MACAQUE MODEL OF PNEUMOCYSTIS CARINII INFECTION: SIV

研究的目的是制定一个 经气管接种卡氏肺孢子虫（P. cariniif.sp.simiae），这将允许需要 可重复的严重感染模型，并使用该模型来测试 研究空气样本中的有机物，以确定可能的 传输因素。 灵长类动物的模型对于 确定药物的治疗和预防效果。 目前 啮齿类动物被用来预测药物对人类的功效， 某些药物（及其对映体）被代谢或活化， 在啮齿类动物和灵长类动物中的差异。 据了解，恒河猴 由于肺孢子虫的爆发，猕猴对肺孢子虫易感 感染SIV的动物也发生过感染。 这些疾病的爆发 是自发的，也需要一个受控的感染模型 作为检测感染的灵敏方法和分型方法 鉴定在动物和空气中发现的肺孢子虫菌株 样品 最初的研究集中在研究受感染的微生物 肺银行在杜兰制定出最好的引物和方法， 聚合酶链反应（PCR）扩增核或 线粒体rRNA。 小部分18S和26S rRNA的研究 基因、整个5.8 rRNA基因和内部间隔区ITS1和ITS2 克隆了猴肺孢子虫的一个标本，并进行了序列测定。 一 还克隆了线粒体大亚基rRNA基因的一部分 和序列测定 在ITS1和ITS2中有显著差异， 猿类卡氏肺孢子虫的线粒体基因序列与 其他宿主的相同序列。 这些研究建立了 方法来扩增特定的基因部分，建立一个独特的 从这只猴子身上分离出卡氏肺孢子虫，并证明 检测和分型其他猕猴的微生物。 为了 确定检测到的类型是否是猕猴特有的， 无论发现的类型是一个范围内的一些，需要研究许多 更多的肺部样本 发现的序列变异性已被 在一篇提交给《临床医学杂志》的文章中报道， 微生物学. 来自杜兰的13个标本目前正在 研究了 迄今为止，已确定了其中3个的序列。 在 其他的，克隆正在进行中。带有插入物的质粒， 通过挑选具有适当 特色 这项工作还需要几个星期才能完成。 将提供序列的比较。 一旦在 确定了13个肺样本，样本中的类型或类型 用于接种可以建立和这种类型或这些类型 在接种动物的肺中定量。 的下一步 该模型的发展是使用免疫抑制剂， 确定的剂量将使淋巴细胞减少50%或更多， 允许接种卡氏肺孢子虫和随后的 感染. 博姆博士正在研发免疫抑制剂， 准备好帮助两只动物进行试验接种程序。 检测感染的发展将需要我们的 合作通过支气管镜检查对动物进行采样（由 博士Bohm）进行组织化学染色（Marilyn Bartlett）和PCR 放大（由李博士执行）。 随着建立 我们可以安排药物治疗试验和空气采样 实验

The purposes of the studies are to develop a transtracheally-inoculated primate model of Pneumocystis carinii (P. carinii f.sp.simiae) that will allow studies which require a reproducible severe infection model and to use that model to test compounds and study organisms in air samples to determine possible transmission factors. The primate model will be very important for determining therapeutic and prophylactic effects of drugs. Currently rodents are used to predict drug efficacy for humans and it is known that some drugs (and their enantiomers) are metabolized or activated differently in rodents than in primates. It is known that rhesus macaques are susceptible to Pneumocystis spp because outbreaks of infection have occurred in SIV-infected animals. The outbreaks have been spontaneous and a controlled model of infection is needed as well as a sensitive method for detection of infection and a typing method to identify strains of Pneumocystis sp found in animals and air samples. Initial studies focused on study of organisms from infected lungs banked at Tulane to work out the best primers and methods for polymerase chain reaction (PCR) amplification of nuclear or mitochondrial rRNA. Studies of small portions of the 18S and 26S rRNA genes, the entire 5.8 rRNA gene and the internal spacers ITS1 and ITS2 of one specimen of simian Pneumocystis were cloned and sequenced. A portion of the large subunit mitochondrial rRNA gene was also cloned and sequenced. There were marked differences in the ITS1 and ITS2 and mitochonrial gene sequences of the simian P. carinii as compared to the same sequences of other hosts. These studies establshed the methods to amplify the specific gene portions, establish a distinct type of P. carinii from this monkey and demonstrate the capability of detecting and typing organisms from other macaques. In order to determine whether the types detected are specific within macaques or whether the types found are some of a range, requires study of many more lung samples. The sequence variabilities found have been reported in an article submitted to the Journal of Clinical Microbiology. Thirteen specimens from Tulane currently are being studied. To date sequences have been established for 3 of these. In the others, cloning is being carried on. Plasmids with inserts are being selected by picking of colonies with appropriate characteristics. The work will require several more weeks before comparisons of sequences will be available. Once types found in the thirteen lung samples are determined, type or types in the specimen used for inoculation can be established and this type or these ypes quantitated in the lungs of inoculated animals. The next step in development of the model is use of an immunosuppressive agent in established doses that will deplete lymphocytes by 50% or greater and allow inoculation of P. carinii and subsequent development of infections. Dr. Bohm is developing the immunosuppression doses and we are prepared to help in a trial inoculation procedure for two animals. Testing for the development of infections will require our collaboration on sampling the animals by bronchoscopy (performed by Dr. Bohm) with histochemical staining (by Marilyn Bartlett) and PCR amplification (performed by Dr. Lee). With establishment of the model, we can schedule both drug treatment trials and air sampling experiments.