PNEUMOCYSTIS CARINII IN CONTINOUS AXENIC CULTURE

连续无菌培养中的卡氏肺囊虫

• 批准号: 2873457
• 负责人: ALLEN B CLARKSON JR
• 金额: $ 29.73万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2002-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2873457
• 关键词: Pneumocystis carinii; acidity /alkalinity; cell motility; electron microscopy; growth media; human tissue; light microscopy; microorganism culture; microorganism reproduction; morphology; polymerase chain reaction; technology /technique development; temperature

DESCRIPTION (Adapted from Investigator's Abstract) Pneumocystis carinii is also a very poorly understood fungal pathogen and P. carinii pneumonia (PcP) remains one of the most common opportunistic infections associated with AIDS despite effective anti-retroviral therapy, improved PcP treatment protocols and widespread PcP prophylaxis. The greatest obstacle in P. carinii research has been the inability to culture this organism other than in short term systems, generally with a co-cultured mammalian cell line. This laboratory has obtained continuous axenic growth of both rat- and human-derived P. carinii.The overall goal of this project is to develop this culture system further and use it to study P. carinii. Conditions will be established allowing for optimal growth in culture. Temperature and pH optima will be determined. Validation will be sought for the supplements now added to the culture medium and other supplements will be tested. Supplement concentrations will be optimized. Minimum medium exchange rates for optimal growth will be determined. A method of growing a P. carinii culture from a single cell will be developed. Cultured P. carinii will be characterized with reference to morphology, movement, and aspects of cell physiology such as respiration and polyamine metabolism. A library of 50 isolates of human-derived P. carinii will be obtained using bronchoalveolar lavage fluid from patients with P. carinii pneumonia. These isolates will be studied in vitro for sensitivity to the combination of trimethoprim + sulfamethoxazole (the mainstay for both treatment of and prophylaxis against P. carinii pneumonia) and to atovaquone (a commonly used alternative drug). In these isolates, we will determine critical sequences in the sulfamethoxazole target enzyme (dihydropteroate synthase) gene and the atovaquone target enzyme (cytochrome b) gene. An attempt will be made to correlate drug sensitivity, gene sequence and patient response to determine if there is resistance to this drug combination in human P. carinii.

描述（改编自研究者摘要）卡氏肺孢子虫也是 一种知之甚少的真菌病原体和卡氏肺孢子虫肺炎（PcP） 艾滋病最常见的机会性感染之一， 有效的抗逆转录病毒疗法，改进的PcP治疗方案， 广泛的PcP预防。卡氏肺吸虫研究的最大障碍是 除了在短期系统中，无法培养这种生物体， 通常与共培养的哺乳动物细胞系。该实验室获得了 大鼠和人源卡氏肺孢子虫的连续无菌生长。 该项目的目标是进一步发展这一文化体系，并将其用于 研究卡氏肺吸虫。将创造条件， 文化将确定最佳温度和pH值。确认将 寻求现在添加到培养基和其他补充剂 补充剂将进行测试。将优化补充浓度。 将确定最佳增长的最低中间汇率。的方法 从单个细胞培养卡氏肺孢子虫的方法。培养 卡氏肺孢子虫将参照形态、运动和 细胞生理学方面，如呼吸和多胺代谢。一 将使用以下方法获得50个人源卡氏肺孢子虫分离株的文库 卡氏肺孢子虫肺炎患者的支气管肺泡灌洗液。这些 将在体外研究分离株对以下组合的敏感性： 甲氧苄啶+磺胺甲恶唑（治疗和 预防卡氏肺孢子虫肺炎）和阿托伐醌（一种常用的 替代药物）。在这些分离株中，我们将确定关键序列， 磺胺甲恶唑靶酶（二氢蝶酸合酶）基因和 阿托伐醌靶酶（细胞色素B）基因。将尝试 将药物敏感性、基因序列和患者反应相关联，以确定 在人卡氏肺孢子虫中存在对该药物组合的抗性。