Proteomic identification and functional characterization of prohibitin 1 and 2 in T cell activation, expansion, survival and disease

Several immune pathologies are the result of aberrant regulation of T lymphocytes. Pronounced T cell proliferation can result in autoimmunity or hematologic malignancy, whereas loss of T cell activity can manifest as immunodeficiency. Thus, there is a critical need to characterize the signal transduction pathways that mediate T cell activation so that novel and rational strategies to detect and effectively control T cell mediated disease can be achieved. ^ The first objective of this dissertation was to identify and characterize novel T cell regulatory proteins that are differentially expressed upon antigen induced activation. Using a functional proteomics approach, two members of the prohibitin (Phb) family of proteins, Phb1 and Phb2, were determined to be upregulated upon activation of primary human T cells. Furthermore, their regulated expression was dependent upon CD3 and CD28 signaling pathways which synergistically increased their expression. In contrast to previous reports of Phb nuclear localization, both proteins were determined to localize to the mitochondrial inner membrane of human T cells. Additionally, novel Phb phosphorylation sites were identified and characterized using mass spectrometry, phosphospecific antibodies and site directed mutagenesis. ^ Prohibitins have been proposed to play important roles in cancer development however the mechanism of action has not been elucidated. The second objective of this dissertation was to define the functional role of Phbs in T cell activity, survival and disease. Compared to levels in normal human T cells, Phb expression was higher in the human tumor T cell line Kit225 and subcellularly localized to the mitochondrion. Ablation of Phb expression by siRNA treatment of Kit225 cells resulted in disruption of mitochondrial membrane potential and significantly enhanced their sensitivity to cell death, suggesting they serve a protective function in T cells. Furthermore, Q-RT-PCR analysis of human oncology cDNA expression libraries indicated the Phbs may represent hematological cancer biomarkers. Indeed, Phb1 and Phb2 protein levels were 6-10 fold higher in peripheral blood mononuclear cells isolated from malignant lymphoma and multiple myeloma patients compared to healthy individuals. ^ Taken together, Phb1 and Phb2 are novel phosphoproteins upregulated during T cell activation and transformation to function in the maintenance of mitochondrial integrity and perhaps energy metabolism, thus representing previously unrecognized intracellular biomarkers and therapeutic targets for regulating T cell activation and hematologic malignancies. ^

HIV prevalence and contextual risk-factors among injection drug users in Harris County, Texas

Injection drug use is the third most frequent risk factor for new HIV infections in the United States. A dual mode of exposure: unsafe drug using practices and risky sexual behaviors underlies injection drug users' (IDUs) risk for HIV infection. This research study aims to characterize patterns of drug use and sexual behaviors and to examine the social contexts associated with risk behaviors among a sample of injection drug users. ^ This cross-sectional study includes 523 eligible injection drug users from Houston, Texas, recruited into the 2009 National HIV Behavioral Surveillance project. Three separate set of analyses were carried out. First, using latent class analysis (LCA) and maximum likelihood we identified classes of behavior describing levels of HIV risk, from nine drug and sexual behaviors. Second, eight separate multivariable regression models were built to examine the odds of reporting a given risk behavior. We constructed the most parsimonious multivariable model using a manual backward stepwise process. Third, we examined whether HIV serostatus knowledge (self-reported positive, negative, or unknown serostatus) is associated with drug use and sexual HIV risk behaviors. ^ Participants were mostly male, older, and non-Hispanic Black. Forty-two percent of our sample had behaviors putting them at high risk, 25% at moderate risk, and 33% at low risk for HIV infection. Individuals in the High-risk group had the highest probability of risky behaviors, categorized as almost always sharing needles (0.93), seldom using condoms (0.10), reporting recent exchange sex partners (0.90), and practicing anal sex (0.34). We observed that unsafe injecting practices were associated with high risk sexual behaviors. IDUs who shared needles had higher odds of having anal sex (OR=2.89, 95%CI: 1.69-4.92) and unprotected sex (OR=2.66, 95%CI: 1.38-5.10) at last sex. Additionally, homelessness was associated with needle sharing (OR=2.24, 95% CI: 1.34-3.76) and cocaine use was associated with multiple sex partners (OR=1.82, 95% CI: 1.07-3.11). Furthermore, twenty-one percent of the sample was unaware of their HIV serostatus. The three groups were not different from each other in terms of drug-use behaviors: always using a new sterile needle, or in sharing needles or drug preparation equipment. However, IDUs unaware of their HIV serostatus were 33% more likely to report having more than three sexual partners in the past 12 months; 45% more likely to report to have unprotected sex and 85% more likely to use drug and or alcohol during or before at last sex compared to HIV-positive IDUs. ^ This analysis underscores the merit of LCA approach to empirically categorize injection drug users into distinct classes and identify their risk pattern using multiple indicators and our results show considerable overlap of high risk sexual and drug use behaviors among the high-risk class members. The observed clustering pattern of drug and sexual risk behavior among this population confirms that injection drug users do not represent a homogeneous population in terms of HIV risk. These findings will help develop tailored prevention programs.^