Arenavirus nucleoproteins prevent activation of nuclear factor kappa B.

Arenaviruses include several causative agents of hemorrhagic fever (HF) disease in humans that are associated with high morbidity and significant mortality. Morbidity and lethality associated with HF arenaviruses are believed to involve the dysregulation of the host innate immune and inflammatory responses that leads to impaired development of protective and efficient immunity. The molecular mechanisms underlying this dysregulation are not completely understood, but it is suggested that viral infection leads to disruption of early host defenses and contributes to arenavirus pathogenesis in humans. We demonstrate in the accompanying paper that the prototype member in the family, lymphocytic choriomeningitis virus (LCMV), disables the host innate defense by interfering with type I interferon (IFN-I) production through inhibition of the interferon regulatory factor 3 (IRF3) activation pathway and that the viral nucleoprotein (NP) alone is responsible for this inhibitory effect (C. Pythoud, W. W. Rodrigo, G. Pasqual, S. Rothenberger, L. Martínez-Sobrido, J. C. de la Torre, and S. Kunz, J. Virol. 86:7728-7738, 2012). In this report, we show that LCMV-NP, as well as NPs encoded by representative members of both Old World (OW) and New World (NW) arenaviruses, also inhibits the nuclear translocation and transcriptional activity of the nuclear factor kappa B (NF-κB). Similar to the situation previously reported for IRF3, Tacaribe virus NP (TCRV-NP) does not inhibit NF-κB nuclear translocation and transcriptional activity to levels comparable to those seen with other members in the family. Altogether, our findings demonstrate that arenavirus infection inhibits NF-κB-dependent innate immune and inflammatory responses, possibly playing a key role in the pathogenesis and virulence of arenavirus.

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.

Identification of a novel 45-kDa cell surface molecule involved in activation of the human Jurkat T cell line.

This report describes a surface molecule, Tp45, which appears to be involved in interleukin 2 production and Ca2+ mobilization by Jurkat cells. The Tp45 molecule was identified by a monoclonal antibody, MX13, on the surface of either T3/TCR+ or T3/TCR- human T cell lines. Biochemical data showed that mAb MX13 precipitated a single polypeptide chain of 45 kDa both under reduced and nonreduced conditions from lysates of 125I-surface-labeled cells. Sequential immunodepletion experiments using lysates of 125I-labeled T3/TCR+ cells showed that Tp45 was distinct from the alpha chain of the TCR complex. However, incubation of such cells with either anti-T3 or anti-TCR monoclonal antibody induced complete modulation of both the T3/TCR complex and Tp45. Conversely, complete modulation of both Tp45 and the T3/TCR complex was observed after incubation with anti-Tp45 antibody. Functional studies showed that anti-Tp45 antibody induced high levels of interleukin 2 production in Jurkat cells. In addition, incubation of these cells with the antibody resulted in Ca2+ mobilization from internal stores. Anti-Tp45 antibody reacted with 3-19% peripheral blood (E-rosette-positive) T cells in individual donors. The magnitude of the proliferative response elicited by anti-Tp45 antibody for peripheral blood T cells was lower than that induced by an anti-T3 antibody. This observation is compatible with the idea that only a subpopulation of T cells is reactive with anti-Tp45. Multicolor flow cytometry analysis showed that the Tp45+ cells belong preferentially to the T8 subset.

A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes.

The amiloride-sensitive epithelial sodium channel (ENaC) constitutes a limiting step in sodium reabsorption across distal airway epithelium and controlling mucociliary clearance. ENaC is activated by serine proteases secreted in the extracellular milieu. In cystic fibrosis lungs, high concentrations of secreted neutrophil elastase (NE) are observed. hNE could activate ENaC and contribute to further decreased mucociliary clearance. The aims of this study were (i) to test the ability of an engineered human neutrophil elastase inhibitor (EPI-hNE4) to specifically inhibit the elastase activation of ENaC-mediated amiloride-sensitive currents (I(Na)) and (ii) to examine the effect of elastase on cell surface expression of ENaC and its cleavage pattern (exogenous proteolysis). Oocytes were exposed to hNE (10-100 microg/ml) and/or trypsin (10 microg/ml) for 2-5 min in the presence or absence of EPI-hNE4 (0.7 microm). hNE activated I(Na) 3.6-fold (p < 0.001) relative to non-treated hENaC-injected oocytes. EPI-hNE4 fully inhibited hNE-activated I(Na) but had no effect on trypsin- or prostasin-activated I(Na). The co-activation of I(Na) by hNE and trypsin was not additive. Biotinylation experiments revealed that cell surface gamma ENaC (but not alpha or beta ENaC) exposed to hNE for 2 min was cleaved (as a 67-kDa fragment) and correlated with increased I(Na). The elastase-induced exogenous proteolysis pattern is distinct from the endogenous proteolysis pattern induced upon preferential assembly, suggesting a causal relationship between gamma ENaC cleavage and ENaC activation, taking place at the plasma membrane.

Dynamic responses of O2 uptake at the onset and end of exercise in trained subjects.

Inconsistencies about dynamic asymmetry between the on- and off-transient responses in .VO2 are found in the literature. Therefore the purpose of this study was to examine .VO2on- and off-transients during moderate- and heavy-intensity cycling exercise in trained subjects. Ten men underwent an initial incremental test for the estimation of ventilatory threshold (VT) and, on different days, two bouts of square-wave exercise at moderate (<VT) and heavy (>VT) intensities. .VO2 kinetics in exercise and recovery were better described by a single exponential model (<VT) or by a double exponential with two time delays (>VT). For moderate exercise, we found a symmetry of .VO2 kinetics between the on- and off-transients (i.e., fundamental component), consistent with a system manifesting linear control dynamics. For heavy exercise, a slow component superimposed on the fundamental phase was expressed in both the exercise and recovery, with similar parameter estimates. But the on-transient values of the time constant were appreciably faster than the associated off-transient, and independent of the work rate imposed (<VT and >VT). Our results do not support a dynamically linear system model of .VO2 during cycling exercise in the heavy-intensity domain.

Addition of inspiratory resistance increases the amplitude of the slow component of O2 uptake kinetics.

The contribution of respiratory muscle work to the development of the O(2) consumption (Vo(2)) slow component is a point of controversy because it has been shown that the increased ventilation in hypoxia is not associated with a concomitant increase in Vo(2) slow component. The first purpose of this study was thus to test the hypothesis of a direct relationship between respiratory muscle work and Vo(2) slow component by manipulating inspiratory resistance. Because the conditions for a Vo(2) slow component specific to respiratory muscle can be reached during intense exercise, the second purpose was to determine whether respiratory muscles behave like limb muscles during heavy exercise. Ten trained subjects performed two 8-min constant-load heavy cycling exercises with and without a threshold valve in random order. Vo(2) was measured breath by breath by using a fast gas exchange analyzer, and the Vo(2) response was modeled after removal of the cardiodynamic phase by using two monoexponential functions. As anticipated, when total work was slightly increased with loaded inspiratory resistance, slight increases in base Vo(2), the primary phase amplitude, and peak Vo(2) were noted (14.2%, P < 0.01; 3.5%, P > 0.05; and 8.3%, P < 0.01, respectively). The bootstrap method revealed small coefficients of variation for the model parameter, including the slow-component amplitude and delay (15 and 19%, respectively), indicating an accurate determination for this critical parameter. The amplitude of the Vo(2) slow component displayed a 27% increase from 8.1 +/- 3.6 to 10.3 +/- 3.4 ml. min(-1). kg(-1) (P < 0.01) with the addition of inspiratory resistance. Taken together, this increase and the lack of any differences in minute volume and ventilatory parameters between the two experimental conditions suggest the occurrence of a Vo(2) slow component specific to the respiratory muscles in loaded condition.

Disparate activation of the coronary sinus and inferior left atrium during atrial tachycardia after persistent atrial fibrillation ablation: prevalence, pitfalls, and impact on mapping.

INTRODUCTION: Persistent atrial fibrillation (AF) ablation may lead to partial disconnection of the coronary sinus (CS). As a result, disparate activation sequences of the local CS versus contiguous left atrium (LA) may be observed during atrial tachycardia (AT). We aimed to evaluate the prevalence of this phenomenon and its impact on activation mapping. METHODS: AT occurring after persistent AF ablation were investigated in 74 consecutive patients. Partial CS disconnection during AT was suspected when double potentials with disparate activation sequences were observed on the CS catheter. Endocardial mapping facing CS bipoles was performed to differentiate LA far-field from local CS potentials. RESULTS: A total of 149 ATs were observed. Disparate LA-CS activations were apparent in 20 ATs after magnifying the recording scale (13%). The most common pattern (90%) was distal to proximal endocardial LA activation against proximal to distal CS activation, the latter involving the whole CS or its distal part. Perimitral macroreentry was more common when disparate LA-CS activations were observed (67% vs 29%; P = 0.002). Partial CS disconnection also resulted in "pseudo" mitral isthmus (MI) block during LA appendage pacing in 20% of patients as local CS activation was proximal to distal despite distal to proximal activation of the contiguous LA. CONCLUSION: Careful analysis of CS recordings during AT following persistent AF ablation often reveals disparate patterns of activation. Recognizing when endocardial LA activation occurs in the opposite direction to the more obvious local CS signals is critical to avoid misleading interpretations during mapping of AT and evaluation of MI block.

Role of Leptin in the Activation of Immune Cells

Adipose tissue is an active endocrine organ that secretes various humoral factors (adipokines), and its shift to production of proinflammatory cytokines in obesity likely contributes to the low-level systemic inflammation that may be present in metabolic syndrome-associated chronic pathologies such as atherosclerosis. Leptin is one of the most important hormones secreted by adipocytes, with a variety of physiological roles related to the control of metabolism and energy homeostasis. One of these functions is the connection between nutritional status and immune competence. The adipocyte-derived hormone leptin has been shown to regulate the immune response, innate and adaptive response, both in normal and pathological conditions. The role of leptin in regulating immune response has been assessed in vitro as well as in clinical studies. It has been shown that conditions of reduced leptin production are associated with increased infection susceptibility. Conversely, immune-mediated disorders such as autoimmune diseases are associated with increased secretion of leptin and production of proinflammatory pathogenic cytokines. Thus, leptin is a mediator of the inflammatory response

Mitochondrial dysfunction and mitophagy activation in blood mononuclear cells of fibromyalgia patients: implications in the pathogenesis of the disease.

Introduction. Fibromyalgia is a chronic pain syndrome with unknown etiology. Recent studies have shown some evidence demonstrating that oxidative stress may have a role in the pathophysiology of fibromyalgia. However, it is still not clear whether oxidative stress is the cause or the effect of the abnormalities documented in fibromyalgia. Furthermore, the role of mitochondria in the redox imbalance reported in fibromyalgia also is controversial. We undertook this study to investigate the role of mitochondrial dysfunction, oxidative stress, and mitophagy in fibromyalgia. Methods. We studied 20 patients (2 male, 18 female patients) from the database of the Sevillian Fibromyalgia Association and 10 healthy controls. We evaluated mitochondrial function in blood mononuclear cells from fibromyalgia patients measuring, coenzyme Q10 levels with high-performance liquid chromatography (HPLC), and mitochondrial membrane potential with flow cytometry. Oxidative stress was determined by measuring mitochondrial superoxide production with MitoSOX™ and lipid peroxidation in blood mononuclear cells and plasma from fibromyalgia patients. Autophagy activation was evaluated by quantifying the fluorescence intensity of LysoTracker™ Red staining of blood mononuclear cells. Mitophagy was confirmed by measuring citrate synthase activity and electron microscopy examination of blood mononuclear cells. Results. We found reduced levels of coenzyme Q10, decreased mitochondrial membrane potential, increased levels of mitochondrial superoxide in blood mononuclear cells, and increased levels of lipid peroxidation in both blood mononuclear cells and plasma from fibromyalgia patients. Mitochondrial dysfunction was also associated with increased expression of autophagic genes and the elimination of dysfunctional mitochondria with mitophagy. Conclusions. These findings may support the role of oxidative stress and mitophagy in the pathophysiology of fibromyalgia.

REPETIDO_A new extract of the plant calendula officinalis produces a dual in vitro effect: cytotoxic anti-tumor activity and lymphocyte activation

BACKGROUND Phytopharmacological studies of different Calendula extracts have shown anti-inflammatory, anti-viral and anti-genotoxic properties of therapeutic interest. In this study, we evaluated the in vitro cytotoxic anti-tumor and immunomodulatory activities and in vivo anti-tumor effect of Laser Activated Calendula Extract (LACE), a novel extract of the plant Calendula Officinalis (Asteraceae). METHODS An aqueous extract of Calendula Officinalis was obtained by a novel extraction method in order to measure its anti-tumor and immunomodulatory activities in vitro. Tumor cell lines derived from leukemias, melanomas, fibrosarcomas and cancers of breast, prostate, cervix, lung, pancreas and colorectal were used and tumor cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of LACE on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in LACE-treated cells. In vivo anti-tumor activity was evaluated in nude mice bearing subcutaneously human Ando-2 melanoma cells. RESULTS The LACE extract showed a potent in vitro inhibition of tumor cell proliferation when tested on a wide variety of human and murine tumor cell lines. The inhibition ranged from 70 to 100%. Mechanisms of inhibition were identified as cell cycle arrest in G0/G1 phase and Caspase-3-induced apoptosis. Interestingly, the same extract showed an opposite effect when tested on PBLs and NKL cell line, in which in vitro induction of proliferation and activation of these cells was observed. The intraperitoneal injection or oral administration of LACE extract in nude mice inhibits in vivo tumor growth of Ando-2 melanoma cells and prolongs the survival day of the mice. CONCLUSION These results indicate that LACE aqueous extract has two complementary activities in vitro with potential anti-tumor therapeutic effect: cytotoxic tumor cell activity and lymphocyte activation. The LACE extract presented in vivo anti-tumoral activity in nude mice against tumor growth of Ando-2 melanoma cells.

Regulation of dendritic development by BDNF requires activation of CRTC1 by glutamate.

Dendritic growth is essential for the establishment of a functional nervous system. Among extrinsic signals that control dendritic development, substantial evidence indicates that BDNF regulates dendritic morphology. However, little is known about the underlying mechanisms by which BDNF controls dendritic growth. In this study, we show that the MAPK signaling pathway and the transcription factor cAMP response element-binding protein (CREB) mediate the effects of BDNF on dendritic length and complexity. However, phosphorylation of CREB alone is not sufficient for the stimulation of dendritic growth by BDNF. Thus, using a mutant form of CREB unable to bind CREB-regulated transcription coactivator (CRTC1), we demonstrate that this effect also requires a functional interaction between CREB and CRTC1. Moreover, inhibition of CRTC1 expression by shRNA-mediated knockdown abolished BDNF-induced dendritic growth of cortical neurons. Interestingly, we found that nuclear translocation of CRTC1 results from activation of NMDA receptors by glutamate, a process that is essential for the effects of BDNF on dendritic development. Together, these data identify a previously unrecognized mechanism by which CREB and the coactivator CRTC1 mediate the effects of BDNF on dendritic growth.

Involvement of microglia-neuron interactions in the tumor necrosis factor-alpha release, microglial activation, and neurodegeneration induced by trimethyltin.

Trimethyltin (TMT) is a neurotoxicant known to induce early microglial activation. The present study was undertaken to investigate the role played by these microglial cells in the TMT-induced neurotoxicity. The effects of TMT were investigated in monolayer cultures of isolated microglia or in neuron-enriched cultures and in neuron-microglia and astrocyte-microglia cocultures. The end points used were morphological criteria; evaluation of cell death and cell proliferation; and measurements of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO) release in culture supernatant. The results showed that, in cultures of microglia, TMT (10(-6) M) caused, after a 5-day treatment, an increased release of TNF-alpha, without affecting microglial shape or cell viability. When microglia were cocultured with astrocytes, TNF-alpha release was decreased to undetectable levels. In contrast, in neuron-microglia cocultures, TNF-alpha levels were found to increase at lower concentrations of TMT (i.e., 10(-8) M). Moreover, at 10(-6) M of TMT, microglia displayed further morphological activation, as suggested by process retraction and by decrease in cell size. No morphological activation was observed in cultures of isolated microglial cells and in astrocyte-microglia cocultures. With regard to neurons, 10(-6) M of TMT induced about 30% of cell death, when applied to neuron-enriched cultures, whereas close to 100% of neuronal death was observed in neuron-microglia cocultures. In conclusion, whereas astrocytes may rather dampen the microglial activation by decreasing microglial TNF-alpha production, neuronal-microglial interactions lead to enhanced microglial activation. This microglial activation, in turn, exacerbates the neurotoxic effects of TMT. TNF-alpha may play a major role in such cell-cell communications.

Control and host-dependent activation of insect toxin expression in a root-associated biocontrol pseudomonad.

Pseudomonas fluorescens CHA0 is a root-associated biocontrol agent that suppresses soil-borne fungal diseases of crops. Remarkably, the pseudomonad is also endowed with systemic and oral activity against pest insects which depends on the production of the insecticidal Fit toxin. The toxin gene (fitD) is part of a virulence cassette encoding three regulators (FitF, FitG, FitH) and a type I secretion system (FitABC-E). Immunoassays with a toxin-specific antibody and transcriptional analyses involving fitG and fitH deletion and overexpression mutants identified LysR family regulator FitG and response regulator FitH as activator and repressor, respectively, of Fit toxin and transporter expression. To visualize and quantify toxin expression in single live cells by fluorescence microscopy, we developed reporters which in lieu of the native toxin protein express a fusion of the Fit toxin with red fluorescent mCherry. In a wild-type background, expression of the mCherry-tagged Fit toxin was activated at high levels in insect hosts, i.e. when needed, yet not on plant roots or in batch culture. By contrast, a derepressed fitH mutant expressed the toxin in all conditions. P. fluorescens hence can actively induce insect toxin production in response to the host environment, and FitH and FitG are key regulators in this mechanism.

Fas ligand-induced proinflammatory transcriptional responses in reconstructed human epidermis. Recruitment of the epidermal growth factor receptor and activation of MAP kinases.

Fas ligand (FasL) exerts potent proapoptotic and proinflammatory actions on epidermal keratinocytes and has been implicated in the pathogenesis of eczema, toxic epidermal necrolysis, and drug-induced skin eruptions. We used reconstructed human epidermis to investigate the mechanisms of FasL-induced inflammatory responses and their relationships with FasL-triggered caspase activity. Caspase activity was a potent antagonist of the pro-inflammatory gene expression triggered by FasL prior to the onset of cell death. Furthermore, we found that FasL-stimulated autocrine production of epidermal growth factor receptor (EGFR) ligands, and the subsequent activation of EGFR and ERK1 and ERK2 mitogen-activated protein kinases, were obligatory extracellular steps for the FasL-induced expression of a subset of inflammatory mediators, including CXCL8/interleukin (IL)-8, ICAM-1, IL-1alpha, IL-1beta, CCL20/MIP-3alpha, and thymic stromal lymphopoietin. These results expand the known physiological role of EGFR and its ligands from promoting keratinocyte mitogenesis and survival to mediating FasL-induced epidermal inflammation.

Interleukin-15 augments oxidative metabolism and fungicidal activity of human monocytes against Paracoccidioides brasiliensis

Interleukin (IL)-15 is a pleiotropic cytokine that regulates the proliferation and survival of many cell types. IL-15 is produced by monocytes and macrophages against infectious agents and plays a pivotal role in innate and adaptive immune responses. This study analyzed the effect of IL-15 on fungicidal activity, oxidative metabolism and cytokine production by human monocytes challenged in vitro with Paracoccidioides brasiliensis (Pb18), the agent of paracoccidioidomycosis. Peripheral blood monocytes were pre-incubated with IL-15 and then challenged with Pb18. Fungicidal activity was assessed by viable fungi recovery from cultures after plating on brain-heart infusion-agar. Superoxide anion (O2-), hydrogen peroxide (H2O2), tumour necrosis factor-alpha (TNF-α), IL-6, IL-15 and IL-10 production by monocytes were also determined. IL-15 enhanced fungicidal activity against Pb18 in a dose-dependent pattern. This effect was abrogated by addition of anti-IL-15 monoclonal antibody. A significant stimulatory effect of IL-15 on O2- and H2O2 release suggests that fungicidal activity was dependent on the activation of oxidative metabolism. Pre-treatment of monocytes with IL-15 induced significantly higher levels of TNF-α, IL-10 and IL-15 production by cells challenged with the fungus. These results suggest a modulatory effect of IL-15 on pro and anti-inflammatory cytokine production, oxidative metabolism and fungicidal activity of monocytes during Pb18 infection.