Demographic data

<p>Demographic data.</p

Alterations in the Chromatin Environment Following the Introduction of DNA Breaks

The presence of DNA breaks has extensive biochemical implications for the integrity of the genome. It is well established that distinct DNA damage response proteins are recruited to, and accumulate at, sites of genomic lesions, including kinases that initiate multiple DNA damage signaling cascades. The repair of DNA breaks is facilitated by the phosphorylation of H2AX, which organizes DNA damage response factors in the vicinity of the lesion. Metabolism of the DNA breaks occurs in a chromatin environment and modulating chromatin structure is necessary for the fidelity of the DNA damage response. We set out to determine in living cells both how chromatin is remodeled in the presence of DNA breaks and whether the establishment of large sub-cellular DNA damage response domains influences other DNA metabolic processes, such as transcription. Using a photoactivatable histone H2B, we examined the mobility and structure of chromatin immediately after the introduction of DNA breaks. We find that chromatin-containing damaged DNA exhibits limited mobility but undergoes an initial energy-dependent local expansion that occurs independently of H2AX and ATM. We also took advantage of the large copy number, tandem gene arrangement, and spatial organization of ribosomal transcription units as a model system to measure the kinetics of transcription in real time in the presence of DNA breaks. We find that RNA polI inhibition is not the direct result of the physical DNA break but mediated by ATM kinase activity and surrogate DNA repair proteins. We propose that the localized opening of chromatin at DNA breaks establishes an accessible biochemically unique sub-nuclear environment that facilitates DNA damage signaling and repair

Enhancement of HIV-1 pathogenesis by plasmodium falciparum : mechanisms and clinical applications

HIV-1 and Plasmodium falciparum malaria cause substantial morbidity in Sub-Saharan Africa, especially as co- infecting pathogens. However, many aspects of this interaction have not been fully delineated. We hypothesize that dual infection causes increased HIV expression and more rapid HIV disease progression and that activation of malaria-specific CD4+ T-cells in the presence of uncontrolled HIV replication leads to depletion of malaria -specific immunity. To better understand the effects of P. falciparum on HIV, we studied the interaction ex vivo using peripheral blood mononuclear cells (PBMCs) from human malaria naïve volunteers experimentally infected with P. falciparum in a malaria challenge trial and we used an in vitro co-culture system to study the mechanisms contributing to increased viral replication. To assess the effects of HIV on P. falciparum, we took advantage of a recently completed clinical trial of antiretroviral therapy to study the relationship between subpatent parasitemia (detection of parasites by methods more sensitive than blood smears, but without clinical malaria) and clinical disease and attempted to assess whether the HIV-1 protease inhibitor (PI) atazanavir reduced the rate of detection of plasma P. falciparum DNA. The ex vivo experiments showed an increase in HIV production and pro- inflammatory cytokine secretion that occurred after the parasitemia and generalized immune activation resolved, suggesting that enhanced HIV production is related to the development of anti-malaria immunity and may be mediated by pro-inflammatory cytokines. We also demonstrated that phagocytic, antigen presenting cells ingest P. falciparum infected red blood cells and stimulate CD4+ T-cells to produce HIV-1 in a cytokine- and contact-dependent manner. Lastly, subpatent parasitemia was not significantly associated with clinical malaria and HIV PI therapy did not decrease the prevalence of parasitemia. Taken together, our results suggest that while subpatent parasitemia may not lead to clinical disease, it may contribute to an increase in plasma HIV-1 RNA levels in untreated patients, especially upon repeat encounters with malaria parasites. It is plausible that any level of parasitemia may contribute to CD4+ T-cell activation, even in the presence of anti-retroviral therapy. Thus, it may be important to suppress parasitemia in HIV seropositive individuals, even if they are asymptomati

Th17 cells are associated with protection from ventilator associated pneumonia

<div><p>Background</p><p>CD4+ T-helper 17 (Th17) cells and Interleukin (IL)-17A play an important role in clearing pathogens in mouse models of pneumonia. We hypothesized that numbers of Th17 cells and levels of IL-17A are associated with risk for nosocomial pneumonia in humans.</p><p>Methods</p><p>We collected bronchoalveolar lavage (BAL) fluid from mechanically ventilated (n = 25) patients undergoing quantitative bacterial culture to evaluate for ventilator associated pneumonia (VAP). We identified Th17 cells by positive selection of CD4+ cells, stimulation with ionomycin and PMA, then staining for CD4, CD45, CCR6, IL-17A, and IFN-γ followed by flow cytometric analysis (n = 21). We measured inflammatory cytokine levels, including IL-17A, in BAL fluid by immunoassay.</p><p>Results</p><p>VAP was detected in 13 of the 25 subjects. We identified a decreased percentage of IL-17A producing Th17 cells in BAL fluid from patients with VAP compared to those without (p = 0.02). However, we found no significant difference in levels of IL-17A in patients with VAP compared to those without (p = 0.07). Interestingly, IL-17A levels did not correlate with Th17 cell numbers. IL-17A levels did show strong positive correlations with alveolar neutrophil numbers and total protein levels.</p><p>Conclusions</p><p>Th17 cells are found at lower percentages in BAL fluid from mechanically ventilated patients with VAP and IL-17A levels correlated with Th17 cell percentages in non-VAP subjects, but not those with VAP. These findings suggest that Th17 cells may be protective against development of nosocomial pneumonia in patients receiving mechanical ventilation and that alveolar IL-17A in VAP may be derived from sources other than alveolar Th17 cells.</p></div

Percentage of Th17 cells reduced in the setting of ventilator associated pneumonia.

<p>Cells were isolated from bronchoalveolar lavage fluid, stained, and percentages of Th17 cells and CD4+ IFN-γ+ positive cells were obtained using flow cytometry. A: Percentage of Th17 cells (CCR6+IL-17A+) of the total CD4+ population in the BAL of patients with VAP or without VAP. B. Percentage of IFN-γ+ cells of the total CD4+ population. C. IL-17A protein concentrations measured by immunoassay in the BAL of subjects with and without VAP. D. We compared the relationship between numbers of Th17 cells (x-axis) and amount of IL-17A protein (y-axis) in subjects with VAP (triangles) and without VAP (circles).</p

Increased levels of pro-inflammatory cytokines in bronchoalveolar lavage samples of patients with ventilator associated pneumonia.

<p>Bronchoalveolar lavage fluid was isolated from patients with and without ventilator associated pneumonia (VAP) and run on a multi-plex immuno assay to measure cytokine protein levels. Subjects with VAP (red triangles) are compared to those without VAP (black circles). All y-values are the log of the concentration in pg/mL. p-values were measured using a 2-tailed T-test. For most pro-inflammatory cytokines, such as A: TNF-α, B: IFN-γ, C: IL-1β, and E: IL-12p 70, and for the chemokine IL-8 (D), there was increased expression in the VAP samples. For IL-6 (F) and IL-10 (G), there was no significant difference between the two groups.</p

Neutrophil numbers in BAL correlate to IL-17A protein levels.

<p>Cytospins were counted for total numbers of neutrophils present in each BAL sample. Correlation between percentage of neutrophils (x-axis) and A) IL-17A protein concentration (log, y-axis) and B) percentage of Th17 cell numbers.</p