Myeloperoxidase Gene Expression Research Articles

Expression of Myeloperoxidase in Patient-Derived Endothelial Colony-Forming Cells-Associations with Coronary Artery Disease and Mitochondrial Function.

Myeloperoxidase (MPO) plays a critical role in the innate immune response and has been suggested to be a surrogate marker of oxidative stress and inflammation, with elevated levels implicated in cardiovascular diseases, such as atherosclerosis and heart failure, as well as in conditions like rheumatoid arthritis and cancer. While MPO is well-known in leukocytes, its expression and function in human endothelial cells remain unclear. This study investigates MPO expression in patient-derived endothelial colony-forming cells (ECFCs) and its potential association with CAD and mitochondrial function. ECFCs were cultured from the peripheral blood of 93 BioHEART-CT patients. MPO expression and associated functions were examined using qRT-PCR, immunochemistry, flow cytometry, and MPO activity assays. CAD presence was defined using CT coronary angiography (CACS > 0). We report MPO presence in patient-derived ECFCs for the first time. MPO protein expression occurred in 70.7% of samples (n = 41) which had nuclear co-localisation, an atypical observation given its conventional localisation in the granules of neutrophils and monocytes. This suggests potential alternative roles for MPO in nuclear processes. MPO mRNA expression was detected in 66.23% of samples (n = 77). CAD patients had a lower proportion of MPO-positive ECFCs compared to non-CAD controls (57.45% vs. 80%, p = 0.04), a difference that persisted in the statin-naïve sub-cohort (53.85% vs. 84.62%, p = 0.02). Non-CAD patients with MPO expression showed upregulated mitochondrial-antioxidant genes (AIFM2, TXNRD1, CAT, PRDX3, PRDX6). In contrast, CAD patients with MPO gene expression had heightened mROS production and mitochondrial mass and decreased mitochondrial function compared to that of CAD patients without MPO gene expression. MPO is present in the nucleus of ECFCs. In non-CAD ECFCs, MPO expression is linked to upregulated mitochondrial-antioxidant genes, whereas in CAD ECFCs, it is associated with greater mitochondrial dysfunction.

The effect of an antioxidant gel compared to chlorhexidine during the soft tissue healing process: An animal study.

Prolonged inflammation and oxidative stress can impede healing. To enhance healing efficiency, many solutions have been employed. This is an in vivo study comparing chlorhexidine (CHX) to a commercial antioxidant gel (AO). Envelope flaps were created in the lower incisor gingival region of 60 Sprague-Dawley rats, and acellular dermal matrix (ADM) was inserted. Animals were randomly assigned to postsurgical treatment application of AO gel or 0.12% CHX twice daily. A control group received no postsurgical treatment. Data collected (before surgery, 24 h, and 72 h) included surgical images, tissue samples, and weights. Blinded scorers assessed images using a wound healing scale. Real-time polymerase chain reaction (RT-PCR) was used for gene expression of tumor necrosis factor-alpha (TNFα), interleukin-1 (IL-1), myeloperoxidase (MPO), and superoxide dismutase (SOD). The AO group scored higher than the CHX and control groups in clinical evaluation (p<0.05). At 24h, TNFα expression was upregulated in the AO group compared to CHX (p=0.027) and controls (p=0.018). The AO group had significantly higher expression of antioxidant enzyme (SOD) at 24h compared to CHX (p=0.021). All animals lost weight in the first 24h. Animals treated with AO or CHX regained more weight at 72h than control animals (p=0.034 and 0.003, respectively). Animals treated with AO healed faster. AO led to earlier upregulation of TNFα and antioxidant enzyme SOD. We hypothesized that AO promoted an earlier inflammatory process while counteracting oxidative stress by increasing antioxidant responses via SOD.

The mitochondrial biliverdin exporter ABCB10 in hepatocytes mitigates neutrophilic inflammation in alcoholic hepatitis

Acute liver failure caused by alcoholic hepatitis (AH) is only effectively treated with liver transplantation. Livers of patients with AH show a unique molecular signature characterized by defective hepatocellular redox metabolism, concurrent to hepatic infiltration of neutrophils that express myeloperoxidase (MPO) and form neutrophil extracellular traps (NETs). Exacerbated NET formation and MPO activity contribute to liver damage in mice with AH and predicts poor prognosis in AH patients. The identification of pathways that maladaptively exacerbate neutrophilic activity in liver could inform of novel therapeutic approaches to treat AH. Whether the redox defects of hepatocytes in AH directly exacerbate neutrophilic inflammation and NET formation is unclear. Here we identify that the protein content of the mitochondrial biliverdin exporter ABCB10, which increases hepatocyte-autonomous synthesis of the ROS-scavenger bilirubin, is decreased in livers from humans and mice with AH. Increasing ABCB10 expression selectively in hepatocytes of mice with AH is sufficient to decrease MPO gene expression and histone H3 citrullination, a specific marker of NET formation. These anti-inflammatory effects can be explained by ABCB10 function reducing ROS-mediated actions in liver. Accordingly, ABCB10 gain-of-function selectively increased the mitochondrial GSH/GSSG ratio and decreased hepatic 4-HNE protein adducts, without elevating mitochondrial fat expenditure capacity, nor mitigating steatosis and hepatocyte death. Thus, our study supports that ABCB10 function regulating ROS-mediated actions within surviving hepatocytes mitigates the maladaptive activation of infiltrated neutrophils in AH. Consequently, ABCB10 gain-of-function in human hepatocytes could potentially decrease acute liver failure by decreasing the inflammatory flare caused by excessive neutrophil activity.

The Effect of Melon Seed Extract on the Transcription of NF-κB and Myeloperoxidase in the Liver Tissue of Mice Poisoned with Ethylene Glycol

Background and Objectives: The liver is exposed to poisoning caused by various chemical compounds. Considering the antioxidant effects found in melon seeds and the role of nuclear factor kappa B (NF-κB) and Myeloperoxidase (MPO) in inflammation caused by oxidative stress, the present study aims to assess the effect of hydroethanolic extract of melon seeds on the transcriptional of NF-κB encoding genes and the MPO gene expression in rats with ethylene glycol-induced toxicity. Methods: Thirty male rats were randomly divided into five groups of healthy control, poisoned control (0.75% ethylene glycol), and three melon extract-treated poisoned groups (0.75% ethylene glycol and 150, 300, or 600 mg/kg body weight of melon extract) and were treated for 38 days. Then, the liver tissue of the rats was removed to evaluate the transcription of NF-κB encoding genes and the MPO level by real-time PCR method. Results: The transcription level of NF-κB encoding genes and the MPO level in the poisoned control group rats significantly increased compared to the healthy group. Treatment with hydroethanolic extract of melon seeds at concentrations of 300 and 600 mg/kg body weight caused a significant decrease in the transcription of NF-κB encoding genes. Treatment with hydroethanolic extracts at concentrations of 150, 300, and 600 mg/kg body weight caused a significant decrease in the MPO level compared to the poisoned control group. Conclusion: The decrease in MPO and NF-κB encoding genes after administration of hydroethanolic extract of melon seeds indicates the therapeutic effect of this extract on liver damage.

The Influence of Age and Gender on the Serum XOR Activity, Leukocyte Gene Expression of XOR and MPO, and Biochemical Parameters in Newborn Foals

One of the initial responses of the host's innate immunity of newborns against pathogens is the use of oxidative enzymes. This study aimed to evaluate changes in serum xanthine oxidoreductase (XOR) activity, the leukocyte myeloperoxidase (MPO) and XOR genes expression, and some biochemical parameters in healthy Darehshuri newborn foals up to 60 days of life. Blood samples were collected from 16 foals at 1, 7, 15, 30, and 60 days and used for detecting XOR activity, biochemical parameters, and also gene expression by real-time RT-PCR. High activity of XOR was observed at birth, explained by physiologic hypoxia during the birth without sex difference. The significant decrease in XOR activity during the following days is probably related to the decreased levels of substrate and feedback inhibition of XOR by uric acid. No correlations were found between XOR activity and uric acid. A positive correlation was observed between XOR mRNA and serum XOR activity in 15 days. The results also indicate higher levels of MPO gene expression at 30 days, which may be associated with their capacity for neutrophil phagocytosis. The concentrations of creatinine, total protein, and albumin were higher at birth, whereas uric acid level was lower (P < 0.05). It can be concluded that XOR activity decreases with age and there is no significant change in its gene expression and MPO expression increases with age and is sex-dependent. There is an influence of age on XOR activity, leukocyte expression of MPO, and biochemical parameters in healthy newborn foals up to 60 days of life.

Arsenic intake-induced gastric toxicity is blocked by grape skin extract by modulating inflammation and oxidative stress in a mouse model

Arsenic (As) is known to induce toxic responses in many organs of human beings and animals. However, research concerning toxicity in the stomach is limited. In this study, arsenic-induced gastric toxicity was investigated in a mouse model, and grape skin extract (GSE) was confirmed to have protective effects against arsenic toxicity. Our experimental results showed that exposure to 10 mg/l arsenic via drinking water for 56 days caused oxidative damage and inflammatory responses. The H2O2 and malondialdehyde (MDA) contents were significantly increased, accompanied by significant decreases in total superoxide dismutase (T-SOD) activity and glutathione (GSH) content in the gastric tissue of arsenic-treated mice. Two inflammatory signalling pathways, i.e., TLR2/MyD88/NF-κB and IL-6/STAT-3, were activated, along with inflammatory cell infiltration and the elevated mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β and IFN-γ) and myeloperoxidase (MPO) in the gastric tissue of mice exposed to arsenic. Meanwhile, the mRNA levels of the ZO-1, ZO-2 and occludin genes, which encode the key components of tight junction (TJ) complexes, were downregulated. However, the application of GSE (300 mg/kg bw) significantly inhibited the arsenic-induced increases in H2O2 and MDA contents and the decreases in T-SOD activity and GSH content. The arsenic-mediated gene expression of pro-inflammatory cytokines (TNF-α, IL-1β and IFN-γ), MPO and IL-6/STAT3 and TLR2/MyD88/NF-κB pathways was found down-regulated. Moreover, the arsenic-induced inflammatory cell infiltration and inhibition of TJ genes transcription were markedly attenuated in the As+GSE (300 mg/kg bw) group. Based on the present findings, arsenic intake appears to cause gastric toxicity via oxidative stress and inflammation, and the application of GSE offers significant protection against arsenic toxicity in a mouse model by attenuating the oxidative stress and inflammatory response. Our results suggest that GSE by oral administration might function as a candidate therapeutic supplement to antagonize arsenic toxicity.

Myeloperoxidase expression in diencephalon is potentially associated with fear-related behavior in chicks of laying hen.

Preventing feather pecking (FP) in adult laying hens is important for the welfare of intensively poultry farming. Fear-related behavior in growing female layer chicks may predict FP in adult hens. In this study, in two representative laying breeds (White Leghorn [WL] and Rhode Island Red [RIR]) that have different FP frequencies, we identified a candidate gene associated with fear-related behavior in chicks and FP in adult hens. In the tonic immobility test and open-field test, the behavioral activity was lower in WL chicks than in RIR chicks (P < 0.01), suggesting that WL chicks were more fearful than RIR chicks. Based on previous studies, 51 genes that have been found to be differentially expressed in the brain between high- and low-FP populations were chosen, and their expression levels were screened in the chick diencephalon. This analysis revealed that myeloperoxidase (MPO) gene expression level was higher in WL chicks than that in RIR chicks (P < 0.05). Furthermore, STRING analysis predicted the gene network including MPO and MPO-related genes and revealed the association of these genes with fear-related behavior. These results suggest that MPO is potentially associated with fear-related behavior in growing female layer chicks and FP in adult hens.

Implication of mucus-secreting cells, acidophilic granulocytes and monocytes/macrophages in the resolution of skin inflammation caused by subcutaneous injection of λ/κ-carrageenin to gilthead seabream (Sparus aurata) specimens.

To date, the mechanisms of inflammation have been poorly studied in fish of commercial interest, due to the lack of development of appropriate experimental models. The current study evaluated a local inflammation triggered by a polymeric carrageenin mixture (a mucopolysaccharide derived from the red seaweed Chondrus crispus) in the skin of gilthead seabream (Sparus aurata). Fish were injected subcutaneously with phosphate-buffered saline (as control) or λ/κ-carrageenin (1%), and skin samples from the injection sites were collected 1.5, 3 and 6hr post-injection, processed for inclusion in paraplast and stained with haematoxylin-eosin, Alcian blue or periodic acid-Schiff. Furthermore, immunohistochemistry and expression analyses of several cells' markers and proinflammatory genes were also analysed in samples of the injected sites. Microscopic results indicated an increased number of skin mucus-secreting cells and acidophilic granulocytes in the skin of fish studied at 1.5hr and 3hr post-injection with carrageenin, respectively, with respect to the data obtained in control fish. Otherwise, both the gene expression of the non-specific cytotoxic cell marker (granzyme B, grb) and the proinflammatory cytokine (interleukin-1β, il-1β) were up-regulated at 1.5hr in the skin of fish injected with carrageenin compared with the control fish, whilst the gene expression of acidophilic granulocyte markers (NADPH oxidase subunit Phox22 and Phox40, phox22 and phox40) was up-regulated at 3 and 6hr in the carrageenin group, compared with the control group. In addition, the gene expression of myeloperoxidase (mpo) was also up-regulated at 6hr in the skin of fish injected with carrageenin in comparison with control samples. The present results indicate the chronological participation of two important immune cells involved in the resolution of the inflammation in the skin of gilthead seabream.

MiR-223-3p levels in the plasma and atherosclerotic plaques are increased in aged patients with carotid artery stenosis; association with HDL-related proteins

Cardiovascular diseases are still the main cause of death worldwide. Our aim was to analyse the link between miR-223-3p levels, dysfunctional HDL and the age of patients with carotid artery stenosis (CAS). Thirty-two CAS patients enrolled for endarterectomy were divided in 2 groups: aged over 65years (n = 19) and under 65years (n = 13). Plasma samples and atherosclerotic plaques from the carotid artery were collected from all patients. Plaque levels of miR-223-3p and its primary transcript (pri-miR-223) were assessed, together with Drosha, Dicer, apolipoprotein (apo)A-I, apoE and myeloperoxidase (MPO) gene expression. In the plasma and plaques, miR-223-3p expression levels were significantly increased in CAS patients over 65years. Positive correlations between plaque miR-223-3p and pri-miR-223 levels with Drosha, apoA-I and MPO expression were observed. Significantly increased miR-223-3p levels in the plasma of CAS patients over 65years were measured. Significant correlations between plasma miR-223-3p levels and HDL-related proteins were determined. The variance of plasma miR-223-3p levels was predicted significantly by the multiple regression models using either age, clinical variables, blood lipids or oxidative and inflammatory parameters. Receiver operator characteristic analysis revealed that plasma miR-223-3p levels and HDL-related proteins (MPO activity/apoA-I ratio, MPO specific activity) were correlated with advanced age. Taken together, these data suggest that plasma levels of miR-223-3p are independently associated with ageing in CAS patients and that, correlated with parameters associated with dysfunctional HDL, could predict the aggravation of CAS in elderly patients.

Expression of Netosis Regulators and Correlation with Inflammatory and Coagulation Biomarkers in Sickle Cell Anemia Patients

Introduction: Sickle cell anemia (SCA) is a multi-systemic disease characterized by inflammation and chronic hemolysis. SCA is also associated with an increased risk of venous thromboembolism (VTE), with recent evidence suggesting that coagulation activation could also be involved in the pathogenesis of progressive organ failure in this condition. The generation of neutrophil extracellular traps, also termed NETosis, has been associated with the pathogenesis of VTE, and is also part of the immunothrombosis process. Furthermore, biomarkers of NETosis have been demonstrated in both animal models and SCA patients. NETosis is a complex process that involves the orchestrated participation of several proteins such as peptidyl arginine deaminase (PADI4), neutrophil elastase (ELANE) and myeloperoxidase (MPO). PADI4 mediates histone critulination, an essential step for NETosis, so that inhibition of PADI4 could be considered a potential therapeutic strategy for conditions in which NETosis play a relevant pathogenic role. Although attractive, investment in this strategy requires a more detailed knowledge of the role of PADI4 in pathogenesis of SCA. Objetives: in this study, we aimed to evaluate the expression of PADI4, ELANE, and MPO in steady state SCA patients, and to correlate these results with markers of inflammation and coagulation activation. Methods: mRNA was obtained from isolated neutrophils, and gene expression of PADI4, ELANE, and MPO were measured by qPCR in 54 steady state SCA patients and 40 healthy volunteers matched by age and geographic origin. Gene expressions levels were then correlated with inflammatory mediators and D-dimer. Results: no significant difference could be observed in the expression of PADI4 (0.55 vs 0.49; P = 0.42) and MPO (0.29 vs 0.39; P = 0.88) between healthy volunteers and patients respectively. In contrast, ELANE was up-regulated when patients compared with controls (1.46 vs 0.16; P <0.0001). PADI4 expression did not correlate with any biomarker of inflammation or coagulation (P>0.05). Interestingly, both ELANE and MPO were positively correlated with D-dimer (R=0.4; P=0.004 and R=0.32; P=0.004, respectively), and Von Willebrand factor (R=0.27; P=0.04 and R=0.30; P=0.04, respectively). ELANE was also correlated with TNF-α (R=0.30; P=0.03). Conclusion: gene expression of ELANE, but not PADI4 is up-regulated in steady state SCA patients, with both ELANE and MPO presenting significant associations with D-dimer and other biomarkers of inflammatory activation. Additional studies are ongoing to evaluate PADI4 expression and activity, both in steady state and during acute episodes in SCA. A more detailed evaluation of the role of PADI4 in the pathogenesis of SCA is warranted for the proper design of clinical trials based on PADI4 inhibition. DisclosuresNo relevant conflicts of interest to declare.

Neutrophil extracellular trap regulators in sickle cell disease: Modulation of gene expression of PADI4, neutrophil elastase, and myeloperoxidase during vaso‐occlusive crisis

BackgroundRecent evidence suggests that generation of neutrophil extracellular traps (NETosis), one of the components of immunothrombosis, is associated with the pathogenesis of both venous thromboembolism and sickle cell disease (SCD). NETosis is a complex process regulated by several proteins such as peptidyl arginine deaminase 4 (PADI4), neutrophil elastase (ELANE), and myeloperoxidase (MPO). Among these regulators, PADI4 is responsible of histone citrullination, an essential step for NETosis. Accordingly, its inhibition has been recently cited as a promising therapeutic strategy for diseases such as SCD. Although attractive, this strategy requires supportive evidence of its role in the pathogenesis of SCD. Patients and MethodsPatients from two independent cohorts were enrolled in this study. Samples were obtained at steady state (53 patients) or during acute episodes of vaso‐occlusive crisis (VOC; 28 patients) in patients from cohort 1. mRNA was extracted from granulocytes to analyze PADI4, ELANE, and MPO expression by qPCR. Furthermore, plasma activity of PADI4 was assessed from an independent cohort in 15 patients, within 24 hours from admission for VOC. Race‐matched healthy individuals from the same geographic regions were used as controls for each cohort. Results and ConclusionsHigher levels of gene expression of PADI4 and ELANE were observed during VOC. Furthermore, plasma activity of PADI4 was higher in acute VOC when compared to healthy individuals. These results demonstrate that NETosis regulators are modulated during acute VOC, and pave the way for studies of PADI4 inhibition as a therapeutic strategy for acute VOC in SCD.

Comparative study on the non-specific immune response and hsp70 gene expression among three strains of koi (Cyprinus carpio) under acute heat stress

A comparative study was performed to examine the effects of acute heat stress on non-specific immune response and heat shock protein 70 (hsp70) gene expression in three strains of koi (Cyprinus carpio), Showa Sanshoku, Kohaku, Ogon. Fish were exposed to increasing water temperature from 25 to 32 °C. Samples were collected at 0, 2, 6, 10, 14, 18, 22, 26 h after heat stress. Data were analysed by two-way ANOVA with strains of koi and time after heat-stressed as fixed factors. The results indicated that different strains had significant effects on whole blood respiratory burst (RB) and plasma superoxide dismutase (SOD) activity (P < 0.01). Different strains, time after heat-stressed and interaction of both factors affected plasma malonaldehyde (MDA), myeloperoxidase (MPO), complement 3 (C3), and heart hsp70 gene expression significantly (P < 0.01). After heat stress, Showa Sanshoku showed increased MDA, MPO, and suppressed C3 (P < 0.05). In Kohaku, MDA firstly raised (P < 0.05) and then fell to the basal level, while MPO, C3 significantly enhanced (P < 0.05). Continuously declined MDA and increased C3 were observed in Ogon (P < 0.05). The expression of hsp70 in the heart of koi was remarkably induced by heat stress and peaked at 2 h. After a sharp increase, the mRNA level of hsp70 recovered to the basal level (P > 0.05) but was induced again by continuous heat stress (P < 0.05). In summary, MPO, C3, and hsp70 played important roles in coping with heat stress. Hsp70 was a sensitive biomarker of heat stress in koi. The three strains of koi responded differently to heat stress. Ogon seemed to have the best ability to cope with heat stress, followed by Kohaku, while the Showa Sanshoku showed continuous oxidative stress and immunosuppression under acute heat stress.

The novel mechanism of valproate to prevent peritoneal adhesion formation.

A novel pharmacological mechanism of valproate was analyzed using a hamster model of adhesion. Valproate or placebo was administered just after cecal injury and adhesion severity scores and histological were analyzed. The adhesion severity scores in the placebo- and valproate-treated groups were 2.67 ± 0.42 and 1.0 ± 0.37, respectively, with a significant difference between the groups. A significant increase in mast cell numbers was observed in the placebo-treated group vs. the sham-operated group; however, the mast cell number in the adhesive lesion was significantly lower in the valproate-treated group than in the placebo-treated group. The number of cells positive for chymase, an enzyme in mast cells, in the adhesive lesion was significantly higher in the placebo-treated group, but its increase was attenuated significantly by treatment with valproate. The myeloperoxidase gene expression level in the cecum was significantly higher in the placebo-treated group than in the sham-operated group, but there was no significant difference in the myeloperoxidase gene expression level between the sham-operated and valproate-treated groups in. In an in vitro experiment, valproate inhibited purified human and hamster chymases dose-dependently. The chymase inhibitory effect of valproate may contribute to prevent adhesion formation after abdominal injury.

Expression of neutrophil elastase and myeloperoxidase mRNA in patients with newly diagnosed type 2 diabetes mellitus

Background and aimsNeutrophil elastase and myeloperoxidase enzymes protect us from infection by killing pathogens. However, exaggerated activities of these enzymes can induce tissue damage, inflammation and oxidative stress. The present study was aimed to explore the expressions of neutrophil elastase and myeloperoxidase mRNA in the peripheral blood leukocytes (PBL) in patients with newly diagnosed type 2 diabetes mellitus. MethodsIn this cross-sectional study, 104 participants including 65 normoglycemic control subjects and 39 newly diagnosed type 2 diabetes patients were recruited. Glycemic and metabolic markers were evaluated from fasting blood samples. The mRNA levels of neutrophil elastase and myeloperoxidase genes in the PBL were quantified by real-time quantitative PCR. ResultsCompared to control subjects, diabetes patients showed a significant down regulation of both neutrophil elastase (p = 0.039) and myeloperoxidase (p = 0.023) mRNA expressions in the PBL. The neutrophil elastase and myeloperoxidase mRNA levels showed a negative trend with fasting glucose levels but did not show any significant correlations with HbA1c, insulin level, insulin resistance or sensitivity status. ConclusionsIt was concluded that type 2 diabetes mellitus is associated with a decrease in neutrophil elastase and myeloperoxidase gene expression in the PBL.

A high-carbohydrate diet induces greater inflammation than a high-fat diet in mouse skeletal muscle.

We previously reported that both the high-carbohydrate diet (HCD) and high-fat diet (HFD) given for two months promote lipid deposition and inflammation in the liver and brain of mice. The results obtained indicate a tissue-specific response to both diets. Herein, we compared the effects of HCD and HFD on fatty acid (FA) composition and inflammation in the gastrocnemius muscle. Male Swiss mice were fed with HCD or HFD for 1 or 2 months. Saturated FA (SFA), monounsaturated FA (MUFA), n-3 polyunsaturated FA (n-3 PUFA), and n-6 PUFA were quantified. The activities of stearoyl-CoA desaturase 1 (SCD-1), Δ-6 desaturase (D6D), elongase 6, and de novo lipogenesis (DNL) were estimated. As for indicators of the inflammatory tissue state, we measured myeloperoxidase (MPO) activity and gene expression of F4/80, tumor necrosis factor-α (TNF-α), interleukin (IL)-4, IL-6, and IL-10. The HCD led to a lower deposition of SFA, MUFA, n-3 PUFA, and n-6 PUFA compared to HFD. However, the HCD increased arachidonic acid levels, SFA/n-3 PUFA ratio, DNL, SCD-1, D6D, and MPO activities, and expression of IL-6, contrasting with the general idea that increased lipid deposition is associated with more intense inflammation. The HCD was more potent to induce skeletal muscle inflammation than the HFD, regardless of the lower lipid accumulation.

Up-Regulation of the mRNA Expression and Plasma Activity of PADI4in Sickle Cell Anemia during Acute Crisis

Introduction: Recent evidence suggests that coagulation activation is involved in the pathogenesis of progressive organ failure in Sickle Cell Anemia (SCA). In addition, generation of neutrophil extracellular traps (NETosis), one of the components of immunothrombosis, has been associated with the pathogenesis of both venous thromboembolism and SCA. NETosis is a complex process that involves the orchestrated participation of several proteins such as peptidyl arginine deaminase (PADI4), neutrophil elastase (ELANE) and myeloperoxidase (MPO). PADI4 mediates histone citrulination, an essential step for NETosis. Accordingly, its inhibition has been recently cited as a potential therapeutic strategy for diseases in which NETosis are thought to play a relevant pathogenic role such as SCA. Although attractive, investment in PADI4 inhibitors in SCA requires gathering of more convincing evidences of the role of this enzyme in its pathogenesis. Herein, we used two cohorts of patients to investigate the expression of NETosis regulators (PADI4, ELANE and MPO) in SCA at steady state and during acute crisis, and to assess whether PADI4 activity is increased in any of these states. Methods: patients were recruited from two different centers in Brazil. Whole blood samples were obtained from patients from cohort 1 at steady state or during acute crisis (within 24 hours from admission). mRNA was obtained from granulocytes isolated by Ficoll gradient and gene expression of PADI4, ELANE, and MPO were measured by qPCR. In patients from cohort 2, PADI4 and MPO activity were measured in samples obtained within 24 hours from admission and after patient discharge (convalescence) using commercial kits in serum (MPO, Myeloperoxidase activity assay kit; ab105136) and plasma (PADI4, PAD4 Inhibitor Screening Assay Kit; Cayman chemical). Healthy individuals from the same geographic region were used as controls for each cohort, independently Results: In total, 54 steady state patients, 27 acute crisis and 40 healthy volunteers were evaluated for mRNA expression of NETosis regulators. Patients in acute crisis expressed higher levels of PADI4, MPO and ELANE compared to both healthy volunteers and patients in steady-state. Furthermore, plasma activity of PADI4 was higher in acute crisis when compared to healthy individuals (7.36x106 vs 5.24x106; P= 0.002), with no decrease after discharge (median of 13.5 days after admission) (7.36x106vs 7.41x106; P= 0.004). No differences were observed in serum MPO activity during acute crisis. Conclusion: we demonstrate that the mRNA expression of NETosis regulators, including PADI4, is increased in SCA; and that this increase is associated with higher levels of PADI4 activity in plasma during acute crisis. These results support the concept of PADI4 inhibition as a therapeutic strategy for acute episodes of SCA, and warrant additional studies in this area. Disclosures No relevant conflicts of interest to declare.

Increase Expression of the Genes PADI4, MPO and ELANE in the Chronic Phase of DVT Patients

Venous thromboembolism (VTE) is a common multifactorial disease, and the knowledge in the pathophysiology can improve prophylaxis and treatment. The myeloid lineage, especially neutrophils, have been implied in the pathophysiology of VTE, but it is still unclear whether its activation is persistent after the acute phase of the disease. In addition to phagocytosis, neutrophils participate of the innate immunity through the release of NETs (Neutrophil Extracellular Traps): Reactive Oxygen Species (ROS)-triggered antimicrobial structures composed of DNA lined with granular components (Brinkmann et al., 2004). NETs also contribute to the coagulation, as they facilitate the interaction between erythrocytes, leukocytes, platelets and endothelium, besides potentially inhibit anticoagulant pathways (Fuchs et al., 2012). The molecular mechanisms underlying the "NETosis" are still being characterized, but it is known that following inflammatory stimuli, the generation of ROS leads to the dissolution of intracellular membranes and translocation of Myeloperoxidase (MPO) and Neutrophil Elastase (ELANE) to nucleus. Histones are then citrullinated by Peptidyl Arginine Deiminase 4 (PADI4) and degraded by ELANE, promoting chromatin decondensation, plasma membrane rupture and NET's release. Previously we showed increased neutrophil activation, adhesive properties and the presence of NETs serum markers in patients with VTE in the chronic phase (Zapponi et al., 2014; Zapponi, manuscript in preparation). However, assessment of NETs activity is challenging and, until now, there is no consensus in markers or gold standards measurements. We decided to investigate if the expression of the genes PADI4, ELANE, MPO, could provide additional information regarding NETosis activity, through an indirect analysis of the process by Real-Time PCR. We analyzed samples from 20 controls and 19 patients previously included in the NET study, respecting the time window in which the NETs plasma markers were assessed (between 3 and 43 months after the TVP event). RNA extraction was performed from peripheral blood leukocytes using Trizol reagent, according to the manufacturer's protocol. Real time-PCR reactions were performed with qPCRBIO SyGreen mix (PCRBio), on RotorGene Q (Qiagen) with a reaction efficiency above 0.99. The relative fold changes (Fc) of the genes PADI4, ELANE, MPO were calculated using the Pfaffl method (Pfaffl, 2001 - Nucleic Acids Res.) and the significance level of Mann-Whitney test was considered if p &lt;0.05. The 39 cDNA samples were amplified in duplicates and a standard sample was included for normalization between different runs. The threshold values (Ct) from PADI4, ELANE, MPO were adjusted for the individual's neutrophil proportion and the geometric means of the housekeeping genes (Actin Beta and Glyceraldehyde-3-Phosphate Dehydrogenase) were used for normalization between samples. The standard deviation was 0.30 between runs and ranged between duplicates from 0 to 1.16 (0.096 on average). The patients showed significant increased expression of PADI4 (p &lt;0.01), MPO and ELANE (p &lt;0.05) when compared to controls (Figure 1). Neutrophils emerges fully differentiated from the bone marrow and ranges from 40 to 70% of circulating leukocytes (Cowland and Borregaard, 2016). Isolating these cells can be challenging and the process itself may influence expression profile. The use of total leukocytes would not be the most appropriate process but considering that the analyzed genes are predominantly expressed in neutrophils, the Ct values were weighted according to the neutrophil to leucocytes proportions of each individual. Our results indicate that the increased neutrophils activation in patients can be represented in PADI4, ELANE and MPO gene expression of peripheral blood leukocytes, which could better represent NETotic activity in these patients. We cannot rule out that the expression of these genes in myeloid precursors could better represent NETotic activity, but our results indicate that this analysis may also be viable in peripheral blood. Disclosures No relevant conflicts of interest to declare.

Role of Myeloperoxidase of northern snakehead (Channa argus) in Aeromonas veronii infection

Myeloperoxidase (MPO) is a ferrous lysosomal protein with many immune functions that belongs to the heme peroxidase enzyme. In this study, the functions of MPO in the northern snakehead (Channa argus) were investigated by cloning an MPO cDNA sequence with a full length of 3181 bp. Homology analysis showed that northern snakehead MPO gene had the highest (81%) homology with mandarin fish (Siniperca chuatsi). In healthy northern snakehead, the MPO gene was expressed in the head-kidney, kidney, heart, gill, spleen, liver, and muscles but not midgut. After the northern snakehead was infected with Aeromonas veronii, the MPO gene expression varied in different tissues with low level in spleen, liver, gill and muscle, fluctuated in kidney and head-kidney and showed high level in heart. The result indicated that MPO might play an important role in the antimicrobial immune response of the northern snakehead.

Reduced gravity contributes to Neutrophil to Lymphocyte Ratio shifting and promotion of the Oxidative Stress Response

Abstract Spaceflight can cause immune system dysfunction, such as elevated white blood cells (WBC) and polymorphonuclear neutrophils (PMN), along with unchanged or reduced lymphocyte counts. A high PMN to lymphocyte ratio (NLR) can acts as a poor prognosis in cancer and a biomarker for subclinical inflammation however, the NLR has not been identified as a predictor of astronaut health during spaceflight. CBC data collected on board the International Space Station (ISS) was repurposed to determine the granulocyte to lymphocyte ratio (GLR) in humans and the NLR in rodents. The results displayed a progressive increase in GLR and NLR during spaceflight and at landing. The mechanism for increased NLR was assessed in vitro using the microgravity-analog, rotating wall vessel (RWV), with human WBCs. The results indicated that simulated microgravity led to increased GLR and NLR profiles, and production of reactive oxygen species (ROS) and myeloperoxidase (MPO). Interestingly, simulated microgravity increased the number of matured PMNs that showed impaired phagocytic function, while treatment with tert-Butyl hydroperoxide (TBHP), also reduced PMN phagocytosis. In addition, 30-days of simulated microgravity (hindlimb unloading) in mice, indicated an increased NLR and MPO gene expression, which were mitigated in mitochondrial catalase overexpressing transgenic mice, suggesting ROS scavenging is essential for maintaining homeostatic immunity. Collectively, we propose that the health status of astronauts during future short- and long-term space missions can be monitored by their NLR profile, in addition to utilizing this measurement as a tool for oxidative stress response countermeasure development to restore homeostatic immunity.

Expression changes of CD177 and MPO as novel biomarkers in lung tissue of CLP model rats

Sepsis is an unregulated systemic response to microbial invasion that can lead to multiple organ failure. This study aims at investigating the relationships among myeloperoxidase (MPO) and CD177 in major organ systems including whole blood, liver, and lung tissues in septic rats. Sepsis was induced by cecal ligation and puncture (CLP) in female Wistar rats. Whole blood, liver, and lung samples were obtained from rats of 3 groups (n = 10 for each group, n total = 30: control as a wild-type group, laparotomy group (LAP), and CLP). Gene expression of MPO and CD177 in targeted tissues was determined by real-time PCR after CLP. MPO activity was also determined by ELISA method for the result validation of the real-time PCR. Expression levels of MPO increased significantly in all targeted organs in the CLP group, while CD177 expression was upregulated only in lung tissue in response to sepsis (P < 0.05). The results obtained with ELISA analysis also show that MPO level was significantly increased in all the targeted tissues in the CLP group (P < 0.05). A high level of MPO as an inflammatory enzyme can be a potentially novel biomarker for sepsis in all organs. On the other hand, CD177 may be a marker in lung tissue.