小鼠肺炎链球菌psaA核酸疫苗的制备及其初免-蛋白加强免疫策略的研究

白加强免疫1次,对照组5只小鼠经pVAX1空载体免疫3次后,生理盐水加强免疫1次,分别采集血清测定两组抗PsaA抗体水平.结果 成功克隆出psaA基因并实现其亚克隆;所构建的psaA核酸疫苗序列正确,同时实现了PsaA蛋白的一步亲和纯化;psaA核酸疫苗可在293T真核细胞中成功表达;核酸疫苗初免后蛋白加强组的抗PsaA抗体滴度高于对照组(t=87.518,P＜0.05).结论 成功构建出psaA核酸疫苗,采用核酸疫苗初免-蛋白加强免疫策略能增强肺炎链球菌psaA核酸疫苗的免疫原性.     

中耳炎与肺炎链球菌疫苗研究现状

中耳炎是耳鼻喉科常见病,多发病,尤其在儿童中发病率更高.在美国就诊的儿科患者中大约有12%是因为中耳炎.大约有75%的儿童在一岁时至少患过一次中耳炎,17%的儿童至少患过三次中耳炎,而中耳炎的发病高峰年龄是在出生后的6到18月之间.     

CTB增强PsaA疫苗对肺炎链球菌急性中耳炎的保护作用

目的研究以霍乱毒素B(CTB)作为PsaA蛋白疫苗黏膜佐剂经鼻腔黏膜途径免疫在BALB/c小鼠体内诱导的特异性免疫应答能力以及对肺炎链球菌急性中耳炎的保护作用。方法 6~8周龄的BALB/c小鼠随机分为3组,分别接受PsaA蛋白免疫(PsaA组,15μg PsaA蛋白),PsaA蛋白及CTB免疫(PsaA/CTB组,15μg PsaA/4μg CTB),CTB免疫(CTB组,4μg CTB),经鼻腔途径免疫,相同剂量和方法每周免疫两次,连续三周,末次免疫后两周收集鼻腔灌洗液、中耳灌洗液及支气管肺泡灌洗液检测特异性IgA抗体反应;收集血清,检测特异性IgG、IgG1及IgG2a抗体反应;末次免疫后2周,小鼠经鼓膜注射14型肺炎链球菌,攻毒后5天组织病理学评估中耳炎症程度。结果PsaA/CTB组鼻腔黏膜免疫获得了较高水平的全面免疫应答:鼻腔、中耳和支气管肺泡灌洗液中特异性IgA抗体水平明显高于PsaA组和CTB组(P<0.05);血清中特异性IgG、IgG1、IgG2a抗体水平明显高于PsaA组和CTB组(P<0.05);攻毒后5天PsaA/CTB组中耳炎症细胞数少于PsaA和CTB组(P<0.05)。结论 CTB是肺炎链球菌蛋白疫苗PsaA有效的黏膜免疫佐剂,黏膜免疫也是PsaA有效的免疫途径,该免疫策略研究为新一代肺炎链球菌疫苗的设计提供了实验基础。     

Toll样受体2和4在小鼠肺炎链球菌中耳急性感染中的作用

目的 探讨Toll样受体2(Toll-like receptor 2,TLR2)和Toll样受体4(Toll-like receptor 4,TLR4)在小鼠肺炎链球菌中耳感染中的作用.方法 野生型(wild type,WT)C57BL/6J小鼠、TLR2缺陷(TLR2-/-)和TLR4缺陷(TLR4-/-)小鼠分别通过中耳鼓膜接种肺炎链球菌悬液[1×106菌落形成单位(colony forming unit,CFU)].在细菌接种前、接种后第3天和第7天分别进行听性脑干反应(ABR)和鼓室声导抗测试,血液及中耳积液中细菌滴度测定,颞骨病理切片形态学观察,反转录聚合酶链反应(RT-PCR)检测不同基因型小鼠炎性细胞因子IκB激酶β(IκB kinase β,IKKβ)、核因子κB(NF-κB)、肿瘤坏死因子α(TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、巨噬细胞炎性蛋白1α(MIP-1α)、黏蛋白/黏液素5AC和5B(MUC5AC和MUC5B)mRNA的表达.结果 中耳接种肺炎链球菌3d后,TLR2-/-小鼠死亡率为58.8％(40/68),TLR4-/-小鼠死亡率为35.6％(21/59),而WT小鼠的死亡率仅为17.3％(9/52).接种7d后,TUR2-/-小鼠死亡率为82.4％(54/68),TLR4-/-小鼠死亡率为54.2％(32/59),而WT小鼠的死亡率仅为23％(12/52),三组之间差异具有统计学意义(P＜0.01).ABR测试结果显示,接种后存活3d及7d的TLR2-/-和TLR4-/-小鼠ABR阈值高于WT小鼠,三组之间的差异具有统计学意义(P＜0.01).颞骨病理发现,TLR2-/-和TLR4-/-小鼠接种肺炎链球菌后第3天中耳出现积液和组织破坏,接种后第7天感染极为严重.TLR2-/-鼠出现严重的耳蜗炎性细胞浸润,内外毛细胞破坏、盖膜肿大和血管纹退变,以及螺旋神经节细胞的损伤;TLR4-/-鼠可见耳蜗内外毛细胞和螺旋神经节细胞的损伤;而WT鼠的耳蜗未见炎性细胞浸润和组织破坏,内外毛细胞基本正常.在接种细菌3d和7d的TLR2-/-鼠中耳黏膜未发现肥大细胞,但在TLR4-/-和WT鼠中耳黏膜发现较多的肥大细胞并有脱颗粒现象.接种肺炎链球菌后3d和7d的TLR2-/-和TLR4-/-小鼠血液中细菌滴度均高于WT小鼠,差异具有统计学意义(P值均＜0.05).接种肺炎链球菌3d后,TLR2-/-小鼠听泡组织中NF-κB、TNFα、IL-1β、MIP-1α、MUC5AC、MUC5B等因子的mRNA表达水平低于TLR4-/-和WT小鼠,差异具有统计学意义(P值均＜0.05).结论 TLR2-/-鼠在肺炎链球菌激惹后产生相对低水平的致炎性细胞因子,中耳清除细菌能力下降,引发脓毒血症,导致高死亡率.TLR2和TLR4是2种重要的小鼠中耳炎性反应的受体和信号转导分子.     

In VitroStreptococcus pneumoniae Biofilm Formation and In Vivo Middle Ear Mucosal Biofilm in a Rat Model of Acute Otitis Induced by S. pneumoniae

Objectives

Streptococcus pneumoniae is one of the most common pathogens of otitis media (OM) that exists in biofilm, which enhances the resistance of bacteria against antibiotic killing and diagnosis, compared to the free-floating (planktonic) form. This study evaluated biofilm formation by S. pneumoniae on an abiotic surface and in the middle ear cavity in a rat model of OM.

Methods

In vitro biofilm formation was evaluated by inoculation of a 1:100 diluted S. pneumoniae cell suspension in a 96-well microplate. Adherent cells were quantified spectrophotometrically following staining with crystal violet by measurement of optical density at 570 nm. The ultrastructure of pneumococcal biofilm was assessed by scanning electron microscopy (SEM). For in vitro biofilm study, S. pneumoniae cell suspensions containing 1×10⁷ colony forming units were injected through transtympanic membrane into the middle ear cavity of Sprague Dawley rats. The ultrastructure of middle ear mucus was observed by SEM 1 and 2 weeks post-inoculation.

Results

The in vitro study revealed robust biofilm formation by S. pneumoniae after 12-18 hours of incubation in high glucose medium, independent of exogenously supplied competence stimulating peptide and medium replacement. Adherent cells formed three-dimensional structures approximately 20-30 µm thick. The in vivo study revealed that ciliated epithelium was relatively resistant to biofilm formation and that biofilm formation occurred mainly on non-ciliated epithelium of the middle ear cavity. One week after inoculation, biofilm formation was high in 50% of the treated rats and low in 25% of the rats. After 2 weeks, biofilm formation was high and low in 25% and 37.5% of rats, respectively.

Conclusion

The results imply that glucose level is important for the S. pneumoniae biofilm formation and S. pneumoniae biofilm formation may play important role in the pathophysiology of OM.     

Acute otitis media develops in the rat after intranasal challenge of Streptococcus pneumoniae

OBJECTIVES/HYPOTHESIS: The rat is a frequently used animal model for middle ear research. To date, acute otitis media (AOM) has been evoked after instillation of bacteria directly into the middle ear cavity or after traumatizing the tympanic membrane. The purpose of the study was to examine whether, with an intact tympanic membrane and middle ear cavity, intranasally deposited bacteria cause AOM and how tympanic membrane stimulation influences this procedure. STUDY DESIGN: In vivo, murine model. METHODS: In a rat model, Streptococcus pneumoniae, type 3, was intranasally inoculated for 5 consecutive days. The tympanic membrane was treated with saline or with compound 48/80 or was left untreated. The development of AOM was evaluated by otomicroscopy, light microscopy, and middle ear culture. RESULTS: Ninety percent of the ears developed AOM. However, when the tympanic membranes were treated with saline or compound 48/80, only 40% and 57%, respectively, developed AOM. In all, 23 of 40 ears developed AOM and 20 ears showed growth of bacteria. CONCLUSION: Repeated intranasal deposition of S. pneumoniae, type 3, causes AOM in the rat. The development of AOM can be influenced by tympanic membrane stimulation.     

Cytokine profiles in a rat model of otitis media with effusion caused by eustachian tube obstruction with and without Streptococcus pneumoniae infection

OBJECTIVE: Cytokine expression was studied in a rat model of otitis media with effusion. METHODS: The left eustachian tube was obstructed (eustachian tube obstruction [ETO]) in 84 rats. Forty-two ears were challenged with, and those rats were treated from day 2 to day 7 with ampicillin. Twelve rats (6 per group) were killed on days 1, 2, 7, 21, 35, 56, and 112; mucosa was harvested and assayed for interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-10 (IL-10), interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), monocyte chemoattractant protein-1 (MCP-1), and interleukin-8 (IL-8) gene expression, and effusion was assayed for IL-1beta, TNF-alpha, IL-6, IL-10, and macrophage inflammatory protein-2 (MIP-2) protein. RESULTS: Most cytokines were detectable in the effusion from infected ears with ETO on days 1 and 2 only. MIP-2 exhibited a biphasic response. Only effusion MIP-2 was consistently detected in uninfected ears with ETO. Three patterns of mucosal cytokine messenger RNA (mRNA) upregulation were observed: isolated early (IL-1beta, IL-8), isolated late (TNF-alpha, IFN-gamma), and biphasic (MCP-1, IL-6, TGF-beta) responses. Early cytokine mRNA upregulations were observed only in the infected ears with ETO, whereas late upregulations were observed in both groups. CONCLUSIONS: Early expression of the assayed cytokines occurred only in ears with active infection. For both groups, a late upregulation of cytokine message but not protein was documented. The profile of cytokine expression during otitis media episodes may be useful in defining etiology, disease stage, and prognosis.     

Serum IgA and IgG functional antibodies and their subclasses to Streptococcus pneumoniae capsular antigen found in two aged‐matched cohorts of children with and without otitis media with effusion

Serum IgA and IgG functional antibodies and their subclasses to Streptococcus pneumoniae capsular antigen found in two aged‐matched cohorts of children with and without otitis media with effusion The relationship between acute otitis media and otitis media with effusion (OME) is uncertain and the aetiology of OME is multifactorial. Otitis media with effusion may be an inflammatory condition; both bacteria and viral infections could play a part in this inflammation. The four bacteria Streptococcus pneumoniae, Haemophilus influenza, Staphylococcus aureus and Branhamella catarrhalis cause 60% of the infections whereas S. pneumoniae accounts for up to 35%. IgA provides the dominant surface response to polysaccharide and lipopolysaccharide antigens, of which IgA₂ is the main subclass. Once the mucosa has been breached, most protection is provided by IgG. IgG₂ acts mainly against bacterial capsular antigens. This study looked at two groups of 50 children with and without OME who were aged between 3 and 10 years. The aims were to determine if, firstly, the levels of the serum immunoglobulins were different in the two groups, secondly whether these children made the appropriate antibody response to the capsular antigen to S. pneumoniae (PCP), and finally if there was a delay in the maturity of the IgA response. The total IgG, IgA and all subclass levels were measured using radial immunodiffusion. Levels of functional IgA and IgG were measured using ELISAs (25 patients in each group). The results were analysed with non‐parametric tests. The immunoglobulin levels were within the normal levels for both groups. There were very good correlations between the IgG total anti‐PCP and the IgG₂ anti‐PCP (R > 0.9, p = 0.001). There was a good correlation between the levels of both IgG total and IgG₂ anti‐PCP against IgA total anti‐PCP in both groups (R > 0.85, p > 0.01). This confirms a normal antibody response between both groups of patients. The ages of the controls and patients (50 samples) were correlated with increasing titres of circulating functional antibodies (P = 0.001). This is highly suggestive of a normal age‐related response. In conclusion, the findings were contradictory to our original hypothesis that there is a subtle difference in surface protection between children with and without OME. We believe that a previous history of recurrent acute otitis media is unrelated to the development of OME after 3 years of age.     

Occurrence of Streptococcus pneumoniae and Haemophilus influenzae in otitis media with effusion

Eighty mucoid effusion samples obtained from 56 patients with otitis media with effusion (OME) were subjected to quantitative and qualitative bacteriological analysis using standard culturing methods, direct microscopy and immunofluorescent assay. 30% of the samples contained culture-positive pathogens (H. influenzae, S. pneumoniae, B. catarrhalis), with counts never exceeding 5 times 10⁵ per ml. In addition, 19% of the samples had dormant H. influenzae and S. pneumoniae, which did not grow on standard agar plates. Viable and dormant bacteria, as well as bacterial remnants, play a crucial role in the pathogenesis of OME and similarities between OME and reactive arthritis, i.e. Lyme arthritis, Reiter's syndrome and rheumatic fever, are evident.     

pVVP3IL-18HN核酸疫苗的构建及其对喉癌细胞杀伤作用的研究

目的通过构建共表达凋亡素基因、新城疫病毒HN基因和白介素18（interleukin-18，IL-18）基因的核酸疫苗，观察联合应用此三种基因对喉癌细胞的杀伤效应。方法在真核表达质粒pVAX1中的CMV启动子下游插入凋亡素基因和含有IL-18HN嵌合基因并携带IRES启动子的基因片段，构建能同时表达三种基因的真核表达质粒pVVP3IL-18HN，经RT—PCR、间接免疫荧光和Western—blot法检测外源基因的表达。采用脂质体介导法将构建的重组质粒pVVP3IL-18HN体外转染Hep-2细胞，运用MTT法检测不同质粒浓度、不同作用时间，该重组质粒对喉癌细胞Hep-2的杀伤率。结果pVVP3IL-18HN可有效杀伤喉癌细胞，且在作用72h、质粒浓度为20μg／ml时杀伤率最高，达61．9％。结论重组质粒pVVP3IL-18HN能有效杀伤Hep-2细胞，可用于喉癌的治疗和研究。