VIRAL LOAD IN CELLULAR SUBCOMPARTMENTS FOLLOWING HAART

联合治疗与新的药物对抗人类免疫缺陷病毒（HIV）显著减缓进展到艾滋病和死亡。这些治疗方法能够将外周血中的病毒降低到检测水平以下。然而，病毒复制的储存库仍然存在，主要存在于淋巴结组织中，这可能会重新点燃HIV的复制。本项目检查了患者在高活性治疗前和治疗期间切除颈部淋巴结组织和外周血的一系列样本。它将建立一种机制，同时从多个隔间获得可重复的样品。结合最先进的流式细胞术和细胞分选，等温核酸序列测定（NASBA） HIV定量和非放射性同位素原位杂交技术将用于测量这些样品中的细胞亚型（淋巴细胞与单核细胞/巨噬细胞）HIV病毒载量。我们将研究细胞亚区室在不同时间和不同区室之间的变化模式，以试图理解治疗失败，并为设计更有效的治疗策略提供新的见解。属于独特反应类别的患者-被称为不协调应答者（15 - 20%的患者在治疗反应中病毒载量和CD4计数均上升，但似乎与具有预期病毒学反应的患者获得相同的临床益处），也将进行研究，以确定对治疗反应的病理生理参数，这些参数超出吸收，依从性，以及表型/基因型抗性模式作为病毒学反应的解释。该项目将研究病毒趋向性和治疗方案在特定淋巴样细胞亚型（包括终末分化细胞和分裂细胞）中的特异性活性的作用。对感染细胞亚群的进一步表征代表了该项目的未来方向，即通过细胞周期蛋白A、B和D分析细胞周期，并将为特定治疗方案如何影响总病毒载量提供独特的见解。然而，这个研究生涯奖的首要目标是为PI提供教学培训，重叠密切监督的临床研究期间，导致PI作为一个可行的独立研究人员建立。

Combination therapy with new agents against the human immunodeficiency virus (HIV) has dramatically slowed the progression to AIDS and death. These treatments are capable of reducing virus in the peripheral blood to below the levels of detection. However, reservoirs of viral replication persist, largely in lymph node tissue, that potentially reignite HIV replication. This project examines serial samples from excisional cervical lymph node tissue and peripheral blood in patients before and during highly active therapy. It will establish a mechanism for obtaining samples from several compartments simultaneously and reproducibly. The combination of state-of-the-art flow cytometry and cell sorting, isothermal Nucleic Acid Sequence Based Assay (NASBA) HIV quantitation, and a non-radioisotope in situ hybridization technique will be used to measure cellular subtype (lymphocyte versus monocyte/macrophage) HIV viral load in these samples. Patterns of changes in cellular subcompartments across time and between the compartments will be examined in an attempt to understand treatment failure and to provide new insights into the design of more effective treatment strategies. Patients who fall into the unique category of response - coined Discordant Responders (the 15 - 20 percent of patients who experience a rise in both viral load and CD4 count in response to therapy yet appear to reap the same clinical benefits as patients with the expected virologic response), will also be studied in order to determine pathophysiologic parameters of response to therapy that go beyond absorption, adherence, and phenotypic/genotypic resistance patterns as explanations for virologic response. This project will investigate the role of viral tropism and the specific activity of treatment regimens in specific lymphoid cell subtypes, including terminally differentiated versus dividing cells. A further characterization of the subset of infected cells represents the future direction of this project, namely analysis of cell cycle by cyclins A, B and D, and will provide unique insights in how specific treatment regimens might affect total viral load. However, the paramount goal of this research career award is to provide the PI with didactic training overlapping a closely supervised period of clinical investigation leading to establishment of the PI as a viable independent researcher.