The biguanide polyamine analog verlindamycin promotes differentiation in neuroblastoma via induction of antizyme.

Deregulated polyamine biosynthesis is emerging as a common feature of neuroblastoma and drugs targeting this metabolic pathway such as DFMO are in clinical and preclinical development. The polyamine analog verlindamycin inhibits the polyamine biosynthesis pathway enzymes SMOX and PAOX, as well as the histone demethylase LSD1. Based on our previous research in acute myeloid leukemia (AML), we reasoned verlindamycin may also unblock neuroblastoma differentiation when combined with all-trans-retinoic acid (ATRA). Indeed, co-treatment with verlindamycin and ATRA strongly induced differentiation regardless of MYCN status, but in MYCN-expressing cells, protein levels were strongly diminished. This process was not transcriptionally regulated but was due to increased degradation of MYCN protein, at least in part via ubiquitin-independent, proteasome-dependent destruction. Here we report that verlindamycin effectively induces the expression of functional tumor suppressor-antizyme via ribosomal frameshifting. Consistent with previous results describing the function of antizyme, we found that verlindamycin treatment led to the selective targeting of ornithine decarboxylase (the rate-limiting enzyme for polyamine biosynthesis) as well as key oncoproteins, such as cyclin D and Aurora A kinase. Retinoid-based multimodal differentiation therapy is one of the few interventions that extends relapse-free survival in MYCN-associated high-risk neuroblastoma and these results point toward the potential use of verlindamycin in this regimen.

Retinoic acid receptor γ is a therapeutically targetable driver of growth and survival in prostate cancer.

BACKGROUND: Prostate cancer (PC) tissue contains all-trans retinoic acid (ATRA) at a very low level (10-9 M), at least an order of magnitude lower than in adjacent normal healthy prostate cells or benign prostate hyperplasia. When this is coupled with deregulated expression of the intracellular lipid-binding proteins FABP5 and CRABP2 that is frequently found in PC, this is likely to result in the preferential delivery of ATRA to oncogenic PPARß/δ rather than retinoic acid receptors (RARs). There are three isotypes of RARs (RARα, RARß, and RARγ) and recent studies have revealed discrete physiological roles. For example, RARα and RARγ promote differentiation and self-renewal, respectively, which are critical for proper hematopoiesis. AIMS: We have previously shown that ATRA stimulates transactivation of RARγ at sub-nanomolar concentrations (EC50 0.24 nM), whereas an 80-fold higher concentration was required for RARα-mediated transactivation (EC50 19.3 nM). Additionally, we have shown that RAR pan-antagonists inhibit the growth of PC cells (at 16-34 nM). These findings, together with the low level of ATRA in PC, led us to hypothesize that RARγ plays a role in PC pathogenesis and that RARγ-selective antagonism may be an effective treatment. METHODS AND RESULTS: We found that concentrations of 10-9 M and below of ATRA promoted survival/proliferation and opposed adipogenic differentiation of human PC cell lines by a mechanism that involves RARγ. We also found that a RARγ-selective antagonist (AGN205728) potently induced mitochondria-dependent, but caspase-independent, cell death in PC cell lines. Furthermore, AGN205728 demonstrated synergism in killing PC cells in combination with cytotoxic chemotherapeutic agents. CONCLUSION: We suggest that the use of RARγ-selective antagonists may be effective in PC (and potentially other cancers), either as a single agent or in combination with cytotoxic chemotherapy.

Socioeconomic and Racial Disparities in Bariatric Surgery.

The purpose of this study was to examine the associations among race and socioeconomic factors (receiving social security disability, insurance type, and income) with undergoing bariatric surgery and weight loss outcomes in a racially diverse, urban cohort of bariatric surgery candidates (N = 314). Patients with private insurance and who identified as Caucasian were more likely to undergo bariatric surgery. Income significantly predicted percentage of excess weight loss 1 year after surgery, although this was no longer significant when accounting for race. Race and socioeconomic factors should be considered during psychosocial evaluations to support patients at risk of surgical attrition and poorer weight loss outcomes. Future research should explore policy solutions to improve access, while qualitative work may help with understanding racial disparities in bariatric surgery.

Entry of Polarized Effector Cells into Quiescence Forces HIV Latency.

The latent HIV reservoir is generated following HIV infection of activated effector CD4 T cells, which then transition to a memory phenotype. Here, we describe an ex vivo method, called QUECEL (quiescent effector cell latency), that mimics this process efficiently and allows production of large numbers of latently infected CD4+ T cells. Naïve CD4+ T cells were polarized into the four major T cell subsets (Th1, Th2, Th17, and Treg) and subsequently infected with a single-round reporter virus which expressed GFP/CD8a. The infected cells were purified and coerced into quiescence using a defined cocktail of cytokines, including tumor growth factor beta, interleukin-10 (IL-10), and IL-8, producing a homogeneous population of latently infected cells. Flow cytometry and transcriptome sequencing (RNA-Seq) demonstrated that the cells maintained the correct polarization phenotypes and had withdrawn from the cell cycle. Key pathways and gene sets enriched during transition from quiescence to reactivation include E2F targets, G2M checkpoint, estrogen response late gene expression, and c-myc targets. Reactivation of HIV by latency-reversing agents (LRAs) closely mimics RNA induction profiles seen in cells from well-suppressed HIV patient samples using the envelope detection of in vitro transcription sequencing (EDITS) assay. Since homogeneous populations of latently infected cells can be recovered, the QUECEL model has an excellent signal-to-noise ratio and has been extremely consistent and reproducible in numerous experiments performed during the last 4 years. The ease, efficiency, and accuracy of the mimicking of physiological conditions make the QUECEL model a robust and reproducible tool to study the molecular mechanisms underlying HIV latency.IMPORTANCE Current primary cell models for HIV latency correlate poorly with the reactivation behavior of patient cells. We have developed a new model, called QUECEL, which generates a large and homogenous population of latently infected CD4+ memory cells. By purifying HIV-infected cells and inducing cell quiescence with a defined cocktail of cytokines, we have eliminated the largest problems with previous primary cell models of HIV latency: variable infection levels, ill-defined polarization states, and inefficient shutdown of cellular transcription. Latency reversal in the QUECEL model by a wide range of agents correlates strongly with RNA induction in patient samples. This scalable and highly reproducible model of HIV latency will permit detailed analysis of cellular mechanisms controlling HIV latency and reactivation.

Th1-like Plasmodium-Specific Memory CD4+ T Cells Support Humoral Immunity.

Effector T cells exhibiting features of either T helper 1 (Th1) or T follicular helper (Tfh) populations are essential to control experimental Plasmodium infection and are believed to be critical for resistance to clinical malaria. To determine whether Plasmodium-specific Th1- and Tfh-like effector cells generate memory populations that contribute to protection, we developed transgenic parasites that enable high-resolution study of anti-malarial memory CD4 T cells in experimental models. We found that populations of both Th1- and Tfh-like Plasmodium-specific memory CD4 T cells persist. Unexpectedly, Th1-like memory cells exhibit phenotypic and functional features of Tfh cells during recall and provide potent B cell help and protection following transfer, characteristics that are enhanced following ligation of the T cell co-stimulatory receptor OX40. Our findings delineate critical functional attributes of Plasmodium-specific memory CD4 T cells and identify a host-specific factor that can be targeted to improve resolution of acute malaria and provide durable, long-term protection against Plasmodium parasite re-exposure.

Mast cells and influenza a virus: association with allergic responses and beyond.

Influenza A virus (IAV) is a widespread infectious agent commonly found in mammalian and avian species. In humans, IAV is a respiratory pathogen that causes seasonal infections associated with significant morbidity in young and elderly populations, and has a large economic impact. Moreover, IAV has the potential to cause both zoonotic spillover infection and global pandemics, which have significantly greater morbidity and mortality across all ages. The pathology associated with these pandemic and spillover infections appear to be the result of an excessive inflammatory response leading to severe lung damage, which likely predisposes the lungs for secondary bacterial infections. The lung is protected from pathogens by alveolar epithelial cells, endothelial cells, tissue resident alveolar macrophages, dendritic cells, and mast cells. The importance of mast cells during bacterial and parasitic infections has been extensively studied; yet, the role of these hematopoietic cells during viral infections is only beginning to emerge. Recently, it has been shown that mast cells can be directly activated in response to IAV, releasing mediators such histamine, proteases, leukotrienes, inflammatory cytokines, and antiviral chemokines, which participate in the excessive inflammatory and pathological response observed during IAV infections. In this review, we will examine the relationship between mast cells and IAV, and discuss the role of mast cells as a potential drug target during highly pathological IAV infections. Finally, we proposed an emerging role for mast cells in other viral infections associated with significant host pathology.

Negative elongation factor is required for the maintenance of proviral latency but does not induce promoter-proximal pausing of RNA polymerase II on the HIV long terminal repeat.

The role of the negative elongation factor (NELF) in maintaining HIV latency was investigated following small hairpin RNA (shRNA) knockdown of the NELF-E subunit, a condition that induced high levels of proviral transcription in latently infected Jurkat T cells. Chromatin immunoprecipitation (ChIP) assays showed that latent proviruses accumulate RNA polymerase II (RNAP II) on the 5' long terminal repeat (LTR) but not on the 3' LTR. NELF colocalizes with RNAP II, and its level increases following proviral induction. RNAP II pause sites on the HIV provirus were mapped to high resolution by ChIP with high-throughput sequencing (ChIP-Seq). Like cellular promoters, RNAP II accumulates at around position +30, but HIV also shows additional pausing at +90, which is immediately downstream of a transactivation response (TAR) element and other distal sites on the HIV LTR. Following NELF-E knockdown or tumor necrosis factor alpha (TNF-α) stimulation, promoter-proximal RNAP II levels increase up to 3-fold, and there is a dramatic increase in RNAP II levels within the HIV genome. These data support a kinetic model for proviral transcription based on continuous replacement of paused RNAP II during both latency and productive transcription. In contrast to most cellular genes, HIV is highly activated by the combined effects of NELF-E depletion and activation of initiation by TNF-α, suggesting that opportunities exist to selectively activate latent HIV proviruses.

Extended interactive voice response telephony (IVR) for relapse prevention after smoking cessation using varenicline and IVR: a pilot study.

BACKGROUND: There is a significant resumption of smoking following smoking cessation using varenicline. Both smoking cessation medications and counseling have been shown to increase smoking quit rates at one year. Thus, the combination of varenicline and interactive voice response (IVR) telephony followed by extended IVR may further improve smoking cessation rates at one and two years. METHODS: 101 participants were recruited from the community via newspaper advertisement. They attended a group counseling session and were given smoking information booklets from the Canadian Cancer Society. After 12 weeks of varenicline and 9 IVR calls, all participants who had quit smoking were randomized into 2 groups matched by levels of motivation and addiction as per baseline questionnaire score. The intervention group continued to receive bi-weekly IVR support for weeks 13-52. The control group no longer received IVR. The primary end-point was self-reported abstinence and exhaled carbon monoxide levels of less than 10 ppm for weeks 12, 52 and 2 years. Data were analyzed by Fisher's exact test or Wilcoxon rank-sum test. RESULTS: Of the 101 participants, 44 (43%) had stopped smoking after 12 weeks of varenicline and 9 IVR calls. Of these, 23 (52%) were randomized to receive IVR calls from weeks 13 to 52.At 52 weeks, 26 (59%) participants remained smoke-free. Of the 23 with IVR, 12 (52.2%) stopped smoking compared to 14 of 21 (66.7%) without IVR. At 2 years, 40 of the 44 (90.9%) randomized participants were contacted and 24 of the 44 (54.5%) came in for testing. Fourteen (13% of the original cohort, 30% who were abstinent at 12 weeks and 53% who were abstinent at 52 weeks) remained smoke-free. Five of the 23 (21.7%) randomized to IVR and 9 of the 21 (42.9%) randomized to no IVR remained smoke-free at 2 years. CONCLUSIONS: In this pilot study of an apparently healthy population, extended IVR did not affect abstinence rates. There was no relapse prevention benefit in offering 9 months of continued IVR to subjects who had stopped smoking after receiving 3 months of varenicline and IVR treatment. TRIAL REGISTRATION: ClinicalTrial.gov: NCT00832806.