应用3种方法诊断弓形虫淋巴结炎

目的探讨研究弓形虫淋巴结炎的病理诊断。方法应用免疫组化、原位杂交及间接原位PCR技术,检测71例（均来自可疑弓形虫感染者）经病理诊断为慢性非特异性淋巴结炎（淋巴结反应性增生）病人组织切片中的弓形虫。结果发现在71例中弓形虫阳性病例分别为17例（23．94％）、35例（49．30％）、41例（57．75％）,免疫组化与原位杂交、间接原位PCR结果差异有统计学意义（P〈0．005）。结论在病理诊断为慢性非特异性淋巴结炎（淋巴结反应性增生）的病人中,部分为漏诊的弓形虫淋巴结炎病例。免疫组化、原位杂交及间接原位PCR均能显示淋巴结组织切片中的弓形虫,但弓形虫检测结果阳性率从高到低依次为间接原位PCR、原位杂交及免疫组化。     

弓形虫P22编码基因真核表达质粒接种小鼠后的细胞免疫效果

目的　观察分析弓形虫表面抗原 P2 2编码基因真核表达质粒 (p BK/P2 2 )接种小鼠诱导的细胞免疫效果。方法　采用肌肉注射免疫接种 BAL B/c小鼠 ,5周后 ,取鼠脾细胞作 Con A刺激淋转试验 (MTT法 )及 T细胞亚群的测定。结果　MTT试验中 ,p BK/P2 2免疫组、空质粒 p BK- CMV对照组与 NS对照组的 Con A孔的光密度值与不加 Con A孔的光密度值的差值疫苗免疫组略高于二对照组 ,统计学分析无明显差异 (P>0 .0 5 ) ;CD+4 细胞数量变化无显著性差异 (P>0 .0 5 ) ,而 CD+8细胞数量则明显增多 ,p BK/P2 2免疫组、空质粒 p BK- CMV均高于 NS对照组 ,其差异有显著性 (P<0 .0 1) ;p BK/P2 2免疫组与空质粒组间无显著性差异 (P>0 .0 5 )。结论　 p BK/P2 2对 Con A刺激的淋巴细胞增殖功能作用不明显 ;T淋巴细胞亚群测定结果表明 ,p BK/P2 2免疫后小鼠 CD+4 淋巴细胞增殖不明显 ,而 CD+8淋巴细胞则显著增殖     

zs株弓形虫P22基因片段在巨噬细胞中的表达

目的　克隆表达ZS株弓形虫表膜抗原P2 2编码基因的巨噬细胞株。方法　扩增P2 2编码基因ORF的全长 5 6 1bp片段 ,经PstI、XbaI双酶切后 ,定向克隆于质粒 pBudCE 4 .1,构建真核重组表达质粒 pBudCE 4 .1/P2 2 ,通过脂质体介导转染入小鼠巨噬细胞RAW2 6 4 .7,以RT PCR方法鉴定目的基因在巨噬细胞中的表达。结果　成功获取pBudCE 4 .1/P2 2转染的巨噬细胞阳性克隆 ,并证实P2 2 mRNA在阳性克隆细胞中的表达 ,为P2 2蛋白对巨噬细胞的免疫调节功能作用的研究奠定基础。     

弓形虫新基因表位疫苗质粒的构建及鉴定

目的构建两个弓形虫新基因2B9G1、7C3-C3的表位疫苗质粒,为阐明表位疫苗的保护性奠定基础。方法采用生物信息学方法对新基因2B9G1、7C3-C3进行表位预测,PCR扩增基因中编码3个表位的片段W2b、W2a和W4a,连接入pcDNA3,转入DH5α,挑阳性菌落进行PCR、酶切和测序鉴定;同法将W2a、W4a分别克隆入pcDNA3-W2b载体中W2b片段下游,再将W4a克隆入pcDNA3-W2b2a载体中W2a片段下游。结果经PCR、酶切鉴定及序列测定,W2b、W2a和W4a 3个片段分别插入pcDNA3预期位置;W2a和W4a片段分别插入pcDNA3-W2b预期位置;W4a片段插入pcDNA3-W2b2a预期位置。结论成功构建单表位、双表位、多表位疫苗质粒pcDNA3-W2b、pcDNA3-W2a、pcDNA3-W4a、pcDNA3-W2b2a、pcDNA3-W2b4a、pcDNA3-W2a2b4a。     

Different effect of Toxoplasma gondii infection on adhesion capacity of fibroblasts and monocytes

This study investigated the effect of infection with the apicomplexan parasite Toxoplasma gondii , in combination with the concomitant cytokine environment (IFN-γ/TNF-α), on adhesion of THP-1 monocytic cells to MRC-5 fibroblasts. Surprisingly, infection of THP-1 cells decreased their adhesion to the MRC-5 cell monolayer. This decrease was compensated by IFN-γ/TNF-α stimulation. In contrast, infection of MRC-5 cells significantly increased adhesion, which was synergistically augmented by cytokine stimulation. Levels of ICAM-1 (CD54) on MRC-5 cells, as well as LFA-1 (CD11a) on THP-1 cells, were not changed by infection, neither in resting, nor in cytokine stimulated cells. These results show that T. gondii infection alters adhesion properties and reactivity to cytokine stimulation in a cell-specific way.     

两种免疫调节剂增强弓形虫W2b2a表位疫苗的小鼠保护作用

目的:探讨匹多莫德、沙利度胺增强弓形虫W2b2a表位疫苗刺激机体产生免疫应答和保护免疫的效果。方法:将匹多莫德、沙利度胺分别与弓形虫W2b2a表位疫苗混合肌注免疫小鼠,检测其细胞免疫和体液免疫,并观察其受到弓形虫攻击感染后的生存时间。结果:pc DNA3-W2b2a加匹多莫德组、pc DNA3-W2b2a加弗氏佐剂组、pc DNA3-W2b2a加沙利度胺组小鼠血清IFN-γ水平显著高于pc DNA3-W2b2a组(P0.05);pc DNA3-W2b2a加匹多莫德组、pc DNA3-W2b2a加弗氏佐剂组Ig G抗体水平、CD4+/CD8+比值、T细胞增殖活性显著高于pc DNA3-W2b2a组和pc DNA3-W2b2a加沙利度胺组(P0.05);小鼠攻击试验表明,免疫组小鼠存活时间明显长于对照组(P0.05)。结论:匹多莫德、沙利度胺可增强弓形虫W2b2a表位疫苗免疫小鼠的保护作用。     

弓形虫PRU株慢性感染小鼠脑部包囊的时空分布特征

目的观察实验小鼠脑内包囊的时空分布特点,为探讨弓形虫感染引起的情志和行为改变提供病理基础。方法弓形虫PRU株经口感染小鼠,经HE染色和免疫组化检测,在显微镜下计算前额、海马、丘脑、小脑和杏仁核部位的包囊个数,然后随机选取上述部位的5个视野拍照,计算包囊的平均密度,并用方差分析比较不同脑组织包囊密度有无统计学差异。结果弓形虫感染30和90d时,HE染色和免疫组化后显微镜观察发现,小鼠不同位置弓形虫包囊密度差异有统计学意义,其中,丘脑的包囊密度最大,其次是前额皮质、海马、杏仁核,小脑的包囊密度最小。丘脑的包囊密度显著高于其他4个脑区（P〈0.01）,小脑的包囊密度显著低于其它4个部位（P〈0.01）,而前额皮质、海马和杏仁核所含的包囊密度差异无统计学意义（P〉0.05）;弓形虫感染1个月时,杏仁核包囊密度明显低于感染3个月（F=18.314,P〈0.001）,但是小脑的包囊密度高于感染3个月（F=18.314,P〈0.001）。结论弓形虫包囊在慢性感染小鼠不同脑组织内的分布具有时空特异性,这可能是其临床表现的病理学基础。     

Vaccination against murine toxoplasmosis using recombinant Toxoplasma gondii SAG3 antigen alone or in combination with Quil A

PURPOSE: Surface antigen 3 (SAG3) of Toxoplasma gondii is very similar in structure to the major surface antigen 1 (SAG1). Although numerous studies have supported the importance of SAG1 in protection against T. gondii infection, few reports exist on SAG3. MATERIALS AND METHODS: Glutathione-S-transferase (GST)-fused SAG3 of T. gondii (rSAG3) were immunized into BALB/c mice alone or in combination with Quil A (rSAG3/Quil A), and then evaluated the protective immunity in vivo and in vitro against murine toxoplasmosis. RESULTS: Immunization with rSAG3 or rSAG3/Quil A resulted in significantly more survival days and fewer brain cysts after challenge with T. gondii compared to an infected control group. Mice immunized with rSAG3 alone or in combination with Quil A produced significantly more specific IgG2a antibody, whereas specific IgG1 antibody titers did not increase. The percentage of CD8+ T cells, IFN-gamma mRNA expression, and nitric oxide production significantly increased in rSAG3- and rSAG3/Quil A-immunized mice. CONCLUSION: These results indicate that vaccination with Toxoplasma rSAG3 results in partial protective immunity against T. gondii infection through induction of a Th1-type immune response, and that protective immunity is accelerated by the modulating effects of Quil A.