Research Projects Abstracts
The Identification Of An Anti-HIV Mechanism: A Proteomic Based ApproachIt is now accepted that restoration of the immune function using only highly active antiretroviral therapy (HAART) is incomplete. Because of the emergence of drug-resistant strains of HIV-1, therapeutic immunization strategies are needed to reinforce HAART in the treatment of HIV disease. We have previously observed that a polyantigenic immunomodulator, known as PAI, which consists of a mixture of inactivated influenza and bacterial vaccine, induces MHC non restricted non-cytolytic anti-HIV-1 activity. Based on our preliminary data documenting viral suppression, we propose to test the hypothesis that PAI induced antiviral activity can be differentially determined by a proteomic approach.
To address this hypothesis, our specific aims are: Specific Aim #1: To determine differential protein expression in intracellular lysates from target T cells treated with supernatant from effector cells (PBMC, CD8+and CD8+ depleted T cells) by performing two-dimensional gel electrophoresis (2D-GE) and image analysis. Specific Aim #2: To identify and compare differentially expressed proteins from target T cells treated with PAI by performing peptide mass finger printing using mass spectrometry (MS), stable-isotope labeling by amino acids in cell culture (SILAC) analysis and database search. Specific Aim #3: To validate by Western blots and/or qRT-PCR the identity of differentially expressed proteins identified by mass spectrometry. Preliminary findings identified PBMC proteins responsive to PAI treatment. Master maps were compared to assess differences in protein expression. This revealed 47 differentially expressed spots in PAI treated PBMC. The altered proteins were analyzed by tandem MS (MS/MS) for protein identification. After querying the MS/MS data against the NCBInr protein database, we have identified 11 differentially expressed protein spots. We believe that the identified proteins will generate a proteomic signature of the PAI-anti HIV-1 response. We will make the proteome reference map of PAI treatment in different cell type with their respective related HIV mechanisms available for others to use for comparative studies.
In addition, in our community, the relationship of stress and addiction to drugs cannot be addressed without considering their implication on HIV infection and on key components of the immune system. Therefore, we will test the hypothesis that high stress has a profound effect on T-lymphocyte protein expression in HIV+ chronic” PDUs, and cytokines that are defined as TH2 type will be increased in PDUs under high stress and this will be exacerbated with HIV infection. We will test whether: (1) high stress produces abnormal changes in cytokine profile and key protein expression of T-lymphocytes from HIV and HCV negative chronic PDUs; (2) high stress produces changes in cytokine profiles, in key protein expression and viral infection in T-lymphocytes from HIV positive chronic PDUs. The specific aims to test this hypothesis are the following: (1) To group chronic PDUs according to their psychological, physiological and immunological characteristics; (2) to determine how high stress alters plasma cytokine profile in HIV-positive and HIV-negative chronic PDUs; (3) to examine the impact of stress on T-lymphocyte intracellular protein expression. This will be accomplished using proteomic analysis of T-lymphocytes isolated from HIV-positive and HIV-negative chronic PDUs.
Stress will be measured by psychometric scaling and cortisol quantification in chronic PDUs. Cellular immune response will be evaluated by quantification of CD4+ and CD8+T-cells. Moreover, since TH1-versus TH2-versus TH3-predominant is a major immunological factor governing the outcome of stress and infectious disease, changes in the cytokine profile will also be assessed. The correlation of the expected deregulation of key cellular immune components with abnormal global protein expression in T-lymphocytes from chronic PDUs under high stress may unveil important markers with health implications for HIV infection. Lymphocyte protein expression will be determined using the powerful and valuable tool of proteomics. This study, we believe, would greatly facilitate designing future targeted proteomic experiments and elucidate health problems associated with immune alterations in proteins response pathways.Determination Of Human Neuronal Responses To HIV Clades By Proteomic Fingerprinting It is suggested that the degree of neuroAIDS vary according to the HIV-1 clade, but the molecular mechanisms behind the differences still remain unclear. We hypothesized that HIV-1 clade B and C induce differential protein profiles on human neurons.
Proteins from neurons treated with HIV-1 clade A and B will be separated with 2 dimensional gel electrophoresis followed by mass spectrometry to establish homologies and dissimilarities in protein expression.