BACKGROUND

Nitric oxide (NO) influences susceptibility to infection and hemodynamic failure (HF) in sepsis. NOS3 and NOS2 SNPs might modify plasma nitrite/nitrate (NOx) levels, sepsis development, hemodynamics and survival.

METHODS

90 severely septic and 91 non-infected ICU patients were prospectively studied. NOS3 (E298D), NOS3 (-786 T/C), NOS3 (27 bp-VNTR), and NOS2A (exon 22) SNPs and plasma NOx levels were assessed.

RESULTS

21 patients (11.6%) died, 7 with sepsis. TT homozygotes and T allele carriers of NOS3 (E298D) and AG carriers of the NOS2A (exon 22) SNPs were more frequent among septic compared to non-infected ICU patients (p < 0.05). Plasma NOx was higher in septic, especially in septic with hemodynamic failure (HF) or fatal outcome (p < 0.006). Plasma NOx was higher in carriers of the T allele of the NOS3 (E298D) SNP (p = 0.006). Sepsis independently associated with HF, increased NOx, peripheral neutrophils, and fibrinogen levels, decreased prothrombin and the presence of the NOS3 (E298D) and NOS2A (exon 22) SNPs. A low APACHE II score was the only variable associated with sepsis survival. NOx was independently associated with sepsis, HF, decreased neutrophils and higher APACHE.

CONCLUSIONS

NOS3 (E298D) and NOS2A (exon 22) SNPs, individually and in combination, and plasma NOx, associated with sepsis development. NOx associated with HF and fatal outcome.

The gene encoding the inducible form of Nitric Oxide Synthase (NOS2A) has been considered with interest in the evaluation of the genetic predisposition to Multiple Sclerosis (MS). The aim of the present study was to address the possible contribution of two microsatellites repeats of the NOS2A promoter region - (CCTTT)(n) and (AAAT)(n) - to MS susceptibility. One hundred and thirteen Italian patients with clinically definite RRMS and 237 age and sex matched healthy controls from Calabria (South Italy) were studied. The distribution analysis of the markers frequencies showed that the (CCTTT)(14) allele was found in 11.5% of the RRMS patients and in 25.3% of the healthy subjects, with a statistically significant difference (chi(2)=8.843, p=0.003). This data seems to confer a significant protection against MS (OR=0.348; 95% CI=0.174-0.693, corrected for age and gender). No association with MS susceptibility was observed for the bi-allelic (AAAT)(n) microsatellite. In conclusion, we found that the NOS2A (CCTTT)(14) allele was detected more frequently in the control group than in the RRMS patients, thus confirming the scientific interest on this marker.

As with other autoimmune diseases, susceptibility to multiple sclerosis (MS) is believed to result from the complex interaction of a number of genes, each with modest effect. Several genome-wide screens have implicated the nitric oxide synthase 2A gene (NOS2A), which encodes the inducible form of nitric oxide synthase, as being potentially associated with MS. To determine whether genetic variants within this gene may constitute a risk factor for causing MS, we investigated 13 single nucleotide polymorphisms in the NOS2A gene, in a case-control group of 214 Italian patients with MS and 121 controls. All these single nucleotide polymorphisms (SNPs) were analyzed using the SNPlex™ Genotyping System (Applied Biosystems). Data were analyzed using Genemapper 4.0 and Haploview 4.1. No significant association between cases and controls (P>0.05) was observed for the alleles of the SNPs. Defining the haplotype blocks also failed to detect any associated haplotypes. Our results suggest that polymorphic variation within the NOS2A gene does not influence the susceptibility to MS in patients of Italian origin.

Leprosy is one of the most neglected infectious tropical diseases of the skin and the nerves caused by the intracellular pathogen Mycobacterium leprae. The inducible NOS isoform encoded by NOS2A plays a vital role in host defence against bacterial infections. The functional promoter polymorphisms in NOS2A are associated with various autoimmune and infectious diseases. We investigated the association of NOS2A variants with progression of leprosy in a Brazilian cohort including 221 clinically classified patients and 103 unrelated healthy controls. We observed a novel variant ss528838018A/G in the promoter region at position -6558. The other functional variants were observed with low frequency of minor allele (<0.005). NOS2A promoter variant (-954G/C) was not observed in Brazilian populations, and the new observed promoter variant (ss528838018A/G) as well as other promoter variants were not associated with any clinical forms of leprosy in the Brazilian populations.

This study aimed to evaluate whether genetic polymorphisms of the inducible nitric oxide synthase (iNOS) gene NOS2A could be associated with a sustained virological response (SVR) among patients infected with hepatitis C virus genotypes 1 and 2 (HCV-1 and HCV-2) who were treated with peginterferon plus ribavirin (PEG-IFNα-RBV). We analyzed the associations between SVR to PEG-IFNα-RBV therapy and two single nucleotide polymorphisms (SNPs) in NOS2A. This study included Taiwanese Chinese patients infected with either HCV-1 (n = 265) or HCV-2 (n = 195) with or without a SVR. Among the NOS2A SNPs examined, the combination of genotypes A/A and A/G of rs2248814 was inversely correlated with SVR in patients infected with HCV-1 (P = 0.0048), particularly in males (P = 0.0281). This effect was not observed in patients infected with HCV-2. The AC NOS2A haplotype comprising two SNPs (rs2248814 and rs2072324) was found to be associated with SVR, and its presence may decrease the chances for a successful outcome of treatment of patients infected with HCV-1 (P = 0.0053). HCV-1 infected patients who carried the A-C diplotype will have a lower success rate of achieving a SVR (P = 0.0117). In addition, a multivariate logistic regression model for predicting a SVR revealed that the presence of the A-C diplotype interactively affected the outcome of PEG-IFNα-RBV treatment. The presence of NOS2A SNPs and the association with SVR showed that NOS2A polymorphisms may influence the therapeutic outcomes of patients infected with HCV-1 under standard of care treatment.

Little is known regarding the molecular mechanisms behind respiratory inflammatory response induced by ozone. We performed a longitudinal panel study with four repeated measurements among 43 young adults in Shanghai, China from May to October in 2016. We collected buccal samples and measured the fractional exhaled nitric oxide (FeNO) after 3-day personal ozone monitoring. In buccal samples, we measured concentrations of inducible nitric oxide synthase (iNOS) and arginase (ARG), and DNA methylation of NOS2A and ARG2. We used linear mixed-effect models to analyze the effects of ozone on FeNO, two enzymes and their DNA methylation. A 10 ppb increase in ozone (lag 0-8 h) was significantly associated with a 3.89% increase in FeNO, a 36.33% increase in iNOS, and a decrease of 0.36 in the average methylation (%5mC) of NOS2A. Ozone was associated with decreased ARG and elevated ARG2 methylation, but the associations were not significant. These effects were more pronounced among allergic subjects than healthy subjects. The effects were much stronger when using personal exposure monitoring than fixed-site measurements. Our study demonstrated that personal short-term exposure to ozone may result in acute respiratory inflammation, which may be mainly modulated by NOS2A hypomethylation in the arginase-nitric oxide synthase pathway.

OBJECTIVE

To investigate whether common variants in the oxidative pathway genes influence inflammatory bowel disease (IBD) risk among Moroccan patients.

METHODS

The distribution of (TAAA)n_rs12720460 and (CCTTT)n _rs3833912 NOS2A microsatellite repeats, HIF-1A_rs11549467 and NFKB1-94ins/delATTG_rs28362491 was analyzed in 507 subjects grouped in 199 IBD and 308 healthy controls. Genotyping was performed with polymerase chain reaction-fluorescent method and the TaqMan® allelic discrimination technology.

RESULTS

The allele and genotype frequencies of HIF1A_ rs11549467, NFKB1_rs28362491 and NOS2A_ (TAAA)n did not differ significantly between patients and controls. Analysis of NOS2A_ (CCTTT)n markers evidenced differences between patients and healthy controls. A preferential presence of the (CCTTT)8 (P = 0.02; OR = 1.71, 95%CI: 1.07-2.74), (CCTTT)14 (P = 0.02; OR = 1.71, 95%CI: 1.06-2.76) alleles in IBD, (CCTTT)8 (P = 0.008; OR = 1.95, 95%CI: 1.17-3.23) in CD and (CCTTT)7 (P = 0.009; OR = 7.61, 95%CI: 1.25-46.08), (CCTTT)11 (P = 0.05; OR = 0.51, 95%CI: 0.25-1.01), (CCTTT)14 (P = 0.02; OR = 2.05, 95%CI: 1.07-3.94), (CCTTT)15 (P = 0.01; OR = 2.25, 95%CI: 1.16-4.35) repeats in UC patients indicated its possible association with higher disease risk which need to be confirmed in a larger sample size.

CONCLUSIONS

Our results suggest that the NOS2A_ (CCTTT)n gene variations may influence IBD susceptibility in the Moroccan population.

To assess the influence of inducible and endothelial nitric oxide synthase gene (NOS2A and NOS3) polymorphisms in susceptibility to Crohn's disease (CD) and ulcerative colitis (UC). A total of 505 inflammatory bowel disease (IBD) patients (221 with UC and 284 with CD) and 332 ethnically matched controls were studied. Patients and controls were genotyped by polymerase chain reaction -based techniques for a multiallelic (CCTTT)(n) repeat and biallelic marker (TAAA)(n) in the promoter region of the NOS2A gene and for a T/C polymorphism at position -786 in the promoter region and a polymorphism in exon 7(298Glu/Asp) of the NOS3 gene. There was not association between NOS2A and NOS3 genotypes, alleles or haplotypes frequencies with UC, CD and controls. Our data suggest that NOS2A and NOS3 polymorphisms do not play a major role in IBD predisposition.

Hearing loss in Ménière's disease (MD) is associated with loss of spiral ganglion neurons and hair cells. In a guinea pig model of endolymphatic hydrops, nitric oxide synthases (NOS) and oxidative stress mediate loss of spiral ganglion neurons. To test the hypothesis that functional variants of NOS1 and NOS2A are associated with MD, we genotyped three functional variants of NOS1 (rs41279104, rs2682826, and a cytosine-adenosine microsatellite repeat in exon 1f) and the CCTTT repeat in the promoter of NOS2A gene (rs3833912) in two independent MD sets (273 patients in total) and 550 controls. A third cohort of American patients was genotyped as replication cohort for the CCTTT repeat. Neither allele nor genotype frequencies of rs41279104 and rs2682826 were associated with MD, although longer alleles of the cytosine-adenosine microsatellite repeat were marginally significant (corrected p = 0.05) in the Mediterranean cohort but not in a second Galicia cohort. Shorter numbers of the CCTTT repeat in NOS2A were significantly more frequent in Galicia controls (OR = 0.37 [CI, 0.18-0.76], corrected p = 0.04), but this finding could not be replicated in Mediterranean or American case-control populations. Meta-analysis did not support an association between CCTTT repeats and risk for MD. Severe hearing loss (>75 dB) was also not associated with any functional variants studied. Functional variants of NOS1 and NOS2A do not confer susceptibility for MD.

This study was designed to investigate possible interference of Xenobiotics with SUMOylation in eukaryotic cells. To begin with, we docked 71 chemical structures from PubChem with human SUMO1 and UBC9 protein structures using Auto Dock 4.2 and Hex 6.3 and selected five compounds for binding studies in Surface Plasmon Resonance (SPR) with human SUMO1. In SPR studies, only endosulfan showed binding to SUMO1 (Kd1.313 × 10-4 M). Further, we treated HePG2 and differentiated 3T3-L1 cells with endosulfan/bisphenol A/perfluorooctanoic acid (PFOA) to test induction of oxidative stress and SUMO isoform/UBC9 expression. Treatment with these compounds resulted in higher levels of nitric oxide (NO), NOS2A mRNA, and reactive oxygen species (ROS) associated with decreased NADPH levels. Additionally, treatment with these chemicals resulted in elevated mRNA levels of IL-6 and IL-1β in 3T3-L1 cells. In HePG2 cells, endosulfan treatment resulted in elevated mRNA levels of SUMO1, 3 and UBC9, whereas, treatment with bisphenol A resulted in increased mRNA of SUMO2, 3 and UBC9. Treatment with PFOA resulted in elevated mRNA levels of SUMO2. Apart from influencing the gene expression, endosulfan caused decrease in SUMO1-Sumoylation of few proteins. We propose that one reason for the severe health consequences of exposure to endosulfan/bisphenol could be due to induction of oxidative stress and modulation in SUMO and UBC9 gene expression.

BACKGROUND

Pre-eclampsia is one of the most frequent complications of pregnancy, however, little is known about its aetiology.

OBJECTIVE

The objective of this study was to investigate the association between inducible nitric oxide synthase (iNOS) genotypes and pre-eclampsia. We also measured the concentrations of tumour necrosis factor-alpha (TNF-alpha), nitric oxide (NO) and superoxide dismutase (SOD) in patients with pre-eclampsia to evaluate their relations to the single nucleotide polymorphisms (SNPs) observed.

METHODS

This cross-sectional study included 30 pregnant women with pre-eclampsia and 30 healthy pregnant women. They were screened at 28th, 36th weeks of gestation and just after delivery (within 48 h), and their blood samples were analysed for NO, SOD, TNF-alpha and iNOS gene polymorphism.

RESULTS

Patients with pre-eclampsia at 36 weeks gestation showed significantly increased serum NO levels (P=0.007), whereas SOD activity was decreased significantly (P=0.004). A doublefold increase was observed in TNF-alpha levels at 36 weeks in patients with pre-eclampsia (P=0.003) which decreased significantly (P=0.001) after delivery. A total of four SNPs were observed, of which two (G300A exon 8 and G274T exon 16) showed statistically significant association with pre-eclampsia. When compared, G274T exon 16 SNP also showed association with TNF-alpha levels and SOD activity in pre-eclamptic patients.

CONCLUSIONS

As pre-eclampsia is a disease of multifactorial aetiopathology, NO, TNF-alpha, SOD activity and NOS2A polymorphism might play an intermingled role in its development.

Perinatal asphyxia in the neonatal brain triggers a robust inflammatory response in which nitric oxide (NO) generation plays a hazardous role. Increased levels of NO can be maintained by the activity of inducible NO synthase (NOS2A) on its own or activated by IL-1beta (IL-1β) gene transcription and positive back stimulation of the NOS2 (CCTTT)n microsatellite by IL-1β, thus potentiating brain injury after ischemic perinatal asphyxia. We investigated whether the risk for cerebral palsy (CP) increases when an expansion of the - 2.5 kb (CCTTT)n microsatellite in the NOS2A gene and a single nucleotide polymorphism (SNP) in -C511T of the IL- IL-1β gene promoter occur in patients after perinatal hypoxic-ischemic encephalopathy. Genomic DNA was purified from peripheral leukocytes of 48 patients with CP and of 57 healthy control children. IL-1β SNP genotypes were established using a real-time PCR technique and fluorogenic probes and were validated by restriction fragment length polymorphism (RFLP) analysis using the AvaI restriction enzyme. The length of the CCTTTn microsatellite in the NOS2 gene promoter was determined by automated sequencing. The 14 repeat-long allele of the CCTTTn NOS2A microsatellite was present in 27% of CP patients vs 12.3% of controls, showing an odds ratio (OR) = 2.6531 and 95% confidence interval (CI) = 0.9612-7.3232 (P < 0.0469). The -511 TT genotype frequency showed an OR = 2.6325 (95% CI = 1.1348-6.1066, P = 0.0189). Interestingly, the haplotype CCTTT14/TT showed an OR = 9.561 (95%, CI = 1.1321-80.753; P = 0.0164). The haplotype (CCTTT)14/TT, formed by the expansion of the - 2.5 kb (CCTTT)n microsatellite in the NOS2A gene promoter and the -511 C➝ T SNP of the IL-1β gene promoter, might be a useful marker to identify patients who are at high risk for developing CP after hypoxic-ischemic encephalopathy.

Background: Hydrogen sulphide (H₂S) is considered as the third member of the gasotransmitter family, along with nitric oxide (NO) and carbon monoxide. H₂S has been reported to induce angiogenesis by promoting the growth, migration and tube-like structure formation of endothelial cells. Those studies were conducted in conditions of cell culture, mouse Matrigel plug assay model, rat wound healing model or rat hindlimb ischaemia model. Recent in vivo studies showed the physiological importance of H₂S in muscle angiogenesis. However, the importance of endogenous H₂S for brain angiogenesis during development remains unknown. We therefore aimed at determining the role of H₂S in brain vascular development.

Methods and results: Both knockdown and knockout of H₂S-producing enzymes, cystathionine β-synthase (cbs) and cystathionine γ-lyase (cth), using morpholino oligonucleotides and clustered regularly interspaced short palindromic repeats/Cas9-mediated mutation, impaired brain vascular development of larval zebrafish. Incubation with the slow-releasing H₂S donor GYY4137 alleviated the defects of brain vascular development in cbs and cth morphants. Quantitative analysis of the midbrain vascular network showed that H₂S enhances angiogenesis without affecting the topological structure of the brain vasculature. Mechanically, nitric oxide synthase 2a (nos2a) expression and NO production were decreased in both cbs and cth morphants. Overexpression of nos2a by coinjection of cbs or cth MO with full-length zebrafish nos2a mRNA alleviated the brain vascular developmental defects in cbs and cth morphants.

Conclusion: We conclude that H₂S promotes brain developmental angiogenesis via the NOS/NO pathway in zebrafish.

Keywords: brain; stroke; vascular malformation.

Ethnopharmacology relevance: Qiwei Putao powder (Uzhumu-7 in Mongolian) is a traditional Mongolian medicine, which has been widely used for alleviating cough and dyspnea, especially in aged individuals in both Inner Mongolia Autonomous Region and Xinjiang Uygur Autonomous Region of China. However, the active ingredients and exact pharmacological mechanism remain unclear.

Materials and methods: The protective effect of Qiwei Putao powder (QPP) on mice with cigarette smoke (CS)- and lipopolysaccharide (LPS)-induced chronic obstructive pulmonary disease (COPD) was assessed by histopathological hematoxylin and eosin staining, lung coefficient determination and measurement of cytokine levels. The bioactive ingredients and potential targets of the QPP were screened and detected with network pharmacology method and ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS). The mechanism and efficacy of active ingredients were further validated in COPD mice with immunohistochemistry tests, cytokine level measurement and RT-PCR. The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) in the nucleus, interleukin (IL)-1β, superoxide dismutase (SOD), malondialdehyde (MDA) and tumor necrosis factor-alpha (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA) kits to evaluate oxidative stress and inflammatory conditions in vivo after treatment. The expression of Nrf2 and downstream genes was detected by PCR.

Results: QPP can alleviate pathological changes in the lung during COPD progression. Sixty-one bioactive molecules were identified in QPP, 42 candidate compounds present in UPLC-Q/TOF-MS and 30 predicted COPD-related targets were generated by in silico analysis. A therapeutic network was constructed with all potential targets to predict the preventive effects of the targets on respiratory disease as well as cardiovascular diseases, nervous system diseases, musculoskeletal diseases and bacterial infections. Targets related to inflammation, immunity and oxidative stress (prostaglandin-endoperoxide synthase two, PTGS2; Nrf2; heat shock protein 90 alpha class A1, HSP90AA1; nitric oxide synthase, NOS2A; etc.) influenced COPD progression the most. We found that Nrf2 promotes a cell antioxidant response and is a key common target in the response to treatment with isoliquiritigenin (ISL), pterostilbene (PTE) and quercetin (QUE), the highly absorbed active ingredients in the formula. The data showed a strong synergistic protective role of these three molecules against the death of human type II alveolar adenocarcinoma (A549) cells through Nrf2 activation following H₂O₂ exposure.

Keywords: Antioxidation; Chronic obstructive pulmonary disease; Nrf2; Oxidative stress; Qiwei Putao powder formula; Systematic pharmacology.

Here we present immunostimulant-loaded nanoliposomes (NL_c) as a strategy to protect zebrafish larvae against bacterial infection. The NL_c encapsulate crude lipopolysaccharide (LPS) from E. coli and polyinosinic:polycytidylic acid (Poly I:C), a synthetic analogue of viral dsRNA. Fluorescently-labeled NL_c were ingested by zebrafish larvae 4 days post fertilization, when administrated by bath immersion, and accumulated in the intestine. RT-qPCR analysis showed the expression of innate immune related genes (tnfα, il1β, nos2a, irf1a and ptgs2a) was significantly upregulated at 48 h post NL_c treatment. A zebrafish larvae infection model for Aeromonas hydrophila was set up by bath immersion, achieving bacterial-dose-dependent significant differences in survival at day 5 post infection in both injured and non-injured larvae. Using this model, NL_c protected non-injured zebrafish larvae against an A. hydrophila lethal infection. In contrast, neither the empty nanoliposomes nor the mixture of immunostimulants could protect larvae against lethal challenges. Our results demonstrate that nanoliposomes could be further developed as an efficient carrier, widening the scope for delivery of other immunostimulants in aquaculture.

Few epidemiological studies have evaluated the respiratory effects of personal exposure to nitrogen dioxide (NO₂), a major traffic-related air pollutant. The biological pathway for these effects remains unknown.

To evaluate the short-term effects of personal NO₂ exposure on lung function, fractional exhaled nitric oxide (FeNO) and DNA methylation of genes involved.

We conducted a longitudinal panel study among 40 college students with four repeated measurements in Shanghai from May to October in 2016. We measured DNA methylation of the key encoding genes of inducible nitric oxide synthase (NOS2A) and arginase (ARG2). We applied linear mixed-effect models to assess the effects of NO₂ on respiratory outcomes.

Personal exposure to NO₂ was 27.39 ± 23.20 ppb on average. In response to a 10-ppb increase in NO₂ exposure, NOS2A methylation (%5 mC) decreased 0.19 at lag 0 d, ARG2 methylation (%5 mC) increased 0.21 and FeNO levels increased 2.82% at lag 1 d; and at lag 2 d the percentage of forced vital capacity, forced expiratory volume in 1 s and peak expiratory flow in predicted values decreased 0.12, 0.37 and 0.67, respectively. The model performance was better compared with those estimated using fixed-site measurements. These effects were robust to the adjustment for co-pollutants and weather conditions.

Our study suggests that short-term personal exposure to NO₂ is associated with NOS2A hypomethylation, ARG2 hypermethylation, respiratory inflammation and lung function impairment. The use of personal measurements may better predict the respiratory effects of NO₂.

Marek's disease (MD) is a complex pathology of chickens caused by MD virus (MDV) 1 and is observed as paralysis, immune suppression, neurologic signs, and the rapid formation of T-cell lymphomas. The incidence of MD in commercial broilers is largely controlled via vaccination, either in ovo or at hatch with live attenuated vaccines, i.e., turkey herpesvirus (HVT) or a bivalent combination of HVT with the MDV 2 strain (SB1). To further extend the protection conferred by bivalent HVT/SB-1, recombinant HVTs encoding transgenes of other avian viruses have similarly been used for in ovo administration. Despite decades of use, the specific mechanisms associated with vaccine-induced protection remain obscure. Additionally, the mechanistic basis for vaccine synergism conferred by bivalent HVT/SB-1, compared with HVT or SB-1 administered alone, is largely unknown. In the present study, we report on temporal changes in innate and acquired immune-patterning gene expression by using ex vivo splenocyte infection and in ovo vaccination models. We report that in the ex vivo splenocyte infection model, by 72 hr postinfection, vaccines induced IFN and IFN-stimulated gene expression, with lesser proinflammatory cytokine induction. For several genes (TLR3, IFN-γ, OASL, Mx1, NOS2A, and IL-1β), the effects on gene expression were additive for HVT, SB1, and HVT/SB1 infection. We observed similar patterns of induction in in ovo-vaccinated commercial broiler embryos and chicks with HVT/SB-1 or recombinant HVT-based bivalent combination (HVT-LT/SB-1). Furthermore, HVT/SB-1 or HVT-LT/SB-1 in ovo vaccination appeared to hasten immune maturation, with expression patterns suggesting accelerated migration of T and natural killer cells into the spleen. Finally, HVT/SB-1 vaccination resulted in a coordinated induction of IL-12p40 and downregulation of suppressors of cytokine signaling 1 and 3, indicative of classical macrophage 1 and T-helper 1 patterning.

OBJECTIVE

To determine whether polymorphism of NOS2A promoter -969G>C is associated with the portal hypertension of liver cirrhosis.

METHODS

A case control study covering 106 patients with liver cirrhosis due to hepatitis B virus(HBV) in comparison with 108 controls was performed using PCR-restriction fragment length polymorphism. The NOS2A mRNA and protein expression in liver cirrhosis tissues were detected by reverse transcription-PCR and Western blot. The recombinant plasmids of NOS2A promoter luciferase reporter gene were constructed and were transfected transiently into HepG2 cells for analyzing the functional activity of the promoter.

RESULTS

The frequencies of the C allele and GC genotype at NOS2A promoter -969G>C were significantly higher in portal hypertension group (16.9%, 33.8%) than in control group(8.8%, 17.6%)(P<0.05), and positive correlation (r=0.18) and association (OR=2.42) were noted. There was no significant difference in frequency distribution between single liver cirrhosis group and control group(P>0.05). The expressions of NOS2A mRNA and protein in liver cirrhosis tissues were more increased in C allele carriers with liver cirrhosis than in G allele carriers with liver cirrhosis, which led to higher functional activity of the promoter. Multivariate logistic regression analysis revealed that NOS2A polymorphism at promoter -969G>C is an independent novel risk factor for the occurrence of portal hypertension in patients with liver cirrhosis.

CONCLUSIONS

The polymorphism of NOS2A promoter -969(G>C) is associated with portal hypertension of liver cirrhosis, which results in functional activity increase of NOS2A promoter and is an independent risk factor for portal hypertension.

Background: Traffic-related air pollution (TRAP) has been associated with increased risk of airway inflammation in children with asthma. While epigenetic changes could potentially modulate TRAP-induced inflammatory responses, few studies have assessed the temporal pattern of exposure to TRAP, epigenetic changes and inflammation in children with asthma. Our goal was to test the time-lag patterns of personal exposure to TRAP, airway inflammation (measured as fractional exhaled nitric oxide, FeNO), and DNA methylation in the promoter regions of genes involved in nitric oxide synthesis among children with asthma.

Methods: We measured personal exposure to black carbon (BC) and FeNO for up to 30 days in a panel of children with asthma. We collected 90 buccal cell samples for DNA methylation analysis from 18 children (5 per child). Methylation in promoter regions of nitric oxide synthase (NOS1, NOS2A, NOS3) and arginase (ARG1, ARG2) was assessed by bisulfite pyrosequencing. Linear-mixed effect models were used to test the associations of BC at different lag periods, percent DNA methylation at each site and FeNO level.

Results: Exposure to BC was positively associated with FeNO, and negatively associated with DNA methylation in NOS3. We found strongest association between FeNO and BC at lag 0-6 h while strongest associations between methylation at positions 1 and 2 in NOS3 and BC were at lag 13-24 h and lag 0-24 h, respectively. The strengths of associations were attenuated at longer lag periods. No significant associations between exposure to TRAP and methylation levels in other NOS and ARG isoforms were observed.

Conclusions: Exposure to TRAP was associated with higher levels of FeNO and lower levels of DNA methylation in the promoter regions of the NOS3 gene, indicating that DNA methylation of the NOS3 gene could be an important epigenetic mechanism in physiological responses to TRAP in children with asthma.

Keywords: Asthma; DNA methylation; Exhaled nitric oxide; Traffic-related air pollution.

Introduction A new emerging role of nitric oxide (NO) in the aetiology of osteoarthritis (OA) has been reported. Inducible NO synthase (iNOS), produced by chondrocytes, is the major source of NO in the osteoarthritic cartilage. The aim of this study is to evaluate the potential association between the -1173C/T (rs9282799), -1026 C/A (rs 2779249) and -954G/C (rs1800482) single nucleotide polymorphisms (SNPs) in the promoter of the iNOS gene (NOS2A) and the incidence of knee OA in Greek population. Methods Ninety-six patients with primary knee OA were included in the study along with 44 controls. Genotypes were identified using polymerase chain reaction (PCR) and DNA sequencing techniques. Allelic and genotypic frequencies were compared between patients and controls. Results None of the -1173C/T, -1026 C/A and -954G/C SNPs were detected in the studied population, either in patients or controls. However, another SNP was identified at the site -1056 at the promoter region, where the initial G allele was substituted by the T allele. Interestingly, the TT genotype was completely absent in controls, but was detected in six patients with a 6.2% observed frequency. The difference between patients and controls was not statistically significant (p-value = 0.18). In male OA patients, the observed frequency of the TT genotype was higher (28.6%) in comparison to the 0% of the male controls (p-value = 0.1). The frequency of the G allele was 0.82 in controls and 0.78 in OA patients (p-value = 0.53). Conclusions The present study demonstrates that the 954G/C, -1026C/A, -1056G/T and -1173C/T SNPs of the NOS2A gene are not a risk factor for primary knee OA in Greek population. Moreover, -954G/C, -1026C/A and -1173C/T are rare, if not completely absent, in the Greek population. Additional research is mandatory in order to investigate the association of these SNPs with OA in different ethnic populations.

Multiple clinical and physiopathological studies as well as genetic analysis, suggest that diabetic retinopathy (DR) is a consequent of interactions between environmental factors, especially hyperglycaemia, and several genetic factors. The genes of aldose reductase (AR), inducible nitric oxide synthase (NOS2A), endothelial nitric oxide synthase (NOS3), vascular endothelial growth factor (VEGF), pigmented epithelium-derived factor (PEDF), protein kinase C-beta (PKC-beta) and receptor for advanced glycation end products (RAGE) implicated in the pathogenesis of DR. The only genetic marker associated with risk of DR in several studies is a microsatellite (A-C)n at 5'end of AR. The synergistic combination of conventional approaches (e.g. candidate gene association studies) with new emerging technologies (e.g. biochips) will be a key factor in the elucidation of the genetic aspects of DR.