Ozone effect on the inflammatory and proteomic profile of human macrophages and airway epithelial cells.

Ozone (O3) is one of the most harmful urban pollutants, but its biological mechanisms have not been fully elucidated yet. Human bronchial epithelial cells (HBEpC) and human macrophage cells (differentiated human monocytic cell line) were exposed to O3 at the concentration of 240 µg/m3 (120 ppb), corresponding to the European Union alert threshold. Cell viability, reactive oxygen species (ROS) production, and pro-inflammatory cytokines release (IL-8 and TNF-α) were evaluated. Results indicated that O3 exposure increases ROS production in both cell types and enhances cytokines release in macrophages. O3 stimulated IL-8 and TNF-α in HBEpC when the cells were pretreated with Lipopolysaccharide, used to mimic a pre-existing inflammatory condition. Proteomics analysis revealed that, in HBEpC, O3 caused the up-regulation of aldo-keto reductase family 1 member B10, a recognized critical protein in lung carcinogenesis. In conclusion, our results show that 120 ppb O3 can lead to potential damage to human health suggesting the need for a revision of the actual alert levels.

Cigarette smoke is associated with up-regulation of inducible NOS and COX-2 protein expression and activity in granulosa cells of women undergoing in vitro fertilization.

Cigarette smoke exposure represents a well-established ovotoxic exogenous stress, but the molecular mechanisms underlying of this effect are still unclear. Cigarette smoke upregulates inflammatory genes in the female reproductive organs, therefore an abnormal inflammation response may contribute to the impairment of female fertility. In this study we investigated for the first time the effect of cigarette smoke exposure on NOS and COX expression and activity and on their transcription factors (CREB and NF-kB) in human GCs and on the release of NO and PGE2 in the FF in smoking and non-smoking patients undergoing IVF treatment. In addition, correlation analysis between AMH serum levels, an index of ovarian reserve, and smoking exposure or iNOS and COX-2 protein expression levels were performed using a Pearson correlation method. Cigarette smoke exposure resulted in a significant increase of iNOS and COX-2 protein expression together with an increase of iNOS activity and PGE2 levels. pNF-kB and pCREB protein expression were upregulated in the GCs of smokers compared to non-smokers. The habit of smoking was negatively correlated with serum AMH levels, and positively correlated with iNOS and COX-2 protein expression levels. The data presented in the current study revealed a novel molecular mechanism underlying the toxic effects of cigarette smoke on fertility. Additional pathways mediating the effects of cigarette smoke exposure in human GCs cannot be excluded and should be investigated in future studies.

The plasmatic and salivary levels of IL-1ß, IL-18 and IL-6 are associated to emotional difference during stress in young male.

Saliva collection is considered a non-invasive method to detect inflammatory markers in response to emotional states within natural social contexts. Numerous studies have prompted an important role of cytokines in modulating distinct aspects of social and emotional behavior. The aim of this study was to investigate the reliability of plasma and saliva as investigative tools for measure some inflammatory marker levels (CRP, IL-1ß, IL-18, and IL-6). At the same time, the relationships between these markers and emotional states in response to a socio-cognitive stress (Academic Exam, AE), were considered. It was demonstrated that the plasma and saliva concentrations of all immune-mediators analyzed were significantly related across the socio-cognitive stress. In addition, when there was a close correlation to AE, the anger state, the IL-1ß, the IL-18 salivary and plasmatic concentrations were significantly higher, while they decreased during the AE. On the other hand, the anxiety state and the IL-6 levels significantly increased throughout the AE. The IL-1ß and IL-6 were positively associated to the anger and the anxiety state, respectively. In conclusion, our data highlight that different immune markers are similarly detectable in plasma and saliva during socio-cognitive stress. Also, they could be related to different emotional responses.

Extremely low-frequency electromagnetic fields accelerates wound healing modulating MMP-9 and inflammatory cytokines.

OBJECTIVES: In our previous reports, we have demonstrated that extremely low-frequency electromagnetic fields (ELF-EMF) exposure enhances the proliferation of keratinocyte. The present study aimed to clarify effects of ELF-EMF on wound healing and molecular mechanisms involved, using a scratch in vitro model. MATERIALS AND METHODS: The wounded monolayer cultures of human immortalized keratinocytes (HaCaT), at different ELF-EMF and Sham exposure times were monitored under an inverted microscope. The production and expression of IL-1ß, TNF-α, IL-18 and IL-18BP were measured by enzyme-linked immunosorbent assay and quantitative real-time PCR. The activity and the expression of matrix metalloproteinases (MMP)-2/9 was evaluated by zymography and Western blot analysis, respectively. Signal transduction proteins expression (Akt and ERK) was measured by Western blot. RESULTS: The results of wound healing in vitro assay revealed a significant reduction of cell-free area time-dependent in ELF-EMF-exposed cells compared to Sham condition. Gene expression and release of cytokines analysed were significantly increased in ELF-EMF-exposed cells. Our results further showed that ELF-EMF exposure induced the activity and expressions of MMP-9. Molecular data showed that effects of ELF-EMF might be mediated via Akt and ERK signal pathway, as demonstrated using their specific inhibitors. CONCLUSIONS: Our results highlight ability of ELF-EMF to modulate inflammation mediators and keratinocyte proliferation/migration, playing an important role in wound repair. The ELF-EMF accelerates wound healing modulating expression of the MMP-9 via Akt/ERK pathway.

Are Gravitational Waves Spinning Down PSR J1023+0038?

The pulsar J1023+0038 rotates with a frequency ν≈592 Hz and has been observed to transition between a radio state, during which it is visible as a millisecond radio pulsar, and a low-mass x-ray binary (LMXB) state, during which accretion powered x-ray pulsations are visible. Timing during the two phases reveals that during the LMXB phase the neutron star is spinning down at a rate of ν[over ˙]≈-3×10^{-15} Hz/s, which is approximately 27% faster than the rate measured during the radio phase, ν[over ˙]≈-2.4×10^{-15} Hz/s, and is at odds with the predictions of accretion models. We suggest that the increase in spin-down rate is compatible with gravitational wave emission, particularly with the creation of a "mountain" during the accretion phase. We show that asymmetries in pycnonuclear reaction rates in the crust can lead to a large enough mass quadrupole to explain the observed spin-down rate, which thus far has no other self-consistent explanation. We also suggest two observational tests of this scenario, involving radio timing at the onset of the next millisecond radio pulsar phase, when the mountain should dissipate, and accurate timing during the next LMXB phase to track the increase in torque as the mountain builds up. Another possibility is that an unstable r mode with an amplitude α≈5×10^{-8} may be present in the system.

Super-oxide anion production and antioxidant enzymatic activities associated with the executive functions in peripheral blood mononuclear cells of healthy adult samples.

Executive Functions (EFs) involve a set of high cognitive abilities impairment which have been successfully related to a redox omeostasis imbalance in several psychiatric disorders. Firstly, we aimed to investigate the relationship between executive functioning and some oxidative metabolism parameters in Peripheral Blood Mononuclear Cells (PBMCs) from healthy adult samples. The Brown Attention-Deficit Disorder Scales were administered to assess five specific facets of executive functioning. Total superoxide anion production, Super Oxide Dismutase (SOD), Catalase (CAT), Glutathione Reductase (GR) and Glutathione Peroxidase (GPx) activities were evaluated on proteins extracted from the PBMCs. We found significant positive correlations between superoxide anion production and the total score of the 'Brown' Scale and some of its clusters. The GPx and CAT activities were negatively associated with the total score and some clusters. In a linear regression analysis, these biological variables were indicated as the most salient predictors of the total score, explaining the 24% variance (adjusted R(2)=0.24, ANOVA, p<.001). This study provides novel evidence that Executive Functions have underpinnings in the oxidative metabolism, as ascertained in healthy subjects.

The SHP-1 expression is associated with cytokines and psychopathological status in unmedicated first episode schizophrenia patients.

BACKGROUND: Recent lines of research have boosted awareness of the immunological facets of schizophrenia. However, associations with protein tyrosine phosphatase regulators have never been reported. The aim of our study was to investigate the expression and promoter status methylation of phosphatase SHP-1, a key negative regulator of the inflammatory process, in Peripheral blood mononuclear cells (PBMCs) of Schizophrenic patients. METHODS: We enrolled fifty-four (28 men and 26 women) unmedicated first episode subjects (SC) who met DSM-IV and thirty-eight (22 men and 16 women) healthy controls (HC). The SC psychopathological status was assessed using the Positive and Negative Syndrome Scale. We evaluated SHP-1 expression by Quantitative Real-time PCR (qPCR) and Western blotting (WB) methods and promoter status methylation through PCR bisulfate. IKK/NFkB signaling was detected by WB, and medium and plasma levels of pro-inflammatory cytokines (IL-1ß, IL-2, and TNF-α) by the ELISA method. SHP-1 was silenced by treating cells with specific siRNA. RESULTS: We found a significantly lower level of SHP-1 gene expression in PBMCs from SC vs. HC, consistently with which the promoter region analyzed presented significant hypermethylation. Silencing of SHP-1 expression induced higher activation of IKK/NF-kB signaling and pro-inflammatory cytokine production in ex vivo PBMCs from both SC and HC. Linear regression among patients generated a model in which SHP-1 expression explained 30% of the clinical negative symptom variance (adjusted R(2)=0.30, ANOVA p<0.001). CONCLUSIONS: Our findings are the first to suggest that impairment of SHP-1 expression is involved in the physiopathology of schizophrenia, opening fruitful new avenues for ameliorating treatment at least of negative symptoms.

Negative feedback interaction of HO-1/INOS in PBMC of acute congestive heart failure patients.

Heart failure (HF) is a common clinical syndrome with frequent exacerbations requiring hospitalization. Among the various mechanisms that underlie the pathogenesis of HF, the activation of the immune system leads to a progressive and redundant release of proinflammatory cytokines responsible for a variety of deleterious effects in heart failure, such as endothelial dysfunction, apoptosis of myocytes, activation of MMPs (Matrix Metallo Proteinases) and oxidative stress, with the result of decreased inotropism and clinical syndrome such as pulmonary edema,. The condition of oxidative stress induces the expression of genes coding for the proteins inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1). Twenty-five hospitalized cardiology patients with symptomatic acute congestive HF (NYHA Class III-IV) and impaired left ventricular (LV) function (ejection fraction less than 35 percent) were included in the study. The aim of this study was to evaluate the cytokines plasma concentrations and the expression and activity of iNOS and HO-1 proteins in peripheral blood mononuclear cell (PBMC) extracted from patients in comparison to control group. In ACHF; left ventricular ejection fraction (LVEF) percent was reduced. Furthermore; iNOS and HO-1 expression and cytokines plasma levels were significantly higher in patients with ACHF as compared to controls group. Moreover the enzyme activity presents an opposite trend compared to that obtained in the analysis of the transcript and proteins. Our studies suggest a negative feedback interaction between iNOS and HO-1 important in the physiopathology of heart failure that could be considered a good candidate as a future therapeutic target for the development of new drugs.

Biological role of interleukin-1beta in defensive-aggressive behaviour.

During the past decade, a great deal of data has accumulated supporting the notion that cytokines interact to regulate several aspects of social and emotional behaviour. There are reports of a positive correlation between cytokine levels and aggressive behaviour in healthy populations, and clinical reports describe an increase of aggressive traits in patients who receive cytokine immunotherapy. Interleukin-1beta released during an immune response acts as messenger that helps to modulate behaviour by influencing relevant neurotransmitter systems, and in some cases, by directly acting within the brain. In this site, IL-1beta exerts its actions by acting through 5-HT2 and IL-1 Type I receptors in hypothalamus or by potentially indirect routes, including activation of sensory afferents, and stimulation of cytokine release by brain endothelial cells. This review reports research investigating the relationship between IL-1beta, and the immune and central nervous systems involving or potentially involving defensive aggressive behaviour.

Kinetic study on the effects of extremely low frequency electromagnetic field on catalase, cytochrome P450 and inducible nitric oxide synthase in human HaCaT and THP-1 cell lines.

Extremely low frequency electromagnetic fields (ELF-EMF) have been found to produce a variety of biological effects. These effects of ELF-EMF depend upon frequency, amplitude, and length of exposure, and are also related to intrinsic susceptibility and responsiveness of different cell types. Although the mechanism of this interaction is still obscure, ELF-EMF can influence cell proliferation, differentiation, cell cycle, apoptosis, DNA replication and protein expression. The aim of this study was to estimate various kinetic constants of catalase, cytochrome P450 and inducible nitric oxide synthase in response to ELF-EMF exposure in human HaCaT and THP-1 cell lines. In order to evaluate the effect of ELF-EMF on the modulation of cellular responses to an inflammatory stimulus, both cell lines were treated with lipopolysaccharide. To the best of our knowledge there is no available report on such type of kinetic study of selected enzymes in response to ELF-EMF in these cell lines. Therefore, the current study may reveal novel mechanism of ELFEMF biological interaction with the enzymological and hormonal systems of living organisms. These new insights may be important for ELF-EMF application particularly for wound healing, tissue regeneration, Parkinson's and Alzheimer's diseases.

Influenza A (H1N1) encephalitis with severe intracranial hypertension.

The swine-origin influenza A (H1N1) virus was responsible for the pandemic infection in 2009. We report a case of encephalitis diagnosed as the H1N1 virus infection in a young child. The H1N1 virus infection can be causative of the encephalitis, as with other influenza virus infections. For patients presenting with influenza-like illness accompanied by mental status changes or seizures, high suspicion for unusual presentations of influenza A virus infection and careful monitoring, including EEG and intracranial pressure monitoring, are essential for reducing complications.

Extremely low frequency electromagnetic fields modulate expression of inducible nitric oxide synthase, endothelial nitric oxide synthase and cyclooxygenase-2 in the human keratinocyte cell line HaCat: potential therapeutic effects in wound healing.

BACKGROUND: Extremely low frequency (ELF) electromagnetic fields (EMF) are known to produce a variety of biological effects. Clinical studies are ongoing using EMF in healing of bone fractures and skin wounds. However, little is known about the mechanisms of action of ELF-EMF. Several studies have demonstrated that expression and regulation of nitric oxide synthase (NOS) and cyclooxygenase-2 (COX-2) are vital for wound healing; however, no reports have demonstrated a direct action of ELF-EMF in the modulation of these inflammatory molecules in human keratinocytes. OBJECTIVES: The present study analysed the effect of ELF-EMF on the human keratinocyte cell line HaCaT in order to assess the mechanisms of action of ELF-EMF and to provide further support for their therapeutic use in wound healing. METHODS: Exposed HaCaT cells were compared with unexposed control cells. At different exposure times, expression of inducible NOS (iNOS), endothelial NOS (eNOS) and COX-2 was evaluated by Western blot analysis. Modulation of iNOS and eNOS was monitored by evaluation of NOS activities, production of nitric oxide (NO) and O(2)(-) and expression of activator protein 1 (AP-1). In addition, catalase activity and prostaglandin (PG) E(2) production were determined. Effects of ELF-EMF on cell growth and viability were monitored. RESULTS: The exposure of HaCaT cells to ELF-EMF increased iNOS and eNOS expression levels. These ELF-EMF-dependent increased expression levels were paralled by increased NOS activities, and increased NO production. In addition, higher levels of AP-1 expression as well as a higher cell proliferation rate were associated with ELF-EMF exposure. In contrast, ELF-EMF decreased COX-2 expression, PGE(2) production, catalase activity and O(2)(-) production. CONCLUSIONS: Mediators of inflammation, such as reactive nitrogen and PGE(2), and keratinocyte proliferation are critical for the tissue regenerative processes. The ability of ELF-EMF to upmodulate NOS activities, thus nitrogen intermediates, as well as cell proliferation, and to downregulate COX-2 expression and the downstream intermediate PGE(2), highlights the potential therapeutic role of ELF-EMF in wound healing processes.

Infections and mast cells.

Mast cells play a role in various physiological functions: innate and acquired immunity, epithelium remodelling and proliferation, angiogenesis, cancer, inflammation and infections. Mast cells are activated by cross-linking of FcERI molecules, which are involved in the binding of multivalent antigens to the attached IgE molecules, resulting in a variety of responses including the immediate release of potent inflammatory mediators. In addition, mast cell biology consists in the capability to secrete preformed mediators which include biogenic amines and newly synthetized mediators, which include lipid-derived mediators and cytokines. It has been reported that parasite infections induce a systemic immunomodulatory network, including regulatory T cells, pro-inflammatory and anti-inflammatory cytokines, which might play a key role in the allergic phenotype. Here, in this article, we revisited the relationship between mast cells and infections.

Anti-inflammatory properties of the plant Verbascum mallophorum.

Verbascum mallophorum is part of a large family of Scrophulariaceae consisting of more than 360 species. Verbascum mallophorums contains diverse polysaccharides, iroid glycosides, flavonoids, saponins, volatile oils and phenylentanoids. Verbascum has been used in popular medicine for treating wounds, chilblains, respiratory ailments, acne and arthritic disturbances. Inducible nitric oxide synthase (iNOS) represents one of the three isoforms that produce nitric oxide using L-arginine as a substrate in response to an increase in superoxide anion activated by NF-kappaB. It is implicated in different pathophysiological events and its expression increases greatly during an inflammatory process due to oxidative stress. In our study we reproduced an inflammatory state by treating THP-1 cells (human myelomonocytic leukaemia) with pro-inflammatory stimuli, such as LPS and IFN-gamma, obtaining an up-regulation both in the expression and in the activity of iNOS. The aim of our work is to investigate the possible antiinflammatory action of verbascoside extract from Verbascum mallophorum using a concentration of 100 muM. Our results show a significant decrease in the expression and activity of iNOS and extracellular O2- when cells were treated with verbascoside. Based on these results we hypothesize that verbascoside extract from Verbascum mallophorum has anti-inflammatory properties since it reduces the production of superoxide radicals and consequently reduces the activity of iNOS.

Biology of neurotensin: revisited study.

The tridecapeptide neurotensin (NT) acts in the mammalian brain as a primary neurotransmitter or neuromodulator of classical neurotransmitters. Morphological and functional in vitro and in vivo studies have demonstrated the existence of close interactions between NT and dopamine both in limbic and in striatal brain regions. Additionally, biochemical and neurochemical evidence indicates that in these brain regions NT also plays a crucial role in the regulation of the aminoacidergic signalling. Immune cells, such as lymphocytes, macrophages and mast cells are reported to be activated by neuropeptides, such as neurotensin; this activation leads to cytokine and immunoglobulin production. In addition, neurotensin increases calcium level and the production of nitric oxide. Therefore neurotensin is deeply involved in immunity and inflammation but its real function still remains to be elucidated.

Reduction of free radicals in multiple sclerosis: effect of glatiramer acetate (Copaxone).

Free radicals have been found in high concentrations within inflammatory multiple sclerosis (MS) lesions. The superoxide anion (O(2)(-)) reacts rapidly with nitric oxide (NO), producing peroxynitrite (ONOO(-)). Glatiramer acetate (GA) is a specific MS immunomodulator that induces the synthesis of Th2 cytokines, and reduces the frequency of relapses and the formation of active brain lesions. Proinflammatory cytokines could play a role in free radicals production in the peripheral immune system as well as in the central nervous system (CNS). The effect of GA on iNOS, superoxide radicals (O(2)(-)) and 3-nitrotyrosine production by peripheral blood adherent mononuclear cells (PBAMs) was assessed. Our findings demonstrate that in vitro GA reduced spontaneous and LPS-induced iNOS, 3-nitrotyrosine, NO and O(2)(-) production, and that similar inhibition can be demonstrated ex vivo in mononuclear cells obtained from GA-treated patients. The inhibition of the production of free radicals in PBAMs may represent a new therapeutic mechanism against inflammation during MS.

Phosphodiesterase type-5 inhibitor and oxidative stress.

Erectile dysfunction (ED) is a common medical condition that affects the sexual life of millions of men worldwide. Numerous physical and psychological factors are involved in normal erectile function, including neurological, vascular, hormonal and cavernous functions. The current therapy for the condition is pharmacological and psychotherapeutic which regulates the erectile function and amplifies the NO-mediated response. The aim of this work is to test the action of three common phosphodiesterase inhibitors: Tadalafil, Sildenafil Citrate and Vardenafil at 0.05 microM on human monocytes, analyzing the expression of iNOS protein and mRNA by Western blot and rt-PCR, and production of NO by conversion of L-(2,3,4,5)-[3H]Arginine to L-(3H) citrulline. We also tested the efficiency of the antioxidant network by spectrophotometer (SOD, CAT, GPx and Gr), under normal conditions and after stimulation with LPS. The results showed an increase in ROS levels, similar for all the molecules with regard to the antioxidant enzymes. In all cases the treatment determines a response to the limited efficiency, arriving at a situation in which phosphodiesterase inhibitors + LPS clearly show oxidative stress.

Localization and activity of iNOS in normal human lung tissue and lung cancer tissue.

Inducible nitric oxide synthase (iNOS) is one of three enzymes generating nitric oxide (NO) from the amino acid L-arginine. iNOS-derived NO plays an important role in several physiological and pathophysiological conditions. NO is a free radical which produces many reactive intermediates that account for its bioactivity. In the human lung, the alveolar macrophage is an important producer of cytokines and this production may be modified by NO. Moreover, high concentrations of NO have been shown to increase nuclear factor kappaB (NF-kB) activation. Recent investigations of NO expression in tumor tissue indicated that, at least for certain tumors, NO may mediate one or more roles during the growth of human cancer. We have studied iNOS in two tissue groups: normal human lung tissue and human lung cancer tissue. We localized iNOS in these tissues by immunohistochemistry and tested the mRNA expression by RT-PCR, the protein level by Western blot, and the protein activity by radiometric analysis. The results demonstrate different expression, localization and activity of iNOS in normal versus tumor tissue. This is suggestive of a role for NO production from iNOS in human lung cancer because high concentrations of this short molecule may transform to highly reactive compounds such as peroxynitrite (ONOO-); moreover, through the upregulator NF-kB, they can induce a chronic inflammatory state representing an elevated risk for cell transformation to cancer.