白介素18启动子区基因多态性与儿童EV71感染相关性研究

目的:研究白介素18基因启动子多态性与儿童EV71感染遗传易感性的关系.方法:收集EV71感染患儿177例,单纯HFMD组127例,HFMD并脑炎组50例,提取外周血DNA,用序列特异性引物-聚合酶链反应(SSP-PCR)技术及基因测序法检测IL-18启动子区-137G/C、-607A/C位点的基因多态性.结果:EV71感染患儿与健康儿童IL-18-607基因型以CA为主,AA、CC次之,EV71感染患儿AA基因型、A等位基因分布频率显著高于健康儿童.EV71感染HFMD并脑炎组患儿AA基因型分布显著高于单纯HFMD组患儿,差异有统计学意义.EV71感染患儿与健康儿童IL-18-137基因型以CC为主,CG次之,GG占少数.该位点基因型及等位基因在EV71感染组与健康儿童、单纯HFMD与HFMD并脑炎组的分布无显著差异.结论:IL-18基因多态性与EV71感染相关,IL-18-607AA基因型、A等位基因携带儿童更易感染EV71病毒,且AA基因型患儿易并发脑炎,-607AA基因型可能为EV71感染的易感基因型.-137C/G位点基因多态性与EV71感染无相关性.     

三个品系角突臂尾轮虫生长和发育的比较研究

以浓度为2．4×10^6cells.ml^-1的斜生栅藻为食物,在25℃下,对采自青岛、广州与芜湖等品系的角突臂尾轮虫的胚胎发育、生殖前期和最小世代时间等个体发育阶段历时以及幼体和成体的个体大小、卵体积和相对卵体积进行了研究．结果表明,轮虫的胚胎发育时间、生殖前期历时和最小世代时间等在品系间均存在显著差异．芜湖品系具有最长的胚胎发育时间,其次为广州品系和青岛品系;青岛品系的生殖前期最长,广州品系最短;而最小世代时间以广州品系最小,青岛品系和芜湖品系次之,后两者之间无显著差异．各品系轮虫胚胎发育时间具有随其相对卵体积的增大而缩短的趋势．同一年龄段各品系间的轮虫幼体体积均有显著差异,且均以芜湖品系为最大．三品系轮虫个体幼体体积与其年龄间均呈曲线相关;刚孵化出的幼体体积与其成体间差异在芜湖品系达244．24％,青岛和广州品系分别为182．89％和156．28％．轮虫成体的个体大小、卵大小和相对卵大小在品系间也存在显著差异．     

2007年北京地区儿童手足口病病原的初步筛查

2007年4～6月儿童手足口病流行期间,对北京地区51例皮损症状典型、伴/不伴发热、无重症合并症的手足口病患儿采样,建立RT-PCR方法,以5'非编码区(5'UTR)肠道病毒通用引物、CA16和EV71 VP1区特异性引物直接对82份临床标本进行了初步筛查,肠道病毒阳性率达70.6%。检测病例中CA16阳性25例(25/51)、EV71阳性4例(4/51)、非CA16和EV71的肠道病毒阳性病例7例(7/51),三者比例约为6:1:2。2007年北京地区儿童轻症手足口病主要病原包括CA16和EV71,同时还存在一定比例其它肠道病毒。部分EV71毒株经测序验证及系统进化分析显示为C4基因亚型。     

Hand foot and mouth disease due to enterovirus 71 in Malaysia

Hand foot and mouth disease is a febrile sickness complex characterized by cutaneous eruption (exanthem) on the palms and soles with simultaneous occurrence of muco-cutanous vesiculo-ulcerative lesions (enanthem) affecting the mouth.The illness is caused by a number of enteroviruses with coxsackievirus A16 and enterovirus 71 as the main causative agents.Human enterovirus 71 (EV71) belongs to the species Human enterovirus A under the genus Enterovirus within the family Picornaviridae.EV71 has been associated with an array of clinical diseases including hand foot and mouth disease (HFMD),aseptic meningitis,encephalitis and poliomyelitis-like acute flaccid paralysis.A large outbreak of HFMD due to highly neurovirulent EV71 emerged in Malaysia in 1997,and caused 41deaths amongst young children.In late 2000,a recurrence of an outbreak of HFMD occurred in Malaysia with S fatalities in peninsular Malaysia.Outbreak of HFMD due to EV71 recurred in 2003 with an unknown number of cases and mortalities.A similar outbreak of HFMD with 2 recorded deaths in young children occurred in peninsular Malaysia in late 2005 and this was followed by a larger outbreak in Sarawak (Malaysian Borneo) with 6 reported fatalities in the early part of 2006.The current on-going outbreak of HFMD started in peninsular Malaysia in epidemiological week 12 of 2010.As with other HFMD outbreaks in Malaysia,both EV71 and CA16 were the main aetiological viruses isolated.In similarity with the HFMD outbreak in 2005,the isolation of CA16 preceded the appearance of EV71.Based on the VP 1 gene nucleotide sequences,4 sub-genogroups of EV71 (C1,C2,B3 and B4) co-circulated and caused the outbreak of hand,foot and mouth disease in peninsular Malaysia in 1997.Two sub-genogroups (C1 and B4) were noted to cause the outbreak in 2000 in both peninsular Malaysia and Sarawak.EV71 of sub-genogroup B5 with smaller contribution from sub-genogroup C1 caused the outbreak in 2003.In the 2005 outbreak,besides the EV71 strains of sub-genogroup C1,EV71 strains belonging to sub-genogroup B5 were isolated but formed a cluster which was distinct from the EV71 strains from the sub-genogroup B5 isolated in 2003.The four EV71 strains isolated from clinical specimens of patients with hand,foot and mouth disease in the Sarawak outbreak in early 2006 also belonged to sub-genogroup B5.Phylogenetic analysis of the VP1 gene suggests that the EV71 strains causing the outbreak in Sarawak could have originated from peninsular Malaysia.Epidemiological and molecular data since 1997 show the recurrence of HFMD due to EV71 in Malaysia every 2 to 4 years.In each of the past outbreaks,more than one sub-genogroup of the virus co-circulate.     

Retroviral transduction of interferon-γ cDNA into a nonimmunogenic murinefibrosarcoma: generation of T cells in draining lymph nodes capable of treating established parental metastatic tumor

Gene modification of tumor cells with the cDNA for interferon (IFN) has been shown to increase the immunogenicity of some tumor cells. In order to explore further the possible therapeutic relevance of these previous findings, two clones of the nonimmunogenic MCA-102 fibrosarcoma of C57BL/6 origin were retrovirally transduced with the cDNA encoding murine IFN: 102.4JK (4JK), a clone with relatively high major histocompatibility complex (MHC) class I expression, and 102.24JK (24JK), a clone with low expression of surface MHC class I molecules. Retroviral transduction of tumor cells with the cDNA encoding for IFN resulted in a substantial up-regulation of MHC class I surface expression in the 24JK clone but little change of class I in the 4JK clone. In an attempt to generate antitumor lymphocytes, these gene-modified cells were inoculated into mouse footpads and draining lymph nodes (DLN) were removed, dispersed, and cultured in vitro for 10 days with irradiated tumor cells and interleukin-2. DLN from mice bearing either unmodified tumor or tumor transduced with cDNA encoding neomycin resistance (Neo ^R) or IFN, were used to treat recipients harboring 3-day pulmonary metastases induced by the parental, unmodified tumor. Treatment with DLN cells obtained following the injection of 24JK tumor cells modified with the gene for IFN significantly reduced the number of pulmonary metastases in four separate experiments, compared to groups treated by DLN cells generated from inoculation of either the unmodified, parental 24JK clone or the same clone transduced with theNeo ^R gene only. In contrast, DLN cells induced either by IFN-transduced 4JK (high expression of MHC class I) or an unmodified 4JK tumor (moderate expression of MHC class I) had significant but equal therapeutic efficacy. Although the in vitro growth rate of tumor cell lines was unaffected by the insertion of the mouse IFN cDNA, their in vivo (s.c.) growth rates were significantly slower than those of the nontransduced tumors. Thus, after retroviral transduction of the murine IFN cDNA into a nonimmunogenic tumor with a very low level of surface expression of MHC class I, modified tumor cells could elicit therapeutic T cells from DLN capable of successfully treating established pulmonary metastases upon adoptive transfer. This strategy significantly confirms previous observations on the potential therapeutic effects of gene modification of tumor cells with IFN and extends the realm of therapeutic possibilities to include the use of DLN cells for the development of T-cell based immunotherapies against nonimmunogenic human tumors.     

Dp71Δ78‐79 dystrophin mutant stimulates neurite outgrowth in PC12 cells via upregulation and phosphorylation of HspB1

PC12 cells acquire a neuronal phenotype in response to nerve growth factor (NGF). However, this phenotype is more efficiently achieved when the Dp71Δ_78‐79 dystrophin mutant is stably expressed in PC12‐C11 cells. To investigate the effect of Dp71Δ_78‐79 overexpression on the protein profile of PC12‐C11 cells, we compared the expression profiles of undifferentiated and NGF‐differentiated PC12‐C11 and PC12 cells by 2DE. In undifferentiated cultures, one protein was downregulated, and five were upregulated. Dp71Δ_78‐79 overexpression had a greater effect on differentiated cultures, with ten proteins downregulated and seven upregulated. The protein with the highest upregulation was HspB1. Changes in HspB1 expression were validated by Western blot and immunofluorescence analyses. Interestingly, the neurite outgrowth in PC12‐C11 cells was affected by a polyclonal antibody against HspB1, and the level of HspB1 and HspB1Ser86 decreased, suggesting an important role for this protein in this cellular process. Our results show that Dp71Δ_78‐79 affects the expression level of some proteins and that the stimulated neurite outgrowth produced by this mutant is mainly through upregulation and phosphorylation of HspB1.     

肠道病毒71型感染前后宿主细胞蛋白质组的二维液相色谱分离和比较

以肠道病毒71型及其宿主细胞为研究主体,建立了一种二维液相色谱分离和分析比较病毒感染前后细胞蛋白表达谱的方法。该方法以高效液相色谱(HPLC)为技术平台,对细胞裂解物先后进行一维色谱聚焦分离和二维反相色谱分离。利用ProteoVue软件将二维色谱数据转换成模拟胶图,再利用DeltaVue软件对感染前后的宿主蛋白表达谱进行比较和分析,找出差异蛋白。二维液相色谱分离法能够根据蛋白的等电点和疏水性建立精确的细胞蛋白表达图谱,每0．2个pH为一个收集区段,在pH8．5～3．9的范围内可见蛋白条带约1200条。该方法良好的重现性、自动化以及结果分析的简易化,使之在细胞表达谱差异显示中的应用潜力巨大,并且为研究病毒与宿主相互作用提供了新的方法和思路。     

Complete sequence analyses of enterovirus 71 strains from fatal and non-fatal cases of the hand,foot and mouth disease outbreak in Singapore (2000)

Enterovirus 71 (EV71) is a major aetiological agent of hand, foot and mouth disease (HFMD). In recent years, several outbreaks in East Asia were associated with neurological complications and numerous deaths. An outbreak in Singapore in October 2000 afflicted thousands of children, resulting in four fatal cases from three of whom EV71 was isolated. The genomes of two representative EV71 strains isolated from a fatal case and a surviving patient were completely sequenced, and their nucleotide and amino acid sequences compared with known EV71 strains. The two outbreak strains were classified under genogroup B, together with those previously isolated in Singapore, Malaysia and Japan. Comparative sequence analysis of the two Singapore strains revealed 99% nucleotide similarity, while their deduced amino acid sequences were almost identical except for residue 1506 in the 3A non-structural region. Given that the outbreak involved closely related genetic variants of EV71, the broad spectrum of disease severity may be attributed to critical factors such as varying viral inoculation doses or differing host immune responses following infection, but is less likely to be due to the emergence of EV71 strains with heightened virulence.