Impact of HAART on T Cell activation in HIV-Infected Adolescents

Every year, HIV-positive adolescents add to the growing number of HIV-infected individuals. In fact, as of 2005, 50% of newly diagnosed persons with HIV worldwide were youth with an estimated 10.3 million between the ages of 15 and 24. Adolescents will live with HIV 20 years longer than their adult counterparts, but disease progression in them is less understood. It is well known that immune activation is prognostic of chronic disease progression in adults and is often reduced when subjects go on therapy. It is unclear whether immune activation is entirely eliminated in adolescents on therapy. To date, studies of HIV-positive adolescents have been limited by design and methodology, resulting in a lack of research addressing the relationship between outcome of antiretroviral therapy in adolescents and immune activation. To this end, we designed a study to examine the relationship between therapeutic outcomes and immune activation in HIV-positive adolescents. We assembled a cohort of 35 HIV-infected and 12 healthy adolescents and measured immune activation (CD38,CD38/HLA-DR, Ki67) in both baseline and longitudinal data. As expected, cell turnover was greatly increased in memory CD4 T cells over time. We found that levels of immune activation in both CD8 and CD4 T cell subsets was elevated in viremic subjects at baseline and after long-term therapy. One clear distinguishing factor of pathogenic HIV-infection is increased microbial translocation, which can be measured by CD14 release by activated monocytes. We report sustained elevation of sCD14 in viremic adolescents, which directly correlated to immune activation. Our results show that adolescents who are not therapy compliant have elevated immune activation and turnover. Collectively, our results show that in the presence of suboptimal therapy measures associated with disease progression are elevated. Therefore, it is necessary to reconsider current treatment guidelines in adolescents to promote optimal adherence and virologic control in this growing population of HIV-infected individuals

Isolation of viable antigen-specific CD8+ T cells based on membrane-bound tumor necrosis factor (TNF)-α expression

Current technology to isolate viable cytokine-producing antigen-specific primary human T cells is limited to bi-specific antibody capture systems, which suffer from limited sensitivity and high background. Here, we describe a novel procedure for isolating antigen-specific human T cells based on their ability to produce tumor necrosis factor (TNF)-α. Unlike many cytokines, TNF-α is initially produced in a biologically active membrane-bound form that is subsequently cleaved by TNF-α converting enzyme (TACE) to release the soluble form of TNF-α. By preventing this cleavage event, we show that TNF-α can be ‘trapped’ on the surface of the T cells from which it originates and directly labeled for viable isolation of these antigen-specific T cells. Together with other existing sorting procedures to isolate activated T cells, this new technique should permit the direct isolation of multi-functional T lymphocytes for further protein and gene expression analyses, as well as a detailed functional assessment of the potential role that TNF-α producing T cells play in the adaptive immune system

Where It’s at Really Matters: In Situ In Vivo Vascular Endothelial Growth Factor Spatially Correlates with Electron Paramagnetic Resonance pO2 Images in Tumors of Living Mice

Purpose: Tumor microenvironments show remarkable tumor pO_{2} heterogeneity, as seen in prior EPR pO_{2} images (EPROI). pO_{2} correlation with hypoxia response proteins is frustrated by large rapid pO2 changes with position. Procedures: To overcome this limitation, biopsies stereotactically located in the EPROI were used to explore the relationship between vascular endothelial growth factor A (VEGF) concentrations in living mouse tumors and the local EPROI pO_{2}. Results: Quantitative ELISA VEGF concentrations correlated (p = 0.0068 to 0.019) with mean pO_{2}, median pO_{2}, and the fraction of voxels in the biopsy volume with pO_{2} less than 3, 6, and 10 Torr. Conclusions: This validates EPROI hypoxic fractions at the molecular level and provides a new paradigm for the assessment of the relationship, in vivo, between hypoxia and hypoxia response proteins. When translated to human subjects, this will enhance understanding of human tumor pathophysiology and cancer response to therapy

Impact of HAART on T cell activation in HIV-infected adolescents

Every year, HIV-positive adolescents add to the growing number of HIV-infected individuals. In fact, as of 2005, 50% of newly diagnosed persons with HIV worldwide were youth with an estimated 10.3 million between the ages of 15 and 24. Adolescents will live with HIV 20 years longer than their adult counterparts, but disease progression in them is less understood. It is well known that immune activation is prognostic of chronic disease progression in adults and is often reduced when subjects go on therapy. It is unclear whether immune activation is entirely eliminated in adolescents on therapy. To date, studies of HIV-positive adolescents have been limited by design and methodology, resulting in a lack of research addressing the relationship between outcome of antiretroviral therapy in adolescents and immune activation. To this end, we designed a study to examine the relationship between therapeutic outcomes and immune activation in HIV-positive adolescents. We assembled a cohort of 35 HIV-infected and 12 healthy adolescents and measured immune activation (CD38,CD38/HLA-DR, Ki67) in both baseline and longitudinal data. As expected, cell turnover was greatly increased in memory CD4 T cells over time. We found that levels of immune activation in both CD8 and CD4 T cell subsets was elevated in viremic subjects at baseline and after long-term therapy. One clear distinguishing factor of pathogenic HIV-infection is increased microbial translocation, which can be measured by CD14 release by activated monocytes. We report sustained elevation of sCD14 in viremic adolescents, which directly correlated to immune activation. Our results show that adolescents who are not therapy compliant have elevated immune activation and turnover. Collectively, our results show that in the presence of suboptimal therapy measures associated with disease progression are elevated. Therefore, it is necessary to reconsider current treatment guidelines in adolescents to promote optimal adherence and virologic control in this growing population of HIV-infected individuals

Perforin and IL-2 upregulation define qualitative differences among highly functional virus-specific human CD8 T cells.

The prevailing paradigm of T lymphocyte control of viral replication is that the protective capacity of virus-specific CD8(+) T cells is directly proportional to the number of functions they can perform, with IL-2 production capacity considered critical. Having recently defined rapid perforin upregulation as a novel effector function of antigen-specific CD8(+) T cells, here we sought to determine whether new perforin production is a component of polyfunctional CD8(+) T cell responses that contributes to the control of several human viral infections: cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza (flu), and adenovirus (Ad). We stimulated normal human donor PBMC with synthetic peptides whose amino acid sequences correspond to defined CTL epitopes in the aforementioned viruses, and then used polychromatic flow cytometry to measure the functional capacity and the phenotype of the responding CD8(+) T cells. While EBV and flu-specific CD8(+) T cells rarely upregulate perforin, CMV-specific cells often do and Ad stimulates an exceptionally strong perforin response. The differential propensity of CD8(+) T cells to produce either IL-2 or perforin is in part related to levels of CD28 and the transcription factor T-bet, as CD8(+) T cells that rapidly upregulate perforin harbor high levels of T-bet and those producing IL-2 express high amounts of CD28. Thus, "polyfunctional" profiling of antigen-specific CD8(+) T cells must not be limited to simply the number of functions the cell can perform, or one particular memory phenotype, but should actually define which combinations of memory markers and functions are relevant in each pathogenic context

Electron paramagnetic resonance oxygen image hypoxic fraction plus radiation dose strongly correlates with tumor cure in FSA fibrosarcomas

PURPOSE: Tumor hypoxia has long been known to produce resistance to radiation. In this study, electron paramagnetic resonance (EPR) oxygen imaging was investigated for its power to predict the success of tumor control depending on tumor oxygenation level and radiation therapy dose. METHODS AND MATERIALS: Thirty-four EPR oxygen images were obtained from the legs of C3H mice bearing 0.5 ml FSa fibrosarcomas under both normal (air breathing) and clamped tumor conditions. Under the same conditions as those during which the images were obtained, tumors were irradiated to a variety doses near the FSa TCD(50). Tumor tissue was distinguished from normal tissue using co-registration of the EPR oxygen images with spin-echo MRI images of the tumor and/or stereotactic localization. Tumor voxel statistics in the EPR oxygen image included mean and median pO(2), and the fraction of tumor voxels below the specified pO(2) values of 3, 6 and 10 torr. Bivariate logistic regression analysis using radiation dose and each of the EPR oxygen image statistics determined which best separated treatment failure from success. RESULTS AND CONCLUSIONS: TCD(50) measurements were similar to those found in the literature for this syngeneic tumor. Bivariate analysis of 34 tumors demonstrated that tumor cure correlated with dose (p=0.004) and with <10 torr hypoxic fraction (p=0.023). Together, radiation dose and EPR image hypoxic fraction separate the population of FSa fibrosarcomas which are cured from those which fail, thus predicting curability