Evaluating a New Class of AKT/mTOR Activators for HIV Latency Reversing Activity Ex Vivo and In Vivo.

An ability to activate latent HIV-1 expression could benefit many HIV cure strategies, but the first generation of latency reversing agents (LRAs) has proven disappointing. We evaluated AKT/mTOR activators as a potential new class of LRAs. Two glycogen synthase kinase-3 inhibitors (GSK-3i's), SB-216763 and tideglusib (the latter already in phase II clinical trials) that activate AKT/mTOR signaling were tested. These GSK-3i's reactivated latent HIV-1 present in blood samples from aviremic individuals on antiretroviral therapy (ART) in the absence of T cell activation, release of inflammatory cytokines, cell toxicity, or impaired effector function of cytotoxic T lymphocytes or NK cells. However, when administered in vivo to SIV-infected rhesus macaques on suppressive ART, tideglusib exhibited poor pharmacodynamic properties and resulted in no clear evidence of significant SIV latency reversal. Whether alternative pharmacological formulations or combinations of this drug with other classes of LRAs will lead to an effective in vivo latency-reversing strategy remains to be determined.IMPORTANCE If combined with immune therapeutics, latency reversing agents (LRAs) have the potential to reduce the size of the reservoir sufficiently that an engineered immune response can control the virus in the absence of antiretroviral therapy. We have identified a new class of LRAs that do not induce T-cell activation and that are able to potentiate, rather than inhibit, CD8+ T and NK cell cytotoxic effector functions. This new class of LRAs corresponds to inhibitors of glycogen synthase kinase-3. In this work, we have also studied the effects of one member of this drug class, tideglusib, in SIV-infected rhesus monkeys. When tested in vivo, however, tideglusib showed unfavorable pharmacokinetic properties, which resulted in lack of SIV latency reversal. The disconnect between our ex vivo and in vivo results highlights the importance of developing next generation LRAs with pharmacological properties that allow systemic drug delivery in relevant anatomical compartments harboring latent reservoirs.

Identification and functional characterization of the distinct plant pectin esterases PAE8 and PAE9 and their deletion mutants.

MAIN CONCLUSION: PAE8 and PAE9 have pectin acetylesterase activity and together remove one-third of the cell wall acetate associated with pectin formation in Arabidopsis leaves. In pae8 and pae9 mutants, substantial amounts of acetate accumulate in cell walls. In addition, the inflorescence stem height is decreased. Pectic polysaccharides constitute a significant part of the primary cell walls in dicotyledonous angiosperms. This diverse group of polysaccharides has been implicated in several physiological processes including cell-to-cell adhesion and pathogenesis. Several pectic polysaccharides contain acetyl-moieties directly affecting their physical properties such as gelling capacity, an important trait for the food industry. In order to gain further insight into the biological role of pectin acetylation, a reverse genetics approach was used to investigate the function of genes that are members of the Pectin AcetylEsterase gene family (PAE) in Arabidopsis. Mutations in two members of the PAE family (PAE8 and PAE9) lead to cell walls with an approximately 20 % increase in acetate content. High-molecular-weight fractions enriched in pectic rhamnogalacturonan I (RGI) extracted from the mutants had increased acetate content. In addition, the pae8 mutant displayed increased acetate content also in low-molecular-weight pectic fractions. The pae8/pae9-2 double mutant exhibited an additive effect by increasing wall acetate content by up to 37 %, suggesting that the two genes are not redundant and act on acetyl-substituents of different pectic domains. The pae8 and pae8/pae9-2 mutants exhibit reduced inflorescence growth underscoring the role of pectic acetylation in plant development. When heterologously expressed and purified, both gene products were shown to release acetate from the corresponding mutant pectic fractions in vitro. PAEs play a significant role in modulating the acetylation state of pectic polymers in the wall, highlighting the importance of apoplastic metabolism for the plant cell and plant growth.

Smoking, mental illness and socioeconomic disadvantage: analysis of the Australian National Survey of Mental Health and Wellbeing.

BACKGROUND: High rates of smoking and lower rates of smoking cessation are known to be associated with common mental disorders such as anxiety and depression, and with individual and community measures of socioeconomic status. It is not known to what extent mental illness and socioeconomic status might be jointly associated with smoking behaviour. We set out to examine the relationship between mental illness, measures of socioeconomic disadvantage and both current smoking and smoking cessation rates. METHODS: We used data from the 2007 Australian National Survey of Mental Health and Wellbeing to examine the relationship between mental illness, socioeconomic status and both current smoking and smoking cessation. We used cross-classified tables and logistic regression to examine the relationship between psychosocial and sociodemographic predictors and current smoking. We also used proportional hazards regression to examine the relationship between the factors and smoking cessation. RESULTS: Both mental illness and socioeconomic status were independently associated with current smoking and with lower likelihood of smoking cessation, with gradients in smoking by mental health status being observed within levels of socioeconomic indicators and vice versa. Having a mental illness in the past 12 months was the most prevalent factor strongly associated with smoking, affecting 20.0% of the population, associated with increased current smoking (OR 2.43; 95% CI: 1.97-3.01) and reduced likelihood of smoking cessation (HR: 0.77; 95% CI: 0.65-0.91). CONCLUSIONS: The association between mental illness and smoking is not explained by the association between mental illness and socioeconomic status. There are strong socioeconomic and psychosocial gradients in both current smoking and smoking cessation. Incorporating knowledge of the other adverse factors in smokers' lives may increase the penetration of tobacco control interventions in population groups that have historically benefitted less from these activities.

Cancer-associated fibroblasts are the main contributors to epithelial-to-mesenchymal signatures in the tumor microenvironment.

Epithelial-to-mesenchymal transition (EMT) is associated with tumor initiation, metastasis, and drug resistance. However, the mechanisms underlying these associations are largely unknown. We studied several tumor types to identify the source of EMT gene expression signals and a potential mechanism of resistance to immuno-oncology treatment. Across tumor types, EMT-related gene expression was strongly associated with expression of stroma-related genes. Based on RNA sequencing of multiple patient-derived xenograft models, EMT-related gene expression was enriched in the stroma versus parenchyma. EMT-related markers were predominantly expressed by cancer-associated fibroblasts (CAFs), cells of mesenchymal origin which produce a variety of matrix proteins and growth factors. Scores derived from a 3-gene CAF transcriptional signature (COL1A1, COL1A2, COL3A1) were sufficient to reproduce association between EMT-related markers and disease prognosis. Our results suggest that CAFs are the primary source of EMT signaling and have potential roles as biomarkers and targets for immuno-oncology therapies.

Can social and community service organisations embrace tobacco control for their disadvantaged clients?

ISSUE ADDRESSED: High smoking rates among the disadvantaged lead to inequalities in health, quality-of-life and financial well-being. Non-government social and community service organisations (SCSO) are a promising setting for tobacco control interventions targeting disadvantaged smokers. METHODS: Financial grants were provided to twenty SCSO in New South Wales to support multi-level changes in service culture, smoking related policies and cessation support between 2007 and 2009. Evaluation was conducted using a mixed-methods approach that included key informant interviews, document analysis and staff survey data. RESULTS: SCSO working with disadvantaged clients can feasibly implement tobacco control activities, including smoking-related policy changes and cessation support. Tobacco control activities were generally acceptable to staff and clients, and staff pessimism regarding their clients' ability to quit reduced, as did the acceptability of staff smoking with their clients. Improvements in levels of organisational support for cessation training and resources, smoking policies and provision of free nicotine replacement therapy (NRT) were reported. Within mental health SCSO there was a positive response from staff to cessation support, financial benefits of quitting and the role of NRT. CONCLUSIONS: The evaluation pointed to the acceptability and feasibility of engaging disadvantaged smokers by SCSO, and that tobacco policy and attitude changes can be achieved by small investments, such as grant programs.

FOXO1 promotes HIV latency by suppressing ER stress in T cells.

Quiescence is a hallmark of CD4+ T cells latently infected with human immunodeficiency virus 1 (HIV-1). While reversing this quiescence is an effective approach to reactivate latent HIV from T cells in culture, it can cause deleterious cytokine dysregulation in patients. As a key regulator of T-cell quiescence, FOXO1 promotes latency and suppresses productive HIV infection. We report that, in resting T cells, FOXO1 inhibition impaired autophagy and induced endoplasmic reticulum (ER) stress, thereby activating two associated transcription factors: activating transcription factor 4 (ATF4) and nuclear factor of activated T cells (NFAT). Both factors associate with HIV chromatin and are necessary for HIV reactivation. Indeed, inhibition of protein kinase R-like ER kinase, an ER stress sensor that can mediate the induction of ATF4, and calcineurin, a calcium-dependent regulator of NFAT, synergistically suppressed HIV reactivation induced by FOXO1 inhibition. Thus, our studies uncover a link of FOXO1, ER stress and HIV infection that could be therapeutically exploited to selectively reverse T-cell quiescence and reduce the size of the latent viral reservoir.

Metabolic reprogramming of human CD8+ memory T cells through loss of SIRT1.

The expansion of CD8+CD28- T cells, a population of terminally differentiated memory T cells, is one of the most consistent immunological changes in humans during aging. CD8+CD28- T cells are highly cytotoxic, and their frequency is linked to many age-related diseases. As they do not accumulate in mice, many of the molecular mechanisms regulating their fate and function remain unclear. In this paper, we find that human CD8+CD28- T cells, under resting conditions, have an enhanced capacity to use glycolysis, a function linked to decreased expression of the NAD+-dependent protein deacetylase SIRT1. Global gene expression profiling identified the transcription factor FoxO1 as a SIRT1 target involved in transcriptional reprogramming of CD8+CD28- T cells. FoxO1 is proteasomally degraded in SIRT1-deficient CD8+CD28- T cells, and inhibiting its activity in resting CD8+CD28+ T cells enhanced glycolytic capacity and granzyme B production as in CD8+CD28- T cells. These data identify the evolutionarily conserved SIRT1-FoxO1 axis as a regulator of resting CD8+ memory T cell metabolism and activity in humans.

SMYD2-Mediated Histone Methylation Contributes to HIV-1 Latency.

Transcriptional latency of HIV is a last barrier to viral eradication. Chromatin-remodeling complexes and post-translational histone modifications likely play key roles in HIV-1 reactivation, but the underlying mechanisms are incompletely understood. We performed an RNAi-based screen of human lysine methyltransferases and identified the SET and MYND domain-containing protein 2 (SMYD2) as an enzyme that regulates HIV-1 latency. Knockdown of SMYD2 or its pharmacological inhibition reactivated latent HIV-1 in T cell lines and in primary CD4+ T cells. SMYD2 associated with latent HIV-1 promoter chromatin, which was enriched in monomethylated lysine 20 at histone H4 (H4K20me1), a mark lost in cells lacking SMYD2. Further, we find that lethal 3 malignant brain tumor 1 (L3MBTL1), a reader protein with chromatin-compacting properties that recognizes H4K20me1, was recruited to the latent HIV-1 promoter in a SMYD2-dependent manner. We propose that a SMYD2-H4K20me1-L3MBTL1 axis contributes to HIV-1 latency and can be targeted with small-molecule SMYD2 inhibitors.

A conceptual schema for government purchasing arrangements for Australian alcohol and other drug treatment.

AIM: The aim of this study was to establish a conceptual schema for government purchasing of alcohol and other drug treatment in Australia which could encompass the diversity and variety in purchasing arrangements, and facilitate better decision-maker by purchasers. There is a limited evidence base on purchasing arrangements in alcohol and drug treatment despite the clear impact of purchasing arrangements on both treatment processes and treatment outcomes. METHODS: The relevant health and social welfare literature on purchasing arrangements was reviewed; data were collected from Australian purchasers and providers of treatment giving detailed descriptions of the array of purchasing arrangements. Combined analysis of the literature and the Australian purchasing data resulted in a draft schema which was then reviewed by an expert committee and subsequently finalised. RESULTS: The conceptual schema presented here was purpose-built for alcohol and other drug treatment, with its overlap between health and social welfare services. It has three dimensions: 1. The ways in which providers are chosen; 2. The ways in which services are paid for; and 3. How price is managed. Distinguishing between the methods for choosing providers (such as competitive or individually negotiated processes) from the way in which organisations are paid for their provision of treatment (such as via a block grant or payment for activity) provides conceptual clarity and enables closer analysis of each mechanism. CONCLUSIONS: Governments can improve health and wellbeing by making informed decisions about the way they purchase and fund alcohol and other drug treatment. Research comparing different purchasing arrangements can provide a vital evidence-base to inform funders; however a first step is to accurately and consistently categorise current approaches against a typology or conceptual schema.

Individual differences underlying susceptibility to addiction: Role for the endogenous oxytocin system.

Recent research shows that the effects of oxytocin are more diverse than initially thought and that in some cases oxytocin can directly influence the response to drugs and alcohol. Large individual differences in basal oxytocin levels and reactivity of the oxytocin system exist. This paper will review the literature to explore how individual differences in the oxytocin system arise and examine the hypothesis that this may mediate some of the individual differences in susceptibility to addiction and relapse. Differences in the oxytocin system can be based on individual factors, e.g. genetic variation especially in the oxytocin receptor, age or gender, or be the result of early environmental influences such as social experiences, stress or trauma. The paper addresses the factors that cause individual differences in the oxytocin system and the environmental factors that have been identified to induce long-term changes in the developing oxytocin system during different life phases. Individual differences in the oxytocin system can influence effects of drugs and alcohol directly or indirectly. The oxytocin system has bidirectional interactions with the stress-axis, autonomic nervous system, neurotransmitter systems (e.g. dopamine, serotonin and GABA/glutamate) and the immune system. These systems are all important, even vital, in different phases of addiction. It is suggested that early life adversity can change the development of the oxytocin system and the way it modulates other systems. This in turn could minimise the negative feedback loops that would normally exist. Individuals may show only minor differences in behaviour and function unless subsequent stressors or drug use challenges the system. It is postulated that at that time individual differences in oxytocin levels, reactivity of the system or interactions with other systems can influence general resilience, drug effects and the susceptibility to develop problematic drug and alcohol use.

Implementing a smoking cessation program in social and community service organisations: a feasibility and acceptability trial.

INTRODUCTION AND AIMS: Novel ways of accessing and engaging smokers who are socially and economically disadvantaged may help reduce socioeconomic disparities in smoking rates. This study assessed the feasibility and acceptability of integrating smoking cessation support into usual care at a social and community service organisation (SCSO). DESIGN AND METHODS: One SCSO providing a Personal Helpers and Mentors program participated. Support workers were provided with training in 5A's, brief motivational interviewing and use of nicotine replacement therapy, and then recruited clients into a 6 month smoking program. Acceptability and feasibility was assessed prior to receiving training and at 3 and 6 month follow up for support workers, and at enrolment into the program and at 4 and 6 month follow up for clients. RESULTS: Six support workers (67%) and 20 of their clients (65%) took part. Overall acceptability of the program was high, particularly among clients. The amount of time spent talking about smoking increased from 3.8 min per visit at baseline to 15.5 min at 6 month follow up. There was a significant reduction in the number of cigarettes smoked from 20.5 cigarettes per day at baseline to 15 cigarettes per day at 6 month follow up (P = 0.04). DISCUSSION AND CONCLUSIONS: SCSOs are both interested in and capable of providing smoking care and the majority of clients found the smoking cessation intervention acceptable and helpful. Given the demonstrated acceptability and feasibility of this approach, further research to determine the effectiveness of this approach is warranted.