6 to 10 million y has been suggested for SIVs based on the fact that they have so far been found only in African, but not Asian, lineages of Old World monkeys (6), certain features of antiviral defense genes suggest that monkeys may have been exposed to lentiviruses long before this (27). Cellular restriction factors, such as APOBEC3G and TRIM5, are exquisitely antiviral and are counteracted by dedicated SIV accessary proteins. These restriction factors have evolved under strong positive selection at sites specifically involved in the interaction with lentiviruses, in both African and Asian monkeys (28, 29). However, if SIV indeed infected the common ancestor of African and Asian monkeys, this would imply numerous subsequent infection losses from multiple host lineages. Thus, it remains unclear when lentiviruses first infected primates.Among lentiviruses, those infecting primates are unique in their use of the CD4 receptor for entry into target cells. The viral envelope glycoprotein (Env) interacts with CD4 and subsequently undergoes conformational changes to expose the coreceptor binding site, which is required for viral–cell membrane fusion (30). CD4 is an immunoglobulin-like integral membrane protein that is expressed on multiple immune cells and stabilizes the interaction of the T cell receptor (TCR) with major histocompatibility complex class II (MHC II) molecules (31, 32). The most outward domain of CD4 (the D1 domain) binds a nonpolymorphic region on MHC II, which enhances TCR signaling (32). Importantly, the D1 domain is also the region that is bound by the HIV/SIV Env glycoprotein (33, 34). In HIV-infected humans and SIVmac-infected macaques, continuous high level viral replication leads to CD4⁺ T cell depletion, systemic immune activation, T cell exhaustion, and the development of AIDS (35, 36). Naturally occurring SIVs can also cause immunodeficiency and disease, as shown for chimpanzees and mandrills (37–40), indicating that these viruses are intrinsically pathogenic (41–43). However, a number of primate species with presumed longstanding SIV infections, such as African green monkeys, sooty mangabeys, and Ugandan red colobus monkeys (Piliocolobus tephrosceles), have evolved unique mechanisms that prevent disease progression despite continuous high viral replication (44–48). While the time required to evolve these adaptations is unknown, such protective mechanisms are absent from hosts that acquire new SIV infections.Unlike restriction factors, which prevent or limit viral replication, CD4 is a dependency factor (i.e., a host protein that is required for successful infection). Since there are many examples of host receptors coevolving with pathogens (49–51), it has been assumed that pressures exerted by pathogenic SIVs are responsible for the rapid diversification of primate CD4 (52–54). However, direct evidence for this hypothesis has been lacking. Examining the functional consequences of CD4 diversity in chimpanzees, we recently found that naturally occurring amino acid replacements in the D1 domain were able to inhibit SIVcpz infection, both in vitro and in vivo (55). Protective mechanisms included charged residues at the CD4–Env interface and steric hindrance between CD4- and Env-encoded glycans, which were effective not only against SIVcpz but also other SIVs that chimpanzees frequently encounter. These results suggested that CD4 diversity protects wild chimpanzee populations from SIV infection, possibly by conferring a heterozygote advantage (55). Since humans lack polymorphisms and glycans in the D1 domain, we asked whether CD4 diversification was a unique adaptation of chimpanzees. Sequencing the D1 domain in members of 36 African primate species, we identified a remarkable degree of CD4 diversity, both within and between species. The observed polymorphisms altered the cell entry of a panel of diverse SIV Envs, with the level of restriction depending on the particular allele and virus strain analyzed. Thus, the diversification of the primate CD4 receptor appears to have resulted from an ancient arms race between primate lentiviruses and their hosts.     

Pannexin 1 channels facilitate communication between T cells to restrict the severity of airway inflammation

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Mutations in the transcription factor FOXO1 mimic positive selection signals to promote germinal center B cell expansion and lymphomagenesis

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Metabolism of non-steroidal anti-inflammatory drugs (NSAIDs) by Streptomyces griseolus CYP105A1 and its variants

In the field of drug development, technology for producing human metabolites at a low cost is required. In this study, we explored the possibility of using prokaryotic water-soluble cytochrome P450 (CYP) to produce human metabolites. Streptomyces griseolus CYP105A1 metabolizes various non-steroidal anti-inflammatory drugs (NSAIDs), including diclofenac, mefenamic acid, flufenamic acid, tolfenamic acid, meclofenamic acid, and ibuprofen. CYP105A1 showed 4′-hydroxylation activity towards diclofenac, mefenamic acid, flufenamic acid, tolfenamic acid, and meclofenamic acid. It should be noted that this reaction specificity was similar to that of human CYP2C9. In the case of mefenamic acid, another metabolite, 3′-hydroxymethyl mefenamic acid, was detected as a major metabolite. Substitution of Arg at position 73 with Ala in CYP105A1 dramatically reduced the hydroxylation activity toward diclofenac, flufenamic acid, and ibuprofen, indicating that Arg73 is essential for the hydroxylation of these substrates. In contrast, substitution of Arg84 with Ala remarkably increased the hydroxylation activity towards diclofenac, mefenamic acid, and flufenamic acid. Recombinant Rhodococcus erythrocyte cells expressing the CYP105A1 variant R84A/M239A showed complete conversion of diclofenac into 4′-hydroxydiclofenac. These results suggest the usefulness of recombinant R. erythropolis cells expressing actinomycete CYP, such as CYP105A1, for the production of human drug metabolites.     

IL-37b作用于树突状细胞CD39/ATP轴抑制类风湿性关节炎大鼠炎症反应的作用及机制

目的探讨白细胞介素37b重组蛋白(rmIL-37b)通过调节CD39/ATP轴抑制树突状细胞(DC)诱导类风湿性关节炎(RA)大鼠炎症反应的机制。方法将SD大鼠随机分为空白对照组(CTL)、CIA模型组、rmIL-37b 5μg/kg组、rmIL-37b 10μg/kg组,每组各10只。除了空白对照组外,其余大鼠采用含有卡介苗的完全弗氏佐剂和牛Ⅱ型胶原混合乳液免疫刺激,建立CIA模型。确定建模成功当天(D0),rmIL-37b组分别尾静脉注射5μg/kg、10μg/kg rmIL-37b;CTL组和CIA模型组注射相同体积的(1 ml/kg)生理盐水,连续给药15 d。免疫组化法检测滑膜组织Nod样受体蛋白3(NRPL3)炎症小体的表达,流式细胞术检测DC表型,另外试剂盒检测血清三磷酸腺苷(ATP)和免疫学指标。结果与CIA模型组相比,rmIL-37b 10μg/kg组大鼠足容积、AI值、NLRP3炎症小体表达量、血清ATP、IL-1β、IL-18、肿瘤坏死因子α(TNF-α)、抗Ⅱ型胶原抗体亚型(anti-ColⅡ-IgG、anti-ColⅡ-IgG2a)水平均降低,同时DC表面CD...