P47phox Research Articles

P47phox deficiency improves cognitive impairment and attenuates tau hyperphosphorylation in mouse models of AD

BackgroundAlzheimer’s disease (AD) is characterized by progressive memory loss and cognitive impairment. The aggregation of amyloid β (Aβ) and hyperphosphorylated tau protein are two major pathological features of AD. Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase, NOX) has been indicated in Aβ pathology; however, whether and how it affects tau pathology are not yet clear.MethodsThe role of NOX2 in cognitive function, amyloid plaque formation, and tau hyperphosphorylation were examined in APP/PS1 transgenic mice mated with p47phox-deficient mice (with deletion of the gene of neutrophil cytosolic factor 1, Ncf1) and/or in p47phox-deficient mice receiving intracerebroventricular (ICV) injection of streptozotocin (STZ). The cognitive and non-cognitive functions in these mice were assessed by Morris water maze, Rotarod test, open field, and elevated plus maze. Aβ levels, amyloid plaques, p47phox expression, and astrocyte activation were evaluated using immunofluorescence staining, ELISA, and/or Western blotting. Cultured primary neuronal cells were treated with okadaic acid or conditioned media (CM) from high glucose-stimulated primary astrocytes. The alteration in tau pathology was determined using Western blotting and immunofluorescence staining.ResultsDeletion of the gene coding for p47phox, the organizer subunit of NOX2, significantly attenuated cognitive impairment and tau pathology in these mice. p47phox deficiency decreased the activation of astrocytes but had no effect on Aβ levels and amyloid plaque formation in the brains of aged APP/PS1 mice, which displayed markedly increased expression of p47phox in neurons and astrocytes. Cell culture studies found that neuronal p47phox deletion attenuated okadaic acid-induced tau hyperphosphorylation at specific sites in primary cultures of neurons. CM from high glucose-treated WT astrocytes increased tau hyperphosphorylation in primary neurons, whereas this effect was absent from p47phox-deficient astrocytes.ConclusionsThese results suggest that p47phox is associated with cognitive function and tau pathology in AD. p47phox expressed in neurons contributes to tau hyperphosphorylation directly, while p47phox in astrocytes affect tau hyperphosphorylation by activating astrocytes indirectly. Our results provide new insights into the role of NOX2 in AD and indicate that targeted inhibition of p47phox may be a new strategy for the treatment of AD.

Upregulation of glutaminase 2 and neutrophil cytosolic factor 2 is associated with the poor prognosis of glioblastoma.

Background: This study aimed to identify glioblastoma prognosis-associated genes with potential diagnosis or prognosis values using integrated bioinformatics analysis. Results: In total, 1831 differentially expressed genes (DEGs) between the glioblastoma and control samples were identified and were clustered into seven weighed gene co-expression network analysis (WGCNA)modules. These DEGs were associated with different functional categories and pathways. Nine prognosis-associated DEGs (including glutaminase 2 [GLS2] and neutrophil cytosolic factor 2 [NCF2]) were identified, and the higher expression levels of GLS2 and NCF2 genes were associated with the poor prognosis of glioblastoma in 'The Cancer Genome Atlas' cohort and a clinical cohort. Conclusion: These results showed that the two genes play novel roles in the etiological and development of glioblastoma.

Transcriptomic analysis of red-spotted grouper nervous necrosis virus infected Asian seabass Lates calcarifer (Bloch, 1790)

Red-spotted grouper nervous necrosis virus (RGNNV) has threatened aquaculture of the Asian seabass (Lates calcarifer) in many countries of the Asia-Pacific for decades. To understand the pathogenesis of RGNNV, a transcriptome analysis of the head kidney, a major fish lymphoid tissue which performs important immune functions and is involved in the hematopoiesis was undertaken. RNA from the head kidney of RGNNV infected fish was sequenced and compared RNA from uninfected control fish at two and 15-days post infection. Functional analysis revealed a number of differentially expressed genes implicated in autophagy (beclin-1 and inositol 1,4,5-trisphosphate receptor), superoxide production (cytochrome b-245 light chain, C3a anaphylatoxin chemotactic receptor and neutrophil cytosol factor 2-like), inflammation (leukocyte surface antigen CD53), antigen presentation (MHC class I alpha chain), antiviral activity (cholesterol 25-hydroxylase) and other immune functions (oxysterol-binding protein 1, dipeptidyl peptidase I, interleukin-5 receptor subunit alpha-like and tyrosine-protein kinase BTK). This is the first description of the change in L. calcarifer head kidney mRNA expression as a consequence of RGNNV infection and the information will serve as the rational basis for future investigations into the pathophysiology of NNV infection of fish.

Taurine Metabolism Aggravates the Progression of Lupus by Promoting the Function of Plasmacytoid Dendritic Cells.

Type I interferons (IFNs) are critical in the development of systemic lupus erythematosus (SLE). Metabolic abnormalities cause dysregulation of multiple immune cells, but the metabolic regulation of type I IFN production is not well clarified in SLE. We undertook this study to define amino acid metabolism features in SLE and to explore the function of disease-relevant metabolites in the control of plasmacytoid dendritic cell (pDC)-mediated type I IFN production and the progression of SLE. Metabolomic profiling of the serum from SLE patients and healthy controls was performed by mass spectrometry. The effects of SLE-related metabolites on type I IFN production were explored in human and mouse pDCs. The reactive oxygen species (ROS) levels of pDCs from wild-type and Ncf1-/- mice were measured by flow cytometry. Mechanisms were investigated by RNA sequencing and immunoblotting. In vivo effects of SLE-relevant metabolites were systemically analyzed in B6.Cg-Sle1NZM2410/Aeg Yaa/DcrJ mice. Taurine was higher in the serum from SLE patients compared to healthy controls (P < 0.001) and rheumatoid arthritis patients (P < 0.001). Taurine content was positively correlated with disease activity and the expression of IFN signature genes. The addition of taurine facilitated IFN regulatory factor 7 phosphorylation and enhanced type I IFN production by reducing the ROS levels in pDCs in a neutrophil cytosolic factor 1-dependent manner. Taurine supplementation promoted expression of type I IFN-induced genes, activated lymphocytes, and increased autoantibodies and proteinuria, leading to more serious nephritis. Taurine metabolism is involved in the development of SLE by enhancing pDC-mediated type I IFN production. Targeted inhibition of taurine or implementation of a taurine-restricted diet has therapeutic potential in SLE.

Association of NCF2, NCF4, and CYBA Gene Polymorphisms with Rheumatoid Arthritis in a Chinese Population.

Objective Recent studies have focused on the special roles of NADPH-oxidase in multiple autoimmune diseases. Nevertheless, the association of genetic variation in NADPH-oxidase genes with rheumatoid arthritis (RA) was not extensively studied in a Chinese population. We performed this study to examine the association of NCF2, NCF4, and CYBA gene polymorphisms with RA susceptibility in a Chinese population. Methods Six single nucleotide polymorphisms (SNPs) (NCF2 rs10911363, NCF4 rs1883112, rs4821544, rs729749, CYBA rs3794624, and rs4673) were genotyped in a cohort composed of 593 RA patients and 596 normal controls. Improved multiple ligase detection reaction (iMLDR) was used for genotyping. Results We observed that NCF4 rs4821544 CT genotype and C allele frequencies in RA patients were significantly decreased when compared to controls (CT vs. TT: P = 0.043; C vs. T: P = 0.031), and rs4821544 polymorphism was significantly associated with an increased RA risk under the dominant model (TT vs. CT+CC: P = 0.031). Our results also indicated that rs729749 CT genotype frequency was significantly lower in RA patients than that in controls (CT vs. CC: P = 0.033). Moreover, the rs729749 CT genotype frequency was also significantly decreased in RA patients in males (CT vs. CC: P = 0.024). No significant association between NCF2 and CYBA gene polymorphisms and RA susceptibility was observed. There were significant associations between rs4821544 TT genotype and T allele frequencies and anti-CCP in male RA patients. Conclusions In summary, NCF4 rs4821544 and rs729749 polymorphisms might contribute to RA susceptibility, while NCF2 and CYBA gene polymorphisms were not associated with RA susceptibility.

Mechanistic computational modeling of the kinetics and regulation of NADPH oxidase 2 assembly and activation facilitating superoxide production

Reactive oxygen species (ROS) play a crucial role in many physiological processes. However, ROS overproduction leads to oxidative stress, which plays a critical role in cell injury/death and the pathogenesis of many diseases. Members of NADPH oxidase (NOX) family, most of which are comprised of membrane and cytosolic components, are known to be the major nonmitochondrial sources of ROS in many cells. NOX2 is a widely-expressed and well-studied NOX family member, which is activated upon assembly of its membrane subunits gp91 phox and p22 phox with its cytosolic subunits p40 phox , p47 phox , p67 phox , and Rac, facilitating ROS production. NOX2 activation is also enhanced by GTP and inhibited by GDP. However, there remains a lack of a mechanistic, quantitative, and integrated understanding of the kinetics and regulation of the assembly of these subunits and their relative contributions toward NOX2 activation and ROS production. Toward this end, we have developed a mechanistic computational model, which incorporates a generalized random rapid equilibrium binding mechanism for NOX2 assembly and activation as well as regulations by GTP (activation), GDP (inhibition), and individual subunits enhancing the binding of other subunits (mutual binding enhancement). The resulting model replicates diverse published kinetic data, including subunit concentration-dependent NOX2 activation and ROS production, under different assay conditions, with appropriate estimates of the unknown model parameters. The model provides a mechanistic, quantitative, and integrated framework for investigating the critical roles of NOX2 subunits in NOX2 assembly and activation facilitating ROS production in a variety of physiological and pathophysiological conditions. However, there is also a need for better quantitative kinetic data based on current understanding of NOX2 assembly and activation in order to test and further develop this model.

Identification of inflammation‑associated circulating long non‑coding RNAs and genes in intracranial aneurysm rupture‑induced subarachnoid hemorrhage.

Ruptured intracranial aneurysm (IA)-induced subarachnoid hemorrhage (SAH) triggers a series of immune responses and inflammation in the brain and body. The present study was conducted to identify additional circulating biomarkers that may serve as potential therapeutic targets for SAH-induced inflammation. Differentially expressed (DE) long non-coding RNAs (lncRNAs; DElncRNAs) and genes (DEGs) in the peripheral blood mononuclear cells between patients with IA rupture-induced SAH and healthy controls were identified in the GSE36791 dataset. DEGs were used for weighted gene co-expression network analysis (WGCNA), and SAH-associated WGCNA modules were identified. Subsequently, an lncRNA-mRNA regulatory network was constructed using the DEGs in SAH-associated WGCNA modules. A total of 25 DElncRNAs and 1,979 DEGs were screened from patients with IA-induced SAH in the GSE36791 dataset compared with the controls. A total of 11 WGCNA modules, including four upregulated modules significantly associated with IA rupture-induced SAH were obtained. The DEGs in the SAH-associated modules were associated with Gene Ontology biological processes such as ‘regulation of programmed cell death’, ‘apoptosis’ and ‘immune response’. The subsequent lncRNA-mRNA regulatory network included seven upregulated lncRNAs [HCG27, ZNFX1 antisense RNA 1, long intergenic non-protein coding RNA (LINC)00265, murine retrovirus integration site 1 homolog-antisense RNA 1, cytochrome P450 1B1-AS1, LINC01347 and LINC02193] and 375 DEGs. Functional enrichment analysis and screening in the Comparative Toxicogenomics Database demonstrated that SAH-associated DEGs, including neutrophil cytosolic factor (NCF)2 and NCF4, were enriched in ‘chemokine signaling pathway’ (hsa04062), ‘leukocyte transendothelial migration’ (hsa04670) and ‘Fc gamma R-mediated phagocytosis’ (hsa04666). The upregulated lncRNAs and genes, including NCF2 and NCF4, in patients with IA rupture-induced SAH indicated their respective potentials as anti-inflammatory therapeutic targets.

Abstract 445: Myristoylated Protein Kinase C Beta II Peptide Inhibitor Attenuates Hypoxia-Reoxygenation Injury in Human Umbilical Vein Endothelial Cells

Endothelial dysfunction as a result of ischemia/reperfusion (I/R) injury contributes to local organ damage in heart attack patients. In vascular cells, NADPH oxidase (NOX) and the mitochondrial electron transport chain are initiating sources of reactive oxygen species (ROS) during I/R injury. Protein kinase C beta II (PKCβII) is an attractive therapeutic target due to its phosphorylation of p66Shc to enhance mitochondrial-derived ROS production and p47 phox to promote ROS release from NOX. In previous studies, a cell-permeable myristoylated PKCβII peptide inhibitor (N-myr-SLNPEWNET; myr-PKCβII-) has been shown to improve post-reperfusion cardiac function and reduce infarct size in rat myocardial I/R injury. The decrease in myocardial I/R injury with myr-PKCβII- may in part be attributed to improved vascular endothelial function. Due to myr-PKCβII- peptide sequence being highly conserved among mammalian species, we hypothesize that myr-PKCβII- will confer protection by directly inhibiting ROS production from NOX and mitochondria in human umbilical endothelial cells (HUVECs) subjected to hypoxia/reoxygenation (H/R) mediated injury. HUVECs, cultured in gelatin-coated 96-well plates, were subjected to 24h hypoxia and 24h reoxygenation in a Billups-Rothenburg chamber with 1% O 2 , 5% CO 2 , and balance nitrogen. Myr-PKCβII- (20 μM) was administered at the beginning of the 24h reoxygenation period. Cell viability was assessed using tetrazolium-salt (WST-8) colorimetric assay with a microplate reader (450 nm) and normalized against the normoxia control group. Data were analyzed using Student-Newman-Keuls post-hoc analysis. At the 24h reoxygenation period, cell viability (%) was significantly reduced to 78±2% (n=5; p&lt;0.05) in the non-treated H/R group compared to normoxia controls (n=5). Myr-PKCβII- significantly improved HUVEC survival (95±4%; n=5) compared to non-treated H/R controls (n=5; p&lt;0.01) which were not significantly different from normoxia controls. The data suggest that PKCβII inhibition promotes cell survival possibly due to directly attenuating NOX and mitochondrial derived ROS in cells subjected to H/R conditions. Further studies are needed to determine cell survival potential under more severe H/R mediated injury.

Diagnostic Modalities Based on Flow Cytometry for Chronic Granulomatous Disease: A Multicenter Study in a Well-Defined Cohort

Chronic granulomatous disease (CGD) is characterized by defective microbial killing due to mutations affecting subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Definitive genetic identification of disease subtypes may be delayed or not readily available. Sought to investigate the role of intracellular staining of NADPH oxidase enzyme subunits in predicting the respective genetic defects in patients with CGD and carriers. Thirty-four patients with genetically inherited CGD, including 12 patients with X-linked CGD (gp91phagocyte oxidase (phox) deficiency due to cytochrome b-245, beta polypeptide [CYBB] mutations) and 22 patients with autosomal-recessive CGD (p22phox, p47phox, and p67phox deficiency due to cytochrome b-245, alpha polypeptide [CYBA], neutrophil cytosolic factor 1 [NCF1] and NCF2 mutations, respectively) were recruited from different immunology centers and followed up prospectively. Dihydrorhodamine testing and NADPH oxidase subunit expression in white blood cells were determined by flow cytometry. gp91phox and p22phox defects, which result in simultaneous loss of both proteins due to their complex formation, were differentiated only by comparative analysis of patients' and mothers' intracellular staining. p47phox and p67phox protein expression was almost undetectable in patients compared with carrier mothers and healthy controls. The expression values of the respective subunits were found to be significantly higher in all controls as compared with carrier mothers, which in turn were higher than those of patients. Analysis of NADPH oxidase enzyme subunits by flow cytometry in patients and carriers is useful in the rapid prediction of the genetic defect of patients with CGD, thus guiding targeted sequencing and aiding in their early diagnosis.

Endothelial prolyl hydroxylase 2 is necessary for angiotensin II-mediated renal fibrosis and injury.

Angiotensin II (ANG II) is the key contributor to renal fibrosis and injury. The present study investigated the role of endothelium prolyl hydroxylase 2 (PHD2) in ANG II-mediated renal fibrosis and injury. In vitro, endothelial cells (ECs) were isolated from PHD2f/f control [wild-type (WT)] mice or PHD2 EC knockout (PHD2ECKO) mice. In vivo, WT and PHD2ECKO mice were infused with ANG II (1,000 ng·kg-1·min-1) for 28 days. Renal fibrosis, reactive oxygen species (ROS), and iron contents were measured. Knockout of PHD2 resulted in a significant increase in the expression of hypoxia-inducible factor (HIF)-1α and HIF-2α in ECs. Intriguingly, knockout of PHD2 significantly reduced expression of the ANG II type 1 receptor (AT1R) in ECs. WT mice infused with ANG II caused increases in renal fibrosis, ROS formation, and iron contents. ANG II treatment led to a downregulation of PHD1 expression and upregulation of HIF-1α and HIF-2α in the renal cortex and medulla. Knockout of PHD2 in EC blunted ANG II-induced downregulation of PHD1 expression. Furthermore, knockout of PHD2 in ECs attenuated ANG II-induced expression of HIF-1α, HIF-2α, transforming growth factor-β1, p47phox, gp91phox, heme oxygenase-1, and ferroportin. This was accompanied by a significant suppression of renal fibrosis, ROS formation, and iron accumulation. In summary, knockout of endothelial PHD2 suppressed the expression of AT1R in ECs and blunted ANG II-induced downregulation of PHD1 and upregulation of HIF-α in the kidney. Our study, for the first time, demonstrates a necessary role of endothelial PHD2 in ANG II-mediated renal fibrosis and injury.

Matrine ameliorates cognitive deficits via inhibition of microglia mediated neuroinflammation in an Alzheimer's disease mouse model.

Amyloid β (Aβ) induced microglial activation and attendant neuroinflammation play pivotal roles in Alzheimer's disease (AD) pathogenesis. Matrine is a natural anti-inflammation compound from the Chinese herbal medicine Sophora flavescens Ait. (Kushen). This study aimed to investigate the effects of matrine on memory deficit and neuroinflammation in an oligomeric Aβ (oAβ)-induced AD mice model. Whether microglial activation and NADPH oxidase were involved in these effects were further studied. Different doses of matrine (10, 20, or 40 mg/kg) were intragastrically administered once a day after intracerebroventricular oAβ injection (2.5 μg/μl, 4 μl). 15 days after the oAβ injection, behavioral experiments including novel object recognition (NOR) test and Morris water maze (MWM) test were performed. 21 days after the oAβ injection, concentration of ROS, TNF-α, IL-1β and IL-6 as well as expression of NADPH oxidase subunits gp91phox and p47phox in mice hippocampal tissues were assessed, and microglial activation were evaluated by Iba-1 immunohistochemical staining. Results of NOR test and MWM test revealed that oAβ injection could remarkably impair learning and memory function in AD mice, and matrine administration could significantly ameliorate the impairment. ROS, TNF-α, IL-1β and IL-6 levels increased after oAβ injection, while matrine could significantly reduce the concentrations of these inflammatory factors. OAβ induced protein expression of NADPH oxidase subunits gp91phox and p47phox were also significantly reduced by matrine. Iba-1 immunohistochemistry results showed less activated microglia in matrine-treated mice brain. These results indicate that matrine could ameliorate learning and memory impairment and neuroinflammation induced by oAβ injection. These effects were found to be mediated through inhibition of microglial activation and NADPH oxidase expression in hippocampal tissue. The results suggest that matrine may be a valuable natural compound with therapeutic potential against AD.

Systemic Redox Imbalance in Patients with Chronic Granulomatous Disease.

The aim of our study was to evaluate redox status, enzymatic and non-enzymatic antioxidant barriers, oxidative damage of proteins, lipids and DNA, as well as concentration of coenzyme Q10 and vitamins A and E in patients with chronic granulomatous disease (CGD). The study was performed on fifteen Caucasian individuals (median age 24 years and seven months) diagnosed with CGD. The mutation in the NCF1 gene was confirmed in ten patients, and in the CYBB gene in five patients. We demonstrated high levels of total oxidant status (TOS) and oxidative stress index (OSI), lipids (↑8-isoprostanes (8-isoP), ↑4-hydroxynonenal (4-HNE)), proteins (↑advanced oxidation protein products (AOPP)) and DNA (↑8-hydroxy-2’-deoxyguanosine (8-OHdG)) oxidation products in CGD individuals as compared to sex- and age-matched healthy controls. We showed enhanced serum enzymatic activity of catalase (CAT) and superoxide dismutase-1 (SOD) and significantly decreased coenzyme Q10 concentration. Our study confirmed redox disturbances and increased oxidative damage in CGD patients, and indicated the need to compare redox imbalance depending on the type of mutation and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The question regarding effectiveness of antioxidant therapy in patients with CGD is open, and the need to establish guidelines in this area remains to be addressed.

Sesame lignans suppress age-related disorders of the kidney in mice.

Sesamin is a functional ingredient in sesame (Sesamum indicum) seeds and has many physiological effects. This study investigated whether sesame lignans, sesamin and episesamin (1:1), can suppress age-related disorders of the kidney. Twenty-month-old mice were divided into three groups, and each group received a regular diet (O-C), diet containing sesame lignans (O-SE), and diet containing sesame lignans and α-tocopherol (VE; O-SE+VE), respectively, for 5 months. Six-month-old young mice (Y-C) were compared to the older mice. Renal lipofuscin deposition was increased in the O-C group compared to that in the Y-C group and its deposition with aging was significantly decreased in both O-SE and O-SE+VE groups. Plasma blood urea nitrogen levels in the O-C group increased compared to those in the Y-C group; however, those in both O-SE and O-SE+VE groups did not differ from those in the Y-C group. The number of podocytes in the O-C group decreased compared to that in the Y-C group and this effect was attenuated in the O-SE and O-SE+VE groups. The effect was strongest in the O-SE+VE group. Histological examinations showed that glomerular hypertrophy accompanied by mesangial hyperplasia and renal tubular degeneration was less severe in the O-SE and O-SE+VE groups than in the O-C group. Moreover, age-related increases in the mRNA expression of NADPH oxidase- and inflammation-related genes, including p67phox, p40phox, TNFα, and IL-6, in the kidney were suppressed in the O-SE and O-SE+VE groups. Sesame lignans might be useful to suppress age-related kidney disorders, and these effects could be enhanced with VE.

Formyl peptide receptor (FPR) agonists promote immune evasion independent of functional Ncf1 derived ROS

Abstract Previous studies have shown that in both the absence of the Ncf1 gene or in the presence of a loss-of-function SNP, mice have a lower tumor burden when compared to wildtype animals. Here, we investigate a formyl peptide receptor (FPR) agonist in this setting and demonstrate that it promotes tumor immune evasion through an immune-Ncf1/ROS-dependent mechanism. We observed an increased colonization of B16F10 tumor cells in the lungs of FPR-agonist-treated wildtype C57BL6N mice compared to vehicle-control-treated mice. A significant reduction in tumor burden was expectedly seen in the Ncf1*/* mutant strain compared to the wildtype. However, FPR-agonist treatment did not promote tumor growth in Ncf1*/* treated mice when compared to vehicle-control-treated Ncf1*/* mice. These results suggest that FPR agonists can promote tumor evasion by acting through FPRs on immune cells in a NOX2/ROS-independent fashion and in a fashion independent of FPRs on the tumor.

Influence of bariatric surgery induced weight loss on oxidative DNA damage

Obesity is associated with elevated cancer risk, which may be represented by elevated genomic damage. Oxidative stress plays a key role in obesity related detrimental health consequences including DNA oxidation damage. The elevated cancer risk in obesity may be a consequence. Weight loss has been shown to reduce genomic damage, but the role of oxidative stress in that has not been clarified.The aim of this study is therefore to investigate the influence of bariatric surgery induced weight loss on DNA oxidation damage in morbidly obese subjects. For this aim, we used cryopreserved peripheral blood mononuclear cells in the FPG comet assay. Advanced protein oxidation products and 3-nitrotyrosine were measured as oxidative and nitrative protein stress markers. Furthermore, expression of oxidative stress related proteins HSP70 and Nrf2 as well as mitochondrial enzyme citrate synthase and NADPH oxidase subunit p22 phox were analysed.Our findings revealed significantly reduced DNA strand breaks, but DNA base oxidation was not reduced. We observed significant reduction in plasma AOPPs and 3-nitrotyrosine, which indicated an improvement in oxidative/nitrative stress. However, expression of HSP70 and Nrf2 were not altered after weight loss. In addition, expression of citrate synthase and p22 phox were also unaltered.Overall, bariatric surgery induced significant reduction in excess body weight and improved the patients’ health status, including reduced DNA strand breaks and slightly improved antioxidant status in some of the investigated endpoints, while cellular ROS formation and DNA oxidation damage stayed unaltered. This complex situation may be due to combined beneficial effects of weight loss and burdening of the body with fat breakdown products. In the future, collecting samples two years after surgery, when patients have been in a weight plateau for some time, might be a promising approach.

CRISPR-Directed Therapeutic Correction at the NCF1 Locus Is Challenged by Frequent Incidence of Chromosomal Deletions

Resurrection of non-processed pseudogenes may increase the efficacy of therapeutic gene editing, upon simultaneous targeting of a mutated gene and its highly homologous pseudogenes. To investigate the potency of this approach for clinical gene therapy of human diseases, we corrected a pseudogene-associated disorder, the immunodeficiency p47phox-deficient chronic granulomatous disease (p47phox CGD), using clustered regularly interspaced short palindromic repeats-associated nuclease Cas9 (CRISPR-Cas9) to target mutated neutrophil cytosolic factor 1 (NCF1). Being separated by less than two million base pairs, NCF1 and two pseudogenes are closely co-localized on chromosome 7. In healthy people, a two-nucleotide GT deletion (ΔGT) is present in the NCF1B and NCF1C pseudogenes only. In the majority of patients with p47phox CGD, the NCF1 gene is inactivated due to a ΔGT transfer from one of the two non-processed pseudogenes. Here we demonstrate that concurrent targeting and correction of mutated NCF1 and its pseudogenes results in therapeutic CGD phenotype correction, but also causes potentially harmful chromosomal deletions between the targeted loci in a p47phox-deficient CGD cell line model. Therefore, development of genome-editing-based treatment of pseudogene-related disorders mandates thorough safety examination, as well as technological advances, limiting concurrent induction of multiple double-strand breaks on a single chromosome.

Independent and inter-dependent immunoregulatory effects of NCF1 and NOS2 in experimental autoimmune encephalomyelitis

BackgroundIncreasing evidence has suggested that a single nucleotide polymorphism in the Ncf1 gene is associated with experimental autoimmune encephalomyelitis (EAE). However, the mechanisms of NCF1-induced immunoregulatory effects remain poorly understood. In this study, we focus on NCF1 deficiency-mediated effects on EAE in NOS2 dependent and independent ways.MethodsTo determine the effects of NCF1 and NOS2 during EAE development, we have established recombinant mouse strains deficient at NCF1 and/or NOS2 in a crossbreeding system. Different strains allow us to examine the entire course of the disease in the Nos2-null mice bearing a Ncf1 gene that encodes a mutated NCF1, deficient in triggering oxidative burst, after immunization with recombinant myelin oligodendrocyte glycoprotein (MOG)79-96 peptides. The peptide-induced innate and adaptive immune responses were analyzed by flow cytometry.ResultsNCF1-deficient mice developed a reduced susceptibility to EAE, whereas NCF1-NOS2 double-deficient mice developed an enhanced EAE, as compared with NOS2-deficient mice. Flow cytometry analyses show that double deficiencies resulted in an increase of neutrophils in the spleen, accompanied with higher release of interleukin-1β in neutrophils prior to EAE onset. The additional deficiency in NCF1 had no added effect on either interleukin-17 or interferon-γ secretion of T cells during the priming phase.ConclusionsThese studies show that NCF1 and NOS2 interact to regulate peptide-induced EAE.

Apoptotic and Oxidative Mechanisms in Liver and Kidney Tissues of Sheep with Fluorosis.

This study was planned to determine the molecular basis and causes of damage to the kidney and the liver, which are the most affected tissues in sheep exposed to chronic fluoride. For this purpose, liver and kidney tissues were obtained from sheep with signs of fluorosis in the age range of 4-6years. The control group consisted of clinically healthy sheep without fluorosis. The apoptotic and oxidative genes expression of target genes was determined using the real qRT-PCR method. According to the control gene (Gapdh) that was detected that in the liver, the apoptotic genes caspase-8, caspase-9, and Bim increased and caspase-3, Bcl-2, and Bak decreased, while in the kidney, caspase-3 and Bax and caspase-8, Bcl-2, Bcl2l-1, Bim, and Bak decreased. According to the 2-ΔCt values of the oxidative stress genes, it was determined that Cygb, Gstp1, and Ncf1 genes increased significantly in the fluorosis group and Gpx1, sod1, and sod2 genes decreased significantly. In the kidney tissue, Cygb and Gpx1 increased in the fluorosis group, while sod1, sod2, Gstp1, Ncf1 and Ccs, and Nos2 were found to decrease significantly. As a result, it was shown that apoptosis and oxidative mechanisms are activated in the liver and the kidney tissues of sheep with fluorosis and these parameters have an important role in understanding the molecular basis of tissue damage in fluorosis.

TIME COURSE CARDIOVASCULAR AND NEUROIMMUNE DYSFUNCTIONS IN A METABOLIC SYNDROME MODEL: IMPACT OF AEROBIC TRAINING

This study analyzed the effects of aerobic exercise training (ET) in the time course of the dysfunctions observed in metabolic syndrome model (SHR + fructose overload). Animals were randomly distributed into two groups: hypertensive + fructose (HF) and hypertensive + fructose + ET (HFT). The fructose overload (100g/l) and the ET (treadmill 1h/d, 5d/wk) was initiated at 30 days of life, and the animals (n=6/group/time) were evaluated after 7, 15, 30 and 60 days. The ET (HFT group) induced an increase in baroreflex sensitivity for bradycardic response at 7 (100%), 15 (86%), 30 (76%) and 60 (74%) days of protocol and for tachycardia response (TR) in 7 (34%), 15 (31%) and 30 (49%) days of protocol as compared to sedentary group (HF). However, the ET did not attenuate the impairment in TR at 60 days of protocol. There was a reduction in the relation of TNF‐a/IL‐10 in fat tissue (40%) and IL‐1β in the spleen (30%) in the HFT group when compared to the HF group in 15 days of protocol. The nitrites in plasma were increased in HFT group as compared to HF group at 7 and 15 days of protocol. Regarding oxidative stress in plasma at 60 days of protocol, there was an increase in SOD activity (74%) and a decrease in lipid peroxidation (46%) and in protein oxidation (60%) in the HFT group when compared to the HF group. The ET reduced blood glucose (15%) and increased insulin sensitivity (31%) at the end of the protocol, although it did not alter blood triglycerides or blood pressure. Fructose consumption increased NFkB and p47 phox protein expression at day 60. However, aerobic exercise training was able to reduce NFkB at day 30 and 60, and p47 phox at day 60. In conclusion, the hemodynamic, autonomic, inflammatory and oxidative stress disorders seem to be time dependent in SHR with fructose overload. On the other hand, aerobic ET is effective in attenuate disorders in this model of metabolic syndrome.Support or Funding InformationFAPESP: 2018/17183‐4

Myristoylated Protein Kinase C Beta II Peptide Inhibitor Improves Cell Survival in Cultured Human Umbilical Vein Endothelial Cells Subjected to Hypoxia/Reoxygenation

Endothelial dysfunction as a result of ischemia/reperfusion (I/R) injury is known to contribute to damage in myocardial infarction and organ transplant patients. In vascular cells, NADPH oxidase (NOX) and the mitochondrial electron transport chain are primary sources of reactive oxygen species (ROS) during I/R injury resulting in reduced nitric oxide bioavailability. Protein kinase C beta II (PKCβII) is an attractive therapeutic target due to its regulation of these downstream mediators of ROS production. PKCβII phosphorylates p66Shc to enhance mitochondrial‐derived ROS production and p47 phox to promote ROS release from NOX. We have previously shown that myristoylated PKCβII peptide inhibitor (N‐myr‐SLNPEWNET; myr‐PKCβII‐) improved post‐reperfusion cardiac function and reduced infarct size in rat myocardial I/R injury compared to non‐treated controls. The decrease in myocardial I/R injury with myr‐PKCβII‐treatment may be attributed to improved vascular endothelial function. The myristoylated peptide sequence (cargo sequence) targets a highly conserved peptide sequence among mammalian species and inhibits PKCβII translocation to protein substrates (e.g. NOX and p66Shc) after second messenger activation. We hypothesize that myr‐PKCβII‐ will confer protection by directly inhibiting ROS production from NOX and mitochondria in human umbilical vein endothelial cells (HUVECs) subjected to hypoxia/reoxygenation (H/R) injury. HUVECs were cultured in gelatin‐coated 96‐well plates and subjected to 24h hypoxia and 24h reoxygenation in Billups‐Rothenburg hypoxia chamber with 1% O2, 5% CO2, and balanced nitrogen. Myr‐PKCβII‐ (20 mM) treatment was administered at the beginning of the 24h reoxygenation period. Cell viability was assessed using tetrazolium‐salt (WST‐8) colorimetric assay with a microplate reader (450 nm) and normalized against the normoxia control group. Data were analyzed using Student‐Newman‐Keuls post‐hoc analysis. At the end of the 24h reoxygenation period, cell viability (%) was significantly reduced to 78±2% (p&lt;0.05; n=5) in the non‐treated H/R group compared to normoxia controls (n=5). Myr‐PKCβII‐significantly improved HUVEC survival (95±4%; p&lt;0.01; n=5) compared to non‐treated H/R controls and was not significantly changed compared to normoxia controls. The data suggests that PKCβII inhibition promotes cell survival subjected to H/R possibly due to directly attenuating NOX and mitochondrial derived ROS. Further studies are needed to control for the myristoylated acid conjugation of the peptide cargo.Support or Funding InformationThis research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at the Philadelphia College of Osteopathic Medicine. Current license is supported by Young Therapeutics, LLC.