肺炎链球菌粘附肺Ⅱ型上皮细胞触发宿主细胞酪氨酸蛋白磷酸化

目的 探讨细胞内信号传导与肺炎球菌侵袭、致病的关系,在体外研究Ⅱ型肺炎链球菌粘附肺Ⅱ型上皮细胞（A549）是否能触发细胞内酪氨酸蛋白激酶（TPK）信号传导途径 ,以及触发该信号传导可能的细菌亚组分。方法 用FTTC荧光标记肺炎链球菌,在体外观察肺炎链球菌粘附肺Ⅱ型上皮细胞的粘附动力学特征;用免疫组织化学和ELISA方法观察完整细菌触发的细胞内酪氨酸蛋白磷酸化,用各种因素预处理肺炎链球菌后,观察触发细胞内酪氨酸蛋白磷酸化可能的细菌亚组分。结果 证实了上述粘附过程存在剂量依赖和时间的依赖关系,而且是特异的过程;细菌粘附使细胞内酪氨酸磷酸化由细菌表面蛋白质介导。结论 肺炎链球菌粘附肺Ⅱ型上皮细胞能触发细胞内信号传导,且细菌表面蛋白质在触发胞内酪氨酸蛋白磷酸化传导中起着重要作用。     

肺炎链球菌毒力蛋白在侵入性感染中的免疫原性评价

目的观察肺炎链球菌表面蛋白A(PspA)、肺炎链球菌表面黏附素A(PsaA)及肺炎链球菌溶菌素(Ply)激发自然途径感染肺炎链球菌机体保护性体液免疫应答的情况,评价这三种毒力蛋白作为治疗性肺炎链球菌疫苗候选抗原的免疫原性。方法采集侵入性肺炎链球菌感染患者(实验组)及同期确诊为侵入性其它细菌感染患者(对照第一组)和同期确诊为非感染性疾病患者(对照第二组)各36例急性期(第0+3天)及恢复期(第21±3天)血清样本,酶联免疫吸附法(ELISA)检测患者血清中肺炎链球菌3种毒力蛋白抗原特异性抗体IgG水平变化。结果实验组与两组对照组间第(0+3)天血清的3种肺炎链球菌毒力蛋白特异性抗体水平无显著性差异(P>0.05)。第一组对照组恢复期较急性期血清中3种毒力蛋白特异性抗体水平无显著变化(P>0.05),而实验组恢复期血清3种肺炎链球菌毒力蛋白特异性抗体水平明显高于急性期血清抗体水平8～30倍(P<0.01),其中PspA和Ply蛋白抗原特异性抗体水平升高更明显,血清中针对PspA家族1(PspA-Faml)特异性抗体水平高于针对PspA家族2(PspA-Fam2)的特异性抗体水平(P<0.01)。结论 PsaA,P...     

肺炎链球菌蛋白疫苗候选蛋白研究进展

肺炎链球菌能够引起肺炎、脑膜炎、中耳炎等疾病,在世界范围内具有很高的致病率和致死率。患者主要集中在婴幼儿、老年人和免疫力低下的人群。据统计,每年大约有一百万的五岁以下儿童死于肺炎链球菌引起的疾病。抗生素是肺炎链球菌引起疾病最常用的治疗药物,但是,耐青霉素和多重耐     

转座子介导的肺炎链球菌耐药水平传播机制

目的 探索转座子介导的耐药基因水平转移在肺炎链球菌(Streptococcus pneumoniae,Sp)耐药传播中的作用.方法 采用长片段聚合酶链反应(long and accurate PCR,LA-PCR)、DNA序列分析、Southern blot、脉冲场凝胶电泳(PFGE)等方法 分析3株不同遗传背景的Sp中转座子的种类、结构,用转化实验分析不同菌株之间耐药性转移方式.结果 (1)Sp中携带mefE的mega元件插入到接合转座子Tn916上形成复合转座子Tn2009-like,其序列与Tn2009具有99%同源性,染色体插入位点完全不同.(2)临床分离的多重耐药Sp(ET86)染色体184 kb和155 kb片段上同时携带Tn1545、Tn917和Tn2009-like,体现出携带耐药决定子的遗传元件集中分布的趋势.另外2株不同耐药表型的Sp分别携带1或2种转座子.(3)转座子上耐药基因可通过转化转移.结论 Sp染色体上转座子可捕获其他耐药元件形成复合转座子,且不同的转座子可在染色体上集中分布、协同作用,促进耐药性的传播和进化.     

粘附蛋白及其在细胞的粘附与聚集中的作用

细胞——细胞,细胞——细胞外基质之间的粘附作用的研究是细胞生物学中重要的课题,这一作用在细胞的迁移、生长、分化中是重要的,在一些生理和病理过程中如伤口愈合和修复、炎症、癌细胞转移等也是重要的,例如血小板之间和血小板与血管壁之间的激活与粘附已成为血栓与止血研究的中心课题,现已初步阐明存在于血浆和基质中的一些蛋白在粘附作用中起重要作用,这些蛋白包括纤维蛋白原(简称Fg),纤维结合蛋白(Fibronectin,简称Fn),Von Wilebrand因子,简称vWF,凝血酶敏感蛋白(Throm-bospondin,简称TSP),Vitronectin(简称Vn),Laminin(简称La),Chon-     

肺炎链球菌感受态缺陷影响细菌对β-内酰胺类抗生素的敏感性

目的　探讨肺炎链球菌在对抗 β 内酰胺类抗生素时是否受感受态形成的影响。方法通过转化得到感受态缺陷菌株 ,采用半定量RT PCR检测相关基因ciaH和cpoA在细菌感受态形成时的表达 ,用肉汤法检测各菌株的最低抑菌浓度 (MIC)。结果　细菌感受态时基因ciaH和cpoA的mRNA表达量增高 (P <0 .0 5 ) ,野生菌感受态缺陷后的MIC值发生改变。结论　肺炎链球菌的感受态形成可诱导ciaH和cpoA的表达增加 ,其感受态缺陷影响到其对 β 内酰胺类抗生素的敏感性 ,但不同菌株的影响不同。     

血浆蛋白对生物材料细菌粘附影响研究进展

防止生物材料细菌粘附是防治生物材料为中心感染(biomaterial centered infect,BCI)的重要环节.研究发现,人体血浆蛋白对生物材料细菌粘附有重要影响.因此,研究血浆蛋白与生物材料细菌粘附关系为防治BCI有重要的意义.本文综述了与生物材料细菌粘附相关的血浆蛋白、血浆蛋白对生物材料细菌粘附影响的有关机制及如何提高血浆蛋白抗细菌粘附作用的展望.     

肺炎链球菌外膜蛋白PspA和PsaA的免疫原性比较

目的 比较肺炎链球菌(Streptococcus pneumoniae)表面黏附素A(PsaA)和表面蛋白A(PspA)的免疫原性.方法 电泳分析Sp6B亚型菌株的两种外膜蛋白基因及所编码蛋白相对分子质量的可变性,采用Western blot方法比较两种重组外膜蛋白对5种亚型菌株相应蛋白抗血清的交叉反应,采用酶联免疫吸附法(ELISA)分析两种外膜蛋白的抗体类型和抗体水平以及抗血清对5种亚型菌株的亲和性,以小鼠保护实验比较两种蛋白对5种亚型菌株的交叉保护作用.结果 两种蛋白产生的抗体亚型水平相当;PspA蛋白与其他亚型蛋白的交叉反应广泛性不如PsaA蛋白,只限于同一支系的蛋白之间;PspA抗体与病原菌具有可接近性,并具备交叉免疫保护作用.可作为一种更有效的抗原成分.结论 PspA对同家族同支系菌株攻击具有交叉免疫保护作用,可作为一种更有效的抗原成分.     

氟喹诺酮体外诱导耐药肺炎克雷伯菌膜孔蛋白表达的变化

目的 探讨氟喹诺酮体外诱导耐药肺炎克雷伯菌(KPn)膜孔蛋白表达变化.方法 取2008年9月至2009年6月本院临床分离对环丙沙星敏感的KPn 20株,分为对照组和实验组,每组10株.对照组直接涂布于含环丙沙星128 mg/L的平板,观察其对环丙沙星的敏感性.实验组应用不同浓度梯度环丙沙星逐级诱导成为高度耐药株,观察应用环丙沙星前后KPn敏感株和耐药株膜孔蛋白表达的差异.结果 对照组10株KPn直接涂布于含环丙沙星128 mg/L的平板培养后无一存活.3对KPn菌株R9、S9、R4、S4、R3、S3环丙沙星诱导前后膜孔蛋白相对表达量分别是3.86±0.11、6.44±0.26、5.46±0.18、9.58±0.34、1.75±0.06和9.78±0.36,诱导耐药的KPn较相应敏感株膜孔蛋白表达明显减少(均P＜0.05).结论 氟喹诺酮体外诱导耐药KPn膜孔蛋白表达减少,可能通过细菌外膜通透性改变在KPn诱导耐药中起重要作用.     

肺炎链球菌减毒活疫苗SPY1激发小鼠巨噬细胞应答的免疫作用机制研究

目的 探讨肺炎链球菌减毒活疫苗SPY1对巨噬细胞引起的天然免疫应答及机制。方法 体外诱导培养小鼠骨髓来源的巨噬细胞(bone marrow derived macrophage,BMDM),SPY1作用巨噬细胞6 h和24 h后,分别用RT-PCR和ELISA检测细胞因子的表达情况;SPY1作用于野生小鼠以及TLR2、TLR4缺陷小鼠的BMDM 24 h后,ELISA检测TNF-α和IL-6的表达;Western blot检测各信号分子的磷酸化水平;MAPK、PI3K和NF-κB抑制剂处理后,检测细胞因子TNF-α和IL-6的表达变化。结果 SPY1作用巨噬细胞后可引起强烈的免疫应答,此过程不依赖于TLR2和TLR4。MAPK、PI3K和NF-κB信号通路参与调控SPY1引起的巨噬细胞天然免疫应答。结论 肺炎链球菌减毒活疫苗SPY1通过MAPK、PI3K和NF-κB通路产生巨噬细胞天然免疫应答,这一过程不依赖于TLR2和TLR4。     

双歧杆菌对肠上皮细胞粘附的研究

用激光共距式细胞仪研究了ＦＩＴＣ标记的双歧杆菌对体外培养肠上皮细胞的粘附。结果表明，双歧杆菌的粘附素是一种蛋白质，由细菌分泌至培养液中；肠上皮细胞上粘附素受体为糖蛋白。光镜及透射电镜观察，双歧杆菌特异性地粘附于Ｌｏｖｏ细胞的刷状缘，被粘附的Ｌｏｖｏ细胞表面结构无破坏。     

肺炎链球菌菌体蛋白LytA和CbpA对感染小鼠的诊断价值

目的 通过基因工程技术获取肺炎链球菌自溶素(autolysin,LytA)和胆碱结合蛋白A(choline binding protein A,CbpA),探讨其对肺炎链球菌感染小鼠的血清学诊断价值.方法 根据GenBank中lytA和cbpA基因保守序列设计合成特异性引物,采用PCR技术从肺炎链球菌基因组中扩增lytA和cbpA保守序列片段;经由IPTG诱导并通过等电点洗脱方法获取纯化的重组蛋白LytA和CbpA;Western blot测定表达蛋白的抗体结合活性.以LytA和CbpA为抗原,建市ELISA反应模式测定感染小鼠血清中相应的IgG和IgM抗体,评价诊断价值.结果 成功构建了原核表达载体pET-32a(+)/lytA和pET-32a(+)/cbpA,表达的重组蛋白LytA和CbpA具有较好的抗体结合活性.实验组小鼠(肺炎链球菌感染组)IgM和IgG类抗LytA抗体滴度高于对照组(止常对照组和乙型链球菌感染对照组),差异有统计学意义(P<0.05),CbpA抗体与正常对照组差异有统计学意义,与乙型链球菌感染组差异无统计学意义(P>0.05).CbpA蛋白对感染小鼠诊断的敏感性较高(IgG:83.3%;lgM:75.0%),而LytA特异性较高(IgG:100%;IgM:100%).结论 协同利用重组蛋白CbpA诊断敏感性及LytA的特异性,对肺炎链球菌感染小鼠的血清学诊断具有一定价值,为进一步用于临床检验奠定基础.

Abstract：

Objective To obtain the pneumococcal autolysin(LytA)and choline binding protein A(CbpA)by prokaryotic expression system and investigate their diagnosis for infection caused by Streptococcus pneumoniae.Methods The specific primers were designed according to lytA and cbpA of Streptococcus pneumoniae gene sequence.lytA and cbpA were amplified by PCR form the pneumococcus genome.After IPTG inducing,the recombinant proteins were purified by electroeluting of bag filter,detected by SDS-PAGE and Western blot.Serum lgG and IgM antibodies accordingly of BALB/c mice infected with Streptococcus pneurnoniae were detected by ELISA.Results The recombinant plasmid pET-32a(+)/lytA and pET-32a (+)/cbpA were constructed successfully.Fusion proteins LytA and CbpA were expressed and displayed expected antigenicity.IgM and IgG antibodies level anti LytA were significantly higher than the control group (infections with B Streptococcus group and healthy mice),(P<0.05),but antibodies level anti CbpA did not increase as compared with group infected with B Streptococcus(P>0.05).Diagnostic sensitivity of CbpA was 83.3%(IgG)and 75.0%(IgM).Diagnostic specificity of LytA was 100%(IgG and IgM).Conclusion The synergistic use of specificity of LytA and sensitivity of CbpA may be worthy of serological diagnosis for Streptococcus pneumoniae infection,and may be used for further clinical test.     

Mechanisms in the serotype-independent pneumococcal immunity induced in mice by intranasal vaccination with the cell wall polysaccharide

We previously reported that cell wall polysaccharide (CWPS) given to mice intranasally with adjuvant induces serotype-independent immunity to pneumococci. Some strains make CWPS with one phosphocholine group (CWPS/1), but most express two per tetrasaccharide repeat unit (CWPS/2). Here, CWPS/1 and CWPS/2 were equally protective against colonization by CWPS/2-type pneumococci, but the related Streptococcus mitis polymer lacking phosphocholine was non-protective. Previously the protection was shown to be CD4 + T cell-dependent, abrogated by antiserum to interleukin (IL)-17A, and demonstrable in antibody-defective mice. Here, CWPS failed to protect IL-17A receptor knockout mice, further indicating IL-17A-dependence. When commercial CWPS/1 was size-fractionated preparatively, the larger exceeded the smaller molecules in their capacity to prime for IL-17A responses, and only the larger protected against pneumococcal colonization. However, a CWPS-tetanus toxoid conjugate – despite raising high titers of phosphocholine antibody – was non-protective, confirming the irrelevance of humoral immunity in this model. The results strengthen the concept that IL-17A-mediated T cell immunity is inducible by zwitterionic polysaccharides with sufficient chain length to provide coiled secondary structure. Coupling CWPS to protein, which paradoxically prevents protection, may occlude this regular linear conformation. We suggest that mucosal immunization with CWPS primes T_H17 cells, which – upon contact with the phosphocholine of colonizing pneumococci – elaborate IL-17A, enhancing phagocytosis.     

Serum antibody responses to pneumococcal colonization in the first 2 years of life: results from an SE Asian longitudinal cohort study

Assessment of antibody responses to pneumococcal colonization in early childhood may aid our understanding of protection and inform vaccine antigen selection. Serum samples were collected from mother-infant pairs during a longitudinal pneumococcal colonization study in Burmese refugees. Maternal and cord sera were collected at birth and infants were bled monthly (1–24 months of age). Nasopharyngeal swabs were taken monthly to detect colonization. Serum IgG titres to 27 pneumococcal protein antigens were measured in 2624 sera and IgG to dominant serotypes (6B, 14, 19F, 19A and 23F) were quantified in 864 infant sera. Antibodies to all protein antigens were detectable in maternal sera. Titres to four proteins (LytB, PcpA, PhtD and PhtE) were significantly higher in mothers colonized by pneumococci at delivery. Maternally-derived antibodies to PiuA and Spr0096 were associated with delayed pneumococcal acquisition in infants in univariate, but not multivariate models. Controlling for infant age and previous homologous serotype exposure, nasopharyngeal acquisition of serotypes 19A, 23F, 14 or 19F was associated significantly with a ≥2-fold antibody response to the homologous capsule (OR 12.84, 7.52, 6.52, 5.33; p <0.05). Acquisition of pneumococcal serotypes in the nasopharynx of infants was not significantly associated with a ≥2-fold rise in antibodies to any of the protein antigens studied. In conclusion, nasopharyngeal colonization in young children resulted in demonstrable serum IgG responses to pneumococcal capsules and surface/virulence proteins. However, the relationship between serum IgG and the prevention of, or response to, pneumococcal nasopharyngeal colonization remains complex. Mechanisms other than serum IgG are likely to have a role but are currently poorly understood.     

The antibacterial effects of ciprofloxacin and trovafloxacin against Streptococcus pneumoniae in an in vitro dynamic model

Objective: To determine whether the newer fluoroquinolone antibiotics such as trovafloxacin posses enhanced activity against Gram-positive organisms, including Streptococcus pneumoniae , because the clinical activity of older quinolones against pneumococci has been questioned.
Methods: In this study, the bactericidal activities of ciprofloxacin and trovafloxacin against six strains of penicillin-resistant and -sensitive strains of Streptococcus pneumoniae were compared using an in vitro model that simulates human pharmacokinetics. Ciprofloxacin was administered at 750 mg every 12 h, higher than the usual daily dose of 500 mg twice a day. Trovafloxacin was administered at 300 mg every 24 h for the six strains and at 200 mg every 24 h for three of the strains.
Results: The single 300-mg dose of trovafloxacin killed five of the six strains in 4 h, with no bacterial regrowth. Ciprofloxacin reduced the initial inoculum by 3–5 logs by 24 h. Although the 300-mg dose of trovafloxacin more rapidly eradicated susceptible strains, the activity of trovafloxacin at 200 mg every 24 h was similar to that of ciprofloxacin at 750 mg every 12 h against the three strains tested.
Conclusion: Trovafloxacin (and ciprofloxacin at high doses) eradicates susceptible strains of pneumococci in an in vitrc dynamic model.     

The human antibody response to pneumococcal capsular polysaccharides is dependent on the CD40-CD40 ligand interaction

Protection against infections with Streptococcus pneumoniae is mediated by antibodies against the capsular polysaccharides (caps-PS). Here we show that in in vitro experiments CD4+ T lymphocytes stimulate and CD8+ T lymphocytes inhibit the human anti-caps-PS antibody response. Using antagonistic anti-CD40 and antagonistic anti-CD40 ligand (CD40L) monoclonal antibodies, we showed that the CD4+ T lymphocyte-mediated stimulation is dependent on the CD40-CD40L interaction. The role of CD40L was further illustrated by the observation that CD4+ T lymphocytes obtained from a patient with hyper-IgM syndrome were unable to enhance the immune response to caps-PS. Furthermore, CD4+ T lymphocytes from cord blood, which did not express CD40L in response to stimulation with caps-PS, failed to stimulate the antibody response of adult B lymphocytes to caps-PS. These in vitro findings were confirmed by in vivo experiments in which SCID/SCID mice were reconstituted with human mononuclear cells. Furthermore, we showed that caps-PS induce production of IL-4, IL-6, IL-10, and IFN-gamma, and that this enhanced production was inhibited by blocking the CD40-CD40L interaction. This is the first demonstration that the human immune response to caps-PS, which is markedly regulated by T lymphocytes, is dependent on the CD40-CD40L interaction.     

Treatment with a monocolonal antibody to IL-8 attenuates the pleocytosis in experimental pneumococcal meningitis in rabbits when given intravenously, but not intracisternally

The role of interleukin (IL)-8 as mediator in the recruitment of leucocytes into the CSF was investigated during experimental pneumococcal meningitis. Rabbits were inoculated intracisternally with approximately 10(6) CFU Streptococcus pneumoniae, and treated (i) intravenously with 5 mg of a monoclonal antibody to IL-8 (n = 7) or 5 mg of an isotype control antibody (n = 6); (ii) intracisternally with anti-IL-8, 100 microg (n = 5), 10 microg (n = 4), 1 microg (n = 4), 0.1 microg (n = 2). Ten rabbits served as untreated control group. Intravenous treatment with anti-IL-8 attenuated the pleocytosis significantly compared to untreated rabbits (P < 0.04) or rabbits treated with an isotype control antibody (P < 0.02). In contrast, intracisternal treatment with anti-IL-8 failed to attenuate the pleocytosis (P > 0.05). These results show, that IL-8 plays an important role in the recruitment of leucocytes during experimental pneumococcal meningitis, and that the functional activity of IL-8 in this process appears to be on the bloodstream side of the microvascular endothelium of the brain.     

Lactobacillus casei addition to a repletion diet-induced early normalisation of cytokine profils during a pneumococcal infection in malnourished mice

This work studied the influence of Lactobacillus casei on cytokine production during repletion of malnourished mice in the face of an infectious challenge. In addition, the number and function of cells involved in the immune response against a respiratory infection was evaluated. Weaned mice were malnourished after consuming a protein-free diet (PFD) for 21 days. Malnourished mice were fed a balanced conventional diet (BCD) for 7 days or BCD for 7 days with L. casei supplementation on day 6 and day 7 (BCD+Lc). The malnourished control group (MNC) received PFD while the well-nourished control (WNC) mice consumed BCD. Mice were challenged intranasally with Streptococcus pneumoniae at the end of each dietary treatment. Malnutrition impaired the levels of serum tumor necrosis factor (TNF)-α and interleukin (IL)-1β, IL-4, IL-6 and IL-10. In addition, neutrophil number and activity, lymphocyte maturation and bone marrow CD4+, CD8+ and CD19+ cells number, were also impaired in the MNC group. Repletion with BCD induced a slight improvement in some of the parameters studied. However, when L. casei was added to the BCD, a normalisation of TNF-α, IL-1β and IL-6 values after infection and an increase in the levels of IL-10 and IL-4 compared to the WNC group was observed. Moreover, BCD+Lc induced a significant improvement in blood and bone marrow cells. Consequently, the use of L. casei as a supplement in a repletion diet was associated with a pattern of inflammatory and anti-inflammatory cytokines that led to an increased number and functionality of the cells that participate in the immune response against a pneumococcal infection.     

重组白细胞介素-17A鼻黏膜免疫抗肺炎链球菌感染的实验研究

目的 观察小鼠重组白细胞介素-17A (rIL-17A)鼻腔黏膜免疫对感染肺炎链球菌(Streptococcus pneumoniae,Sp)小鼠防御素β-2(Defb2)、巨噬细胞炎症蛋白(MIP)等表达的影响,探讨rIL-17A抗肺炎链球菌感染的机制.方法 SPF级BALB/c小鼠随机分为3组:肺炎组、干预组、对照组,以鼻腔接种的方法建立肺炎模型,采用鼻黏膜免疫的方法进行rIL-17A干预.以real-time荧光定量PCR法检测肺组织Defb2、MIP-1α、MIP-2β mRNA的表达,ELISA法检测支气管肺泡灌洗液(BALF)、脾细胞以及纵膈淋巴结细胞培养上清中MIP-1α、MIP-2β、IFN-γ、IL-4的浓度,并对BALF进行白细胞分类计数和Sp菌落计数,对肺组织进行病理分析.结果 rIL-17A干预组BALF中Sp菌落数较肺炎组明显降低(2.18±0.94 vs 4.37+0.57,P＜0.01),中性粒细胞及巨噬细胞数量显著高于肺炎组(P＜0.01);干预组肺组织Defb2、MIP-1α mRNA表达上调,其中Defb2 mRNA表达量为对照组的53.93倍,与肺炎组比较差异有统计学意义(53.93±4.80 vs 14.49±5.84,P＜0.01);rILL-17A干预组与肺炎组比较,淋巴结细胞培养上清中MIP-1α浓度增高明显(431.80±31.57 vs 291.10±5.62,P＜0.01);MIP-2β浓度在脾细胞和淋巴结细胞培养上清中较肺炎组显著增高(246.20±11.50 vs 183.70±10.64,508.50+20.26 vs 290.90+15.20,P＜0.01),而肺泡灌洗液中浓度变化不显著(P＞0.05);IFN-γ浓度在BALF和细胞培养上清中均较肺炎组显著升高;ILM除淋巴结细胞培养上清中外,BALF和脾细胞培养上清中均较肺炎组显著升高(92.42±3.82 vs 80.68±4.83,106.80±8.07 vs 73.57+7.43,P＜0.01);干预组与肺炎组小鼠支气管及血管周围炎症细胞浸润无显著差异,但干预组组织损伤较轻.结论rIL-17A可通过促进Ddb2以及MIP、IFN-y、IL-4等炎症因子的表达,增加炎症部位白细胞募集,提高Sp清除率来增强肺炎小鼠的抗感染能力.