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Human immunodeficiency virus type 1 (HIV-1) evolves in vivo under selective pressure from CD8؉ Tlymphocyte (CTL) responses, which are in turn determined by host and viral genetic factors, such as restricting major histocompatibility complex molecules and the available viral epitope sequences. However, CTL are derived stochastically through the random gene rearrangements to produce T-cell receptors (TCR), and the relative impact of genetic versus stochastic processes on CTL targeting of HIV and immune-driven viral evolution is unclear. Here we evaluate identical twins infected with HIV-1 as neonates from a common blood transfusion, with subsequently similar environmental exposures, thereby allowing controlled comparisons of CTL targeting and viral evolution. Seventeen years after infection, their CTL targeting of HIV-1 was remarkably similar. In contrast, their overall TCR profiles were highly dissimilar, and a dominant epitope was recognized by distinctly different TCR in each twin. Furthermore, their viral epitopes had diverged, and there was ongoing viral phylogenetic divergence between the twins between 12 and 17 years after infection. These results indicate that while CTL targeting is predominately genetically determined, stochastic influences render the interaction of HIV-1 and host immunity, and therefore viral escape and CTL efficacy, unpredictable.

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Vascular calcification impairs vessel compliance and increases the risk of cardiovascular events. We found previously that liver X receptor agonists, which regulate intracellular cholesterol homeostasis, augment PKA agonist-or high phosphateinduced osteogenic differentiation of vascular smooth muscle cells. Because cholesterol is an integral component of the matrix vesicles that nucleate calcium mineral, we examined the role of cellular cholesterol metabolism in vascular cell mineralization. The results showed that vascular smooth muscle cells isolated from LDL receptor null (Ldlr ؊/؊ ) mice, which have impaired cholesterol uptake, had lower levels of intracellular cholesterol and less osteogenic differentiation, as indicated by alkaline phosphatase activity and matrix mineralization, compared with WT cells. PKA activation with forskolin acutely induced genes that promote cholesterol uptake (LDL receptor) and biosynthesis (HMG-CoA reductase). In WT cells, inhibition of cholesterol uptake by lipoprotein-deficient serum attenuated forskolin-induced matrix mineralization, which was partially reversed by the addition of cell-permeable cholesterol. Prolonged activation of both uptake and biosynthesis pathways by cotreatment with a liver X receptor agonist further augmented forskolin-induced matrix mineralization. Inhibition of either cholesterol uptake, using Ldlr ؊/؊ cells, or of cholesterol biosynthesis, using mevastatin-treated WT cells, failed to inhibit matrix mineralization due to up-regulation of the respective compensatory pathway. Inhibition of both pathways simultaneously using mevastatintreated Ldlr ؊/؊ cells did inhibit forskolin-induced matrix mineralization. Altogether, the results suggest that up-regulation of cholesterol metabolism is essential for matrix mineralization by vascular cells.Vascular calcification is frequently found in advanced atherosclerotic lesions and is an independent predictor of cardiovascular morbidity and mortality in patients with chronic kidney disease (1-3). Long considered to be a passive process, there now exists much evidence to suggest that vascular calcification is an active cell-mediated phenomenon that is highly regulated through complex mechanisms under active investigation (4). In vitro and in vivo studies have implicated numerous positive and negative regulatory factors in the pathogenesis of calcification (5-8).A common complication of chronic kidney disease is hyperparathyroidism. Interestingly, parathyroid hormone, an activator of the PKA pathway, has been shown to have a stimulatory role in vascular calcification both in vitro and in vivo. When parathyroid hormone levels are continuously elevated, they induce vascular calcification in rat models, irrespective of renal function (9). Additionally, parathyroid hormone-related protein, also a PKA activator, has been found in calcified atherosclerotic lesions (10). Accordingly, we and others have demonstrated previously that mineralization of vascular smooth muscle cells (VSMCs) 3 is induced by PKA agonists in vitro ...

Vascular calcification is a predictor of cardiovascular mortality and is prevalent in patients with atherosclerosis and chronic renal disease. It resembles skeletal osteogenesis, and many bone cells as well as bone-related factors involved in both formation and resorption have been localized in calcified arteries. Previously, we showed that aortic medial cells undergo osteoblastic differentiation and matrix calcification both spontaneously and in response to PKA agonists. The PKA signaling pathway is also involved in regulating bone resorption in skeletal tissue by stimulating osteoblast-production of osteoclast regulating cytokines, including receptoractivator of nuclear B ligand (RANKL) and interleukins. Therefore, we investigated whether PKA activators regulate osteoclastogenesis in aortic smooth muscle cells (SMC). Treatment of murine SMC with the PKA agonist forskolin stimulated RANKL expression at both mRNA and protein levels. Forskolin also stimulated expression of interleukin-6 but not osteoprotegerin (OPG), an inhibitor of RANKL. Consistent with these results, osteoclastic differentiation was induced when monocytic preosteoclasts (RAW264.7) were cocultured with forskolin-treated aortic SMC. Oxidized phospholipids also slightly induced RANKL expression in T lymphocytes, another potential source of RANKL in the vasculature. Because previous studies have shown that RANKL treatment alone induces matrix calcification of valvular and vascular cells, we next examined whether RANKL mediates forskolin-induced matrix calcification by aortic SMC. RANKL inhibition with OPG had little or no effect on osteoblastic differentiation and matrix calcification of aortic SMC. These findings suggest that, as in skeletal tissues, PKA activation induces bone resorptive factors in the vasculature and that aortic SMC calcification specifically induced by PKA, is not mediated by RANKL.

BackgroundFalls are the leading cause of hip fracture in older women, with important public health implications. Fall risk increases with age and other clinical factors, and varies by race/ethnicity. International studies suggest that fall risk is lower in Asians, although data are limited in U.S. populations. This study examines racial/ethnic differences in fall prevalence among older U.S. women within a large integrated healthcare delivery system.MethodsThis cross-sectional study used data from 6277 women ages 65–90 who responded to the 2008 or 2011 Kaiser Permanente Northern California Member Health Survey (KPNC-MHS). The KPNC-MHS is a mailed questionnaire sent to a random sample of adult members stratified by age, gender, and geographic location, representing a population estimate of >200,000 women age ≥65 years. Age, race/ethnicity, self-reported health status, presence of diabetes, arthritis or prior stroke, mobility limitations and number of falls in the past year were obtained from the KPNC-MHS. The independent association of race/ethnicity and recent falls was examined, adjusting for known risk factors.ResultsThe weighted sample was 76.7% non-Hispanic white, 6.2% Hispanic, 6.8% black and 10.3% Asian. Over 20% reported having fallen during the past year (28.5% non-Hispanic white, 27.8% Hispanic, 23.4% black and 20.1% Asian). Older age was associated with greater fall risk, as was having diabetes (OR 1.24, CI 1.03–1.48), prior stroke (OR 1.51, CI 1.09–2.07), arthritis (OR 1.61, CI 1.39–1.85) and mobility limitations (OR 2.82, CI 2.34–3.39), adjusted for age. Compared to whites, Asian (OR 0.64, CI 0.50–0.81) and black (OR 0.73, CI 0.55–0.95) women were much less likely to have ≥1 fall in the past year, adjusting for age, comorbidities, mobility limitation and poor health status. Asians were also less likely to have ≥2 falls (OR 0.62, CI 0.43–0.88).ConclusionsAmong older women, the risk of having a recent fall was substantially lower for black and Asian women when compared to white women. This may contribute to their lower rates of hip fracture. Future studies should examine cultural and behavioral factors that contribute to these observed racial/ethnic differences in fall risk among U.S. women.

Cell motility is likely to play a pivotal role in HIV infection by promoting the dissemination of infected cells. On the basis of observations indicating an interaction between HIV-1 Gag and target cell filamentous actin, we hypothesized that these interactions would promote cell motility of HIV-infected cells. Indeed, we have found that HIV-1 infection enhances the chemotactic response of macrophages. To specifically investigate the significance of the interactions between Gag and cellular actin, we transfected NIH 3T3 fibroblasts and HeLa cells with a construct that permits the expression of HIV-1 Gag in the absence of any other viral protein. Fractionation experiments showed that Gag was present in cytoskeletal fraction containing long actin filaments and in a high-speed postcytoskeletal fraction with short actin filaments. We have also localized HIV-1 Gag to the lamellipodia of chemoattractant-stimulated cells. Significantly, the motility of Gag-expressing cells was enhanced in chemotaxis assays. In vitro mutagenesis experiments showed that HIV-1 Gag binds filamentous actin through the nucleocapsid domain (NC). An NC-green fluorescent protein fusion had the same cellular distribution as the complete protein, and its expression increased cell motility. These data suggest that interactions between HIV-1 Gag and actin in infected cells enhance cell motility. Ultimately this enhanced motility of infected cells could promote the dissemination of virus into the brain and other tissues.