ROS production and mitochondrial dysfunction driven by PU.1-regulated NOX4-p22phox activation in Aß-induced retinal pigment epithelial cell injury.

Rationale: Amyloid ß (Aß) deposition, an essential pathological process in age-related macular degeneration (AMD), causes retinal pigment epithelium (RPE) degeneration driven mostly by oxidative stress. However, despite intense investigations, the extent to which overoxidation contributes to Aß-mediated RPE damage and its potential mechanism has not been fully elucidated. Methods: We performed tandem mass-tagged (TMT) mass spectrometry (MS) and bioinformatic analysis of the RPE-choroid complex in an Aß1-40-induced mouse model of retinal degeneration to obtain a comprehensive proteomic profile. Key regulators in this model were confirmed by reactive oxygen species (ROS) detection, mitochondrial ROS assay, oxygen consumption rate (OCR) measurement, gene knockout experiment, chromatin immunoprecipitation (ChIP), and luciferase assay. Results: A total of 4243 proteins were identified, 1069 of which were significantly affected by Aß1-40 and found to be enriched in oxidation-related pathways by bioinformatic analysis. Moreover, NADPH oxidases were identified as hub proteins in Aß1-40-mediated oxidative stress, as evidenced by mitochondrial dysfunction and reactive oxygen species overproduction. By motif and binding site analyses, we found that the transcription factor PU.1/Spi1 acted as a master regulator of the activation of NADPH oxidases, especially the NOX4-p22phox complex. Also, PU.1 silencing impeded RPE oxidative stress and mitochondrial dysfunction and rescued the retinal structure and function. Conclusion: Our study suggests that PU.1 is a novel therapeutic target for AMD, and the regulation of PU.1 expression represents a potentially novel approach against excessive oxidative stress in Aß-driven RPE injury.

Avances en el diagnóstico de la enfermedad granulomatosa crónica: primer estudio familiar en Cuba / Advances in the diagnosis of chronic granulomatous disease: the first family study in Cuba

Introducción: La enfermedad granulomatosa crónica es una inmunodeficiencia primaria causada por mutaciones en la enzima NADPH oxidasa. Esta compromete la producción de especies reactivas del oxígeno, que son importantes contra patógenos. La prueba de la oxidación de la dihidrorodamina es un método eficaz para diagnosticar la enfermedad. Objetivo: Demostrar la utilidad de la prueba de la oxidación de la dihidrorodamina y del patrón de herencia en la confirmación del diagnóstico de la enfermedad granulomatosa crónica de un paciente. Métodos: Estudio de caso de una familia con diagnóstico de enfermedad granulomatosa crónica. Se tomó muestra de sangre periférica para citometría de flujo a tres individuos. Se realizó la prueba de la oxidación de la dihidrorodamina bajo estímulo con acetato de forbolmiristato y se evaluaron las subpoblaciones linfocitarias. Las muestras se leyeron en un citómetro GALLIOS, Beckman Coulter. Los datos obtenidos se analizaron en el programa informático Kaluza. Resultados: El paciente masculino tuvo un valor de oxidación de la dihidrorodamina positiva de 0,87 por ciento, que confirmó un patrón de herencia ligado al cromosoma X; mientras que la madre y hermana gemela portadoras tuvieron valores de 46,76 por ciento y 37,32 por ciento, respectivamente. Se encontraron alteraciones en las subpoblaciones linfocitarias. Conclusiones: La prueba de la oxidación de la dihidrorodamina es un método muy efectivo, rápido y sencillo que confirma el diagnóstico de la enfermedad granulomatosa crónica y determina el patrón de herencia y fenotipo de la enfermedad. Además, permite identificar a las mujeres portadoras según la distribución de los neutrófilos normales y los que tienen el gen CYBB mutado(AU)

Introduction: Chronic granulomatous disease is a primary immunodeficiency caused by mutations in the NADPH oxidase enzymes. This compromises the production of oxygen reactive species, which are important against pathogens. The dihydrorhodamine oxidation test is an effective method for diagnosing the disease. Objective: To demonstrate the usefulness of the dihydrorhodamine oxidation test and the inheritance pattern in confirming the diagnosis of chronic granulomatous disease in a patient. Methods: A case study of a family with a diagnosis of chronic granulomatous disease. A peripheral blood sample was taken from three individuals and by flow cytometry. The dihydrorhodamine oxidation test was performed under stimulation with phorbolmyristate acetate, and lymphocyte subpopulations were evaluated. The samples were read on a GALLIOS, Beckman Coulter cytometer. The data obtained were analyzed using the computer program Kaluza. Results: The male patient had a positive dihydrorhodamine oxidation value of 0.87 percent, which confirmed an inheritance pattern linked to the X chromosome; while the carrier mother and twin sister had values 8203;8203;of 46.76 percent and 37.32 percent, respectively. Alterations were found in the lymphocyte subpopulations. Conclusions: The dihydrorhodamine oxidation test is a very effective, fast and simple method that confirms the diagnosis of chronic granulomatous disease and determines the inheritance pattern and phenotype of the disease. In addition, it allows the identification of female carriers according to the distribution of normal neutrophils and those with the CYBB mutation(AU)

Cleaved N-terminal histone tails distinguish between NADPH oxidase (NOX)-dependent and NOX-independent pathways of neutrophil extracellular trap formation.

OBJECTIVES: Neutrophil extracellular traps (NETs) act in various rheumatic diseases. Although NET formation was originally described as a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-dependent pathway, it appears that there are also NOX-independent pathways of NET release. Currently, no tools are available that can discriminate between both NET-forming pathways. We aimed to develop a serological method allowing the discrimination between NETs generated through NOX-dependent or NOX-independent pathways. METHODS: Histones from in vitro generated NOX-dependent and NOX-independent NETs were characterised with a panel of lupus-derived antibodies against N-terminal histone tails using immunofluorescence microscopy, western blot and ELISA. NETs in patients with NET-associated diseases, that is, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), psoriatic arthritis (PsA) and sepsis, were characterised in sandwich ELISAs employing antibodies against myeloperoxidase (MPO) and N-terminal histone tails as detecting and capturing antibodies, respectively. Functional responses of endothelial cells to NOX-dependent and NOX-independent NETs were assessed as well. RESULTS: Neutrophil elastase cleaves the N-terminal tails of core histones during NOX-dependent, but not during NOX-independent NET formation. Consequently, the detection of MPO-histone complexes with antibodies against N-terminal histone tails allows discrimination between NETs formed through a NOX-dependent or NOX-independent manner. Characterisation of in vivo circulating NETs revealed the presence of NOX-independent NETs in RA, SLE and sepsis, but NOX-dependent NETs in PsA. NOX-independent NETs displayed an increased capacity to activate endothelial cells when compared with NOX-dependent NETs. CONCLUSIONS: These results indicate heterogeneity in NET-forming pathways in vivo and highlight the need for disease-specific strategies to prevent NET-mediated pathology.

Oxidative Stress in Aortas of Patients with Advanced Occlusive and Aneurysmal Diseases.

BACKGROUND: Aortoiliac occlusive disease (AOD) and abdominal aortic aneurysm (AAA) are very important cardiovascular diseases that present different aspects of pathophysiology; however, oxidative stress and inflammatory response seem be relevant in both of them. Our objective was to evaluate oxidative damage and degree of inflammatory infiltrate in aortas of patients surgically treated for AOD and AAA. MATERIALS AND METHODS: Levels of reactive oxygen species (ROS), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and myeloperoxidase (MPO) expression as well as nitrite levels and superoxide dismutase (SOD) and catalase (CAT) activities were evaluated in aortas of patients with AOD (n = 16) or AAA (n = 14), while the control group was formed by cadaveric organ donors (n = 10). We also analyzed the degree of inflammatory infiltrate in these aortas. RESULTS: There was an increase in ROS levels and NADPH oxidase activity in patients with AOD and AAA when compared with the control group, and the AOD group demonstrated higher ROS production and NADPH oxidase activity and also nitrite levels when compared with the AAA group (P < 0.001). On the other hand, an increase of SOD activity in the AOD group and CAT activity in the AAA group was observed. Inflammatory infiltrate and MPO expression were higher in the AOD group when compared with the control group (P < 0.05). CONCLUSIONS: Oxidative stress is relevant in both AOD and AAA, though AOD presented higher ROS levels and NADPH activity. Increased activities of antioxidant enzymes may be a compensatory phenomenon which occurs in aortas of patients with AOD and AAA. Perhaps, a relationship between oxidative stress and degree of inflammatory infiltrate may exist in the pathophysiology of AOD and AAA.

Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells.

BACKGROUND Streptococcus agalactiae can causes sepsis, pneumonia, and meningitis in neonates, the elderly, and immunocompromised patients. Although the virulence properties of S. agalactiae have been partially elucidated, the molecular mechanisms related to reactive oxygen species (ROS) generation in infected human endothelial cells need further investigation. OBJECTIVES This study aimed to evaluate the influence of oxidative stress in human umbilical vein endothelial cells (HUVECs) during S. agalactiae infection. METHODS ROS production during S. agalactiae-HUVEC infection was detected using the probe CM-H2DCFDA. Microfilaments labelled with phalloidin-FITC and p47phox-Alexa 546 conjugated were analysed by immunofluorescence. mRNA levels of p47phox (NADPH oxidase subunit) were assessed using Real Time qRT-PCR. The adherence and intracellular viability of S. agalactiae in HUVECs with or without pre-treatment of DPI, apocynin (NADPH oxidase inhibitors), and LY294002 (PI3K inhibitor) were evaluated by penicillin/gentamicin exclusion. Phosphorylation of p47phox and Akt activation by S. agalactiae were evaluated by immunoblotting analysis. FINDINGS Data showed increased ROS production 15 min after HUVEC infection. Real-Time qRT-PCR and western blotting performed in HUVEC infected with S. agalactiae detected alterations in mRNA levels and activation of p47phox. Pre-treatment of endothelial cells with NADPH oxidase (DPI and apocynin) and PI3K/Akt pathway (LY294002) inhibitors reduced ROS production, bacterial intracellular viability, and generation of actin stress fibres in HUVECs infected with S. agalactiae. CONCLUSIONS ROS generation via the NADPH oxidase pathway contributes to invasion of S. agalactiae in human endothelial cells accompanied by cytoskeletal reorganisation through the PI3K/Akt pathway, which provides novel evidence for the involvement of oxidative stress in S. agalactiae pathogenesis.

Targeting the Myofibroblastic Cancer-Associated Fibroblast Phenotype Through Inhibition of NOX4.

Background: Cancer-associated fibroblasts (CAFs) are tumor-promoting and correlate with poor survival in many cancers, which has led to their emergence as potential therapeutic targets. However, effective methods to manipulate these cells clinically have yet to be developed. Methods: CAF accumulation and prognostic significance in head and neck cancer (oral, n = 260; oropharyngeal, n = 271), and colorectal cancer (n = 56) was analyzed using immunohistochemistry. Mechanisms regulating fibroblast-to-myofibroblast transdifferentiation were investigated in vitro using RNA interference/pharmacological inhibitors followed by polymerase chain reaction (PCR), immunoblotting, immunofluorescence, and functional assays. RNA sequencing/bioinformatics and immunohistochemistry were used to analyze NAD(P)H Oxidase-4 (NOX4) expression in different human tumors. NOX4's role in CAF-mediated tumor progression was assessed in vitro, using CAFs from multiple tissues in Transwell and organotypic culture assays, and in vivo, using xenograft (n = 9-15 per group) and isograft (n = 6 per group) tumor models. All statistical tests were two-sided. Results: Patients with moderate/high levels of myofibroblastic-CAF had a statistically significant decrease in cancer-specific survival rates in each cancer type analyzed (hazard ratios [HRs] = 1.69-7.25, 95% confidence intervals [CIs] = 1.11 to 31.30, log-rank P ≤ .01). Fibroblast-to-myofibroblast transdifferentiation was dependent on a delayed phase of intracellular reactive oxygen species, generated by NOX4, across different anatomical sites and differentiation stimuli. A statistically significant upregulation of NOX4 expression was found in multiple human cancers (P < .001), strongly correlating with myofibroblastic-CAFs (r = 0.65-0.91, adjusted P < .001). Genetic/pharmacological inhibition of NOX4 was found to revert the myofibroblastic-CAF phenotype ex vivo (54.3% decrease in α-smooth muscle actin [α-SMA], 95% CI = 10.6% to 80.9%, P = .009), prevent myofibroblastic-CAF accumulation in vivo (53.2%-79.0% decrease in α-SMA across different models, P ≤ .02) and slow tumor growth (30.6%-64.0% decrease across different models, P ≤ .04). Conclusions: These data suggest that pharmacological inhibition of NOX4 may have broad applicability for stromal targeting across cancer types.

The Changing Paradigm of Management of Liver Abscesses in Chronic Granulomatous Disease.

Background: Chronic granulomatous disease (CGD) is a rare genetic disorder causing recurrent infections. More than one-quarter of patients develop hepatic abscesses and liver dysfunction. Recent reports suggest that disease-modifying treatment with corticosteroids is effective for these abscesses. Comparison of corticosteroid therapy to traditional invasive treatments has not been performed. Methods: Records of 268 patients with CGD treated at the National Institutes of Health from 1980 to 2014 were reviewed. Patients with liver involvement and complete records were included. We recorded residual reactive oxygen intermediate (ROI) production by neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase germline mutation status, laboratory values, imaging characteristics, time to repeat hepatic interventions, and overall survival among 3 treatment cohorts: open liver surgery (OS), percutaneous liver-directed interventional radiology therapy (IR), and high-dose corticosteroid management (CM). Results: Eighty-eight of 268 patients with CGD suffered liver involvement. Twenty-six patients with a median follow-up of 15.5 years (8.5-32.9 years of follow-up) had complete records and underwent 100 standard interventions (42 IR and 58 OS). Eight patients received a treatment with high-dose corticosteroids only. There were no differences in NADPH genotype, size, or number of abscesses between patients treated with OS, IR, or CM. Time to repeat intervention was extended in OS compared with IR (18.8 vs 9.5 months, P = .04) and further increased in CM alone (median time to recurrence not met). Impaired macrophage and neutrophil function measured by ROI production correlated with shorter time to repeat intervention (r = 0.6, P = .0019). Conclusions: Treatment of CGD-associated liver abscesses with corticosteroids was associated with fewer subsequent hepatic interventions and improved outcome compared to invasive treatments.

Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells

BACKGROUND Streptococcus agalactiae can causes sepsis, pneumonia, and meningitis in neonates, the elderly, and immunocompromised patients. Although the virulence properties of S. agalactiae have been partially elucidated, the molecular mechanisms related to reactive oxygen species (ROS) generation in infected human endothelial cells need further investigation. OBJECTIVES This study aimed to evaluate the influence of oxidative stress in human umbilical vein endothelial cells (HUVECs) during S. agalactiae infection. METHODS ROS production during S. agalactiae-HUVEC infection was detected using the probe CM-H2DCFDA. Microfilaments labelled with phalloidin-FITC and p47phox-Alexa 546 conjugated were analysed by immunofluorescence. mRNA levels of p47phox (NADPH oxidase subunit) were assessed using Real Time qRT-PCR. The adherence and intracellular viability of S. agalactiae in HUVECs with or without pre-treatment of DPI, apocynin (NADPH oxidase inhibitors), and LY294002 (PI3K inhibitor) were evaluated by penicillin/gentamicin exclusion. Phosphorylation of p47phox and Akt activation by S. agalactiae were evaluated by immunoblotting analysis. FINDINGS Data showed increased ROS production 15 min after HUVEC infection. Real-Time qRT-PCR and western blotting performed in HUVEC infected with S. agalactiae detected alterations in mRNA levels and activation of p47phox. Pre-treatment of endothelial cells with NADPH oxidase (DPI and apocynin) and PI3K/Akt pathway (LY294002) inhibitors reduced ROS production, bacterial intracellular viability, and generation of actin stress fibres in HUVECs infected with S. agalactiae. CONCLUSIONS ROS generation via the NADPH oxidase pathway contributes to invasion of S. agalactiae in human endothelial cells accompanied by cytoskeletal reorganisation through the PI3K/Akt pathway, which provides novel evidence for the involvement of oxidative stress in S. agalactiae pathogenesis.

Eros is a novel transmembrane protein that controls the phagocyte respiratory burst and is essential for innate immunity.

The phagocyte respiratory burst is crucial for innate immunity. The transfer of electrons to oxygen is mediated by a membrane-bound heterodimer, comprising gp91phox and p22phox subunits. Deficiency of either subunit leads to severe immunodeficiency. We describe Eros (essential for reactive oxygen species), a protein encoded by the previously undefined mouse gene bc017643, and show that it is essential for host defense via the phagocyte NAPDH oxidase. Eros is required for expression of the NADPH oxidase components, gp91phox and p22phox Consequently, Eros-deficient mice quickly succumb to infection. Eros also contributes to the formation of neutrophil extracellular traps (NETS) and impacts on the immune response to melanoma metastases. Eros is an ortholog of the plant protein Ycf4, which is necessary for expression of proteins of the photosynthetic photosystem 1 complex, itself also an NADPH oxio-reductase. We thus describe the key role of the previously uncharacterized protein Eros in host defense.

A novel missense mutation in the NADPH binding domain of CYBB abolishes the NADPH oxidase activity in a male patient with increased susceptibility to infections.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the five structural genes (CYBB, CYBA, NCF1, NCF2, and NCF4) that typically results in a decrease in function or inability to generate a respiratory burst, leading to defective killing of pathogens, including fungi and intracellular bacteria. Mutations in CYBB, encoding the gp91phox (also known as NOX2) result in X-linked CGD account for approximately 65% of CGD cases. Here, we aimed the characterization of a novel missense mutation c.1226C > A/p.A409E in the CYBB gene in a patient with X-linked CGD. Relevant clinical data of a male patient whose family was positive for XCGD was reviewed. Oxidative burst and NADPH protein expression was evaluated by flow cytometry, while Genetic analysis was performed by Sanger sequencing. Monocyte-derived macrophages (MDMs) were evaluated for their capacity for phagocytosis and growth suppression of the intracellular Mycobacterium tuberculosis (M. tuberculosis). We thus report the absence of an oxidative burst in the phagocytes of the patient. Flow cytometry evaluation revealed a normal expression of NADPH oxidase components in neutrophils and genetic analysis proved the existence of a novel missense c.1226C > A mutation in the CYBB gene resulting in p.A409E. Further, we have showed that the patient's MDMs were unhindered in their ability to take up mycobacteria normally. Instead, the MDMs failed to control the intracellular proliferation of M. tuberculosis, a phenotype that improved in the presence of recombinant human interferon-gamma (rhIFN-γ). This work expands the genetic spectrum of X-linked CGD and demonstrates improvement in macrophage function in X91+CGD patient by rhIFN-γ.

Effects of tongxinluo on angiogenesis in the carotid adventitia of hyperlipidemic rabbits.

Atherosclerosis, as a common arterial disease with high morbidity rate, is reported to be closely associated with adventitia angiogenesis. The present study aimed to investigate the effect of tongxinluo (TXL) on angiogenesis in the carotid adventitia of hyperlipidemic rabbits and the underlying mechanism. A total of 90 experimental rabbits were randomly assigned into the following six groups (n=15 per group): Normal group, model group, low­dose TXL group, moderate-dose TXL group, high­dose TXL group and atorvastatin group. The normal group was fed with a standard diet. The model and treatment groups were on a high cholesterol diet for 4 weeks. The serum lipid level of the model group was significantly higher compared with the normal group. TXL serum lipid level compared with the model group. Hematoxylin and eosin, and CD31 staining demonstrated that TXL inhibited adventitia angiogenesis in a dose­dependent manner. The dihydroethidium probe and fluorescence in situ hybridization results indicated that TXL reduced O2­ level and positive signal of gp91phox and p22phox mRNA in adventitia. Reverse transcription­polymerase chain reaction and western blot analysis determined that TXL treatment significantly downregulated the expression levels of the gp91phox, p22phox genes and the vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2 (VEGFR-2) proteins compared with the model group. TXL exhibited a dose­dependent inhibitory effect on angiogenesis in the carotid adventitia of hyperlipidemic rabbits. This may be associated with the downregulation of reactive oxygen species generation in the adventitia and the suppression of VEGF/VEGFR-2 expression.

NADPH Oxidase 1 and NADPH Oxidase 4 Have Opposite Prognostic Effects for Patients with Hepatocellular Carcinoma after Hepatectomy.

BACKGROUND/AIMS: Nicotinamide adenine dinucleotide phosphate oxidase (NOX)-mediated reactive oxygen species contribute to various liver diseases, including hepatocellular carcinoma (HCC). Uncertainties remain regarding the prognostic relevance of NOX1 and NOX4 protein expression in HCC. METHODS: NOX1 and NOX4 protein expression was examined by using immunohistochemistry in tumor tissue from 227 HCC patients who underwent hepatectomy. RESULTS: High immunoreactivity for NOX1 was observed in 197 (86.8%) of the 227 HCC cases and low immunoreactivity for NOX4 in 112 (49.3%). NOX1 and NOX4 proteins had opposite prognostic effects. High NOX1 expression was an independent predictor of both shorter recurrence-free survival (RFS) (p<0.01) and shorter overall survival (OS) (p=0.01). Low NOX4 expression was an independent predictor of both shorter RFS (p<0.01) and shorter OS (p=0.01). Subgroup analysis showed that, among patients with normal α-fetoprotein levels, patients with tumor size ≤5.0 cm and patients in Barcelona Clinic Liver Cancer stage A, high NOX1 expression had unfavorable effects on RFS, whereas low NOX4 expression had unfavorable effects on both RFS and OS. CONCLUSIONS: These findings demonstrated that NOX1 and NOX4 protein expression had opposite prognostic effects for HCC patients. Moreover, both proteins had prognostic value in HCC patients with normal α-fetoprotein levels or with early-stage HCC.

Short-term in vivo modifications of platelet NADPH oxidase 2 (NOX2) and prostaglandin F2α in HIV-1 patients on abacavir-based therapies.

OBJECTIVES: The aim of the study was to investigate the in vivo effect of abacavir (ABC) on platelet oxidative stress. METHODS: We performed a randomized pilot study including 39 HIV-1-infected patients, 17 on zidovudine/lamivudine (ZDV/3TC) and 22 on tenofovir/emtricitabine (TDF/FTC). Ten patients on ZDV/3TC and eight patients on TDF/FTC were randomly allocated to switching the nucleoside backbone to ABC/3TC. At baseline and after 6 months, platelet oxidative stress was assessed by platelet NADPH oxidase 2 (NOX2)-derived peptide (sNOX2-dp), a marker of NOX2 activation, and platelet prostaglandin F2α (8-iso-PGF2α ). Platelet activation was measured by soluble CD40L (sCD40L). RESULTS: At baseline, no differences between ZDV/3TC or TDF/FTC recipients were found. After 6 months, patients switching from ZDV/3TC showed a decrease of sNOX2-dp (from 20.9±5.7 to 12.5±3.8 pg/ml, p=0.002) and 8-iso-PGF2α (from 154.3±41.9 to 122.9±28.0 pmol/l, p=0.025). No effects on platelet oxidative stress biomarkers were observed in subjects from TDF/FTC, who showed a significant increase in blood glucose (p=0.043) and total cholesterol (p=0.027). ABC showed no effect on sCD40L levels in both groups. CONCLUSIONS: ABC reduced platelet sNOX2-dp and 8-iso-PGF2α in HIV-1 subjects switching from ZDV/3TC but not in those from TDF/FTC after 6 months. No changes in platelet activation were found in both groups.

Prognostic significance of NADPH oxidase-4 as an indicator of reactive oxygen species stress in human retinoblastoma.

BACKGROUND: Reactive oxygen species (ROS) have been shown to enhance the proliferation of cancer cells. NADPH oxidases (NOX4) are a major intracellular source of ROS and are found to be associated with cancer and tumor cell invasion. Therefore, the purpose of this study is to evaluate the expression of NOX4 protein in human retinoblastoma. METHODS: Immunohistochemical expression of NOX4 protein was analyzed in 109 specimens from prospective cases of retinoblastoma and then correlated with clinicopathological parameters and patient survival. Western blotting confirmed and validated the immunoreactivity of NOX4 protein. RESULTS: In our study we found a male preponderance (55.9 %), and 25/109 (22.9 %) were bilateral. Massive choroidal invasion was the histopathological high-risk factor (HRF) most frequently observed, in 42.2 % of the cases. NOX4 protein was expressed in 67.88 % (74/109) of primary retinoblastoma cases and was confirmed by Western blotting. NOX4 was statistically significant with massive choroidal invasion and pathological TNM staging. There was a statistically significant difference in overall survival in patients with NOX4 expression (p = 0.0461). CONCLUSION: This is the first study to show the expression of NOX4 protein in retinoblastoma tumors. Hence, a retinoblastoma tumor may exhibit greater ROS stress. This protein may prove to be useful as a future therapeutic target for improving the management of retinoblastoma.

NOX1 mediates chemoresistance via HIF1α/MDR1 pathway in gallbladder cancer.

NADPH oxidase 1 (NOX1) plays a key role in tumorigenesis and metastasis through generating reactive oxygen species (ROS), an important intracellular signaling molecule. However, how it is expressed in gallbladder cancer (GBC) tissue sample and whether it associates with GBC chemoresistance have never been investigated. Our study analyzed the relationship between NOX1 expression and cisplatin-sensitivity both in vivo and in vitro. We found that reduced NOX1 expression promoted cisplatin efficiency in GBC-SD cells, whereas overexpression of which potentially inhibited the sensitivity of cisplatin in SGC-996 cells. Further study into the mechanism we found that increased NOX1 expression elevated intracellular ROS levels, which then activated HIF-1α/MDR1 pathway. These findings established NOX1 a novel accelerant of chemoresistance in GBC, and NOX1-targeted therapeutics might be exploited as a strategy for increasing the efficacy of cisplatin treatment.

Bone-Conditioned Medium Changes Gene Expression in Bone-Derived Fibroblasts.

PURPOSE: Autologous bone is used for augmentation in the course of oral implant placement. Bone grafts release paracrine signals that can modulate mesenchymal cell differentiation in vitro. The detailed genetic response of the bone-derived fibroblasts to these paracrine signals has remained elusive. Paracrine signals accumulate in bone-conditioned medium (BCM) prepared from porcine cortical bone chips. MATERIALS AND METHODS: In this study, bone-derived fibroblasts were exposed to BCM followed by a whole genome expression profiling and downstream quantitative reverse transciptase polymerase chain reaction of the most strongly regulated genes. RESULTS: The data show that ADM, IL11, IL33, NOX4, PRG4, and PTX3 were differentially expressed in response to BCM in bone-derived fibroblasts. The transforming growth factor beta (TGF-ß) receptor 1 antagonist SB431542 blocked the effect of BCM on the expression of the gene panel, except for IL33. CONCLUSION: These in vitro results extend existing evidence that cortical bone chips release paracrine signals that provoke a robust genetic response in mesenchymal cells that is not exclusively mediated via the TGF-ß receptor. The present data provide further insights into the process of graft consolidation.

Inhibition of inflammation may enhance nitric oxide availability in patients undergoing bariatric surgery for weight loss.

BACKGROUND: Weight loss surgery is the most effective treatment for morbid obesity. The mechanisms underlying the beneficial cardiovascular effects are poorly understood, although inhibition of inflammatory markers has been demonstrated. We hypothesized that anti-inflammatory and antioxidative stress reactions are responsible for the beneficial effects of bariatric surgery that have been shown in clinical trials. METHODS: The inflammatory system was studied by measuring mRNA levels of E-selectin, tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and in a cell line (HUVEC-CS) of human umbilical vein endothelial cells that were incubated for 4 h with pools of serum, collected before and 3 months after surgery, from 20 women who underwent bariatric surgery for weight loss. The oxidative stress pathway was examined by mRNA expression of NADPH oxidase (P22(phox) ), paraoxonase (PON2), superoxide dismutase 2 (SOD2), glutathione peroxidase (GPx) and catalase following incubation of the cells for 4 h with serum pools. The nitric oxide (NO) pathway was studied by measuring mRNA levels of inducible NOS and endothelial NOS and by determining nitrite and nitrate levels. To study the functional behaviour of endothelial cells under stress, primary human umbilical vein endothelial cells (PECs) were incubated with the serum pools for 48 h, with lipopolysaccharide (LPS) for the last 4 h. RESULTS: The inflammatory system: incubation of HUVEC-CS cells with serum from women who underwent bariatric surgery led to a significant decrease in mRNA expression of E-selectin and IL-6 postsurgery. Stimulation of PECs with LPS in the presence of serum from women who underwent bariatric surgery caused a more significant difference in E-selectin and TNF-α mRNA expression before and after surgery. The antioxidant system: incubation of HUVEC-CS cells with serum from women who underwent bariatric surgery did not lead to any difference in mRNA expression of P22(phox) , PON2, SOD2, GPx or catalase. Stimulation of PECs with LPS showed that obese women had higher levels of P22(phox) , PON2 and the antioxidant enzymes SOD2, GPx and catalase before and after surgery, compared to the control group. The NO pathway: HUVEC-CS cells incubated with serum from women who underwent bariatric surgery secreted higher nitrite/nitrate levels compared to presurgery serum (P = 0.04). CONCLUSIONS: Inhibition of inflammation and enhanced availability of NO 3 months after bariatric surgery could partly explain the beneficial effects of surgery for weight loss.

Relationship between expression of NADPH oxidase 2 and invasion and prognosis of human gastric cancer.

AIM: To assess the expression and prognostic value of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) in gastric cancer, and its correlation with vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). METHODS: Tumor and adjacent tissues were obtained from 123 patients who underwent radical surgery for gastric cancer at Renmin Hospital of Wuhan University from 2008-2009. The expression of NOX2, VEGF, EGFR and CD68 in tumor tissues was detected by immunohistochemistry. The expression of NOX2 in gastric cancer and adjacent tissues was detected by Western blot analysis. Spearman(')s correlation was performed to elucidate the relationship of NOX2 with VEGF and EGFR. The Kaplan-Meier method was used to calculate survival time, and the log-rank test was used to evaluate differences in survival. Cox's proportional hazards regression model was applied in a stepwise manner to analyze the independent prognostic factors. RESULTS: NOX2 exhibited positive expression in 47.2% (58/123) of the gastric cancer tissues. Western blot analysis revealed that NOX2 was up-regulated in tumor tissues compared to the adjacent tissue [39.0% (48/123)]. Immunohistochemistry staining revealed that CD68, which is a specific marker of macrophages, and NOX expression presented a similar localization and staining intensity. The expression of NOX2 was positively correlated with that of VEGF and EGFR. Comparison of the 5-year survival rates of the NOX2 positive and NOX2 negative groups showed that the NOX2 positive group presented a poor prognosis. CONCLUSION: NOX2 positively correlates with the levels of VEGF and EGFR. NOX2 may be used as a new biomarker and a potential therapeutic target for gastric cancer.

Conditioned medium of demineralized freeze-dried bone activates gene expression in periodontal fibroblasts in vitro.

BACKGROUND: Demineralized bone matrix (DBM) is used for the treatment of osseous defects. Conditioned medium from native bone chips can activate transforming growth factor (TGF)-ß signaling in mesenchymal cells. The aim of this study is to determine whether processing of native bone into DBM affects the activity of the conditioned medium. METHODS: Porcine cortical bone blocks were subjected to defatting, different concentrations of hydrochloric acid, and various temperatures. DBM was lyophilized, ground, and placed into culture medium. Human gingiva and periodontal fibroblasts were exposed to the respective conditioned medium obtained from DBM (DBCM). Changes in the expression of TGF-ß target genes were determined. RESULTS: DBCM altered the expression of TGF-ß target genes (e.g., adrenomedullin, pentraxin 3, KN motif and ankyrin repeat domains 4, interleukin 11, NADPH oxidase 4, and BTB [POZ] domain containing 11) by at least five-fold. The response was observed in fibroblasts from both sources. Defatting lowered the activity of DBCM. The TGF-ß receptor type I kinase inhibitor SB431542 [4-(4-(benzo[d][1,3]dioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl)benzamide] but not the inhibitor of bone morphogenetic protein receptor dorsomorphin, blocked the effects of DBCM on gene expression. Moreover, conditioned medium obtained from commercial human DBM modulated the expression of TGF-ß target genes. CONCLUSION: The findings suggest that the DBCM can activate TGF-ß signaling in oral fibroblasts.

Diabetes mellitus tipo 1 e disfunções tireoidianas / Diabetes Mellitus type 1 and thyroid dysfunction

Os pacientes com diabetes mellitus (DM) apresentam maior prevalência de doenças tireoidianas que a população geral. A autoimunidade certamente é um fator-chave na relação entre essas disfunções endócrinas. Entretanto, outros mecanismos, como redução da captação de iodeto, da atividade tireoperoxidase e aumento do estresse oxidativo na glândula tireoide, também parecem contribuir para este fato. O presente trabalho visa rever aspectos importantes na relação entre DM e doenças tireoidianas, com especial ênfase nos mecanismos envolvidos no aumento do estresse oxidativo na glândula tireoide decorrente do DM...

Diabetes mellitus (DM) patients show a greater prevalence of thyroid disorders than general population. Autoimmunity is a key factor in the relation between these endocrine diseases. However, additional mechanisms, such as reduction of iodide uptake and thyroperoxidase activity, besides increased oxidative stress in the thyroid gland seem to contribute for this fact. The present work aims to review important aspects in the relation between DM and thyroid disease, with special emphasis in the mechanisms involved in the increased oxidative stress in the thyroid gland due to DM...