Dual Oxidase Research Articles

DUOX2 hotspots variants and outcomes of patients with congenital hypothyroidism suspected thyroid dyshormonogenesis

Objective To investigate prospectively molecular and clinical characteristics of infants with congenital hypothyroidism (CH) caused by DUOX2 mutations in Guangzhou. Methods A population-based cohort of 83 patients with CH were recruited based on newborn screening results among 108 899 newborns who were born in Guangzhou between April 1 and September 30 in 2015.Genetic analysis of DUOX2 hotspots(including 11 exons)by PCR-direct sequencing was performed in 52 patients with suspected thyroid dyshormonogenesis (SDH) according to thyroid ultrasound at diagnosis.All the patients were followed up for 3 years.The data of this cohort study(prevalence of CH, detection rate of DUOX2, clinical features) were compared with those of 96 patients with SDH in 2011-2012. Results (1) The incidence of CH in 2015 was 1∶1 312, and 73.5%(61/83 cases) of CH patients were classified as SDH.Compared with those founded in 2011-2012, the incidence of CH was increased (1∶1 312 vs.1∶2 779), and the difference was significant (P 0.05). (3) There were no significant differences in the levels of thyrotropin (bsTSH), serum TSH (sTSH), free thyroxine (FT4) and thyroglobulin in neonates with dry blood spot at diagnosis between children with DUOX2 and without DUOX2 variants cases(all P>0.05). Among 27 cases, 24(88.9%) patients with DUOX2 mutation were transient CH, and 3 cases were permanent CH. Conclusions The incidence of CH was increased in last few years in Guangzhou.Most of them were SDH, and 51.9% of SDH cases had DUOX2 hotspots variants.Temporary CH is the main clinical outcome.The p. K530X was the most common mutation in this cohort population. Key words: Congenital hypothyroidism; Thyroid dyshormonogenesis; DUOX2 gene; Newborn screening

Neurotensin receptors regulate transactivation of the EGFR and HER2 in a reactive oxygen species-dependent manner

Neurotensin is a 13 amino acid peptide which is present in many lung cancer cell lines. Neurotensin binds with high affinity to the neurotensin receptor 1, and functions as an autocrine growth factor in lung cancer cells. Neurotensin increases tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and the neurotensin receptor 1 antagonist SR48692 blocks the transactivation of the EGFR. Here the effects of reactive oxygen species on the transactivation of the EGFR and HER2 were investigated. Using non-small cell lung cancer (NSCLC) cell lines, neurotensin receptor 1 mRNA and protein were present. Using NCI–H838 cells, neurotensin or neurotensin8−13 but not neurotensin1−8 increased EGFR, ERK and HER2 tyrosine phosphorylation which was blocked by SR48692. Neurotensin addition to NCI–H838 cells increased significantly reactive oxygen species which was inhibited by SR48692, Tiron (superoxide scavenger) and diphenylene iodonium (DPI inhibits the ability of NADPH oxidase and dual oxidase enzymes to produce reactive oxygen species). Tiron or DPI impaired the ability of neurotensin to increase EGFR, ERK and HER2 tyrosine phosphorylation. Neurotensin stimulated NSCLC cellular proliferation whereas the growth was inhibited by SR48692, DPI or lapatinib (lapatinib is tyrosine kinase inhibitor of the EGFR and HER2). Lapatinib inhibited the ability of the neurotensin receptor 1 to transactivate the EGFR and HER2. The results indicate that neurotensin receptor 1 regulates the transactivation of the EGFR and HER2 in a reactive oxygen species-dependent manner.

High throughput proteomic analysis and machine learning algorithm identifies DUOX2 (dual oxidase 2) as a novel biomarker for response prediction of concurrent chemoradiotherapy for locally advanced rectal cancer.

71 Background: Although many efforts to predict treatment response of concurrent chemoradiotherapy (CCRT) for locally advanced rectal cancer (LARC) have been made, no molecular has proved to be a robust biomarker. Methods: We performed mass spectrometry-based quantitative proteomic analysis of pretreatment Formalin-fixed, Paraffin-embedded (FFPE) biopsy samples of 13 patients with LARC, who were treated with CCRT followed by curative surgery. Based on pathologic report of surgical specimens, we divided thirteen patients as two response groups: complete response (CR) and non-complete response (nCR) groups. Results: A total of 3,637 proteins were identified and 498 proteins were confirmed as expressed at significantly different levels (DEPs; differently expressed proteins) between these two groups. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were also performed: the result showed that up-regulated DEPs enriched in biological processes (BP) were significantly different between two groups; immune response, cell migration & motility, protein transport in CR group; amide/peptide biosynthetic process, translation, posttranscriptional regulation of gene expression and detoxification in nCR group. To identify the best classifier to evaluate predictive power of signatures, we employed for different machine learning algorithms to classify samples between CR and nCR groups. As a result, we identified the predictive relevance of dual oxidase 2 (DUOX2) as the strongest predictive biomarker. Conclusions: This study identified a new biomarker, DUOX2, applicable to discrimination between CR and nCR after NACRT for LARC. To our knowledge, the present study provides the first identification of a clinical biomarker for response prediction based on in-depth proteomics and machine learning algorithms.

Oxidative stress pathway gene transcription after bovine respiratory syncytial virus infection in vitro and ex vivo

Studies in mouse and lamb models indicate important roles of reactive oxygen species (ROS) in the pathology and immune response to respiratory syncytial virus (RSV). The role of ROS in bovine RSV (BRSV) infection of calves remains unclear. BRSV naturally infects calves, leading to similar disease course, micro- and macro-lesions, and symptomology as is observed in RSV infection of human neonates. Furthermore, humans, lambs, and calves, but not mice, have an active lung oxidative system involving lactoperoxidase (LPO) and the dual oxidases (DUOX) 1 and 2. To gain insight into the role of ROS in the BRSV-infected lung, we examined gene expression in infected bovine cells using qPCR. A panel of 19 primers was used to assay ex vivo and in vitro BRSV-infected cells. The panel targeted genes involved in both production and regulation of ROS. BRSV infection significantly increased transcription of five genes in bovine respiratory tract cells in vitro and ex vivo. PTGS2 expression more than doubled in both sample types. Four transcripts varied significantly in lung lesions, but not non-lesion samples, compared with uninfected lung. This is the first report of the transcriptional profile of ROS-related genes in the airway after BRSV infection in the natural host.

Cell-culture growth conditions resulting in the oxidation of a recombinant antigen-binding fragment

Use of Quality-by-Design (QbD) tools is becoming an important part of the bioprocessing industry when developing a process for manufacturing operations to ensure the robustness and reproducibility of the biologic product. In the present study, a QbD tool, Design of Experiments (DOE), was utilized to optimize a bioprocess for the production of a CHO recombinant antigen-binding fragment (rFab) in small-scale bioreactors. DOE studies evaluated percent dissolved oxygen, temperature, and feeding strategy specific to this Chinese Hamster Ovary (CHO) clone. It was determined that these factors influenced cell viability, yield of the recombinant protein, and metabolic byproduct formation. To ensure the quality of the target molecule in the cell-culture process, small-scale purifications and analytical evaluation of the target molecule were completed prior to cell-culture scale-up to ensure that oxidation of the rFab, presence of free light chain, and truncation of thiol group were not observed. Analysis of the purified rFab by mass spectrometry indicated that rFab oxidation occurred under poor cell-culture conditions. PCR profile array results also revealed increased transcription of the oxidative genes Superoxide Dismutase 3, Myeloperoxidase, Dual Oxidase Like 2, Nuclear Receptor Coactivator 7, NADPH Oxidase Organizer 1, Mitochondria Uncouple Protein 3, Eosinophil Peroxidase, Lactoperoxidase Like, Serum Albumin Like, and Glutathione S-Transferase Pi 1 in this CHO strain. The present study suggests a mechanism and pathway for the oxidation of an rFab molecule during cell-culture bioprocess optimization. The present study also demonstrated the importance of utilizing the QbD tool of DOE to optimize the cell-culture bioprocess prior to scaling up into the large-scale production bioreactor.

The Dual Oxidase Duox2 stabilized with DuoxA2 in an enzymatic complex at the surface of the cell produces extracellular H2O2 able to induce DNA damage in an inducible cellular model

Thyroid hormone synthesis requires H2O2, produced by two NADPH oxidases, Duox1 and Duox2. To be fully active at the apical pole of the thyrocytes, these enzymes need additional maturation factors DuoxA1 and DuoxA2. The proteins have been shown to be localized at the cell surface, suggesting that they could form a complex with Duox counterparts. We have generated multiple HEK293 Tet-On3G cell lines that express various combinations of DuoxA upon doxycycline induction, in association with a constitutive expression of the Duox enzyme. We compared Duox specific activity, Duox/DuoxA cell surface interactions and the cellular consequences of sustained H2O2 generation. By normalizing H2O2 extracellular production by Duox or DuoxA membrane expression, we have demonstrated that the most active enzymatic complex is Duox2/DuoxA2, compared to Duox1/DuoxA1. A direct cell surface interaction was shown between Duox1/2 and both DuoxA1 and DuoxA2 using the Duolink® technology, Duox1/DuoxA1 and Duox2/DuoxA2 membrane complexes being more stable than the unpaired ones. A significant increase in DNA damage was observed in the nuclei of Duox2/DuoxA2 expressing cells after doxycycline induction and stimulation of Duox catalytic activity. The maturation and activity of Duox2 were drastically impaired when expressed with the glycosylation-defective maturation factor DuoxA2, while the impact of the unglycosylated DuoxA1 mutant on Duox1 membrane expression and activity was rather limited. The present data demonstrate for the first time that H2O2 produced by the Duox2/DuoxA2 cell surface enzymatic complex could provoke potential mutagenic DNA damage in an inducible cellular model, and highlight the importance of the co-expressed partner in the activity and stability of Duox/DuoxA complexes.

Novel pendrin inhibitor attenuates airway hyperresponsiveness and mucin expression in experimental murine asthma

Novel pendrin inhibitor attenuates airway hyperresponsiveness and mucin expression in experimental murine asthma

Oxidative stress and related gene expression effects of cyfluthrin in human neuroblastoma SH-SY5Y cells: Protective effect of melatonin

This study was designed to assess oxidative stress induction in human neuroblastoma SH-SY5Y cells in response to cyfluthrin exposure. Cell viability MTT assay was carried out to assess cyfluthrin cytotoxicity; IC30 and IC50 values for cyfluthrin were calculated to be 4.81 ± 0.92 μM and 19.39 ± 3.44 μM, respectively. Cyfluthrin induced a significant increase in ROS generation, lipid peroxides measured as malondialdehyde (MDA) and nitric oxide (NO) production and a significant decrease in NQO1 activity. The antioxidant activity of melatonin (MEL), Trolox, N-acetylcysteine (NAC) and Sylibin against cyfluthrin-induced oxidative stress was examined. Cyfluthrin increased significantly gene expressions of apoptosis, proinflammation and oxidative stress (Bax, Bcl-2, Casp-3, BNIP3, AKT1, p53, APAF1, NFκB1, TNFα and Nrf2) mediators. In the most genes, the mRNA levels induced by cyfluthrin were partially reduced by MEL (1 μM). Cyfluthrin effects on gene expression profiling of oxidative stress pathway by Real-Time PCR array analysis showed that of the 84 genes examined, (fold change > 1.5) changes in mRNA levels were detected in 31 genes: 13 upregulated and 18 down-regulated. A fold change >3.0 fold was observed on upregulated CYBB, DUOX1, DUOX2, AOX1, BNIP3, HSPA1A, NOS2, and NQO1 genes. The greater fold change reversion (2.5 fold) by MEL (1 μM) was observed on cyfluthrin-upregulated CYBB, AOX1, BNIP3 and NOS2 genes. These results demonstrated that oxidative stress is a key element in cyfluthrin induced neurotoxicity as well as MEL may play a role in reducing cyfluthrin-induced oxidative stress.

Abstract 514: DUOX2-related H2O2 production contributes to pro-inflammatory cytokine-related DNA damage and tumor cell growth in models of pancreatic ductal adenocarcinoma

Abstract Chronic inflammation is an important risk factor for the development of pancreatic ductal adenocarcinoma (PDAC). Dual oxidase 2 (DUOX2), one of the seven NADPH oxidase (NOX) family members, by generating extracellular H2O2, is intimately involved in both host defense and chronic inflammation in the gastrointestinal tract. Previously, we demonstrated that short (24 h) exposures of several human pancreatic cancer cell lines to pro-inflammatory cytokines (IFN-γ, IL-4/IL-13, IL-17A) can effectively enhance expression of DUOX2 and its maturation factor DUOXA2. To better mimic chronic inflammation in vivo, a panel of human PDAC cell lines was exposed to the TH2 cytokine IL-4 (50ng/ml) for longer times (7 days); and the effect of cytokine treatment on Stat6 activation and DUOX2 mRNA and protein expression was explored. In BxPC-3 and AsPC-1 cell lines, prolonged IL-4 stimulation results in a sustained activation of Stat6, dramatic induction of DUOX2 mRNA and protein expression, a 3-5-fold increase in extracellular H2O2 production compared to solvent-treated cells, and–most importantly–a robust DNA double strand break (DSB) response indicated by elevated levels of γH2AX. In marked contrast, for two JAK-Stat6 signaling defective PDAC lines, PANC-1 and CFPAC-1, no enhancement of DUOX2 expression with concomitant DSBs was observed under the same conditions. Moreover, when the anti-inflammatory drug dexamethasone (Dex) was co-administered with IL-4 (for 48h), cytokine-related DUOX2 induction and DNA damage in BxPC-3 and AsPC-1 cells was significantly diminished. Furthermore, in the KPC GEMM of PDAC, RT-PCR and immunohistochemistry revealed that DUOX2 mRNA and protein expression in pancreatic tumor tissues were significantly increased compared to adjacent normal tissues and were associated with areas of chronic inflammation. Finally, xenografts established with BxPC-3 cell clones in which DUOX2 mRNA was silenced by stable expression of DUOX2-specific shRNAs demonstrated severely delayed growth in vivo compared to clones stably expressing scrambled shRNAs. These studies, coupled with our previous data demonstrating elevated DUOX protein expression in pancreatitis, pancreatic intra-epithelial neoplasia (PanIN), and frank pancreatic cancers in humans, suggest that ROS- derived from cytokine-mediated DUOX2 up-regulation may initiate and promote pancreatic cancer progression through ROS-induced DNA damage. Citation Format: Yongzhong Wu, Jiamo Lu, Smitha Antony, Jennifer L. Meitzler, Agnes Juhasz, Guojian Jiang, Iris Dahan, James H. Doroshow. DUOX2-related H2O2 production contributes to pro-inflammatory cytokine-related DNA damage and tumor cell growth in models of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 514.

Radiation-Induced Dual Oxidase Upregulation in Rat Heart Tissues: Protective Effect of Melatonin.

Background: Radiation-induced heart injury can lead to increased risk of heart failure, attack, and ischemia. Some studies proposed IL-4 and IL-13 as two important cytokines that are involved in late effects of ionizing radiation. On the other hand, these cytokines may, through upregulation of Duox1 and Duox2, induce chronic oxidative stress, inflammation, and fibrosis. In this study, we evaluated the upregulation of Duox1 and Duox2 pathways in hearts following chest irradiation in rats and then detected possible attenuation of them by melatonin. Materials and Methods: Twenty male Wistar rats were divided into four groups: (1) control; (2) melatonin treated (100 mg/kg); (3) radiation (15 Gy gamma rays); (4) melatonin treated before irradiation. All rats were sacrificed after 10 weeks and their heart tissues collected for real-time PCR (RT-PCR), ELISA detection of IL-4 and IL-13, as well as histopathological evaluation of macrophages and lymphocytes infiltration. Results: Results showed an upregulation of IL-4, IL4ra1, Duox1, and Duox2. The biggest changes were for IL4ra1 and Duox1. Treatment with melatonin before irradiation could attenuate the upregulation of all genes. Melatonin also caused a reduction in IL-4 as well as reverse infiltration of inflammatory cells. Conclusion: Duox1 and Duox2 may be involved in the late effects of radiation-induced heart injury. Also, via attenuation of these genes, melatonin can offer protection against the toxic effects of radiation on the heart.

TGF-β Signaling Interferes With the Drosophila Innate Immune and Metabolic Response to Parasitic Nematode Infection.

The common fruit fly, Drosophila melanogaster, is an outstanding model to study the molecular basis of anti-pathogen immunity. The parasitic nematode Heterorhabditis gerrardi, together with its mutualistic bacteria Photorhabdus asymbiotica, infects a wide range of insects, including D. melanogaster. Recently, we have shown that transforming growth factor-β (TGF-ß) signaling in D. melanogaster is regulated in response to parasitic nematode infection. In the current study, we investigated the contribution of two TGF-ß signaling branches, the activin and the bone morphogenetic protein (BMP), to D. melanogaster immune function against H. gerrardi. We used D. melanogaster larvae carrying mutations in the genes coding for the TGF-ß extracellular ligands daw and dpp. We have demonstrated that the number of circulating hemocytes in uninfected daw and dpp mutants decreases twofold compared to background controls, yet no significant changes in hemocyte numbers and survival of the TGF-ß mutants are observed upon nematode infection. However, we have shown that nematode-infected daw mutants express Dual oxidase at higher levels and phenoloxidase activity at lower levels compared to their background controls. To elucidate the contribution of TGF-ß signaling in the metabolic response of D. melanogaster to parasitic nematodes, we estimated lipid and carbohydrate levels in daw and dpp mutant larvae infected with H. gerrardi. We have found that both nematode-infected mutants contain lipid droplets of larger size, with daw mutant larvae also containing elevated glycogen levels. Overall, our findings indicate that the regulation of activin and BMP branches of TGF-ß signaling can alter the immune and metabolic processes in D. melanogaster during response to parasitic nematode infection. Results from this study shed light on the molecular signaling pathways insects activate to regulate mechanisms for fighting potent nematode parasites, which could lead to the identification of novel management strategies for the control of damaging pests.

The depletion of p38alpha kinase upregulates NADPH oxidase 2/NOX2/gp91 expression and the production of superoxide in mouse embryonic stem cells

P38alpha kinase plays an important role in the regulation of both cell stress response and cell fate. In this study, we report that p38alpha kinase-deficient embryonic stem cells exhibit a higher production of reactive oxygen species (ROS) in contrast to their wild-type counterpart. Analysis of the expressions of NADPH oxidases (NOXs) and dual oxidases, crucial enzymes involved in intracellular ROS formation, shows NOX2/gp91phox is over-expressed in p38alpha deficient cells. The particular increase in superoxide formation was confirmed by the specific detection of hydroethidine derivate 2-hydroxyethidium. ROS formation decreased when the level of NOX2 was silenced by siRNA in p38alpha deficient cells. These data suggest the importance of p38alpha kinase in the regulation of ROS metabolism in embryonic stem cells and the significance of the observed phenomena of cancer cell-like phenotypes, which is discussed.

DUOX2/DUOXA2 Mutations Frequently Cause Congenital Hypothyroidism that Evades Detection on Newborn Screening in the United Kingdom.

Background: The etiology, course, and most appropriate management of borderline congenital hypothyroidism (CH) are poorly defined, such that the optimal threshold for diagnosis with bloodspot screening thyrotropin (bsTSH) measurement remains controversial. Dual oxidase 2 (DUOX2) mutations may initially cause borderline elevation of bsTSH, which later evolves into significant hypothyroidism on venous blood measurement. It was hypothesized that mutations in both DUOX2 and its accessory protein DUOXA2 may occur frequently, even in patients with borderline bsTSH elevation, such that higher diagnostic thresholds in bsTSH screening may fail to detect such cases, with consequent risk of undiagnosed neonatal hypothyroidism of sufficient magnitude to require thyroxine therapy. This study aimed to investigate the frequency and characteristics of DUOX2 and DUOXA2 mutations in a borderline CH cohort.Methods: A cross-sectional study of patients with borderline CH was undertaken at Great Ormond Street Hospital, a tertiary British pediatric center. DUOX2 was sequenced in 52 patients with a bsTSH of 6–19.9 mIU/L, venous TSH of >25 mIU/L, and eutopic thyroid gland in situ. DUOXA2 was sequenced in DUOX2 mutation-negative cases, and novel DUOXA2 mutations were functionally characterized.Results: A total of 26 (50%) patients harbored likely pathogenic mutations in DUOX2 (n = 20; 38%) or DUOXA2 (n = 6; 12%), including novel gene variants (DUOX2, n = 3; DUOXA2, n = 7). Two recurrent DUOX2 mutations (p.Q570L, p.F966Sfs*29) occurred frequently in population databases (MAF ≥0.01). Despite bsTSH being <10 mIU/L in 46% of DUOX2 and DUOXA2 mutation-positive cases, venous free thyroxine levels in these patients were in the moderate CH range (M = 9.3 pmol/L, range <3.9–15.8 pmol/L),Conclusions: Targeted DUOX2 and DUOXA2 sequencing in a borderline CH cohort has a high diagnostic yield. These findings might argue for a lowering of bsTSH thresholds, but follow-up studies are required to assess whether cases with borderline bsTSH harboring DUOX2/DUOXA2 mutations will benefit from an early diagnosis and subsequent levothyroxine treatment.

Dysregulation of DNA methylation patterns may identify patients with breast cancer resistant to endocrine therapy: A predictive classifier based on differentially methylated regions.

Endocrine therapy (ET) is one of a number of targeted therapies for estrogen receptor-positive breast cancer (BRCA); however, resistance to ET has become the primary issue affecting treatment outcome. In the present study, a predictive classifier was created using a DNA methylation dataset to identify patients susceptible to endocrine resistance. DNA methylation and RNA sequencing data, and the clinicopathological features of BRCA, were obtained from The Cancer Genome Atlas. Stringent criteria were set to select and classify patients into two groups, namely those resistant to ET (n=11) and sensitive to ET (n=21) groups. Bump hunting analysis revealed that 502 out of 135,418 genomic regions were differentially methylated between these two groups; these regions were differentially methylated regions (DMRs). The majority of the CpG sites contained in the DMRs mapped to the promoter region. Functional enrichment analyses indicated that a total of 562 specific genes encompassing these DMRs were primarily associated with ‘biological progress of organ morphogenesis and development’ and ‘cell-cell adhesion’ gene ontologies. Logistic regression and Pearson's correlation analysis were conducted to construct a predictive classifier for distinguishing patients resistant or sensitive to ET. The highest areas under the curve and relatively low Akaike information criterion values were associated with a total of 60 DMRs; a risk score retained from this classifier was revealed to be an unfavorable predictor of survival in two additional independent datasets. Furthermore, the majority of genes (55/63) exhibited a statistically significant association between DNA methylation and mRNA expression (P<0.05). The association between the mRNA expression of a number of genes (namely calcium release activated channel regulator 2A, Schlafen family member 12, chromosome 3 open reading frame 18, zinc finger protein 880, dual oxidase 1, major histocompatibility complex, class II, DP β1, C-terminal binding protein 1, ALG13 UDP-N-acetylglucosaminyltransferase subunit and RAS protein activator like 2) and the prognosis of patients with estrogen receptor-positive BRCA and ET resistance was determined using Kaplan-Meier Plotter. In summary, the predictive classifier proposed in the present study may aid the identification of patients sensitive or resistant to ET, and numerous genes maybe potential therapeutic targets to delay the development of resistance to ET.

Serum NADPH oxidase concentrations and the associations with iron metabolism in relapsing remitting multiple sclerosis

BackgroundOverproduction of reactive oxygen species (ROS) and impaired iron metabolism are considered to be possible factors in the pathogenesis of Multiple sclerosis (MS). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are the primary sources of regulated ROS production. The NADPH oxidase (NOX) family consists of seven catalytic homologues, NOX1–5 and two dual oxidases. NOX1 and NOX5 are associated with endothelial dysfunction and inflammation but NOX4 has a protective effect on vascular function. The aims of this study were to investigate the status of NOX1, NOX4 and NOX5 and its relationship with serum iron metabolism biomarkers in relapsing-remitting MS patients. MethodsThe study included 53 RRMS patients and 45 control subjects. Serum NOX1,4,5, ferritin, iron, unbound-iron binding capacity, C-reactive protein (CRP), white blood count (WBC) and erythrocyte sedimentation rate (ESR) levels were measured in all the study subjects. ResultsHigher serum NOX5 (p < 0.0001), CRP (p = 0.014), ferritin (p = 0.040) and lower serum NOX4 (p < 0.0001) and iron (p = 0.013) concentrations were found in the patients than in controls. No correlation was found between NOXs, CRP, WBC, ESR and iron metabolism biomarkers in patients. ConclusionOur data suggest that increased NOX5 expression and decreased levels of NOX4 might be related with oxidative stress related vascular changes in MS patients. These findings provide future opportunities to combat MS with separately target individual NOX isoforms.

H2O2 Metabolism in Normal Thyroid Cells and in Thyroid Tumorigenesis: Focus on NADPH Oxidases.

Thyroid hormone synthesis requires adequate hydrogen peroxide (H2O2) production that is utilized as an oxidative agent during the synthesis of thyroxin (T4) and triiodothyronine (T3). Thyroid H2O2 is generated by a member of the family of NADPH oxidase enzymes (NOX-es), termed dual oxidase 2 (DUOX2). NOX/DUOX enzymes produce reactive oxygen species (ROS) as their unique enzymatic activity in a timely and spatially regulated manner and therefore, are important regulators of diverse physiological processes. By contrast, dysfunctional NOX/DUOX-derived ROS production is associated with pathological conditions. Inappropriate DUOX2-generated H2O2 production results in thyroid hypofunction in rodent models. Recent studies also indicate that ROS improperly released by NOX4, another member of the NOX family, are involved in thyroid carcinogenesis. This review focuses on the current knowledge concerning the redox regulation of thyroid hormonogenesis and cancer development with a specific emphasis on the NOX and DUOX enzymes in these processes.

Dual oxidase1 ameliorates survival, viral clearance and pulmonary pathology during influenza infection

Abstract Influenza virus infections cause severe morbidity and mortality, especially in high-risk patient populations. Bronchoepithelial cells orchestrate an oxidative antimicrobial system present in the airway liquid consisting of lactoperoxidase, the thiocyanate ion, and hydrogen peroxide. Dual Oxidase 1 (Duox1), an enzyme highly expressed in these cells, is the source of hydrogen peroxide. While in vitro studies suggest an anti-influenza, and a general antimicrobial role of Duox1, its antiviral role in vivo has not been addressed so far. We hypothesized that Duox1 has a protective effect in vivo against influenza. To test this hypothesis, we infected wild-type C57BL/6 and Duox1-deficient mice intranasally with the mouse-adapted A/Puerto Rico/8/1934 H1N1 (PR8) influenza virus strain. To evaluate the clinical and immunological significance of Duox1 in influenza infection, multicolor flow cytometry, multiplex ELISA, viral titration, histology and immunostaining techniques were used. Our results indicate that Duox1-deficient mice have increased mortality following influenza infection. Weight loss of Duox1-deficient mice post-infection was also significantly higher than in Duox1-expressing animals. Duox1 also limited influenza virus replication in the lung. Characterization of infiltrated leukocyte subsets following influenza infection revealed impaired natural killer cell recruitment in Duox1-deficient animals while other lymphoid or myeloid leukocyte subsets were not affected In summary, our results provide the first evidence for the in vivo antiviral role of Duox1, and also implicate that targeting the Duox1-based system has the potential to provide novel treatment or prophylactic strategies against influenza.

Essential Role of NADPH Oxidase–Dependent Production of Reactive Oxygen Species in Maintenance of Sustained B Cell Receptor Signaling and B Cell Proliferation

Reactive oxygen species (ROS) are not only toxic substances inducing oxidative stress but also play a role as a second messenger in signal transduction through various receptors. Previously, B cell activation was shown to involve prolonged ROS production induced by ligation of BCR. However, the mechanisms for ROS production and ROS-mediated activation in B cells are still poorly understood. In this study, we demonstrate that BCR ligation induces biphasic ROS production in both mouse spleen B cells and the mouse B cell line BAL17; transient and modest ROS production is followed by sustained and robust ROS production at 2-6 h after BCR ligation. ROS production in the late phase but not in the early phase augments activation of signaling pathways, such as the NF-κB and PI3K pathways, and is essential for B cell proliferation. ROS production in the late phase appears to be mediated by NADPH oxidases (NOXes) because prolonged ROS production is inhibited by various NOX inhibitors, including the specific inhibitor VAS2870. BCR ligation-induced ROS production is also inhibited by CRISPR/Cas9-mediated deletion of either the Cyba gene encoding p22phox, the regulator of NOX1-4 required for their activation, or NOX3, whereas ROS production is not affected by double deficiency of the DUOXA1 and DUOXA2 genes essential for the activation of the NOX isoforms DUOX1 and DUOX2. These results indicate that NOXes play a crucial role in sustained but not early BCR signaling and suggest an essential role of NOX-dependent sustained BCR signaling in B cell activation.

MON-270 Diagnostic Exome Sequencing in Children with Permanent Congenital Hypothyroidism

Introduction: Congenital hypothyroidism can be caused by aplasia or hypoplasia of the thyroid gland, ectopic thyroid, or dyshormonogenesis. Thyroid function test, thyroid ultrasonography, and thyroid scintigraphy may be helpful in diagnosis. Molecular genetic approach can be used to identify the genetic causes. In this study, diagnostic exome sequencing (DES) including 23 genes associated with congenital hypothyroidism was performed to identify the genetic causes of congenital hypothyroidism. Methods: The patients who were diagnosed as permanent congenital hypothyroidism and agreed to the genetic test were included in the study. We reviewed the clinical information, laboratory tests, and imaging findings based on medical records, retrospectively. Genetic test was performed with an informed consent. Results: A total of 26 children were included in the study. Twelve (46%) were boys and 14 (54%) were girls (M:F 1:1.16). Morphologically, there were 1 (3%) hypoplasia and 1 (3%) agenesis. Twelve (46%) showed ectopic thyroid, and 12 (46%) had normally positioned thyroid. Eighteen (69%) patients were diagnosed within one month of age, and 8 (31%) were diagnosed after one month later. Initial thyroid function test revealed 112.68±55 ng/dL of T3, 0.71±0 ng/dL of fT4, and 77.26±112uIU/mL of TSH. Eighteen (69%) showed overt hypothyroidism and 8 (31%) patients were in subclinical hypothyroidism. On recent follow-up, the mean age was 10.85 years. All of them are taking levothyroxine and the mean levothyroxine dose is 2.15ug/kg/day. In four (15%) patients, pathogenic mutations were found. There were heterozygous mutations of the gene TSHR in 2 patients, compound heterozygous mutation of TG gene in 1 patient, and compound heterozygous mutation of DUOX2 gene in 1 patient. In addition, fifteen (57%) patients showed several VUS (variants of uncertain significance) in at least one of TRH, TRHR, TSHR, SLC26A4, PAX8, TG, TPO, DUOX2, DUOXA2, and POU1F1 genes by DES. Conclusion: We could find pathogenic mutations in 4 patients (15%) among 26 patients with permanent congenital hypothyroidism by DES.

Calcium‐dependent dual oxidase 2 is a novel source of reactive oxygen species implicated in glomerular mesangial cell fibrotic response to angiotensin II

The vasoactive peptide angiotensin II (Ang II) contributes to the initiation and the progression of glomerular fibrosis via activation of glomerular mesangial cells (MCs) and subsequent extracellular matrix expansion. We have previously shown that oxidative stress is critical for MC fibrotic response to Ang II. Here, we demonstrate that Dual oxidase 2 (Duox2), a member of the Nox/Duox family of NADPH oxidases, is present in MCs and that its protein expression is upregulated by Ang II.Small interfering RNA (siRNA)‐mediated downregulation of Duox2 significantly reduces Ang II‐induced increase in reactive oxygen (ROS) generation and prevents the stimulatory effect of Ang II on MC fibrotic injury (as assessed by measuring a‐smooth muscle actin and fibronectin expression).Duox2 activation is Ca2+‐dependent. Next, we investigated the role of Ca2+ in Ang II‐mediated Duox2 activation and the resulting fibrotic response. We show that the extracellular Ca2+ chelator BAPTA prevented Ang II‐induced ROS generation and the stimulation of MC fibrotic injury by Ang II. Moreover, treatment of MCs with ionomycin resulted in increased ROS production and enhanced MC fibrotic injury. These effects were abrogated by siRNA targeting Duox2. These data indicate that Ang II‐stimulated Duox2 activation and ROS generation subsequently lead to MC fibrotic injury.In summary, we have identified a novel role for Duox2 as a major source of ROS in response to Ang II and established the significance of Duox2 in Ang II‐mediated MC activation and fibrotic injury. Therapeutic targeting of this pathway may prevent or reverse pathophysiologic manifestations of renal fibrotic diseasesSupport or Funding InformationThis project was funded by the University of the Incarnate Word, Office of Research and Graduate Studies and the following grants: NRSA 5T32HL007446‐32, NIH 1RO1DK07999601A2, and NIH UL1 TR001120/IIMS Pilot ProjectThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.