胸腺基质淋巴生成素受体及其抗体在哮喘小鼠气道炎症反应中的作用

目的 探讨胸腺基质淋巴生成素受体(TSLPR)及其抗体在实验性哮喘小鼠气道炎症反应中的作用以及对气道树突状细胞(DCs)成熟和活化的影响.方法 BALB/c小鼠随机分成A、B、C三组.B和C组小鼠腹腔注射卵清白蛋白(OVA)致敏,A组腹腔注射PBS作为正常对照.B和C组小鼠在OVA激发哮喘发作前分别吸入非特异性IgG和TSLPR IgG.采集各组小鼠支气管肺泡灌洗液(BALF)进行细胞分类计数,采用ELISA定量检测BALF中IL-4、IL-5、IFN-γ和IL-10浓度.采集各组小鼠肺组织标本进行病理学检查,采用流式细胞术分别检测各组小鼠淋巴结和肺组织中DCs数量和表型.结果 与A组小鼠比较,8和C组小鼠BALF中各种细胞因子水平均明显升高(P<0.01).C组小鼠BALF中IL-4和IL-5水平低于8组(P<0.05,P<0.01),IFN-γ和IL-10水平高于8组(P<0.05,P<0.01).C组小鼠BALF中细胞总数、嗜酸性粒细胞和淋巴细胞数也明显低于B组(P<0.01).B组小鼠支气管周围有大量炎性细胞浸润以及杯状细胞增生,黏液分泌增强,C组小鼠仅见微弱的炎性细胞浸润和杯状细胞增生.B组小鼠膈淋巴结中DCs数量以及肺组织中DCs的I-Ad、CD40、CD80和CD86表达水平均高于C组小鼠(P<0.05).结论 TSLP/TSLPR具有促哮喘效应并与其调节气道DCs活性的作用密切相关,TSLPR抗体干预可明显减弱TSLP/TSLPR上述作用,故有作为抗哮喘药物的前景.     

中药预防流感作用与黏膜免疫相关性研究

目的:研究中药预防流感的作用与黏膜免疫相关性。方法:(1)不同预防方药预防给药对正常小鼠黏膜免疫的影响:将BALB/c小鼠随机分6组。设空白对照组(生理盐水)、利巴韦林组、银翘散制剂、玉屏风散制剂、冰香散滴鼻组、冰香散雾化组。给药第12天,杀剖小鼠,空白对照组称体重和肺重测定肺指数。各组均采血分离血清,收集鼻咽冲洗液、支气管肺泡灌洗液、肠道冲洗液进行ELISA法检测IgA含量。(2)不同预防方药预防给药对流感病毒感染小鼠黏膜免疫的影响:将BALB/c小鼠随机分6组,设病毒对照组及药物试验组(同前)。试验药物在滴鼻感染15 LD50流感病毒液感染攻击前提前给药一周,攻击后继续给药5天。共给药12天,每天一次给药。感染后5天,杀剖小鼠,称体重和肺重测定肺指数。采血分离血清,收集鼻咽冲洗液、支气管肺泡灌洗液、肠道冲洗液进行ELISA法检测IgA含量。结果:(1)不同预防方药预防给药对正常小鼠黏膜免疫的影响:连续给予正常小鼠12天试验药物,利巴韦林、银翘散制剂、玉屏风散制剂、冰香散对流感病毒鼠肺指数均有显著的改善作用,肺指数抑制率达到25.46%～41.61%。与空白对照组相比,银翘散制剂口服与冰香散雾化给药血清IgA水平显著提高;冰香散滴鼻给药鼻咽部sIgA水平显著提高;利巴韦林组给药支气管-肺泡sIgA水平下降;银翘散、玉屏风散、冰香散给药肠道sIgA水平有一定的提升作用,其中玉屏风散制剂作用明显。(2)不同预防方药预防给药对流感病毒感染小鼠黏膜免疫的影响:病毒感染小鼠攻击前预先连续给药7天,病毒感染第5天,与病毒对照组相比,利巴韦林给药血清IgA水平显著降低、冰香散雾化给药血清IgA水平显著提高;冰香散滴鼻给药能使鼻咽部sIgA水平显著提高;利巴韦林组给药支气管-肺泡sIgA水平下降,玉屏风散与冰香散雾化给药支气管-肺泡sIgA水平显著提高;玉屏风散制剂与冰香散雾化给药肠道sIgA水平显著提高。结论:利巴韦林长期预防给药(100 mg/kg)对黏膜免疫IgA分泌有一定抑制作用,不宜推荐作为流感病毒的预防性长期用药。中药预防流感的三种给药方式:口服、烟薰、芳香佩戴等均可作用于呼吸道或消化道黏膜,激发局部黏膜免疫产生分泌型IgA,从而起到预防和治疗的效果。中药预防流感的作用与黏膜免疫有一定的相关性。     

肺炎支原体P1C-IL-2融合基因疫苗鼻饲对小鼠免疫效果的检测

目的 研究肺炎支原体(Mp) P1C-IL-2融合基因疫苗经鼻饲免疫小鼠后的免疫应答水平和免疫保护作用,了解IL-2对P1C核酸疫苗的免疫佐剂效应.方法 将构建的P1C-IL-2核酸疫苗鼻饲免疫BALB/c鼠,ELISA检测免疫小鼠血清IgG滴度、IgG亚类和支气管肺泡灌洗液中IgA及IFN-γ、IL-4的水平;建立小鼠Mp感染模型,观察Mp攻击后小鼠肺组织炎症情况和支气管肺泡灌洗液中Mp菌落数的变化.结果 P1C-IL-2双基因疫苗组小鼠血清中的总IgG、IgG1、IgG2a亚类和支气管肺泡灌洗液中IFN-γ和IL-4水平均较PIC疫苗组小鼠显著增高(P＜0.05),但两组支气管肺泡灌洗液IgA差异无显著性(P＞0.05).用Mp滴鼻感染免疫小鼠,第1、3、6天P1C-IL-2双基因融合疫苗组小鼠肺组织炎症病理评分显著高于P1C单基因疫苗免疫组小鼠,两组小鼠支气管肺泡灌洗液中的Mp菌落数差异无显著性(P＞0.05).结论 IL-2能显著增强PIC疫苗的免疫应答水平,但在感染早期也激发了较强的肺组织炎症.     

T-bet质粒基因转染对哮喘小鼠支气管肺泡灌洗液白细胞介素13和内皮素1的影响

目的 观察气道内T-bet质粒基因转染对哮喘小鼠气道肺泡灌洗液白细胞介素13(IL13)、内皮素1(ET-1)的影响.方法 C57BL/6J小鼠32只,完全随机分为4组(n=8),即哮喘模型组(A组)、正常对照组(B组)、空质粒干预组(C组)和模型T-bet质粒干预组(D组).用卵白蛋白(OVA)抗原溶液小鼠腹腔注射致敏,滴鼻造模.B组用生理盐水代替卵白蛋白,C组和D组卵白蛋白激发48 h前,分别经鼻滴入50μg空质粒或重组T-bet质粒.免疫印迹检测和酶联免疫吸附法(ELISA)检测各组小鼠的支气管肺泡灌洗液中IL-13、ET-1的水平.结果 哮喘模型组小鼠支气管肺泡灌洗液(BALF)中Th2因子IL-13和ET-1水平比正常对照组升高[(2.32±0.40)ng/L比(0.38±0.16)ng/L,(40.07±6.52)ng/L比(14.16±0.51)ng/L,均P＜0.05];模型T-bet质粒干预组BALF中Th2因子IL-13和ET-1水平[(1.16±0.19)和(23.08±2.59)ng/L]比哮喘模型组降低(均P＜0.05).结论 气道内转染T-bet质粒能有效下调小鼠BALF中Th2因子IL-13及ET-1水平.     

标准化粉尘螨疫苗免疫治疗哮喘小鼠气道炎症的实验研究

目的建立小鼠粉尘螨过敏性哮喘模型,探讨标准化粉尘螨疫苗免疫治疗的疗效及机理。方法 32只Balb/c小鼠随机分为阴性对照组(A组)、哮喘模型组(B组)、粉尘螨疫苗低剂量治疗组(C组)、粉尘螨疫苗高剂量治疗组(D组)。A组始终给予生理盐水,B、C、D组腹腔注射50μg粉尘螨提取液和2 mg Al(OH)3免疫小鼠致敏。然后分别用PBS(50μl)、100μg、1 mg的粉尘螨粗提液皮下注射进行免疫治疗,各组用粉尘螨抗原(50μg/50μl)滴鼻激发,末次激发后24 h用气道高反应仪检测小鼠气道反应性;处死小鼠,收集支气管肺泡灌洗液(BALF)、血清以及取出小鼠的脾细胞进行后续检测。结果 B组、C组肺泡灌洗液中细胞总数和嗜酸性细胞数显著升高,气道反应性明显增强,两组相比无显著性差异。与之相比,D组气道反应性明显下降,肺泡灌洗液中细胞总数和嗜酸性细胞数显著降低(P<0.01)、Der.f特异性抗体IgE显著降低、支气管肺泡灌洗液上清中IL-4降低(P<0.05),IL-10、IFN-r的含量显著增高(P<0.05),细胞增殖反应显示D组受抑制。结论粉尘螨疫苗可以有效抑制哮喘模型肺部炎症变化和降低气道高反应性,其疗效与剂量有关,机制可能与调节Th1/Th2平衡有关。     

白介素17单克隆抗体对变应性鼻炎小鼠气道炎症的作用

目的 探讨白介素17单克隆抗体（IL-17mAb）的不同给予剂量及方式在变应性鼻炎小鼠气道炎症中的作用。方法 将48只小鼠采用随机数字表法分为A、 B、 C、 D、 E、 F组,每组8只。分别于第0、 7、 14 d将20 μg卵清蛋白（OVA）加2 mg铝佐剂腹腔注射处理A、C、D、E及F组小鼠,间隔7 d,第22天开始进行鼻腔激发,每天每侧鼻孔各给予OVA 10 μl（共500 μg）滴鼻,连续7 d。A、C、D、E组小鼠于每次OVA鼻腔激发前1 h分别给予生理盐水、100 ng IL-17mAb、500 ng IL-17mAb、5 μg IL-17mAb滴鼻,F组小鼠于每次OVA鼻腔激发前4 h给予5 μg IL-17mAb腹腔注射,B组小鼠于相同时间点给予等量生理盐水腹腔注射及滴鼻。所有小鼠于最后1次激发后评估鼻部症状学变化,Diff-Quik染色观察鼻腔灌洗液（NLF）中嗜酸性粒细胞浸润情况,ELISA方法检测血清及NLF中IL-6、IL-10水平,鼻黏膜组织行甲苯胺蓝染色观察肥大细胞。结果 ４周末A组所有小鼠症状学评分均＞5分,提示造模成功。F组小鼠的挠鼻及喷嚏次数均少于A组（P＜0.05）;F组小鼠NLF中嗜酸性粒细胞数、血清IL-6水平低于A组,血清及NLF中IL-10水平均高于A组（P＜0.05）;E组小鼠血清中IL-10水平高于A组（P＜0.05）;A组小鼠鼻黏膜组织中肥大细胞数多于B组,统计学意义显著（P＜0.01）;F组小鼠鼻黏膜组织中肥大细胞数少于A组,但差异无统计学意义（P＞0.05）;F组小鼠鼻黏膜组织中肥大细胞数与B组比较,差异无统计学意义（P＞0.05）。结论 高剂量的（5 μg）IL-17mAb腹腔注射处于激发阶段的变应性鼻炎小鼠促使小鼠变应性鼻炎症状明显减轻,鼻腔灌洗液嗜酸性粒细胞减少。促使变应性鼻炎小鼠血清中IL-6表达降低,血清中及鼻腔灌洗液中IL-10表达升高,因此推测这些细胞因子的变化可能抑制Th17/促进Treg的分化,进而对变态反应产生抑制作用。     

黏膜免疫壳聚糖-多胺转运蛋白D(PotD)DNA纳米微粒对小鼠鼻咽部肺炎链球菌定植的保护作用研究

目的 探讨以壳聚糖包裹的PotD DNA(Chitosan-potD)纳米微粒经鼻黏膜免疫小鼠对肺炎链球菌鼻咽部定植的保护作用.方法 将制备的Chitosan-potD微粒、裸pVAX1-potD重组质粒(裸potD)及pVAX1分别鼻腔免疫小鼠,收集黏膜和血清标本用以检测特异性抗体水平;分离并培养脾淋巴细胞,检测IL-17A、IL-4及IFN-γ的分泌水平;于免疫小鼠鼻腔进行肺炎链球菌攻击,通过菌落计数评估各免疫组对小鼠肺炎链球菌鼻咽部定植的保护作用.结果 Chilosan-potD纳米微粒在小鼠体内诱导产生的抗PotD特异性血清IgG、IgG1、IgG2a、黏膜IgA抗体水平及IL-17A、IL-4、IFN-γ水平与裸potD组及pVAX1组相比均明显升高,且差异具有统计学意义(P<0.05);菌落计数结果 显示,Chitosan-potD微粒组在鼻咽部定植的肺炎链球菌数量显著减少(P<0.05).结论 壳聚糖包裹PotD DNA微粒对肺炎链球菌鼻咽部定植具有免疫保护作用.     

蛋白体佐剂及用于鼠疫F1-Ⅴ抗原的免疫效果

目的以蛋白体(proteosomes)佐剂,非共价结合鼠疫F1-V重组蛋白为免疫原,探讨滴鼻免疫BALB/c小鼠后诱导的免疫应答和免疫保护效果。方法佐剂与鼠疫F1-V重组蛋白为免疫原非共价结合,滴鼻免疫BALB/c小鼠4次后,采用间接ELISA检测血清特异性抗F1-V的IgG和IgA抗体及抗体亚型分类,并检测鼻咽、肺、小肠及阴道灌洗液中特异性抗F1-V的黏液分泌型IgA;并用流式细胞术检测鼻相关淋巴组织淋巴细胞、脾淋巴细胞、肠系膜淋巴结及小肠PP结T淋巴细胞表型的变化。第4次免疫后7d,用100 LD_(50)的鼠疫141强毒株进行腹腔攻毒。结果(1)以蛋白体为佐剂的鼠疫F1-V抗原与单纯的F1-V组相比,蛋白体疫苗组诱导血清IgG、IgA抗体显著升高(P<0.01),同时蛋白体疫苗组能诱导鼻咽、肺、小肠和阴道内多个黏膜部位特异性IgA抗体的产生,尤其是肺和生殖道冲冼液内抗体升高极为显著(P<0.01);(2)蛋白体疫苗组主要引起IgG1型抗体,主要诱导T_H2型免疫反应;(3)蛋白体疫苗组NALT和SP中CD4~ /CD8~ 比值比PBS对照有显著增高(P<0.01),MLN和PP中CD4~ /CD8~ 比值与PBS对照差异无统计学意义(P>0.05)。(4)小鼠在100 LD_(50)的鼠疫141强毒株腹腔攻毒后鼠疫F1-V重组蛋白组小鼠免疫保护率为0,而蛋白体佐剂疫苗组小鼠免疫保护率为67%。结论以自制的蛋白体为鼠疫F1-V抗原的佐剂滴鼻免疫小鼠,蛋白体不仅提高鼠疫F1-V抗原的系统免疫应答,而且能诱导小鼠呼吸道、消化道和生殖道局部黏膜免疫应答。蛋白体佐剂鼠疫疫苗对100 LD_(50)的鼠疫141强毒株腹腔攻毒具有一定的免疫保护作用,这为鼠疫黏膜疫苗的研制提供候选材料,也为鼠疫黏膜疫苗深入研究奠定了基础。     

HMGB1对小鼠腹腔巨噬细胞吞噬和I-A/E表达的影响

目的:探讨HMGB1对巨噬细胞免疫功能的影响.方法:梯度浓度HMGB1处理小鼠腹腔巨噬细胞, 或将小鼠随机分为生理盐水对照、 24 h和48 h高与低剂量HMGB1注射组, 腹腔注射0.2 μg或20 μg HMGB1, 或生理盐水.检测巨噬细胞吞噬功能和I-A/E表达.结果:10 μg/L HMGB1刺激6～12 h, 巨噬细胞吞噬功能较其他剂量组明显增强(P<0.05或P<0.01).HMGB1对培养巨噬细胞I-A/-E表达无影响.高剂量HMGB1攻击小鼠24 h, 其腹腔巨噬细胞吞噬能力明显降低(P<0.05); 低剂量HMGB1攻击48 h, 巨噬细胞I-A/-E表达明显上调(P<0.05或P<0.01).结论:高剂量HMGB1抑制巨噬细胞吞噬功能, 低剂量HMGB1增强巨噬细胞免疫功能.     

巨噬细胞源性趋化因子对NTHiP6蛋白疫苗免疫效果的影响

观察巨噬细胞源性趋化因子(MDC)佐剂对NTHiP6蛋白疫苗免疫效果的影响。将原核表达质粒PGEX-6P2/P6转入E.coli XL1-Blue,IPTG诱导P6蛋白的表达并进行纯化。将BALB/c小鼠随机分为A-D四组,分别为PBS对照组、MDC对照组、P6蛋白组、P6蛋白联合MDC组。分别于0、14、28d经黏膜免疫,末次免疫后14d,每组12只小鼠取血和肺泡灌洗液,ELISA检测血清中IgG抗体和肺泡灌洗液中IgA抗体水平。每组取3只小鼠,制备脾淋巴细胞,ELISA检测IL-4、IL-17和IFN-γ水平。用10LD50NTHi攻击每组剩余15只小鼠,观察免疫保护作用。在大肠杆菌中成功表达P6蛋白。第三次免疫后,D组诱导的IgG抗体、IgA抗体、IL-4、IL-17和IFN-γ水平显著高于其他各组(P0.05)。经NTHi攻击后,D组生存率达80%,与A组、B组相比差异有统计学意义(P0.05),但C组、D组之间无显著性差异(P0.05)。MDC作为佐剂可以使NTHiP6疫苗获得较好的免疫效果。     

Comparison of protective efficacy of subcutaneous versus intranasal immunization of mice with a Brucella melitensis lipopolysaccharide subunit vaccine

Groups of mice were immunized either subcutaneously or intranasally with purified Brucella melitensis lipopolysaccharide (LPS) or with LPS as a noncovalent complex with Neisseria meningitidis group B outer membrane protein (LPS-GBOMP). Control mice were inoculated with sterile saline. Two doses of vaccine were given 4 weeks apart. Mice were challenged intranasally with virulent B. melitensis strain 16M 4 weeks after the second dose of vaccine. Sera, spleens, lungs, and livers of mice were harvested 8 weeks after challenge. The bacterial loads in the organs were determined by culture on brucella agar plates. Protective efficacy was determined by comparing the clearance of bacteria from organs of immunized mice with the clearance of bacteria from organs of control mice. At 8 weeks postchallenge there was significant protection from disseminated infection of spleens and livers of mice intranasally immunized with either vaccine compared to infection of control mice (P < 0.01). There was no significant difference in clearance of bacteria from the lungs of immunized mice and control mice. However, mice immunized subcutaneously with either LPS or LPS-GBOMP vaccine showed significant protection against infection of the spleen (P < 0.001), liver (P < 0.001), and lungs (P < 0.05). These results show that intranasal immunization of mice with either vaccine provided significant protection against disseminated infection of the spleen and liver but subcutaneous immunization of mice with the vaccines conferred significant protection against infection of the spleen, liver, and lungs.     

Passive immunization in experimental Herpesvirus hominis infection of newborn mice.

Infection of newborn mice with Herpesvirus hominis type 2(HVH-2) was used as an experimental model of disseminated HVH infection in newborn humans. Mice were challenged with 103 plaque-forming units of HVH-2 intranasally and were given 0.2 ml of rabbit serum intraperitoneally. Passive immunizations with rabbit anti-HVH-2 serum resulted in a significant decrease in mortality and prolongation of survival time. This effect correlated with the neutralizing antibody titer of the serum against HVH-2 and was more pronounced when immune serum was administered 1 h after infection as compared with 24 h. These results suggest that administration of high-titer anti-HVH-2 immunoglobulins shortly after delivery could afford significant protection to the newborn of a mother with genital HVH-2 infection.     

Protection of mice against brucellosis by intranasal immunization with Brucella melitensis lipopolysaccharide as a noncovalent complex with Neisseria meningitidis group B outer membrane protein

Intranasal immunization of mice with purified Brucella melitensis lipopolysaccharide (LPS) as a noncovalent complex with Neisseria meningitidis group B outer membrane protein (GBOMP) elicited a high-titer anti-LPS systemic antibody response and a significant mucosal antibody response. The anti-LPS immunoglobulin G (IgG) antibody was predominantly of the IgG1 subtype, although there was some response of the IgG2a, IgG2b, and IgG3 subtypes. The antibody titer remained high for 16 weeks postimmunization. Immunized mice and sham-immunized control mice were challenged intranasally with 10(4) CFU of virulent B. melitensis strain 16 M 4 weeks after the second dose of vaccine. The numbers of bacteria in lungs, livers, and spleens at 3 days, 9 days, and 8 weeks postchallenge were determined. Bacteria were found in lungs of all mice on day 3, but there was no disseminated infection of liver or spleen. By day 9, 40% of the mice had infected spleens and livers. At 8 weeks postchallenge, spleens of 25 of 62 immunized mice were infected, compared to 61 of 62 control mice (P < 0.0001). The livers of 12 of 43 immunized mice were infected, compared to 22 of 36 control mice (P = 0.005). In contrast, the lungs of 26 of 46 immunized mice were still infected, compared to 27 of 44 control mice. The numbers of bacterial CFU in lungs of immunized and control animals were identical. These studies show that intranasal immunization with B. melitensis LPS-GBOMP subunit vaccine significantly protects mice against intranasal challenge with virulent B. melitensis. Vaccination reduces bacterial dissemination to spleen and liver but has no effect on the course of lung infection.     

Murine hypersensitivity pneumonitis: bidirectional role of interferon-gamma

C57BL/6 mice were instilled intranasally with optimal doses [150 micrograms of antigen 3 days a week) of the actinomycete Faeni rectivirgula to induce an experimental hypersensitivity pneumonitis. Some control mice received normal rat IgG as controls, whereas other mice received 1 mg weekly of rat anti-murine interferon gamma (IFN-gamma) antibody by the intraperitoneal route and 200 micrograms by the intranasal route given 2 days before and during the challenge period before each instillation. Control mice developed a clear hypersensitivity pneumonitis characterized by an early neutrophilic response at 3 days and a later influx of mononuclear cells (nine- to tenfold increase in cell number. P less than 0.001 vs saline instilled mice at 4 weeks post-treatment). F. rectivirgula instillation determined a sharp increase in the lung index (80% increase in lung weight, P less than 0.005 vs saline treated mice), as well as a significant fibrosis at 4 weeks (twofold increase in lung hydroxyproline levels). Cytokine measurements showed that tumour necrosis factor alpha (TNF alpha) was present in the broncho-alveolar lavage (BAL) of challenged mice at 4 weeks when the BAL was obtained 8 hr after the last challenge (130 U/ml). Treatment of mice with the monoclonal antibody against IFN-gamma was associated with very few changes in the number of cells in the BAL of challenged mice. The lung index of challenged mice was significantly reduced by infusion of the anti-IFN-gamma antibody. Anti-IFN-gamma treatment resulted in decreased levels of TNF alpha in the BAL of F. rectivirgula after 4 weeks of treatment (56 U/ml, P less than 0.01). Moreover, depletion of endogenous IFN-gamma in F. rectivirgula-instilled mice resulted in a diminished lung fibrotic response (P less than 0.01 vs mice treated with F. rectivirgula and control antibody). We also studied the effect of exogenous IFN-gamma adminstration on the development of lung disease. Groups of mice received recombinant gamma interferon (IFN-gamma) (1000 U) intraperitoneally just before the first treatment and also daily, whereas controls received saline or IFN-gamma alone (no F. rectivirgula challenge). After 4 weeks of treatment, mice were killed and various markers of the disease were evaluated. As mentioned before, bronchoalveolar lavage (BAL) cell number was increased tenfold in mice treated with F. rectivirgula, whereas mice given F. rectivirgula and IFN-gamma had only a threefold increase in BAL cell number, determined mostly by a decrease in alveolar macrophage recruitment in the lungs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evaluation of formalin-inactivated Clostridium difficile vaccines administered by parenteral and mucosal routes of immunization in hamsters.

Clostridium difficile produces toxins that cause inflammation, necrosis, and fluid in the intestine and is the most important cause of nosocomial antibiotic-associated diarrhea and colitis. We evaluated C. difficile antigens as vaccines to protect against systemic and intestinal disease in a hamster model of clindamycin colitis. Formalin-inactivated culture filtrates from a highly toxigenic strain were administered by mucosal routes (intranasal, intragastric, and rectal) with cholera toxin as a mucosal adjuvant. A preparation of culture filtrate and killed whole cells was also tested rectally. The toxoid was also tested parenterally (subcutaneously and intraperitoneally) and by a combination of three intranasal immunizations followed by a combined intranasal-intraperitoneal boost. Serum antibodies against toxins A and B and whole-cell antigen were measured by enzyme-linked immunosorbent assay, neutralization of cytotoxic activity, and bacterial agglutination. The two rectal immunization regimens induced low antibody responses and protected only 20% of hamsters against death and 0% against diarrhea. The intragastric regimen induced high antibody responses but low protection, 40% against death and 0% against diarrhea. Hamsters immunized by the intranasal, intraperitoneal, and subcutaneous routes were 100% protected against death and partially protected (40, 40, and 20%, respectively) against diarrhea. Among the latter groups, intraperitoneally immunized animals had the highest serum anticytotoxic activity and the highest agglutinating antibody responses. Hamsters immunized intranasally and revaccinated intraperitoneally were 100% protected against both death and diarrhea. Protection against death and diarrhea correlated with antibody responses to all antigens tested. The results indicate that optimal protection against C. difficile disease can be achieved with combined parenteral and mucosal immunization.     

Intranasal immunization with autolysin (LytA) in mice model induced protection against five prevalent <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> serotypes in China

In order to evaluate immunogenicity and protective efficacy of LytA from Streptococcus pneumoniae, we subcloned the full-length lytA-encoded autolysin (LytA) from 5 major pathogenic serotype isolates in China and obtained purified rLytA. Bioinformatics analysis showed that sequences of LytA were highly conserved in all strains we used in this work, and western blot analysis demonstrated that rLytAs from heterogeneous serotypes were cross-recognized by serum of mice infected with 23F strain SH137. Mice were intranasally immunized with purified rLytA, and serum anti-rLytA IgG, IgA and secretory IgA were elicited. More importantly, rLytA intranasal-immunized mice showed a significantly higher survival rate and lower bacterial carriage in response to infection by Streptococcus pneumoniae. The fact that mice immunized with rLytA from strain SH137 also had a higher survival rate after intraperitoneal injection of other four serotype strains of living S. pneumoniae suggested that it possessed cross-protection effect. Our study revealed that intranasal immunization with rLytA may protect mice against mucosal and systemic pneumococcal infection; hence, it was an attractive vaccine candidate.     

Protection against experimental Bordetella bronchiseptica infection in mice by active immunization with killed vaccine.

Mice immunized with a killed vaccine of phase I Bordetella bronchiseptica were challenged with various numbers of virulent B. bronchiseptica by intraperitoneal, intracerebral, or intranasal routes. The course of infection was compared among these routes, and the protective effect of vaccination was quantitatively analyzed. In ddN mice infected intraperitoneally with 1.8 X 10(8) cells (ca. 80 times the 50% lethal dose [LD50]) the organisms rapidly increased in the intraperitoneal fluid, spleen, and liver within few days and caused splenic atrophy, septicemia, and death. However, immunizations with 5 X 10(9) cells gave the mice a high agglutinin titer and suppressed the increase in the number of organisms. With four immunizations, the lungs and livers were clear within 3 days, and with one or two immunizations, they were clear within 7 days. These immunizations effectively protected the mice from death but did not protect them from splenic atrophy. In the intracerebral infection with 1.4 X 10(6) cells (ca. 1.2 X 10(5) LD50), the number of organisms rapidly increased in the brain and caused encephalitis, splenic atrophy, and death. However, four or five immunizations completely suppressed the increase in the brain and protected the mice from death and splenic atrophy. After intranasal infection with 4 X 10(6) cells (ca. 25 LD50), the organisms rapidly increased in the nasal cavity and lungs and caused pneumonia and death. Immunization with 5 X 10(9) cells was effective in clearing the organisms from the lungs and in protecting against death and splenic atrophy. However, the organisms were not cleared from the nasal cavity for 60 to 150 days after the challenge with as little as 10(2) cells, even in the mice with an agglutinin titer as high as 1:10,000.     

T-bet基因转染对哮喘小鼠气道炎症的影响

目的： 观察气道内T-bet基因转染对哮喘小鼠气道炎症的影响。方法: C57BL/6小鼠40只,随机分为4组,每组10只,分别为正常对照组（A组）、哮喘模型组（B组）、空质粒干预组(C组) 和T-bet质粒干预组(D组)。卵白蛋白(OVA) 抗原溶液腹腔注射致敏,滴鼻造模。正常对照组用生理盐水代替OVA,空质粒干预组和T-bet质粒干预组OVA激发48 h前,分别经鼻滴入50 μg空质粒和重组T-bet质粒。观察各组实验小鼠的肺组织炎症以及BALF中各类炎症细胞以及IL-4、IFN-γ水平的变化。 结果: Western blotting检测发现,小鼠气道转染pcDNA3-T-bet质粒48 h后肺组织T-bet蛋白表达显著增加。pcDNA3-T-bet质粒转染能较好抑制给药后48 h OVA激发的哮喘小鼠气道炎症（包括炎症细胞浸润,上皮细胞损伤、黏液分泌、血管壁水肿及管腔缩窄）;下调小鼠BALF中Th2因子IL-4并上调Th1因子IFN-γ水平。 结论: 气道内转染T-bet质粒能有效改善哮喘小鼠的气道炎症。     

霍乱毒素佐剂联合弓形虫ESA鼻内免疫小鼠抗弓形虫感染作用

目的 观察霍乱毒素（cholera toxin,CT）佐剂和弓形虫排泄-分泌抗原（ESA）鼻内免疫小鼠诱导的抗弓形虫感染作用。方法 6周龄BALB/c小鼠60只,随机分为3组,每组20只。分别用PBS 20μl、ESA 20μg或CT 1.0μg＋ESA 20μg每只滴鼻免疫2次,间隔2周。末次免疫后14 d,用4×104个弓形虫速殖子每只灌胃攻击所有小鼠,观察小鼠健康及死亡情况。速殖子攻击后30 d,计数肝、脑组织内弓形虫速殖子。结果 CT作为佐剂联合弓形虫ESA滴鼻免疫小鼠的健康状况明显好于PBS组和ESA组,存活率（95%）也显著高于PBS组（55%）。与PBS组相比,CT＋ESA组肝和脑组织内速殖子数分别减少了80.19%（P〈0.001）和78.24%（P〈0.005）。CT作为佐剂联合ESA滴鼻免疫小鼠诱导了高水平的黏膜免疫应答和系统免疫应答。结论 CT作为佐剂联合弓形虫ESA滴鼻免疫小鼠诱导的免疫应答可有效抵抗弓形虫速殖子攻击。