Alveolar Macrophage Host Defenses

描述（由申请方提供）：肺泡巨噬细胞（AM）是肺中人类免疫缺陷病毒-1（HIV-1）感染的关键储存库。在试图改变人类AM的遗传库以理解HIV-1与这些细胞相互作用的研究过程中，我们进行了新的观察，即在HIV- 1感染之前用修饰的腺病毒基因转移载体（Ad）处理人类AM阻断随后的HIV- 1复制，即使在没有Ad转基因的情况下，本研究的目的是从分子水平上了解Ad抑制HIV-1在人AM中复制的机制。该提案的具体目的是了解：1。HIV-1生命周期中被Ad抑制的步骤; 2.负责引起抑制的Ad组分;以及3. AM中的细胞内途径被调节以介导Ad对HIV-1复制的这种作用。为了实现这些目标，将进行实验以评估与HIV-1生命周期中各个步骤的特异性抑制剂相比，相对于HIV-1感染的AM的Ad治疗时机对抑制作用的影响。在各种抑制剂的条件下，将HIV-1 LTR的转录与p24 ELISA的结果进行比较。将使用TAQMAN PCR试验定量HIV-1特异性DNA的合成。为了确定负责的Ad组分，将使用Ad变体如空衣壳和缺乏特定Ad基因如E4的载体。负责介导Ad对HIV-1复制的抑制作用的细胞内途径将在缺乏相关信号转导分子如STAT-1的细胞系中建模。将通过基因芯片技术检测AM基因表达的变化进行相关性分析，并通过北方和西方分析进行确认。总之，这些研究将提供深入了解Ad抑制HIV-1在人类AM中复制的机制。

DESCRIPTION (provided by applicant): Alveolar macrophages (AM) are a key reservoir of human immunodeficiency virus-1 (HIV-1) infection in the lung. In the course of studies attempting to alter the genetic repertoire of human AM in order to understand HIV-1 interaction with these cells, we made the novel observation that treatment of human AM with modified adenovirus gene transfer vectors (Ad) prior to HIV- 1 infection blocks subsequent HIV- 1 replication, even in the absence of an Ad transgene, The purpose of this proposal is to understand the mechanism of Ad inhibition of HIV-1 replication in human AM at the molecular level. The specific aims of the proposal are to understand: 1. The step(s) in the HIV-1 lifecycle that are being inhibited by Ad; 2. The Ad component(s) that are responsible for causing the inhibition; and 3. The intracellular pathway(s) in AM that are being modulated to mediate this effect of Ad on HIV-1 replication. To accomplish these goals, experiments will be performed to assess the effect of timing of Ad treatment of AM relative to HIV-1 infection on the inhibitory effect in comparison with specific inhibitors of various steps in the HIV-1 life cycle. Transcription from the HIV-1 LTR will be compared with the results of p24 ELISA under conditions of the various inhibitors. A TAQMAN PCR assay will be used to quantify the synthesis of HIV-1 specific DNA. To ascertain the Ad components responsible, Ad variants such as empty capsids and vectors deficient in specific Ad genes such as E4 will be used. The intracellular pathways responsible for mediating the inhibitory effect of Ad on HIV-1 replication will be modeled in cell lines deficient in relevant signal transduction molecules such as STAT-1. Correlation will be made with changes in AM gene expression detected by gene chip technology and confirmed with Northern and Western analysis. Taken together, these studies will provide insight into the mechanism of Ad inhibition of HIV-1 replication in human AM.