A族乙型溶血性链球菌emm基因测序分型的研究进展

emm测序分型是根据编码M蛋白的emm基因高变区核苷酸序列特异性将A族乙型溶血性链球菌分型的分子生物学技术,与传统血清学分型比较,具有准确、快速的特点,实验室间可方便、快捷地通过网络进行资料比较.在多价疫苗的构建和效能检测上将有很好的发展前景.     

应用emm基因序列分析对104株A群链球菌分型

目的　通过emm基因序列分析对A群链球菌 (GAS)进行分型 ,探讨T型与emm型的对应关系。方法　用引物以PCR法扩增GASM蛋白的N末端并进行测序 ,确定GAS的emm型 ,并与T分型对照比较。结果　 (1) 10 4株菌株中 ,emm1所占比例最大 ,为 2 9.8% ,然后依次是emm18(9.6 % ) ,emm12 (7.7% ) ,和emm6 9(5 .8% )等。 (2 )T1、T12和T14 4 9与emm基因对应关系好 ,T116株全部对应emm1型 (10 0 % ) ,T12对应emm12 ,符合率 83.3% ,T14 4 92株均对应emm4 9型 ;其它菌型对应关系差 ;同一T型可对应数个emm基因型 ,同一emm基因型可对应不同的T型。 (3)发现 1株新的emm型菌株 ,序列已递交GenBank ,注册号为AY3472 5 9。结论　emm基因分型方法具有准确、快速、分辨力高的优点 ;我国部分地区A群链球菌emm基因分型主导型为emm1、18、12、6 9、110等菌型。     

牛IgG Fc受体Ⅲ的分子克隆和序列分析

目的 研究牛ＩｇＧＦｃ受体的结构与功能的关系。方法 在构建了牛肺巨噬细胞ＣＤＮＡ文库的基础上,用ＰＣＲ方法获得了码牛ＩｇＧＦｃ受体Ⅲ的全长编码基因。结果 通过分子克隆和序列分析,发现其编码基因为５０７ｂｐ,共编码１６８个氨基酸,其中含有信号肽序列,穿膜区和胞内结构域以及３个Ｎ－连接糖基化位点。与ＣＯＬＬＩＮＳ等克隆的ｂｏＦｃγＲⅢ相比,在它的胞外区中缺失了８２个氨基酸,仅有一个胞外结构域。结论 我     

Fcγ和Fcε受体在小鼠三叉神经节的表达

目的 检测正常小鼠三叉神经节(TG)是否表达免疫球蛋白G(IgG)和免疫球蛋白E(IgE) Fc段Fcγ受体和Fcε受体,及其在过敏小鼠TG中的变化.方法 通过腹腔注射OVA和铝剂,建立小鼠过敏性结膜炎(ACJ)模型.EHSA检测血清总IgE.用Westernblot和免疫荧光检测Fcγ受体和Fcε受体的表达.结果 小鼠TG表达Fcγ受体和Fcε受体.其中IgG激活型高亲和力受体FcγRI和抑制型低亲和力受体FcγRⅡ只表达在小鼠TG神经元上,而IgG激活型低亲和力受体FcγRⅢ表达在小鼠TG中的卫星胶质细胞上.IgE激活型高亲和力受体FcεR Ⅰ表达在小鼠TG神经元上,而IgE低亲和力受体FcεRⅡ同时表达在小鼠TG神经元和卫星胶质细胞上.与正常小鼠比较,ACJ小鼠的血清总IgE水平升高,TG的FcεR Ⅰ和FcγRⅡ表达增加(P＜0.05).而ACJ小鼠的FcεRⅡ和FcγRⅠ表达下降(P＜0.05).FcγRⅢ在正常和ACJ小鼠TG中的表达无显著差别.结论 小鼠TG表达的Fcγ和Fcε受体可能参与ACJ及其他过敏性疾病的发生和发展.     

IgG Fc受体及其与系统性红斑狼疮发病机制的研究进展

IgG Fc受体(FcγRs)是一类重要的膜表面糖蛋白,是免疫反应的重要调节者,表达于几乎所有的免疫细胞.本文对FcγRs的免疫调节作用及其与系统性红斑狼疮(SLE)等自身免疫性疾病的关系进行综述.     

ABC—ELISA方法检测细胞膜表面IgG Fc段受体

为开辟更具实用价值的Fc受体研究的新途径,在ELISA方法基础上引入ABC技术,检测Namalwa细胞表面IgG Fc段受体。用生物素直接标记酰基,与胞膜相应受体结合后再联结ABC 复合物,敏感性大大提高,P/N达4以上,D—N值达0.8左右。     

转铁蛋白受体和免疫球蛋白A/M Fc受体并非人系膜细胞主要的IgA1受体

目的：探讨转铁蛋白受体(TfR)和免疫球蛋白A/M Fc受体(Fcα/μR)是否为人系膜细胞(HMC)主要的免疫球蛋白A(IgA)受体。方法： 亲和层析提取血清IgA1，热聚合并用［125I］标记，RT-PCR法检测TfR与Fcα/μR mRNA在HMC及人系膜细胞系(HMCL)中的表达，放射配基结合法检测聚合IgA1(aIgA1)与HMCL的结合动力学特征，Western blot方法观察IgAN患者aIgA1与正常人aIgA1刺激HMCL引起的细胞外信号调节激酶(ERK)蛋白磷酸化水平的异同。结果： TfR和Fcα/μR mRNA在原代培养的HMC均有表达，在HMCL未见表达。aIgA1与HMCL之间的结合具有特异性和饱和性，最高结合量为(496.7±200.3)fmol/105细胞，结合位点为(3.0±1.2)×106/细胞，解离常数Kd值为(6.4±1.7)×10-7mol/L。IgAN患者aIgA1与正常人aIgA1均呈时间依赖性诱导ERK蛋白磷酸化，但IgAN患者aIgA1的作用明显强于正常人aIgA1(P＜0.01)，且持续时间更长(P＜0.05)。结论： HMCL细胞存在特异性IgA1受体，但该受体并非TfR和Fcα/μR。     

A族链球菌表面蛋白FbaA单克隆抗体FbaAmAb2的免疫功能研究

目的 通过侵袭抑制及攻毒试验检测A族链球菌表面蛋白FbaA的单克隆抗体FbaAmAb2的免疫功能,为进一步探索A族链球菌的致病机制和感染后的治疗策略奠定基础.方法 通过亚克隆、全菌ELISA方法筛选稳定分泌高效价FbaAmAb2的杂交瘤细胞,并将其接种子小鼠腹腔制备腹水.通过侵袭试验对FbaAmAb2能否阻止H因子与A族链球菌的结合进行研究.而后将获得的腹水倍比稀释为1:1、1:2、1:4、1:8、1:16行试管凝集试验,选定合适的稀释度被动免疫动物后用致死量A族链球菌(FbaA+)标准菌株(7.5×107个)攻击,连续观察15 d计算保护率.结果 侵袭试验中FbaAmAb2能够竞争性抑制H因子与A族链球菌表面蛋白FbaA的结合,减少了H因子介导的A族链球菌侵入上皮细胞的数量.与全菌结合的ELISA效价为1:1600,试管凝集效价为1:8.在单抗被动免疫试验中,腹水原液、1:2稀释组的动物保护率高达66.67％,1:4稀释组保护率为50％,经SPSS10.0统计软件分析各实验组与PBS组保护牢差异有统计学意义(P＜0.05).结论 FbaAmAb2有望用于紧急预防及治疗A族链球菌感染,并为深入研究A族链球菌的致病机制、治疗策略及疫苗预防方面提供新的靶标.     

乙脑病毒prME蛋白与BALB/c鼠IgG Fc段编码基因联合构建DNA免疫研究

目的 研究IgG Fc编码基因对流行性乙型脑炎(Japanese encephalitis,JE)DNA疫苗免疫增强效应的影响.方法 巢式RT-PCR法从BALB/c鼠脾组织获取IgG Fc段编码基因,用限制性内切酶从含流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)prME蛋白基因重组子获取prME蛋白基因,分别插入同-真核表达载体pcDNA3.1(+)不同酶切位点,构建蘑组子pJME/IgG Fc并经酶切及DNA测序分析.脂质体法将pJMrY/IgG Fc转染CHO细胞.免疫荧光、Western blot法检测转染的CHO细胞中融合蛋白分布与表达.将pJME/IgG Fc肌注免疫BALB/c鼠,检测小鼠脾特异性细胞毒T细胞(CTL)杀伤活性和中和抗体滴度.结果 pJME/IgG Fc经BamH Ⅰ/EcoR Ⅰ和BamH Ⅰ/Not Ⅰ酶切释出的插入子大小,(2001 bp,2730 bp)分别与预期结果相符合.所编码的融合蛋白相对分子质量(Mr)为101×103,主要分布于胞浆,少最分布于胞膜,pJME/IgG Fc转染CHO细胞经32次传代仍可表达融合蛋白.pJME/IgGFc免疫组中和抗体滴度与CTL活性较pJME及灭活疫苗组均升高(P<0.05).结论 pJME/IgG Fc成功构建,转染的CHO细胞可稳定表达融合蛋白,IgG Fc段编码基因能够增强JEV DNA疫苗的细胞和体液免疫应答.     

Predominance of two M-types among erythromycin-resistant group A Streptococci from Greek children

In order to investigate the potential relationship between erythromycin resistance and specific M-serotypes among clinical isolates of Streptococcus pyogenes from children in Greece, we randomly selected a total of 49 erythromycin-resistant (EryR) and 21 erythromycin-susceptible (EryS) isolates from the 1158 S. pyogenes isolates from the two main children's hospitals of Athens during the period October 1997 to October 1998. The isolates were further characterized by M-serotyping, examined for their susceptibility to penicillin, vancomycin and clindamycin, and categorized into resistance phenotypes. A total of 248 (21%) S. pyogenes isolates in the two main children's hospitals of Athens during the study period were resistant to erythromycin. All 49 EryR and 21 EryS isolates were susceptible to penicillin and vancomycin. With respect to erythromycin and clindamycin resistance, phenotypes M and IR MLS_B dominated, with 30 and 17 isolates, respectively, two isolates belonged to the CR MLS_B phenotype. Among the erythromycin resistant isolates, two M serotypes were dominant: M22 (30%) and M84 (41%). More specifically, M22 and M84 were most prevalent in resistance phenotypes IR MLS_B (65%) and M (63%), respectively. In the susceptible group, no isolate belonged to these two M-serotypes, nor was a predominant serotype found. In contrast to susceptible isolates, two distinct M-serotypes were highly represented among EryR S. pyogenes isolates and predominantly associated with two distinct phenotypes.     

IgA Fc受体研究进展

IgA Fc受体(FcαRI,CD89)属于免疫受体家族成员,主要表达于单核-巨噬细胞、中性粒细胞、嗜酸性粒细胞和树突状细胞等免疫效应细胞表面。FcαRI能特异的与血清型和分泌型IgA结合,并通过γ链介导一系列免疫反应,包括抗体依赖细胞介导的细胞毒性作用(ADCC)、补体依赖的细胞毒性(CDC)及细胞吞噬作用等。FcαRI是一种双功能受体,在不同的生理条件下可以介导免疫系统的活化和抑制反应。FcαRI在机体免疫防御和在维持系统免疫平衡方面扮演着重要的角色,有望成为治疗人类疾病的新靶点。本文对FcαRI的结构、功能及其应用等研究现状进行综述。     

Myocardial tissue damage in rabbits injected with group A streptococci, types M1 and M22. Role of bacterial immunoglobulin G-binding surface proteins

Acute rheumatic fever (ARF) and acute poststreptococcal glomerulonephritis (APSGN), two important sequelae of streptococcal throat or skin infections, according to current concepts may be elicited by autoimmune mechanisms due to molecular mimicry between group A streptococci (GAS) and human tissue. In the case of APSGN, however, our experimental data have indicated that GAS immunoglobulin-binding surface proteins (IgG BPs) might be of pathogenic significance by triggering anti-IgG production and immune complex formation leading to renal damage. Thus, rabbits injected with IgG-binding, as opposed to non-binding, GAS strains were found to develop renal deposition of IgG and complement factor C3 and inflammatory and degenerative glomerular changes resembling the picture seen in APSGN. In the present study, cardiac tissue material from rabbits injected with GAS was investigated. After 8 or more weeks of intravenous (i.v.) injections, minimal changes were seen in those animals receiving an IgG non-binding GAS strain, type T27, whereas those animals receiving either of two IgG-binding GAS strains, types M1 or M22, developed strong inflammatory and degenerative myocardial changes accompanied by deposition of IgG and C3. Furthermore, on injecting rabbits with defined mutants of a type M22 strain, the development of myocardial tissue damage proved to be dependent on the presence of streptococcal IgG-binding activity. Our results demonstrate that myocardial tissue changes may be induced in the rabbit by i.v. injection of whole heat-killed GAS of at least two M serotypes. Conceivably, induction of immune complexes by bacterial IgG BPs may lead to myocardial deposition of IgG, in turn triggering a series of events, involving the complement system and proinflammatory cytokines, with resulting tissue damage. Though many virulence factors may be involved in the development of ARF and APSGN, and a given GAS strain will never cause both, our results may suggest a new pathogenetic mechanism common to these two major non-suppurative complications.     

An extended family of Fc receptor relatives

A surprising number of Fc receptor (FcR) relatives have been recognized recently with the potential capacity to modulate innate and adaptive immune responses. The six human FcR homologs (FcRH1-6), which belong to a phylogenetically conserved gene family, have variable numbers of extracellular immunoglobulin domains of five different subtypes. FcRH immunoregulatory potential is implicated by the presence of consensus tyrosine-based activation or inhibition motifs in their cytoplasmic tails. All but one of these new receptors, FcRH6, are expressed on B cells at different stages in differentiation. Their ligands, function, and prospective roles as diagnostic B cell markers and therapeutic targets are topics of intense interest.     

Effect of Mouse Passage on Fc Receptor Expression by Group A Streptococci

The expression and stability of receptors for the Fc region of human IgG on the surface of group A streptococci was studied. Two strains were sequentially passed in mice 22 times. The Fc-receptor expression on one group A strain, 529, was unaltered while the expression of Fc receptor on a second, 64, was enhanced and approached the level of Fc-receptor expression of the protein A-rich Staphylococcus aureus Cowan strain. The level of Fc-receptor expression on this organism remained stable for over 18 months of laboratory subculture. Mouse passage did not result in the production of a soluble Fc receptor from either of the streptococcal strains. Heat extraction of the Fc-receptor-positive group A strain resulted in solubilization of an Fc-receptor activity which was functionally distinct from either staphylococcal protein A or the Fc receptor isolated from a group C streptococcus.     

IgG Fc receptor polymorphisms and association with autoimmune disease

The aim of this study was to investigate whether a genetic polymorphism of Fc gammaRIII exists in mice, which could explain the different susceptibility to pathogenic IgG anti-collagen type II (CII) antibodies in mice carrying the collagen-induced arthritis (CIA)-susceptible H-2q haplotype. The gene for Fc gammaRIII was sequenced in 11 common mouse strains, and the results revealed three different haplotypes of mouse Fc gammaRIII: Fc gammaRIII:V, Fc gammaRIII:H and Fc gammaRIII:T. To study the consequences of this polymorphism, we generated mice carrying the Fc gammaRIII:H haplotype from the CIA-susceptible, H-2q-positive DBA/1 mouse or the Fc gammaRIII:V haplotype from the CIA-resistant, H-2q-positive SWR mouse. After CII immunization or transfer of IgG anti-CII antibodies, Fc gammaRIII:H-expressing mice, but not Fc gammaRIII:V-expressing mice, developed progressively severe arthritis. We also investigated if C5, in addition to Fc gammaRIII polymorphism, could affect the susceptibility to the pathogenic IgG anti-CII antibodies in H-2q-positive mice. Here we show that SWR mice, naturally deficient in C5, can develop CIA when supplemented with C5 and that anti-C5 antibody treatment of Fc gammaRIII:H-expressing mice inhibits arthritis development. These data demonstrate for the first time a genetic polymorphism of Fc gammaRIII in mice that may, together with C5, regulate induction of autoimmune disease.     

Critical role for complement receptor 3 (CD11b/CD18), but not for Fc receptors, in killing of Streptococcus pyogenes by neutrophils in human immune serum

During phagocytosis, surface receptors on neutrophils interact with pathogens opsonized with complement factor C3b/iC3b and in some cases with antibodies. In human immune sera antibodies directed against surface-bound M proteins mediated killing of Streptococcus pyogenes by neutrophils. Surprisingly, blocking of the Fc receptors had little effect on the killing. In contrast, inhibition of C3b/iC3b generation, or blocking of the major neutrophil iC3b receptor CD11b/CD18, enabled S. pyogenes to grow efficiently in immune sera. Inhibition of CD11b/CD18, but not of CD32, the major neutrophil signaling Fc receptor, prevented Streptococcus-induced NADPH oxidase-dependent respiratory burst, and blocking of C3b/iC3b formation inhibited Streptococcus-induced activation of Cdc42, a small GTPase critically involved in transmitting pro-inflammatory signals to the cytoskeleton. Consequently, ligation of CD11b/CD18 by bacteria-bound iC3b is necessary for inducing a neutrophil response leading to elimination of S. pyogenes in immune human serum.     

Reanalysis of the role of pronase treatment of B cells in the flow cytometric crossmatch assay: Fc receptor is not the primary target

Pronase, a mixture of nonspecific bacterial proteases, is used to pretreat human lymphocytes to prevent false-positive B cell results in the flow cytometric crossmatch (FCXM) assay. The target of pronase has been reported to be B cell-expressed Fc receptors, which nonspecifically bind IgG. As pronase use in FCXM can induce other complications, including degradation of HLA leading to inappropriate FCXM results, and false-positive T cell results when testing serum from HIV-positive patients, we tested whether specifically blocking Fc receptor CD32 could replace pronase. Anti-CD32 mAb 6C4 was superior to pronase for blocking binding of aggregated IgG to B cells. However, 6C4 was unable to replace pronase in clinical FCXM, as it did not prevent false-positive B cell FCXM results, or enhance sensitivity of the assay. We conclude that the functional targets of pronase in the FCXM assay are poorly understood, and that B cell-expressed Fc receptor plays an insignificant role.