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.

Nitric oxide synthase isoenzyme expression in human oral lichen planus.

The roles of nitric oxide (NO) synthase (NOS) enzyme in pathological mechanisms of the oral cavity are still incompletely understood. The aim of this study was to investigate the expression of the endothelial, neuronal and inducible isoforms of NOS (eNOS, nNOS and iNOS) in oral lichen planus (OLP) development in humans. OLP and healthy oral mucosa biopsies were taken for mRNA and protein analysis of NOS isoenzymes by RT-PCR, western blot and immunohistochemistry. The mRNA and protein levels of eNOS and nNOS were present in all samples, with a significant increase only for eNOS in OLP. The normal oral mucosa exhibited only small amounts of iNOS mRNA and protein, while it showed a significant rise in OLP samples. These results were confirmed by immunohistochemical analysis. Our findings suggest that NO produced by increased eNOS and iNOS expression may have circulatory and immune functions in the development of OLP.

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.

Allergic inflammation: role of cytokines with special emphasis on IL-4.

This review examines recent articles on the relationship of cytokines to allergy and inflammation with particular emphasis on interleukin (IL)-4. The objective of this article is therefore to review published studies to identify cytokines consistently involved in allergic inflammation. Proinflammatory cytokines, including IL-4, IL-5, IL-13 and GM-CSF along with TNF-alpha play a role in allergen-induced airway leukocyte recruitment and these cytokines can be generated by T mast cells and other cells. In addition, IL-9, IL-25, IL-33, IL-17, IL-27 and IFN-gamma are deeply involved in the regulation of asthma. Blocking the effect of these proinflammatory cytokines might provide new therapeutic approaches for the control of allergy and inflammation.

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.

IL-31 a Th2 cytokine involved in immunity and inflammation.

Cytokines are immunal regulatory proteins, however they also play a relevant role in inflammatory diseases. IL-31 is a newly discovered cytokine expressed primarily in TH2 cells, introduced by activated CD4+ T cells. IL-31 is capable of inducing chemokines and other cytokines in several inflammatory diseases via its surface receptor. This cytokine is also produced by mast cells and mast cell line, suggesting a role in allergic diseases. In this editorial we revisit the biological role of IL-31 in immunity and inflammation.

Vitamins and mast cells.

The immune system is a highly complex, intricately regulated group of cells whose integrated function is essential to health. The mast cell inflammatory response is characterized by an early phase with massive discharge of mediators stored in cytoplasmic secretory granules. Through multigranular/compound exocytosis and a late phase that involves generation of arachidonic acid metabolites and de novo synthesis of cytokines/chemokines and growth factors. Vitamins have been shown to have a protective effect on the body's immune cells. Vitamin C and E are necessary in allergic disease treatment where mast cells are involved. In addition, ascorbic acid and pyridoxine are useful compounds for the treatment of inflammatory disorder of the respiratory airways. Here we revisited the inter-relationship between vitamins and mast cells.

VEGF, substance P and stress, new aspects: a revisited study.

Mast cells play an essential role in diverse physiological and pathological processes, such as atherosclerosis, malignancy, asthma, pulmonary fibrosis and arthritis, directly interact with bacteria, and appear to play a vital role in host defense against pathogens. Mast cells could be recruited in the inflammatory site, by MCP-1, RANTES and SCF, to selectively secrete proinflammatory molecules; these could include growth factors, histamine, which is mitogenic (H1) and an immunosuppressant (H2), neovascularization agents, such as heparin, IL-8, and VEGF, as well as proteases that could permit new blood vessel formation. Neurogenic inflammation involves vasodilation and plasma protein extravasation in response to neural stimulation. Upon stimulation, sensory neurons release Substance P and other neuropeptides and activate neurokinin-1 receptors leading to plasma protein extravasation from post-capillary venules. Substance P is a neuropeptide that is released from nerve endings in many tissues and plays an important role in immunological and inflammatory states, and it is also a mediator of tissue injury, asthma, arthritis, allergy and autoimmune diseases. SP-positive nerve fibers and mast cell contacts are increased by acute stress in mice leading to dermal mast cell degranulation. VEGF is produced by flammatory cells. IL-33 is the newest inflammatory member of the IL-1 cytokine family and we show here that SP can induce VEGF secretion from mast cells and IL-33 augments the effect of SP in VEGF transcription and translation protein.

Impact of RANTES, MCP-1 and IL-8 in mast cells.

Chemokines are cytokines with chemotactic properties on inflammatory cells and other cell types. RANTES, MCP-1 and related molecules, constitute the C-C class of chemokine supergene family and a group of cytokines produced by hematopoietic cells, while IL-8 constitute the C-X-C class. The roles of most of these chemokines are not well known, although members of the chemokine family are inflammatory agents. The C-C chemokine plays a role in regulating Th-cell cytokine production and leukocyte trafficking. In this study we clearly show that RANTES and MCP-1 are mediators of acute inflammatory responses. Our report describes additional biological activities for RANTES, MCP-1, and IL-8, suggesting that these chemokines play a fundamental role in histamine and serotonin generation and cell function in mast cells.

Autism and immunity: revisited study.

Autism spectrum disorder is of interest neurochemically because it represents a relatively homogeneous disorder with regard to disease development, abnormal cognitive development and intellectual development disturbance. A consistent finding in autistic children is a high number of mast cells and a high level of serotonin which is also found at elevated concentrations in the urine of autistic patients. In addition, a dysfunction of clinical conditions, such as gastrointestinal and immunological symptoms, is frequently noted in autistic children, however, IgE does not appear to be prevalent in these children but probably an increase of cytokines/chemokines produced by mast cells at an early age may play an important role. Therefore an immune hypothesis, involving also autoimmunity, is one possible pathogenetic mechanism in autism. In conclusion, mast cell activation could contribute to immune and neuroinflammatory abnormalities that are evident in patients with autism spectrum disorders.

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.

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.

IL-32: a newly-discovered proinflammatory cytokine.

IL-32, a newly-discovered proinflammatory cytokine that activates the p38MAPK and NF-kappaB pathways, is an important player in innate and adaptive immune response. IL-32, a cytokine produced mainly by T, natural killer, and epithelial cells induces significant amounts of TNFalpha and MIP-2 and increases the production of both cytokines in a dose-dependent manner. IL-32 has been implicated in inflammatory disorders, mycobacterium tuberculosis infections, inflammatory bowel disease, and influenza A virus infection, as well as in some autoimmune diseases, such as rheumatoid arthritis, ulcerative colitis and Crohn?s disease and in human stomach cancer, human lung cancer and breast cancer tissues. Moreover, it has been reported that IL-32 has pro-inflammatory effects on myeloid cells and causes the differentiation of osteoclast precursors into multinucleated cells expressing specific osteoclast markers. We recently found that human IL-32 has the capacity to provoke histamine release in human-derived cord blood mast cells (HDCBMC), but not in LAD 2 cells nor in rat peritoneal mast cells (RPMC), showing that IL-32 may be specie specific and act more in mature human mast cells (HDCBMC) than in transformed mast cells (LAD 2 cells). Certainly, IL-32 is another potent proinflammatory cytokine, however, the specific role of this newly-discovered protein in the network of cytokine biology remains to be determined.

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.

Neuropeptide substance P induces mRNA expression and secretion of CXCL8 chemokine, and HDC in human umbilical cord blood mast cells.

PURPOSE: Mast cells play an important role in innate and acquired immunity and are thought to be the cellular origin of most proteases and cytokines. Substance P (SP) and its receptor, NK-1R, play critical roles in immune regulation in human and animal models of inflammation. METHODS: We used mature human cord blood mast cells (HCBMC) differentiated from cord blood CD34+ precursor activated with SP in culture. RESULTS: Our data indicate that Substance P strongly activates mature HCBMC in releasing CXCL8 expression and secretion ( CONTROL: 1.200 +/- 1.0; SP: 4.10 +/- 0.90; P < 0.01). Moreover, in a RT-PCR, HCBMC expressed CXCL8 mRNA after Substance P activation. Since calcium ionophore A23187 is a pharmacological activator that raises cytosolic free calcium ion concentraion and stimulates mast cells in the production and secretion of proinflammatory compounds, it was used as positive control. In addition, we found that HCBMCs generate the transcription of histidine decarboxylase (HDC), the enzyme responsible for the generation of histamine from histidine, after SP treatment. Since CXCL8 is a member of the CXC chemokine subfamily with potent chemotactic activity and is a primary inflammatory cytokine we conclude that our results, obtained from HCBMC cultures, a good and valid model in vitro, support the concept that the neurogenic system modulates inflammatory events by Substance P-mediated HCBMC chemokine CXCL8 release. CONCLUSION: The expression, synthesis and release of CXCL8 suggest an increase of inflammatory process in vivo mediated by the recruitment and infiltration of inflammatory cells in inflamed tissues.

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.

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.

Plasmatic markers of muscular stress in isokinetic exercise.

In this paper we examined the variations of plasmatic concentrations of hypoxanthine and xanthine, and their relation with other important indicators of muscular stress creatine-kinase (CK), myoglobin, uric acid, leucocytes, in prolonged, isokynetic physical exercise, performed in a concentric mode at different joint excursion. Twenty healthy male subjects performed isokinetic exercises in concentric-concentric mode, with joint excursion of 30, 60, 90 deg/sec. Blood samples were drawn at rest, immediately after exercise and after 45 min of recovery. The plasmatic concentration of hypoxanthine increased at the end of physical exercise, compared to the rest value of about 1,5 micromol/L, up to a level of greater than 19 micromol/L; the values were higher after a period of recovery of 45 min and the increase varies considerably according to the type of exercise that was performed. Myoglobin has a slight but sensible increment too, with the same trend as hypoxanthine, while CK increase without correlation to the type of exercises. The relation with other indicators of muscular activity demonstrates that in none of the different isokinetic exercises, performed at concentric mode, was there ultrastructural damage, while it is possible to come across a considerable metabolic stress, which is dissimilar in the different kinds of exercises. The results suggest that hypoxanthine can be useful in monitoring the effectiveness of a work load and the metabolic stress consequences on the muscle tissue in training or rehabilitation programs. The results also suggest that even myoglobin, at small concentrations, can have the same function.

iNOS activity in the aged rat liver tissue.

Free radical damage to many cellular components has been proposed as the main mechanism underlying the aging process. In the liver, NO can be generated by iNOS, but also by the constitutively expressed endothelial NOS (eNOS). iNOS enzyme appears to be expressed in liver disease such as cirrhosis and fulminant hepatitis, while the eNOS is expressed in physiological conditions. Ten young and ten old Wistar rats were sacrificed and their livers were excised. Liver sections were incubated with an anti-iNOS antibody of rabbit origin. RT-PCR and Western blot analysis were performed and nitric oxide activity was calculated. A significant increase of iNOS immunoreactivity was seen in the aged liver sections versus young liver sections. iNOS protein is expressed in greater quantities in the aged group, compared to the young group. In this study we show, for the first time, that aging in the rat liver is accompanied by a spontaneous induction of iNOS mRNA, high levels of iNOS protein and immunohistochemistry/image analysis.