Role of retroviral restriction factors in the interferon-α-mediated suppression of HIV-1 in vivo.

The antiviral potency of the cytokine IFN-α has been long appreciated but remains poorly understood. A number of studies have suggested that induction of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 (APOBEC3) and bone marrow stromal cell antigen 2 (BST-2/tetherin/CD317) retroviral restriction factors underlies the IFN-α-mediated suppression of HIV-1 replication in vitro. We sought to characterize the as-yet-undefined relationship between IFN-α treatment, retroviral restriction factors, and HIV-1 in vivo. APOBEC3G, APOBEC3F, and BST-2 expression levels were measured in HIV/hepatitis C virus (HCV)-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated IFN-α/ribavirin (IFN-α/riba) combination therapy. IFN-α/riba therapy decreased HIV-1 viral load by -0.921 (±0.858) log(10) copies/mL in HIV/HCV-coinfected patients. APOBEC3G/3F and BST-2 mRNA expression was significantly elevated during IFN-α/riba treatment in patient-derived CD4+ T cells (P < 0.04 and P < 0.008, paired Wilcoxon), and extent of BST-2 induction was correlated with reduction in HIV-1 viral load during treatment (P < 0.05, Pearson's r). APOBEC3 induction during treatment was correlated with degree of viral hypermutation (P < 0.03, Spearman's ρ), and evolution of the HIV-1 accessory protein viral protein U (Vpu) during IFN-α/riba treatment was suggestive of increased BST-2-mediated selection pressure. These data suggest that host restriction factors play a critical role in the antiretroviral capacity of IFN-α in vivo, and warrant investigation into therapeutic strategies that specifically enhance the expression of these intrinsic immune factors in HIV-1-infected individuals.

Suppression of Arabidopsis protophloem differentiation and root meristem growth by CLE45 requires the receptor-like kinase BAM3.

Peptide signaling presumably occupies a central role in plant development, yet only few concrete examples of receptor-ligand pairs that act in the context of specific differentiation processes have been described. Here we report that second-site null mutations in the Arabidopsis leucine-rich repeat receptor-like kinase gene barely any meristem 3 (BAM3) perfectly suppress the postembryonic root meristem growth defect and the associated perturbed protophloem development of the brevis radix (brx) mutant. The roots of bam3 mutants specifically resist growth inhibition by the CLAVATA3/ENDOSPERM SURROUNDING REGION 45 (CLE45) peptide ligand. WT plants transformed with a construct for ectopic overexpression of CLE45 could not be recovered, with the exception of a single severely dwarfed and sterile plant that eventually died. By contrast, we obtained numerous transgenic bam3 mutants transformed with the same construct. These transgenic plants displayed a WT phenotype, however, supporting the notion that CLE45 is the likely BAM3 ligand. The results correlate with the observation that external CLE45 application represses protophloem differentiation in WT, but not in bam3 mutants. BAM3, BRX, and CLE45 are expressed in a similar spatiotemporal trend along the developing protophloem, up to the end of the transition zone. Induction of BAM3 expression upon CLE45 application, ectopic overexpression of BAM3 in brx root meristems, and laser ablation experiments suggest that intertwined regulatory activity of BRX, BAM3, and CLE45 could be involved in the proper transition of protophloem cells from proliferation to differentiation, thereby impinging on postembryonic growth capacity of the root meristem.

INK4a/Arf is required for suppression of EGFR/DeltaEGFR(2-7)-dependent ERK activation in mouse astrocytes and glioma.

Amplification of the epidermal growth factor receptor (EGFR) or expression of its constitutively activated mutant, DeltaEGFR(2-7), in association with the inactivation of the INK4a/Arf gene locus is a frequent alteration in human glioblastoma. The notion of a cooperative effect between these two alterations has been demonstrated in respective mouse brain tumor models including our own. Here, we investigated underlying molecular mechanisms in early passage cortical astrocytes deficient for p16(INK4a)/p19(Arf) or p53, respectively, with or without ectopic expression of DeltaEGFR(2-7). Targeting these cells with the specific EGFR inhibitor tyrphostin AG1478 revealed that phosphorylation of ERK was only abrogated in the presence of an intact INK4a/Arf gene locus. The sensitivity to inhibit ERK phosphorylation was independent of ectopic expression of DeltaEGFR(2-7) and independent of the TP53 status. This resistance to downregulate the MAPK pathway in the absence of INK4a/Arf was confirmed in cell lines derived from our mouse glioma models with the respective initial genetic alterations. Thus, deletion of INK4a/Arf appears to keep ERK in its active, phosphorylated state insensitive to an upstream inhibitor specifically targeting EGFR/DeltaEGFR(2-7). This resistance may contribute to the cooperative tumorigenic effect selected for in human glioblastoma that may be of crucial clinical relevance for treatments specifically targeting EGFR/DeltaEGFR(2-7) in glioblastoma patients.

Pyoluteorin production by Pseudomonas fluorescens strain CHA0 is involved in the suppression of Pythium damping-off of cress but not of cucumber

Pseudomonas fluorescens strain CHA0 is an effective biocontrol agent of various soilborne pathogens. It controls damping-off or root rot caused byPythium ultimum on cucumber, wheat and cress. Strain CHA0 synthesizes several antibiotic metabolites such as hydrogen cyanide, 2,4-diacetylphloroglucinol, and pyoluteorin. The role of pyoluteorin in the suppression of damping-off was investigated. Two Tn5 mutants (CHA660 and CHA661) of strain CHA0 were isolated which had lost the capacity to produce pyoluteorin but still produced 2,4-diacteylphloroglucinol and HCN. These mutants still inhibitedP. ultimum on malt agar (which favours the production of 2,4-diacetylphloroglucinol) but had partially lost the ability to inhibit this pathogen on King's B agar (which favours the production of pyoluteorin). The two pyoluteorin-negative mutants showed a reduced capacity to suppress damping-off of cress caused byP. ultimum but were as effective in the protection of cucumber against this pathogen as the wild-type strain. These results indicate that, depending on the plant, pyoluteorin production plays a role in the suppression of damping-off by strain CHA0 without being a major mechanism in disease suppression. We suggest that the contribution of pyoluteorin to the biocontrol activity of strain CHA0 is determined by the quantity of this antibiotic produced in the rhizosphere, which might depend on the root exudates of the host plant.

Suppression of tumor angiogenesis through the inhibition of integrin function and signaling in endothelial cells: which side to target?

Tumor angiogenesis is an essential step in tumor progression and metastasis formation. Suppression of tumor angiogenesis results in the inhibition of tumor growth. Recent evidence indicates that vascular integrins, in particular alpha V beta 3, are important regulators of angiogenesis, including tumor angiogenesis. Integrin alpha V beta 3 antagonists, such as blocking antibodies or peptides, suppress tumor angiogenesis and tumor progression in many preclinical tumor models. The potential therapeutic efficacy of extracellular integrin antagonists in human cancer is currently being tested in clinical trials. Selective disruption of the tumor vasculature by high doses of tumor necrosis factor (TNF) and interferon gamma (IFN-gamma), and the antiangiogenic activity of nonsteroidal anti-inflammatory drugs are associated with the suppression of integrin alpha V beta 3 function and signaling in endothelial cells. Furthermore, expression of isolated integrin cytoplasmic domains disrupts integrin-dependent adhesion, resulting in endothelial cell detachment and apoptosis. These results confirm the critical role of vascular integrins in promoting endothelial cell survival and angiogenesis and suggest that intracellular targeting of integrin function and signaling may be an alternative strategy to extracellular integrin antagonists for the therapeutic inhibition of tumor angiogenesis.

Suppression of short interfering RNA-mediated gene silencing by the structural proteins of hepatitis C virus.

Viruses have evolved strategies to overcome the antiviral effects of the host at different levels. Besides specific defence mechanisms, the host responds to viral infection via the interferon pathway and also by RNA interference (RNAi). However, several viruses have been identified that suppress RNAi. We addressed the question of whether hepatitis C virus (HCV) suppresses RNAi, using cell lines constitutively expressing green fluorescent protein (GFP) and inducibly expressing HCV proteins. It was found that short interfering RNA-mediated GFP gene silencing was inhibited when the entire HCV polyprotein was expressed. Further studies showed that HCV structural proteins, and in particular envelope protein 2 (E2), were responsible for this inhibition. Co-precipitation assays demonstrated that E2 bound to Argonaute-2 (Ago-2), a member of the RNA-induced silencing complex, RISC. Thus, HCV E2 that interacts with Ago-2 is able to suppress RNAi.

Cognitive dysfunction in HIV patients despite long-standing suppression of viremia.

OBJECTIVE:: To determine the prevalence of cognitive complaints and HIV-associated neurocognitive disorders (HANDs) in a cohort of aviremic HIV-positive patients. To evaluate the relevance of the HIV dementia scale to detect HANDs. DESIGN:: Assessment of HANDs with neuropsychological tests. METHODS:: Two hundred HIV-infected patients with undetectable HIV-1 RNA concentrations in the plasma, no history of major opportunistic infection of the central nervous system in the past 3 years, no current use of intravenous drugs, and no major depression answered a questionnaire designed to elicit cognitive complaints. Cognitive functions of 50 complaining and 50 noncomplaining HIV-positive patients were assessed. RESULTS:: Patients had undetectable HIV-1 RNA concentrations for a median time of 48 months (range 3.2-136.6). The prevalence of cognitive complaints was 27%. The prevalence of HANDs was 84% among patients with cognitive complaints (asymptomatic neurocognitive impairment 24%, mild neurocognitive disorders 52%, and HIV-associated dementia 8%) and 64% among noncomplainers (asymptomatic neurocognitive impairment 60%, mild neurocognitive disorders 4%, and HIV-associated dementia 0%; P < 0.001). A score of 14 points or less on the HIV dementia scale yielded a positive predictive value of HANDs of 92% in complainers and 82% in noncomplainers. CONCLUSION:: The prevalence of HANDs is high even in long-standing aviremic HIV-positive patients. However, HANDs without functional repercussion in daily life (asymptomatic neurocognitive impairment) is the most frequent subtype observed. In this population, the HIV dementia scale with a cutoff of 14 points or less seems to provide a useful tool to screen for the presence of HANDs.

Proximal tibia volumetric bone mineral density is correlated to the magnitude of local acceleration in male long distance runners.

Introduction: The beneficial effect of physical exercise on bone mineral density (BMD) is at least partly explained by the forces exerted directly on the bones. Male runners present generally higher BMD than sedentary individuals. We postulated that the proximal tibia BMD is related to the running distance as well as to the magnitude of the shocks (while running) in male runners. Methods: A prospective study (three yearly measurements) included 81 healthy male subjects: 16 sedentary lean subjects and three groups of runners (5-30 km/week, n=19; 30-50 km/week, n=29; 50-100 km/week, n=17). Several measurements were performed at the proximal tibia level: volumetric BMD (vBMD), cortical index (CI) i.e. an index of cortical bone thickness and peak accelerations (an index of shocks during heel strike) while running (measured by a 3-D accelerometer). A general linear model assessed the prediction of vBMD or CI by a) simple effects (running distance, peak accelerations, time) and b) interactions (for instance if vBMD prediction by peak acceleration depends on running distance). Results: CI and vBMD a) increase with running distance to reach a plateau over 30 km/wk, b) are positively associated with peak accelerations over 30 km/week. Discussion: Running may be associated with high peak accelerations in order to have beneficial effects on BMD. More important strains are needed to be associated with the same increase in BMD during running sessions of short duration than those of long duration. Conclusion: CI and vBMD are associated with the magnitude of the shocks during heel strike in runners. Key words: Bone mineral density, strains, physical exercise, running distance.

Dietary obesity-associated Hif1α activation in adipocytes restricts fatty acid oxidation and energy expenditure via suppression of the Sirt2-NAD+ system.

Dietary obesity is a major factor in the development of type 2 diabetes and is associated with intra-adipose tissue hypoxia and activation of hypoxia-inducible factor 1α (HIF1α). Here we report that, in mice, Hif1α activation in visceral white adipocytes is critical to maintain dietary obesity and associated pathologies, including glucose intolerance, insulin resistance, and cardiomyopathy. This function of Hif1α is linked to its capacity to suppress β-oxidation, in part, through transcriptional repression of sirtuin 2 (Sirt2) NAD(+)-dependent deacetylase. Reduced Sirt2 function directly translates into diminished deacetylation of PPARγ coactivator 1α (Pgc1α) and expression of β-oxidation and mitochondrial genes. Importantly, visceral adipose tissue from human obese subjects is characterized by high levels of HIF1α and low levels of SIRT2. Thus, by negatively regulating the Sirt2-Pgc1α regulatory axis, Hif1α negates adipocyte-intrinsic pathways of fatty acid catabolism, thereby creating a metabolic state supporting the development of obesity.

Selective suppression of cytokine secretion in whole blood cell cultures of patients with colorectal cancer.

We have investigated the secretion of interferon alpha (IFN-alpha), IFN-gamma, interleukin-1alpha (IL-1alpha), IL-1beta, IL-2 and tumour necrosis factor alpha (TNF-alpha) in whole blood cell cultures (WBCCs) of colorectal cancer patients upon mitogen stimulation. Whereas the values for IL-1beta and TNF-alpha remained virtually unchanged in comparison with healthy control subjects, WBCCs of colorectal cancer patients secreted significantly lower amounts of IFN-alpha (P < 0.005), IFN-gamma (P < 0.0001), IL-1alpha (P < 0.0001) and IL-2 (P < 0.05). This reduction correlated with the progression of the disease. The total leucocyte and monocyte population were almost identical in both groups. In contrast, a dramatic depletion of lymphocytes was observed in colorectal cancer patients, which affected both lymphocyte counts (P < 0.0005) and their distribution (P < 0.0001). Our results suggest a selective suppression of cytokines in colorectal cancer patients that is related to tumour burden. Several mechanisms might account for this phenomenon, one of which might be lymphocyte depletion.

Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation-dependent signaling pathways.

BACKGROUND: Recent evidence indicates that zoledronate, a nitrogen-containing bisphosphonate used to treat conditions of increased bone resorption, may have anti-angiogenic activity. The endothelial cells signaling events modulated by zoledronate remain largely elusive. OBJECTIVES: The aim of this work was to identify signaling events suppressed by zoledronate in endothelial cells and responsible for some of its biological effects. METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed to zoledronate, isoprenoid analogs (i.e. farnesol and geranylgeraniol) and various inhibitors of signaling, and the effect on adhesion, survival, migration, actin cytoskeleton and signaling events characterized. RESULTS: Zoledronate reduced Ras prenylation, Ras and RhoA translocation to the membrane, and sustained ERK1/2 phosphorylation and tumor necrosis factor (TNF) induced JNK phosphorylation. Isoprenoid analogs attenuated zoledronate effects on HUVEC adhesion, actin stress fibers and focal adhesions, migration and survival. Isoprenoid analogs also restored Ras prenylation, RhoA translocation to the membrane, sustained FAK and ERK1/2 phosphorylation and prevented suppression of protein kinase B (PKB) and JNK phosphorylation in HUVEC exposed to TNF in the presence of zoledronate. Pharmacological inhibition of Rock, a RhoA target mediating actin fiber formation, phosphatidylinositol 3-kinase, an activator of PKB, MEK1/2, an activator of ERK1/2, and JNK, recapitulated individual zoledronate effects, consistent with the involvement of these molecules and pathways and their inhibition in the zoledronate effects. CONCLUSIONS: This work has demonstrated that zoledronate inhibits HUVEC adhesion, survival, migration and actin stress fiber formation by interfering with protein prenylation and has identified ERK1/2, JNK, Rock, FAK and PKB as kinases affected by zoledronate in a prenylation-dependent manner.

Zoledronate sensitizes endothelial cells to tumor necrosis factor-induced programmed cell death: evidence for the suppression of sustained activation of focal adhesion kinase and protein kinase B/Akt.

Bisphosphonates are potent inhibitors of osteoclast function widely used to treat conditions of excessive bone resorption, including tumor bone metastases. Recent evidence indicates that bisphosphonates have direct cytotoxic activity on tumor cells and suppress angiogenesis, but the associated molecular events have not been fully characterized. In this study we investigated the effects of zoledronate, a nitrogen-containing bisphosphonate, and clodronate, a non-nitrogen-containing bisphosphonate, on human umbilical vein endothelial cell (HUVEC) adhesion, migration, and survival, three events essential for angiogenesis. Zoledronate inhibited HUVEC adhesion mediated by integrin alphaVbeta3, but not alpha5beta1, blocked migration and disrupted established focal adhesions and actin stress fibers without modifying cell surface integrin expression level or affinity. Zoledronate treatment slightly decreased HUVEC viability and strongly enhanced tumor necrosis factor (TNF)-induced cell death. HUVEC treated with zoledronate and TNF died without evidence of enhanced annexin-V binding, chromatin condensation, or nuclear fragmentation and caspase dependence. Zoledronate inhibited sustained phosphorylation of focal adhesion kinase (FAK) and in combination with TNF, with and without interferon (IFN) gamma, of protein kinase B (PKB/Akt). Constitutive active PKB/Akt protected HUVEC from death induced by zoledronate and TNF/IFNgamma. Phosphorylation of c-Src and activation of NF-kappaB were not affected by zoledronate. Clodronate had no effect on HUVEC adhesion, migration, and survival nor did it enhanced TNF cytotoxicity. Taken together these data demonstrate that zoledronate sensitizes endothelial cells to TNF-induced, caspase-independent programmed cell death and point to the FAK-PKB/Akt pathway as a novel zoledronate target. These results have potential implications to the clinical use of zoledronate as an anti-angiogenic or anti-cancer agent.