Estudo do papel da proteína multifuncional APE1/Ref-1 sobre a resposta inflamatória na meningite bacteriana

Despite advances in antibiotic therapy, bacterial meningitis (BM) remains with high mortality and morbidity rates in worldwide. One important mechanism associated to sequels during disease is the intense inflammatory response which promotes an oxidative burst and release of reactive oxygen species, consequently leading to cell death. Activation of DNA repair enzymes during oxidative stress has been demonstrated in several neurological disorders. APE1/Ref-1 is a multifunctional protein involved in DNA repair and plays a redox function on transcription factors such as NFkB and AP-1.The aim of this study was assess the role of APE1/Ref-1 on inflammatory response and the possibility of its modulation to reduce the sequels of the disease. Firstly it was performed an assay to measure cytokine in cerebrospinal fluid of patients with BM due to Streptococcus pneumoniae and Neisseriae meningitides. Further, a cellular model of inflammation was used to observe the effect of the inhibition of the endonuclease and redox activity of APE1/Ref-1 on cytokine levels. Additionally, APE1/Ref-1 expression in cortex and hippocampus of rat with MB after vitamin B6 treatment was evaluated. Altogether, results showed a similar profile of cytokines in the cerebrospinal fluid of patients from both pathogens, although IFNy showed higher expression in patients with BM caused by S. pneumoniae. On the other hand, inhibitors of APE1/Ref-1 reduced cytokine levels, mainly TNF-α. Reduction of oxidative stress markers was also observed after introduction of inhibitors in the LPS-stimulated cell. In the animal model, BM increased the expression of the protein APE1/Ref-1, while vitamin B6 promoted reduction. Thereby, this data rise important factors to be considered in pathogenesis of BM, e.g., IFNy can be used as prognostic factor during corticosteroid therapy, APE1/Ref-1 can be an important target to modulate the level of inflammation and VIII oxidative stress, and vitamin B6 seems modulates several proteins related to cell death. So, this study highlights a new understanding on the role of APE1/Ref-1 on the inflammation and the oxidative stress during inflammation condition

Oxidative stress and inflammatory mediators contribute to endothelial dysfunction in high-fat diet-induced obesity in mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Interleukin-4 and interleukin-13 inhibit the expression of leukemia inhibitory factor and interleukin-11 in fibroblasts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ebselen and cytokine-induced nitric oxide synthase expression in insulin-producing cells

Interleukin-1 (IL-1) may be a mediator of β-cell damage in insulin-dependent diabetes mellitus (IDDM). The IL-1 mechanism of action on insulin-producing cells probably includes activation of the transcription nuclear factor κB (NF-κB), increased transcription of the inducible form of nitric oxide synthase (iNOS) and the subsequent production of nitric oxide (NO). Reactive oxygen intermediates, particularly H2O2, have been proposed as second messengers for NF-κB activation. In the present study, we tested whether ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a glutathione peroxidase mimicking compound, could counteract the effects of IL-1β, H2O2 and alloxan in rat pancreatic islets and in the rat insulinoma cell line RINm5F (RIN cells). Some of these experiments were also reproduced in human pancreatic islets. Ebselen (20 μM) prevented the increase in nitrite production by rat islets exposed to IL-1β for 6 hr and induced significant protection against the acute inhibitory effects of alloxan or H2O2 exposure, as judged by the preserved glucose oxidation rates. However, ebselen failed to prevent the increase in nitrite production and the decrease in glucose oxidation and insulin release by rat islets exposed to IL-1β for 24 hr. Ebselen prevented the increase in nitrite production by human islets exposed for 14 hr to a combination of cytokines (IL-1β, tumor necrosis factor-α and interferon-γ). In RIN cells, ebselen counteracted both the expression of iNOS mRNA and the increase in nitrite production induced by 6 hr exposure to IL-β but failed to block IL-1β-induced iNOS expression following 24 hr exposure to the cytokine. Moreover, ebselen did not prevent IL-1β-induced NF-κB activation. As a whole, these data indicate that ebselen partially counteracts cytokine-induced NOS activation in pancreatic β-cells, an effect not associated with inhibition of NF-κB activation.

Infliximab prevents increased systolic blood pressure and upregulates the AKT/eNOS pathway in the aorta of spontaneously hypertensive rats

High systolic blood pressure caused by endothelial dysfunction is a comorbidity of metabolic syndrome that is mediated by local inflammatory signals. Insulin-induced vasorelaxation due to endothelial nitric oxide synthase (eNOS) activation is highly dependent on the activation of the upstream insulin-stimulated serine/threonine kinase (AKT) and is severely impaired in obese, hypertensive rodents and humans. Neutralisation of circulating tumor necrosis factor-α (TNFα) with infliximab improves glucose homeostasis, but the consequences of this pharmacological strategy on systolic blood pressure and eNOS activation are unknown. To address this issue, we assessed the temporal changes in the systolic pressure of spontaneously hypertensive rats (SHR) treated with infliximab. We also assessed the activation of critical proteins that mediate insulin activity and TNFα-mediated insulin resistance in the aorta and cardiac left ventricle. Our data demonstrate that infliximab prevents the upregulation of both systolic pressure and left ventricle hypertrophy in SHR. These effects paralleled an increase in AKT/eNOS phosphorylation and a reduction in the phosphorylation of inhibitor of nuclear factor-κB (Iκβ) and c-Jun N-terminal kinase (JNK) in the aorta. Overall, our study revealed the cardiovascular benefits of infliximab in SHR. In addition, the present findings further suggested that the reduction of systolic pressure and left ventricle hypertrophy by infliximab are secondary effects to the reduction of endothelial inflammation and the recovery of AKT/eNOS pathway activation. © 2012 Elsevier B.V.

Estresse oxidativo e sinalização pelas vias MAPK e NF-кB no músculo sóleo de ratos com insuficiência cardíaca crônica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Non-hematopoietic PAR-2 is essential for matriptase-driven pre-malignant progression and potentiation of ras-mediated squamous cell carcinogenesis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Gastrin-Releasing Peptide Receptor Antagonism Induces Protection from Lethal Sepsis: Involvement of Toll-like Receptor 4 Signaling

In sepsis, toll-like receptor (TLR)-4 modulates the migration of neutrophils to infectious foci, favoring bacteremia and mortality. In experimental sepsis, organ dysfunction and cytokines released by activated macrophages can be reduced by gastrin-releasing peptide (GRP) receptor (GRPR) antagonist RC-3095. Here we report a link between GRPR and TLR-4 in experimental models and in sepsis patients. RAW 264.7 culture cells were exposed to lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha and RC-3095 (10 ng/mL), Male Wistar rats were subjected to cecal ligation and puncture (CLP), and RC-3095 was administered (3 mg/kg, subcutaneously); after 6 h, we removed the blood, bronchoalveolar lavage, peritoneal lavage and lung. Human patients with a clinical diagnosis of sepsis received a continuous infusion with RC-3095 (3 mg/kg, intravenous) over a period of 12 h, and plasma was collected before and after RC-3095 administration and, in a different set of patients with systemic inflammatory response syndrome (SIRS) or sepsis. GRP plasma levels were determined. RC-3095 inhibited TLR-4, extracellular-signal-related kinase (ERK)-1/2, Jun NH2-terminal kinase (JNK) and Akt and decreased activation of activator protein 1 (AP-1), nuclear factor (NF)-kappa B and interleukin (IL)-6 in macrophages stimulated by LPS. It also decreased IL-6 release from macrophages stimulated by TNF-alpha. RC-3095 treatment in CLP rats decreased lung TLR-4, reduced the migration of cells to the lung and reduced systemic cytokines and bacterial dissemination. Patients with sepsis and systemic inflammatory response syndrome have elevated plasma levels of GRP which associates with clinical outcome in the sepsis patients. These findings highlight the role of GRPR signaling in sepsis outcome and the beneficial action of GRPR antagonists in controlling the inflammatory response in sepsis through a mechanism involving at least inhibition of TLR-4 signaling. Online address: http://www.molmed.org doi: 10.2119/molmed.2012.00083

GM-CSF Priming Drives Bone Marrow-Derived Macrophages to a Pro-Inflammatory Pattern and Downmodulates PGE(2) in Response to TLR2 Ligands

In response to pathogen recognition by Toll-like receptors (TLRs) on their cell surface, macrophages release lipid mediators and cytokines that are widely distributed throughout the body and play essential roles in host responses. Granulocyte macrophage colony-stimulating factor (GM-CSF) is important for the immune response during infections to improve the clearance of microorganisms. In this study, we examined the release of mediators in response to TLR2 ligands by bone marrow-derived macrophages (BMDMs) primed with GM-CSF. We demonstrated that when stimulated with TLR2 ligands, non-primed BMDMs preferentially produced PGE(2) in greater amounts than LTB4. However, GM-CSF priming shifted the release of lipid mediators by BMDMs, resulting in a significant decrease of PGE(2) production in response to the same stimuli. The decrease of PGE(2) production from primed BMDMs was accompanied by a decrease in PGE-synthase mRNA expression and an increase in TNF-alpha and nitric oxide (NO) production. Moreover, some GM-CSF effects were potentiated by the addition of IFN-gamma. Using a variety of TLR2 ligands, we established that PGE(2) release by GM-CSF-primed BMDMs was dependent on TLR2 co-receptors (TLR1, TLR6), CD14, MyD88 and the nuclear translocation of NF kappa B but was not dependent on peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activation. Indeed, GM-CSF priming enhanced TLR2, TLR4 and MyD88 mRNA expression and phospho-I kappa B alpha formation. These findings demonstrate that GM-CSF drives BMDMs to present a profile relevant to the host during infections.

An in vitro study showing the three-dimensional microenvironment influence over the behavior of head and neck squamous cell carcinoma

Objectives: The Head and Neck Squamous Cell Carcinoma (HNSCC) ranks sixth worldwide. The mechanisms of growth, invasion and metastasis of this pathology are extensively studied and generally related to specific variations in signaling pathways like the PI3K-Akt; however most of these competent studies have been performed bidimensionally, which may hide important questions. This study sought to analyze the influence of the microenvironment upon the behavior of HNSCC. Study Design: The status of pAkt, NF-kappa B and Cyclin D1 proteins was accessed through immunofluorescence and western blot methods in HNSCC cell lines originating from tongue, pharynx and metastatic lymph node when submitted to a three-dimensional culture model utilizing a matrix system. A bidimensional culture model (monolayer) was used as control. Results: The HNSCC cell lines cultured three-dimensionally exhibited a growth pattern characterized by small isolated islands, different from the control group. When the three-dimensional model was applied, two of the studied cell lines showed the same expression pattern as the bidimensional model regarding nuclear or cytoplasmatic localization, as well as reduction of all protein levels; however, the cell line originated from tongue, which specially has the epidermal growth factor receptor constitutively activated, demonstrated nuclear translocation of pAkt and also an increase in the levels of Cyclin D1. Conclusions: The results suggest the influence of the microenvironment upon the behavior of HNSCC cells due to the changed expression of proteins related to tumor growth and cellular invasion. Furthermore, intrinsically genetic conditions also played important roles over the cells, despite the culture model employed.

Differential Gene Expression Profiles May Differentiate Responder and Nonresponder Patients with Rheumatoid Arthritis for Methotrexate (MTX) Monotherapy and MTX plus Tumor Necrosis Factor Inhibitor Combined Therapy

Objective. We aimed to evaluate whether the differential gene expression profiles of patients with rheumatoid arthritis (RA) could distinguish responders from nonresponders to methotrexate (MTX) and, in the case of MTX nonresponders, responsiveness to MTX plus anti-tumor necrosis factor-alpha (anti-TNF) combined therapy. Methods. We evaluated 25 patients with RA taking MTX 15-20 mg/week as a monotherapy (8 responders and 17 nonresponders). All MTX nonresponders received intliximab and were reassessed after 20 weeks to evaluate their anti-TNF responsiveness using the European League Against Rheumatism response criteria. A differential gene expression analysis from peripheral blood mononuclear cells was performed in terms of hierarchical gene clustering, and an evaluation of differentially expressed genes was performed using the significance analysis of microarrays program. Results. Hierarchical gene expression clustering discriminated MTX responders from nonresponders, and MTX plus anti-TNF responders from nonresponders. The evaluation of only highly modulated genes (fold change > 1.3 or < 0.7) yielded 5 induced (4 antiapoptotic and CCL4) and 4 repressed (4 proapoptotic) genes in MTX nonresponders compared to responders. In MTX plus anti-TNF nonresponders, the CCL4, CD83, and BCL2A1 genes were induced in relation to responders. Conclusion. Study of the gene expression profiles of RA peripheral blood cells permitted differentiation of responders from nonresponders to MTX and anti-TNF. Several candidate genes in MTX non-responders (CCL4, HTRA2, PRKCD, BCL2A1, CAV1, TNIP1 CASP8AP2, MXD1, and BTG2) and 3 genes in MTX plus anti-TNF nonresponders (CCL4, CD83, and BCL2A1) were identified for further study. (First Release July 1 2012; J Rheumatol 2012;39:1524-32; doi:10.3899/jrheum.120092)

Clove and eugenol in noncytotoxic concentrations exert immunomodulatory/anti-inflammatory action on cytokine production by murine macrophages

Objectives The extract and essential oil of clove (Syzygium aromaticum) are widely used because of their medicinal properties. Eugenol is the most important component of clove, showing several biological properties. Herein we have analysed the immunomodulatory/anti-inflammatory effect of clove and eugenol on cytokine production (interleukin (IL)-1 beta, IL-6 and IL-10) in vitro. Methods Macrophages were incubated with clove or eugenol (5, 10, 25, 50 or 100 mg/well) for 24 h. Concentrations that inhibited the production of cytokines were used before or after incubation with lipopolysaccharide (LPS), to verify a preventive or therapeutic effect. Culture supernatants were harvested for measurement of cytokines by enzyme-linked immunosorbent assay. Key findings Clove (100 mg/well) inhibited IL-1 beta, IL-6 and IL-10 production and exerted an efficient action either before or after LPS challenge for all cytokines. Eugenol did not affect IL-1 beta production but inhibited IL-6 and IL-10 production. The action of eugenol (50 or 100 mg/well) on IL-6 production prevented efficiently effects of LPS either before or after its addition, whereas on IL-10 production it counteracted significantly LPS action when added after LPS incubation. Conclusions Clove exerted immunomodulatory/anti-inflammatory effects by inhibiting LPS action. A possible mechanism of action probably involved the suppression of the nuclear factor-kB pathway by eugenol, since it was the major compound found in clove

Signaling transduction analysis in gingival epithelial cells after infection with Aggregatibacter actinomycetemcomitans

Periodontal diseases result from the interaction of bacterial pathogens with the hosts gingival tissue. Gingival epithelial cells are constantly challenged by microbial cells and respond by altering their transcription profiles, inducing the production of inflammatory mediators. Different transcription profiles are induced by oral bacteria and little is known about how the gingival epithelium responds after interaction with the periodontopathogenic organism Aggregatibacter actinomycetemcomitans. In the present study, we examined the transcription of genes involved in signaling transduction pathways in gingival epithelial cells exposed to viable A.actinomycetemcomitans. Immortalized gingival epithelial cells (OBA-9) were infected with A.actinomycetemcomitans JP2 for 24 h and the transcription profile of genes encoding human signal transduction pathways was determined. Functional analysis of inflammatory mediators positively transcribed was performed by ELISA in culture supernatant and in gingival tissues. Fifteen of 84 genes on the array were over-expressed (P < 0.01) after 24 h of infection with viable A.actinomycetemcomitans. Over-expressed genes included those implicated in tissue remodeling and bone resorption, such as CSF2, genes encoding components of the LDL pathway, nuclear factor-?B-dependent genes and other cytokines. The ELISA data confirmed that granulocytemacrophage colony-stimulating factor/colony-stimulating factor 2, tumor necrosis factor-a and intercellular adhesion molecule-1 were highly expressed by infected gingival cells when compared with control non-infected cells, and presented higher concentrations in tissues from patients with aggressive and chronic periodontitis than in tissues from healthy controls. The induction in epithelial cells of factors such as the pro-inflammatory cytokine CSF2, which is involved in osteoclastogenesis, may help to explain the outcomes of A.actinomycetemcomitans infection.

AMR-Me inhibits PI3K/Akt signaling in hormone-dependent MCF-7 breast cancer cells and inactivates NF-κB in hormone-independent MDA-MB-231 cells

AMR-Me, a C-28 methylester derivative of triterpenoid compound Amooranin isolated from Amoora rohituka stem bark and the plant has been reported to possess multitude of medicinal properties. Our previous studies have shown that AMR-Me can induce apoptosis through mitochondrial apoptotic and MAPK signaling pathways by regulating the expression of apoptosis related genes in human breast cancer MCF-7 cells. However, the molecular mechanism of AMR-Me induced apoptotic cell death remains unclear. Our results showed that AMR-Me dose-dependently inhibited the proliferation of MCF-7 and MDA-MB-231 cells under serum-free conditions supplemented with 1 nM estrogen (E2) with an IC50 value of 0.15 µM, 0.45 µM, respectively. AMR-Me had minimal effects on human normal breast epithelial MCF-10A + ras and MCF-10A cells with IC50 value of 6 and 6.5 µM, respectively. AMR-Me downregulated PI3K p85, Akt1, and p-Akt in an ERα-independent manner in MCF-7 cells and no change in expression levels of PI3K p85 and Akt were observed in MDA-MB-231 cells treated under similar conditions. The PI3K inhibitor LY294002 suppressed Akt activation similar to AMR-Me and potentiated AMR-Me induced apoptosis in MCF-7 cells. EMSA revealed that AMR-Me inhibited nuclear factor-kappaB (NF-κB) DNA binding activity in MDA-MB-231 cells in a time-dependent manner and abrogated EGF induced NF-κB activation. From these studies we conclude that AMR-Me decreased ERα expression and effectively inhibited Akt phosphorylation in MCF-7 cells and inactivate constitutive nuclear NF-κB and its regulated proteins in MDA-MB-231 cells. Due to this multifactorial effect in hormone-dependent and independent breast cancer cells AMR-Me deserves attention for use in breast cancer prevention and therapy

Constitutive NF-kappaB and NFAT activation leads to stimulation of the BLyS survival pathway in aggressive B-cell lymphomas.

B-lymphocyte stimulator (BLyS), a relatively recently recognized member of the tumor necrosis factor ligand family (TNF), is a potent cell-survival factor expressed in many hematopoietic cells. BLyS binds to 3 TNF-R receptors, TACI, BCMA, BAFF-R, to regulate B-cell survival, differentiation, and proliferation. The mechanisms involved in BLYS gene expression and regulation are still incompletely understood. In this study, we examined BLYS gene expression, function, and regulation in B-cell non-Hodgkin lymphoma (NHL-B) cells. Our studies indicate that BLyS is constitutively expressed in aggressive NHL-B cells, including large B-cell lymphoma (LBCL) and mantle cell lymphoma (MCL), playing an important role in the survival and proliferation of malignant B cells. We found that 2 important transcription factors, NF-kappaB and NFAT, are involved in regulating BLyS expression through at least one NF-kappaB and 2 NFAT binding sites in the BLYS promoter. We also provide evidence suggesting that the constitutive activation of NF-kappaB and BLyS in NHL-B cells forms a positive feedback loop associated with lymphoma cell survival and proliferation. Our findings indicate that constitutive NF-kappaB and NFAT activations are crucial transcriptional regulators of the BLyS survival pathway in malignant B cells that could be therapeutic targets in aggressive NHL-B.