Alveolar Macrophage Host Defenses

描述（由申请人提供）：肺泡巨噬细胞（AM）是肺部人类免疫缺陷病毒-1 （HIV-1）感染的关键储存库。在研究的过程中试图改变人类的遗传曲目是为了了解HIV - 1与这些细胞,我们小说观察治疗人类是修改前腺病毒基因转移载体(广告)后续HIV - 1感染HIV - 1块复制,即使没有转基因的广告,这个建议的目的是理解广告的机制抑制HIV - 1复制人类是在分子水平上。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成分；和3。AM中的细胞内通路被调节以介导Ad对HIV-1复制的影响。为了实现这些目标，将进行实验，以评估与HIV-1感染相关的AM的Ad治疗时机对抑制效果的影响，并与HIV-1生命周期中各个步骤的特定抑制剂进行比较。HIV-1 LTR的转录将与p24 ELISA在不同抑制剂条件下的结果进行比较。TAQMAN PCR检测将用于定量HIV-1特异性DNA的合成。为了确定Ad的致病成分，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复制抑制作用的细胞内通路将在缺乏相关信号转导分子（如STAT-1）的细胞系中进行建模。通过基因芯片技术检测AM基因表达变化，并通过Northern和Western分析证实。综上所述，这些研究将为人类AM中Ad抑制HIV-1复制的机制提供见解。

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.