Investigating the impact of chronic inflammation on monocyte function in HIV+ individuals and the elderly

Background: Advances in effective combination antiretroviral therapy (cART) have dramatically reduced the incidence of AIDS in HIV+ individuals; however, relatively young HIV+ individuals continue to have higher risk of inflammatory diseases associated with ageing, such as atherosclerosis. In the elderly these diseases are associated with a chronic inflammatory state known as inflamm-ageing. Younger treated HIV+ individuals also show increased low grade inflammation suggesting that this may contribute to disease pathogenesis in a similar manner. Monocytes, key innate immune cells, may contribute to chronic inflammation due to response to increased presence of pathogenic material such as bacterial lipopolysaccharide (LPS) by producing proinflammatory cytokine such as IL-6 and TNF. However, whether this mechanism drives inflammation and age-related disease pathogenesis in HIV+ individuals and the elderly, and whether HIV infection accelerates or potentiates age-related changes to monocytes in the elderly, is unknown. Therefore, the hypothesis underlying this thesis is that chronic monocyte activation drives chronic inflammation and age-related disease pathogenesis in both HIV+ individuals and the elderly. Methods: Monocyte phenotype and markers of monocyte activation and response to LPS were measured in monocytes from HIV+ individuals and the elderly primarily using flow cytometry and gene expression analysis. Viremic or cART treated virologically suppressed (VS) HIV+ individuals were recruited from the Alfred Hospital, Melbourne and monocytes were compared to those from age-matched and older HIV- individuals recruited from the community. The propensity of monocytes to form foam cells, lipid-laden macrophage associated with atherosclerotic plaque progression was measured using a novel in vitro model of early atherogenesis. Results: Linear regression analyses identified that the percentage of monocyte subsets (intermediate and non-classical) and biomarkers of monocyte activation (CXCL-10, neopterin, sCD163) in HIV+ individuals increased with age at a similar rate as those from HIV- individuals; however, levels of these biomarkers were higher, indicating that HIV infection accentuates age-related changes to monocytes. Monocyte basal activation state and response to LPS in viremic and VS HIV+ individuals was shown to be higher (primed) in comparison to young HIV- controls, and levels of intracellular IL-6 and TNF produced by monocytes were shown to be similar to older HIV- men. However, the mechanism governing priming in HIV+ individuals and the elderly differed, suggesting HIV infection does not prematurely induce age-related changes to monocytes. Finally, monocytes from older HIV- men were shown to have a higher propensity to form foam cells in vivo through both intrinsic and extrinsic mechanisms which may lead to heightened development of atherosclerosis, similar to our previous findings in HIV+ men. Discussion: The findings presented in this thesis provide key insight into how monocytes contribute to chronic inflammation, immune activation and age-related disease in young viremic and VS HIV+ individuals and the elderly. Furthermore, these findings raise doubt that HIV infection simply accelerates age-related changes in younger HIV+ individuals. Identifying the mechanism of primed monocyte response to LPS in HIV+ individuals will provide further insight into how chronic inflammation may drive age-related diseases in these individuals

HIV integration and the establishment of latency in CCL19-treated resting CD4(+) T cells require activation of NF-κB

BACKGROUND: Eradication of HIV cannot be achieved with combination antiretroviral therapy (cART) because of the persistence of long-lived latently infected resting memory CD4(+) T cells. We previously reported that HIV latency could be established in resting CD4(+) T cells in the presence of the chemokine CCL19. To define how CCL19 facilitated the establishment of latent HIV infection, the role of chemokine receptor signalling was explored. RESULTS: In resting CD4(+) T cells, CCL19 induced phosphorylation of RAC-alpha serine/threonine-protein kinase (Akt), nuclear factor kappa B (NF-κB), extracellular-signal-regulated kinase (ERK) and p38. Inhibition of the phosphoinositol-3-kinase (PI3K) and Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/ERK signalling pathways inhibited HIV integration, without significant reduction in HIV nuclear entry (measured by Alu-LTR and 2-LTR circle qPCR respectively). Inhibiting activation of MEK1/ERK1/2, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1) and NF-κB, but not p38, also inhibited HIV integration. We also show that HIV integrases interact with Pin1 in CCL19-treated CD4(+) T cells and inhibition of JNK markedly reduced this interaction, suggesting that CCL19 treatment provided sufficient signals to protect HIV integrase from degradation via the proteasome pathway. Infection of CCL19-treated resting CD4(+) T cells with mutant strains of HIV, lacking NF-κB binding sites in the HIV long terminal repeat (LTR) compared to infection with wild type virus, led to a significant reduction in integration by up to 40-fold (range 1-115.4, p = 0.03). This was in contrast to only a modest reduction of 5-fold (range 1.7-11, p > 0.05) in fully activated CD4(+) T cells infected with the same mutants. Finally, we demonstrated significant differences in integration sites following HIV infection of unactivated, CCL19-treated, and fully activated CD4(+) T cells. CONCLUSIONS: HIV integration in CCL19-treated resting CD4(+) T cells depends on NF-κB signalling and increases the stability of HIV integrase, which allow subsequent integration and establishment of latency. These findings have implications for strategies needed to prevent the establishment, and potentially reverse, latent infection

Family support network for adolescent cannabis users.

[Note. The Cannabis Youth Treatment Series is no longer available on the SAMHSA website. It is here as an archival record]. This is volume 3 of a series of 5 treatment guides of interventions for adolescents with cannabis use problems devised in the US (see related publication links below). This guide describes a twelve session intervention for families for adolescent cannabis users. It is designed to be used alongside the interventions described in Vols 1 & 2 which are focused on the adolescent. The twelve sessions combine home visits and group sessions for family members. The aim is to support the family network to help maintain changes to the young person’s cannabis use. The guide contains many useful handouts and resources in addition to the manual for the individual family and group sessions. This intervention can be carried out by drug workers with additional competences and appropriate supervision

Inflammation-induced foam cell formation in chronic inflammatory disease

Atherosclerosis is the leading cause of cardiovascular disease and is both a metabolic and inflammatory disease. Two models describe early events initiating atherosclerotic plaque formation, whereby foam cells form in response to hyperlipidaemia or inflammation-associated stimuli. Although these models are inextricably linked and not mutually exclusive, identifying the unique contribution of each in different disease settings remains an important question. Circulating monocytes are key mediators of atherogenesis in both models as precursors to lipid-laden foam cells formed in response to either excess lipid deposition in arteries, signalling via pattern-associated molecular patterns or a combination of the two. In this review, we assess the role of monocytes in each model and discuss how key steps in atherogenesis may be targeted to enhance clinical outcomes in patients with chronic inflammatory disease

NBC's GEnesis Broadcast Automation System: . . .

GEnesis is a system in use at the NBC television network for automating the composition and distribution of video. It works in a mission critical environment; a system failure could potentially result in a substantial loss of revenue for the network. Tcl/Tk has been an integral part of the operator interface and data handling portions of the GEnesis system from the earliest stages of prototyping. We originally planned to replace the system prototype based on Tcl/Tk with a production system built in a compiled, object-oriented language and using commercial component software. After the prototype phase was completed, the developers and management together decided to keep numerous system components in Tcl, while migrating some complex and performance-critical functions from Tcl to a C++ message passing architecture. This paper discusses that decision and presents our experience with converting the prototype into a fully functional system

Quantification of monocyte transmigration and foam cell formation from individuals with chronic inflammatory conditions

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the transmigration of innate immune monocytes to inflammatory sites of deposited lipid called fatty streaks, which are present in arterial walls of medium to large arteries. The key pathogenic feature of lesions at this early stage of atherosclerosis is the maturation of monocytes which migrate into arteries to form foam cells or lipid-laden macrophages. Considerable evidence supports the hypothesis that risk of atherosclerosis is increased by chronic inflammatory conditions accompanying diseases such as rheumatoid arthritis and HIV, as well as general ageing, and that this risk is predicted by monocyte activation. While mouse models provide a good platform to investigate the role of monocytes in atherogenesis in vivo, they require genetic alteration of natural cholesterol metabolism and drastic alteration of normal mouse diets, and have limited suitability for the study of atherogenic influences of human comorbid diseases. This motivated us to develop a human in vitro model to measure the atherogenic potential of monocytes isolated from individuals with defined disease states. Currently, human in vitro models are limiting in that they evaluate monocyte transmigration and foam cell formation in isolation. Here we describe a protocol in which monocytes isolated from patient blood transmigrate across human endothelial cells into a type 1 collagen matrix, and their propensity to mature into foam cells in the presence or absence of exogenous lipid is measured. The protocol has been validated for the use of human monocytes purified from individuals with HIV infection and elderly HIV uninfected individuals. This model is versatile and allows monocyte transmigration and foam cell formation to be evaluated using either microscopy or flow cytometry as well as allowing the assessment of ather

Monocytes as regulators of inflammation and HIV-related comorbidities during cART

Combined antiretroviral therapy (cART) extends the lifespan and the quality of life for HIV-infected persons but does not completely eliminate chronic immune activation and inflammation. The low level of chronic immune activation persisting during cART-treated HIV infection is associated with the development of diseases which usually occur in the elderly. Although T-cell activation has been extensively examined in the context of cART-treated HIV infection, monocyte activation is only beginning to be recognized as an important source of inflammation in this context. Here we examine markers and sources of monocyte activation during cART-treated HIV infection and discuss the role of monocytes during cardiovascular disease, HIV-associated neurocognitive disorder, and innate immune aging