Modulation of the oxidative plasmatic state in gastroesophageal reflux disease with the addition of rich water molecular hydrogen: A new biological vision.

Gastroesophageal reflux disease (GERD), a clinical condition characterized by reflux of gastroduodenal contents in the oesophagus, has proved to demonstrate a strong link between oxidative stress and the development of GERD. Proton pump inhibitors (PPIs) have been universally accepted as first-line therapy for management of GERD. The potential benefits of electrolysed reduced water (ERW), rich in molecular hydrogen, in improving symptoms and systemic oxidative stress associated with GERD was assessed. The study was performed on 84 GERD patients undergoing control treatment (PPI + tap water) or experimental treatment (PPI + ERW) for 3 months. These patients were subjected to the GERD-Health Related Quality of Life Questionnaire as well as derivatives reactive oxigen metabolites (d-ROMs) test, biological antioxidant potential (BAP) test, superoxide anion, nitric oxide and malondialdehyde assays, which were all performed as a proxy for the oxidative/nitrosative stress and the antioxidant potential status. Spearman's correlation coefficient was used to evaluate the correlation between scores and laboratory parameters. Overall results demonstrated that an optimal oxidative balance can be restored and GERD symptoms can be reduced rapidly via the integration of ERW in GERD patients. The relative variation of heartburn and regurgitation score was significantly correlated with laboratory parameters. Thus, in the selected patients, combination treatment with PPI and ERW improves the cellular redox state leading to the improvement of the quality of life as demonstrated by the correlation analysis between laboratory parameters and GERD symptoms.

A Novel Biological Role of α-Mangostin in Modulating Inflammatory Response Through the Activation of SIRT-1 Signaling Pathway.

Several studies have shown that xanthones obtained from Garcinia Mangostana (GM) have remarkable biological activities. α-mangostin (α-MG) is the main constituent of the fruit hull of the GM. Several findings have suggested that SIRT-1, a nuclear histone deacetylase, could influence cellular function by the inhibition of NF-kB signaling. ROS can inhibit SIRT-1 activity by initiating oxidative modifications on its cysteine residues, and suppression of SIRT-1 enhances the NF-κB signaling resulting in inflammatory responses. The goals of the present study were to evaluate the quantity of α-MG in the methanolic extract of GM (Vithagroup Spa) and to investigate the activity of this xanthone in U937 cell line and in human monocytes from responsive to inflammatory insult analyzing the possible changes on the activation of SIRT-1 protein via NF-Kb. Cells were treated with the methanolic extract of GM and/or LPS. The chromatographic separation of α-MG was performed by an HPLC analysis. EX 527, a specific SIRT-1 inhibitor, was used to determine if SIRT-1/NfkB signaling pathway might be involved in α-MG action on cells. Our results show that α-MG inhibits p65 acetylation and down-regulates the pro-inflammatory gene products as COX-2, iNOS via SIRT-1 activation. Cells treated with EX 527 showed an up-regulation of NFkB acetylation and an over expression of inducible enzymes and their product of catalysis (NO and PGE2). These results suggest that α-MG may be useful for the development of alternative pharmacological strategies aimed at reducing the inflammatory process. J. Cell. Physiol. 231: 2439-2451, 2016. © 2016 Wiley Periodicals, Inc.

New Approach in Translational Medicine: Effects of Electrolyzed Reduced Water (ERW) on NF-κB/iNOS Pathway in U937 Cell Line under Altered Redox State.

It is known that increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) can exert harmful effects, altering the cellular redox state. Electrolyzed Reduced Water (ERW) produced near the cathode during water electrolysis exhibits high pH, high concentration of dissolved hydrogen and an extremely negative redox potential. Several findings indicate that ERW had the ability of a scavenger free radical, which results from hydrogen molecules with a high reducing ability and may participate in the redox regulation of cellular function. We investigated the effect of ERW on H2O2-induced U937 damage by evaluating the modulation of redox cellular state. Western blotting and spectrophotometrical analysis showed that ERW inhibited oxidative stress by restoring the antioxidant capacity of superoxide dismutase, catalase and glutathione peroxidase. Consequently, ERW restores the ability of the glutathione reductase to supply the cell of an important endogenous antioxidant, such as GSH, reversing the inhibitory effect of H2O2 on redox balance of U937 cells. Therefore, this means a reduction of cytotoxicity induced by peroxynitrite via a downregulation of the NF-κB/iNOS pathway and could be used as an antioxidant for preventive and therapeutic application. In conclusion, ERW can protect the cellular redox balance, reducing the risk of several diseases with altered cellular homeostasis such as inflammation.

Verbascoside down-regulates some pro-inflammatory signal transduction pathways by increasing the activity of tyrosine phosphatase SHP-1 in the U937 cell line.

Polyphenols are the major components of many traditional herbal remedies, which exhibit several beneficial effects including anti-inflammation and antioxidant properties. Src homology region 2 domain-containing phosphatase-1 (SHP-1) is a redox sensitive protein tyrosine phosphatase that negatively influences downstream signalling molecules, such as mitogen-activated protein kinases, thereby inhibiting inflammatory signalling induced by lipopolysaccharide (LPS). Because a role of transforming growth factor ß-activated kinase-1 (TAK1) in the upstream regulation of JNK molecule has been well demonstrated, we conjectured that SHP-1 could mediate the anti-inflammatory effect of verbascoside through the regulation of TAK-1/JNK/AP-1 signalling in the U937 cell line. Our results demonstrate that verbascoside increased the phosphorylation of SHP-1, by attenuating the activation of TAK-1/JNK/AP-1 signalling. This leads to a reduction in the expression and activity of both COX and NOS. Moreover, SHP-1 depletion deletes verbascoside inhibitory effects on pro-inflammatory molecules induced by LPS. Our data confirm that SHP-1 plays a critical role in restoring the physiological mechanisms of inducible proteins such as COX2 and iNOS, and that the down-regulation of TAK-1/JNK/AP-1 signalling by targeting SHP-1 should be considered as a new therapeutic strategy for the treatment of inflammatory diseases.

Novel aminobenzyl-acetamidine derivative modulate the differential regulation of NOSs in LPS induced inflammatory response: role of PI3K/Akt pathway.

BACKGROUND: Previous reports suggest that NO may contribute to the pathophysiology of septic shock. Recently, we have synthesized and characterized a series of benzyl- and dibenzyl derivative of N-(3-aminobenzyl)acetamidine, a potent and selective inhibitor of iNOS, in vitro assay. We evaluated the molecular mechanisms by which these compounds are involved in the regulation of NOSs expression. METHODS: H9c2 cells were stimulated with lipopolysaccharide (LPS) in the presence or absence of acetamidine-derivative. The NOSs mRNA and protein, and activation of signaling pathways (Akt and NF-κB) were assayed. RESULTS: The induction of endotoxic shock in H9c2 with LPS caused an increase of inducible NOS and a down-regulation of constitutive NOS. The molecular mechanism involved in the modulation of NOSs expression in H9c2 cells upon LPS stimulation resulted in the modification of the redox state responsible for NF-kB nuclear translocation via NIK -IKKα/ß-IkBα, simultaneously to the inactivation of the PI3K/Akt pathway. The compounds acted as an anti-inflammatory modulator. CONCLUSION: These results suggest that LPS regulates the opposite NOS expression in H9c2 cells by modifying the redox state of these cells responsible for the NF-kB nuclear translocation via NIK-IKKα/ß-IkBα, simultaneous to the inactivation of the PI3K/Akt pathway. The new molecule acts as an anti-inflammatory modulator in LPS-induced inflammation in H9c2 cells by the restoration of eNOS and nNOS expressions, mechanistically involving the PI3K/Akt pathway. GENERAL SIGNIFICANCE: This study delineates the underlying mechanisms of opposite NOSs expression in H9c2 cells stimulated with LPS.

Analysis of genomic methylation level using micellar electrokinetic chromatography with UV detection.

Analytical methods for quantification of 5'-methylcytosine in genomes are important tools to investigate epigenetic changes in gene expression during development, differentiation, aging, or cancer. Here, we report a novel genomic methylation content assay based on enzymatic hydrolysis of DNA and MEKC separation of 5'-deoxyribonucleoside monophosphates (dNMP) using the cationic surfactant CTAB as pseudostationary phase. Calf Thymus DNA was used during method development to determine electrophoretic parameters and electrolyte composition for a complete separation between 2'-deoxycytosine-5'-monophosphate and 2'-deoxy-5'-methylcytosine 5'-monophosphate (d5mCMP). Methylated and not methylated oligonucleotides were used to confirm the identity of each peak and evaluate analytical parameters of the method. The LOD of the method was found to be 12.5 pmol/µL for d5mCMP.

Positive correlation between serum interleukin-1ß and state anger in rugby athletes.

Recently, several studies reported a relationship between immune system activation and anger expression. Consequently, the aim of this study was to explore immunitary molecular mechanisms that potentially underlie anger expression. To this end, we applied the Frustration-Aggression Theory in a contact sport model, utilizing the nearing of sporting events to trigger anger feelings. In parallel, we evaluated the activation of immune system at mRNA levels. We enrolled 20 amateur rugby players (age ± SD, 27.2 ± 4.5) who underwent psychological assessment to evaluate anger, with the State-Trait Anger Expression Inventory-2 (STAXI-2), before rugby matches; at the same time blood samples were taken to analyze the variations of gene expression by microarray. During the 2 hr before each game, a significant increase was verified in the Rage State (RS) score compared to the score ascertained 72 hr before. At the same time, we found modulation in expression profile, in particular increased expression of gene that encodes interleukin l-ß (IL-1ß). In a regression analysis, RS score was related to IL-1ß, and the potential risk factors age, body mass index, smoking, and drinking. The levels of cytokine were positively and independently related to RS score. Our results suggest that the nearing of sporting event can trigger anger state feelings and activate immune system in rugby players. We propose the IL-1ß as a potential biological marker of anger. However, further research is necessary to clarify the correlation between cytokine and anger.

Activity of matrix metallo proteinases (MMPs) and the tissue inhibitor of MMP (TIMP)-1 in electromagnetic field-exposed THP-1 cells.

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) are the main determinants of tissue remodeling in both physiological and pathological processes. Metabolic processes, which generate oxidants and antioxidants can be influenced by environmental factors such as electromagnetic fields (EMF). We analyzed the effects of EMF on the activity and expression of MMPs in THP-1 cells. Cells were exposed to a 50 Hz, 1 mT EMF for 24 h and incubated with or without LPS. Our data indicate that THP-1 cells exposed to EMF causes a reduction of anti-oxidant enzyme activity and an enhancement of nitrogen intermediates involving the iNOS pathway. We then analyzed the role of nitration of TIMP-1 in increasing the activity of MMPs in EMF exposed cells. Molecular modeling tools were employed to identify the most plausible sites in the active conformation of TIMP-1; at least two protein sites, Y120 and Y38 and/or Y72 were identified. Reactive nitrogen species (RNS) may affect protein targets, such as TIMP-1, which are crucial for the regulation of MMP activities by oxidation of sulfydryl groups, or by nitration of tyrosine residues. These results may suggest a pathway connecting an imbalance of MMPs and their cognate inhibitor TIMP-1.

The role of inducible nitric oxide synthase and haem oxygenase 1 in growth and development of dental tissue'.

In this study, the activity of the antioxidant enzyme network was assessed spectrophotometrically in samples of dental pulp and dental papilla taken from third-molar gem extracts. The production of nitric oxide by the conversion of l-(2,3,4,5)-[3H] arginine to l-(3H) citrulline, the activity of haem oxygenase 1 (HO-1) through bilirubin synthesis and the expression of inducible nitric oxide synthase (iNOS), HO-1 proteins and messenger RNA by Western blot and reverse-transcribed polymerase chain reaction were also tested. The objective of this study was to evaluate the role of two proteins, iNOS and HO-1, which are upregulated by a condition of oxidative stress present during dental tissue differentiation and development. This is fundamental for guaranteeing proper homeostasis favouring a physiological tissue growth. The results revealed an over-expression of iNOS and HO-1 in the papilla, compared with that in the pulp, mediated by the nuclear factor kappa B transcription factor activated by the reactive oxygen species that acts as scavengers for the superoxide radicals. HO-1, a metabolically active enzyme in the papilla, but not in the pulp, seems to inhibit the iNOS enzyme by a crosstalk between the two proteins. We suggest that the probable mechanism through which this happens is the interaction of HO-1 with haem, a cofactor dimer indispensible for iNOS, and the subsequent suppression of its metabolic activity.

Antiinflammatory effects in THP-1 cells treated with verbascoside.

Verbascum thapsus commonly known as 'mullein' is part of a large family of Scrophulariaceae consisting of more than 360 species. From antiquity Verbascum thapsus has been used as a medicinal herb, it contains diverse polysaccharides, iroid glycosides, flavonoids, saponins, volatile oils and phenylentanoids. 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-kB. It is implicated in different pathophysiological events and its expression increases greatly during an inflammatory process, due to oxidative stress and the activation of the enzymes of the antioxidant network such as SOD, CAT and GPx.In this study an inflammatory state was reproduced 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 the work was to investigate the antiinflammatory action of verbascoside using a concentration of 100 mum. The results show a significant decrease of the expression and activity of iNOS, extracellular O(2) (-) production, SOD, CAT and GPx activity when the cells were treated with verbascoside. Based on these results it is hypothesized that verbascoside has antiinflammatory properties since it reduces the production of superoxide radicals and consequently reduces the activity of iNOS.

Aging-Related Oxidative Stress: Positive Effect of Memory Training.

The cognitive impairment characterizing the phenotype of older adults has been related to the efficiency of the antioxidant system. This study aimed at investigating the effect of memory training (MT) on memory, global cognitive functioning, and the oxidant and antioxidant capacity of plasma. We recruited 52 healthy subjects aged over 60. Twenty-nine subjects were submitted to 6-months of MT (Experimental Group, EG), and 23 were used as a Control Group (CG). Global cognitive functioning was assessed by the Mini-Mental State Examination (MMSE) and Short- and Long-Term Memory (STM and LTM, respectively) by the Rey Auditory Verbal Learning Test (RAVLT) at baseline (T0) and after 6-months (T1). Meanwhile, Reactive Oxygen Metabolites derivative compounds (d-ROMs), Biological Antioxidant Potential (BAP), and their ratio were evaluated on plasma. Results showed that the MMSE and RAVLT scores improved in EG at T1. At the same time, the d-ROMs levels significantly decreased, while the BAP and BAP/d-ROMs ratio showed an opposite trend. In both groups, the MMSE and LTM scores were negatively associated with d-ROMs levels, and positively correlated with BAP levels and the BAP/d-ROMs ratio. When we considered the Δvalue (Δvariable = variable post-MT minus variable pre-MT) in EG, the ΔMMSE and ΔLTM scores were negatively associated to Δd-ROMs, and positively to ΔBAP and ΔBAP/dROM. In conclusion, our results suggest that MT improves memory and global cognitive functioning. These processes were significantly associated to increase in resistance against oxidative stress at the plasma level in healthy older adults.

Role of mast cells in tumor growth.

The growth of malignant tumors is determined in large part by the proliferative capacity of the tumor cells. Clinical observations and animal experiments have established that tumor cells elicit immune responses. Histopathologic studies show that many tumors are surrounded by mononuclear cell and mast cell infiltrates. Mast cells are ubiquitous in the body and are critical for allergic reactions. Increasing evidence indicates that mast cells secrete proinflammatory cytokines and are involved in neuro-inflammatory processes and cancer. Mast cells accumulate in the stroma surrounding certain tumors, especially mammary adenocarcinoma, and the molecules they secrete can benefit the tumor. However, mast cells can also increase at the site of tumor growth and participate in tumor rejection. Mast cells may be recruited by tumor-derived chemoattractants and selectively secrete molecules such as growth factors, histamine, heparin, VEGF, and IL-8, as well as proteases that permit the formation of new blood vessels and metastases. Tumor mast cell intersections play regulatory and modulatory roles affecting various aspects of tumor growth. Discovery of these new roles of mast cells further complicates the understanding of tumor growth. This review focuses on the strategic importance of mast cells to the progression of tumors, and proposes a revised immune effector mechanism of mast cell involvement in tumor growth.

The NF-kB regulates the SHP-1 expression in monocytes in congestive heart failure.

It has been shown that functional recovery of patients with acute congestive heart failure (ACHF) after treatment with conventional drugs (CD) is mediated by suppression of inflammation in peripheral blood mononuclear cells. Here, we analyzed gene expression profiles of monocytes from symptomatic ACHF patients (NYHA Class III-IV) before and after pharmacological treatment with CD. The treatment was associated with selective down-regulation of "TNFR signaling" and pro-inflammatory mediators CCL5, MIP-1α receptor, CD14, ITGAM, and significant up-regulation of "TNFR signaling" as evidenced by increase in anti-inflammatory factors including NF-kBIA, TNFAIP3 and SHP-1. In monocyte TNF-alpha-stimulated there is a down-regulation of the phosphatase SHP-1 which induces a significant activation of TAK-1/IKK/NF-kB signaling. These findings suggest that the therapeutic impact of CD treatment in symptomatic ACHF includes negative regulation of the NF-kB signaling in monocytes and the improvement of the SHP-1 activity.

Chronic treatment with rosuvastatin modulates nitric oxide synthase expression and reduces ischemia-reperfusion injury in rat hearts.

OBJECTIVE: Due to reported modulatory effects of statins on nitric oxide synthase (NOS) expression, we tested the hypothesis of protective effects of in vivo chronic treatment with rosuvastatin, a novel 3-hydroxy-3-methyl-glutaryl coenzyme A-reductase inhibitor, on ischemia-reperfusion injury, and investigated mechanisms involved. METHODS: After 3 weeks of in vivo treatment with rosuvastatin (0.2-20 mg/kg/day) or placebo, excised hearts from Wistar rats were subjected to 15 min global ischemia and 22-180 min reperfusion. We evaluated creatine-phosphokinase and nitrite levels in the coronary effluent, heart weight changes, microvascular permeability (extravasation of fluoresceine-labeled albumin), ultrastructural alterations, and the expression of endothelial (e) and inducible (i) nitric oxide synthase (NOS) (by reverse-transcription polymerase chain reaction and Western blotting). RESULTS: Rosuvastatin 0.2 and 2 mg/kg/day significantly reduced myocardial damage and vascular hyperpermeability, concomitant with a reduction in endothelial and cardiomyocyte lesions. At 2 mg/kg/day, rosuvastatin significantly increased eNOS mRNA and protein compared with untreated hearts, and conversely decreased iNOS mRNA and protein, as well as nitrite production after ischemia-reperfusion. The addition of the NOS inhibitor N(omega)-nitro-L-arginine methylester (L-NAME, 30 micromol/L) significantly reduced cardioprotection against ischemia-reperfusion. CONCLUSIONS: Chronic treatment with rosuvastatin before ischemia reduces ischemia-reperfusion injury and prevents coronary endothelial cell and cardiomyocyte damage by NO-dependent mechanisms.

mTOR Activation by PI3K/Akt and ERK Signaling in Short ELF-EMF Exposed Human Keratinocytes.

Several reports suggest that ELF-EMF exposures interact with biological processes including promotion of cell proliferation. However, the molecular mechanisms by which ELF-EMF controls cell growth are not completely understood. The present study aimed to investigate the effect of ELF-EMF on keratinocytes proliferation and molecular mechanisms involved. Effect of ELF-EMF (50 Hz, 1 mT) on HaCaT cell cycle and cells growth and viability was monitored by FACS analysis and BrdU assay. Gene expression profile by microarray and qRT-PCR validation was performed in HaCaT cells exposed or not to ELF-EMF. mTOR, Akt and MAPKs expressions were evaluated by Western blot analysis. In HaCaT cells, short ELF-EMF exposure modulates distinct patterns of gene expression involved in cell proliferation and in the cell cycle. mTOR activation resulted the main molecular target of ELF-EMF on HaCaT cells. Our data showed the increase of the canonical pathway of mTOR regulation (PI3K/Akt) and activation of ERK signaling pathways. Our results indicate that ELF-EMF selectively modulated the expression of multiple genes related to pivotal biological processes and functions that play a key role in physio-pathological mechanisms such as wound healing.

Effect of chronic hypoxia on inducible nitric oxide synthase expression in rat myocardial tissue.

The purpose of our study was to evaluate the effect of chronic exposure to low cellular oxygen tension (90% N2 and 10% O2 for 14 days) in inducing apoptosis and activation of transcription and translation of inducible nitric oxide (NO) synthase (iNOS) in rat hearts tissue. Rats were divided into four groups: normoxic, hypoxic, rats maintained in normoxic condition for 7 days and subjected to hypoxic conditions for another 7 days, and rats maintained in hypoxic condition for 7 days and subjected to normoxic conditions for another 7 days. At the 7th and 14th days, five rats from each group were sacrificed. Immunohistochemical and Western blot analysis were performed on myocardial tissue to reveal the presence of iNOS. Expression of iNOS was determined by RT-PCR. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and by detection of internucleosomal DNA fragmentation by electrophoresis. Electrophoretic analysis of DNA showed oligonucleosomal fragmentation in the hypoxic groups, but no ladder was observed in the other groups. This data was confirmed through end labeling with streptavidin-biotin (biotin d-UTP). iNOS expression was evaluated through immunohistochemical techniques (Ab anti-iNOS) and Western blotting, and the results were quantified with a computerized imaging analysis. The expression of iNOS protein was greater in the hypoxic groups; in the normoxic groups, only a nonspecific background was detected. This data was supported with results obtained through RT-PCR, which showed the specific transcription of mRNA for iNOS in the same experimental conditions. In addition, the iNOS activity was also evaluated and was found to be more active in the hypoxic groups (0.1 +/- 0.01 vs 0.02 +/- 0.003). The present study shows that exposure to low oxygen tension is capable of inducing programmed cell death and activating iNOS.

Inducible nitric oxide synthase and heme oxygenase-1 in rat heart: direct effect of chronic exposure to hypoxia.

Hypoxia is a potent regulator of various biological process. Mammalian cells respond to hypoxia by increased expression of several genes. The aim of this study was to evaluate the effects of chronic exposure to low oxygen tension on the induction of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) in rat heart. Male Wistar rats were assigned randomly to 4 groups: (A) control rats maintained in normoxic conditions for 7 and 14 days; (B) rats maintained in hypoxic conditions for 7 and 14 days; (C) rats maintained in normoxic conditions for 7 days and then transferred to hypoxic conditions for 7 days; and (D) rats maintained in hypoxic conditions for 7 days and then transferred to normoxic conditions for 7 days. In Group A, iNOS and HO-1 immunoreactivities were not evident; in Group B these immunoreactivities increased from day 7 to 14; in Group C the immunoreactivities decreased on day 7, compared to day 14; and in Group D, the immunoreactivities increased on day 7, compared to day 14. These findings were confirmed by Western blot analyses of the respective proteins and by rt-PCR assays of the corresponding mRNAs. The results indicate that the adaptive response to hypoxia involves up-regulation of HO-1 through iNOS activation in cardiac cells. HO-1 helps to regulate vascular tone via CO and thereby participates in an important cardiac defense mechanism.

Astaxanthin treatment confers protection against oxidative stress in U937 cells stimulated with lipopolysaccharide reducing O2- production.

Recently, astaxanthin (ASTA) studies have focused on several biological functions such as radical scavenging, singlet oxygen quenching, anti-carcinogenesis, anti-diabetic, anti-obesity, anti-inflammatory, anti-melanogenesis, and immune enhancement activities. In this study, we investigated the potential role protective of ASTA, an antioxidant marine carotenoid, in restoring physiological conditions in U937 cells stimulated with LPS (10 µg/ml). Our results show that pre-treatment with ASTA (10 µM) for 1 h attenuates the LPS-induced toxicity and ROS production. The beneficial effect of ASTA is associated with a reduction intracellular O2 (-) production by restoring the antioxidant network activity of superoxide dismutase (SOD) and catalase (CAT), which influence HO-1 expression and activity by inhibiting nuclear translocation of Nrf2. We accordingly hypothesize that ASTA has therapeutic properties protecting U937 cells from LPS-induced inflammatory and oxidative stress.

Effect of erythropoietin on primed leucocyte expression profile.

Resistance to erythropoietin (EPO) affects a significant number of anaemic patients with end-stage renal disease. Previous reports suggest that inflammation is one of the major independent predictors of EPO resistance, and the effects of EPO treatment on inflammatory mediators are not well established. The aim of this study was to investigate EPO-induced modification to gene expression in primary cultured leucocytes. Microarray experiments were performed on primed ex vivo peripheral blood mononuclear cells (PBMCs) and treated with human EPO-α. Data suggested that EPO-α modulated genes involved in cell movement and interaction in primed PBMCs. Of note, EPO-α exerts anti-inflammatory effects inhibiting the expression of pro-inflammatory cytokine IL-8 and its receptor CXCR2; by contrast, EPO-α increases expression of genes relating to promotion of inflammation encoding for IL-1ß and CCL8, and induces de novo synthesis of IL-1α, CXCL1 and CXCL5 in primed cells. The reduction in MAPK p38-α activity is involved in modulating both IL-1ß and IL-8 expression. Unlike the induction of MAPK, Erk1/2 activity leads to upregulation of IL-1ß, but does not affect IL-8 expression and release. Furthermore, EPO-α treatment of primed cells induces the activation of caspase-1 upstream higher secretion of IL-1ß, and this process is not dependent on caspase-8 activation. In conclusion, our findings highlight new potential molecules involved in EPO resistance and confirm the anti-inflammatory role for EPO, but also suggest a plausible in vivo scenario in which the positive correlation found between EPO resistance and elevated levels of some pro-inflammatory mediators is due to treatment with EPO itself.

Modulation of MCP-1 and iNOS by 50-Hz sinusoidal electromagnetic field.

The purpose of this study was to investigate whether overnight exposure to 1 mT-50 Hz extremely low-frequency sinusoidal electromagnetic field (EMF) affects the expression and production of inducible nitric oxide synthase (iNOS) and monocyte chemotactic protein-1 (MCP-1) in human monocytes. RT-PCR and Western blot analysis demonstrate that EMF exposure affects the expression of iNOS and MCP-1 in cultured human mononuclear cells at the mRNA level and protein synthesis. Interestingly, the effects of EMF exposure clearly differed with respect to the potentiation and inhibition of iNOS and MCP-1 expression. Whereas iNOS was down-regulated both at the mRNA level and at the protein level, MCP-1 was up-regulated. These results provide helpful information regarding the EMF-mediated modulation of the inflammatory response in vivo. However, additional studies are necessary to demonstrate that EMF acts as a nonpharmacological inhibitor of NO and inducer of MCP-1 in some diseases where the balance of MCP-1 and NO may be important.