Genetic Epidemiology of Glucose-6-Dehydrogenase Deficiency in the Arab World

A systematic search was implemented using four literature databases (PubMed, Embase, Science Direct and Web of Science) to capture all the causative mutations of Glucose-6-phosphate dehydrogenase (G6PD) deficiency (G6PDD) in the 22 Arab countries. Our search yielded 43 studies that captured 33 mutations (23 missense, one silent, two deletions, and seven intronic mutations), in 3,430 Arab patients with G6PDD. The 23 missense mutations were then subjected to phenotypic classification using in silico prediction tools, which were compared to the WHO pathogenicity scale as a reference. These in silico tools were tested for their predicting efficiency using rigorous statistical analyses. Of the 23 missense mutations, p.S188F, p.I48T, p.N126D, and p.V68M, were identified as the most common mutations among Arab populations, but were not unique to the Arab world, interestingly, our search strategy found four other mutations (p.N135T, p.S179N, p.R246L, and p.Q307P) that are unique to Arabs. These mutations were exposed to structural analysis and molecular dynamics simulation analysis (MDSA), which predicting these mutant forms as potentially affect the enzyme function. The combination of the MDSA, structural analysis, and in silico predictions and statistical tools we used will provide a platform for future prediction accuracy for the pathogenicity of genetic mutations.QUST-CAS-FALL-15/16-24 gran

Fast, ultrasensitive detection of reactive oxygen species using a carbon nanotube based-electrocatalytic intracellular sensor

Herein, we report a highly sensitive electrocatalytic sensor-cell construct that can electrochemically communicate with the internal environment of immune cells (e.g., macrophages) via the selective monitoring of a particular reactive oxygen species (ROS), hydrogen peroxide. The sensor, which is based on vertically aligned single-walled carbon nanotubes functionalized with an osmium electrocatalyst, enabled the unprecedented detection of a local intracellular “pulse” of ROS on a short second time scale in response to bacterial endotoxin (lipopolysaccharide-LPS) stimulation. Our studies have shown that this initial pulse of ROS is dependent on NADPH oxidase (NOX) and toll like receptor 4 (TLR4). The results suggest that bacteria can induce a rapid intracellular pulse of ROS in macrophages that initiates the classical innate immune response of these cells to infection

Proteasome Dysfunction Associated to Oxidative Stress and Proteotoxicity in Adipocytes Compromises Insulin Sensitivity in Human Obesity

AIMS: Obesity is characterized by a low-grade systemic inflammatory state and adipose tissue (AT) dysfunction, which predispose individuals to the development of insulin resistance (IR) and metabolic disease. However, a subset of obese individuals, referred to as metabolically healthy obese (MHO) individuals, are protected from obesity-associated metabolic abnormalities. Here, we aim at identifying molecular factors and pathways in adipocytes that are responsible for the progression from the insulin-sensitive to the insulin-resistant, metabolically unhealthy obese (MUHO) phenotype. RESULTS: Proteomic analysis of paired samples of adipocytes from subcutaneous (SC) and omental (OM) human AT revealed that both types of cells are altered in the MUHO state. Specifically, the glutathione redox cycle and other antioxidant defense systems as well as the protein-folding machinery were dysregulated and endoplasmic reticulum stress was increased in adipocytes from IR subjects. Moreover, proteasome activity was also compromised in adipocytes of MUHO individuals, which was associated with enhanced accumulation of oxidized and ubiquitinated proteins in these cells. Proteasome activity was also impaired in adipocytes of diet-induced obese mice and in 3T3-L1 adipocytes exposed to palmitate. In line with these data, proteasome inhibition significantly impaired insulin signaling in 3T3-L1 adipocytes. INNOVATION: This study provides the first evidence of the occurrence of protein homeostasis deregulation in adipocytes in human obesity, which, together with oxidative damage, interferes with insulin signaling in these cells. CONCLUSION: Our results suggest that proteasomal dysfunction and impaired proteostasis in adipocytes, resulting from protein oxidation and/or misfolding, constitute major pathogenic mechanisms in the development of IR in obesity.IMIBIC/Universidad de Córdoba-SCAI (ProteoRed, PRB2-ISCIII)MINECO/FEDERJunta de Andalucía/FEDERCIBERobn(Instituto de Salud Carlos III

Effects of Exendin-4 on human adipose tissue inflammation and ECM remodelling

Subjects with type-2 diabetes are typically obese with dysfunctional adipose tissue (AT). Glucagon-like peptide-1 (GLP-1) analogues are routinely used to improve glycaemia. Although, they also aid weight loss that improves AT function, their direct effect on AT function is unclear. To explore GLP-1 analogues’ influence on human AT’s cytokine and extracellular matrix (ECM) regulation, we therefore obtained and treated omental (OMAT) and subcutaneous (SCAT) AT samples with Exendin-4, an agonist of the GLP-1 receptor (GLP-1R)Final publishe

Butyrate Attenuates Lipopolysaccharide-Induced Inflammation in Intestinal Cells and Crohn's Mucosa through Modulation of Antioxidant Defense Machinery

Oxidative stress plays an important role in the pathogenesis of inflammatory bowel disease (IBD), including Crohn's disease (CrD). High levels of Reactive Oxygen Species (ROS) induce the activation of the redox-sensitive nuclear transcription factor kappa-B (NF-κB), which in turn triggers the inflammatory mediators. Butyrate decreases pro-inflammatory cytokine expression by the lamina propria mononuclear cells in CrD patients via inhibition of NF-κB activation, but how it reduces inflammation is still unclear. We suggest that butyrate controls ROS mediated NF-κB activation and thus mucosal inflammation in intestinal epithelial cells and in CrD colonic mucosa by triggering intracellular antioxidant defense systems. Intestinal epithelial Caco-2 cells and colonic mucosa from 14 patients with CrD and 12 controls were challenged with or without lipopolysaccaride from Escherichia Coli (EC-LPS) in presence or absence of butyrate for 4 and 24 h. The effects of butyrate on oxidative stress, p42/44 MAP kinase phosphorylation, p65-NF-κB activation and mucosal inflammation were investigated by real time PCR, western blot and confocal microscopy. Our results suggest that EC-LPS challenge induces a decrease in Gluthation-S-Transferase-alpha (GSTA1/A2) mRNA levels, protein expression and catalytic activity; enhanced levels of ROS induced by EC-LPS challenge mediates p65-NF-κB activation and inflammatory response in Caco-2 cells and in CrD colonic mucosa. Furthermore butyrate treatment was seen to restore GSTA1/A2 mRNA levels, protein expression and catalytic activity and to control NF-κB activation, COX-2, ICAM-1 and the release of pro-inflammatory cytokine. In conclusion, butyrate rescues the redox machinery and controls the intracellular ROS balance thus switching off EC-LPS induced inflammatory response in intestinal epithelial cells and in CrD colonic mucosa

A combination of ascorbic acid and α-tocopherol to test the effectiveness and safety in the fragile X syndrome: study protocol for a phase II, randomized, placebo-controlled trial

BACKGROUND: Fragile X syndrome (FXS) is an inherited neurodevelopmental condition characterised by behavioural, learning disabilities, phisical and neurological symptoms. In addition, an important degree of comorbidity with autism is also present. Considered a rare disorder affecting both genders, it first becomes apparent during childhood with displays of language delay and behavioural symptoms. Main aim: To show whether the combination of 10 mg/kg/day of ascorbic acid (vitamin C) and 10 mg/kg/day of α-tocopherol (vitamin E) reduces FXS symptoms among male patients ages 6 to 18 years compared to placebo treatment, as measured on the standardized rating scales at baseline, and after 12 and 24 weeks of treatment. Secondary aims: To assess the safety of the treatment. To describe behavioural and cognitive changes revealed by the Developmental Behaviour Checklist Short Form (DBC-P24) and the Wechsler Intelligence Scale for Children–Revised. To describe metabolic changes revealed by blood analysis. To measure treatment impact at home and in an academic environment. METHODS/DESIGN: A phase II randomized, double-blind pilot clinical trial. Scope: male children and adolescents diagnosed with FXS, in accordance with a standardized molecular biology test, who met all the inclusion criteria and none of the exclusion criteria. Instrumentation: clinical data, blood analysis, Wechsler Intelligence Scale for Children–Revised, Conners parent and teacher rating scale scores and the DBC-P24 results will be obtained at the baseline (t0). Follow up examinations will take place at 12 weeks (t1) and 24 weeks (t2) of treatment. DISCUSSION: A limited number of clinical trials have been carried out on children with FXS, but more are necessary as current treatment possibilities are insufficient and often provoke side effects. In the present study, we sought to overcome possible methodological problems by conducting a phase II pilot study in order to calculate the relevant statistical parameters and determine the safety of the proposed treatment. The results will provide evidence to improve hyperactivity control and reduce behavioural and learning problems using ascorbic acid (vitamin C) and α-tocopherol (vitamin E). The study protocol was approved by the Regional Government Committee for Clinical Trials in Andalusia and the Spanish agency for drugs and health products. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01329770 (29 March 2011

Modulation of IgE-dependent COX-2 gene expression by reactive oxygen species in human neutrophils

Cyclooxygenase (COX) is a key enzyme in prostaglandin (PG) synthesis. Up-regulation of its COX-2 isoform is responsible for the increased PG release, taking place under inflammatory conditions, and also, is thought to be involved in allergic and inflammatory diseases. In the present work, we demonstrate that COX-2 expression becomes highly induced by anti-immunoglobulin E (IgE) antibodies and by antigens in human neutrophils from allergic patients. This induction was detected at mRNA and protein levels and was accompanied by a concomitant PGE2 and thromboxane A2 release. We also show evidence that inhibitors of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, such as 4-(2-aminoethyl)benzenesulphonyl fluoride and 4-hydroxy-3-methoxyaceto-phenone, completely cancelled anti-IgE-induced COX-2 protein up-regulation, suggesting that this process is mediated by reactive oxygen species (ROS) derived from NADPH oxidase activity. Moreover, the mitogen-activated protein kinases (MAPKs), p38 and extracellular signal-regulated kinase, and also, the transcription factor, nuclear factor (NF)-κB, are involved in the up-regulation of COX-2 expression, as specific chemical inhibitors of these two kinases, such as SB203580 and PD098059, and of the NF-κB pathway, such as N(α)-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal, abolished IgE-dependent COX-2 induction. Evidence is also presented, using Fe2+/Cu2+ ions, that hydroxyl radicals generated from hydrogen peroxide through Fenton reactions could constitute candidate modulators able to directly trigger anti-IgE-elicited COX-2 expression through MAPK and NF-κB pathways. Present results underscore a new role for ROS as second messengers in the modulation of COX-2 expression by human neutrophils in allergic conditions

Histamine production by human neutrophils

Histamine is an important mediator in the development of allergic reactions. Only a small subset of human cell types is able to produce histamine. No previous studies have shown that human neutrophils are among them. The present work was undertaken to analyze whether human neutrophils produce histamine, and to determine what agonists are involved in histamine production by human neutrophils. The expression of histidine decarboxylase in human neutrophils was established by quantitative PCR, Western blotting, and flow cytometry analysis. The activity of the enzyme was determined by ELISA, which measured histamine in the culture supernatant of neutrophils stimulated with a set of classical agonists. Human neutrophils are bona fide histamine-producing cells. Neutrophils store ∼0.29 pg/cell and release ∼50% of the histamine content in an antigen-dependent manner and on stimulation with other neutrophil agonists. Basal expression of histidine decarboxylase, the rate-limiting enzyme in histamine production, is higher in neutrophils from patients with allergies than from healthy donors. Our results cannot be ascribed to cell contamination for several reasons. LPS failed to induce histamine release by basophils, whereas it induced histamine release by neutrophils; and we did not detect basophils, monocytes, or lymphocytes in our neutrophil preparations. Eosinophils, albeit detected, were only 0.001-0.004% of the final cell population, and they did not store or release histamine on antigen or LPS stimulation. Antigens to which patients with allergies were sensitized stimulated release of histamine from neutrophils. These observations represent a novel view of neutrophils as possible source of histamine in the allergic diseases.—Alcañiz, L., Vega, A., Chacón, P., El Bekay, R., Ventura, I., Aroca, R., Blanca, M., Bergstralh, D. T., Monteseirin, J. Histamine production by human neutrophils