A potential diagnostic serum immunological marker panel to differentiate between primary and secondary knee osteoarthritis.

Inflammation contributes to knee osteoarthritis (KOA) where many immunological mediators participate in its initiation and progression. Most clinicians manage primary (pKOA) and secondary osteoarthritis (sKOA) alike. Whether immunological profiles of pKOA and sKOA differ remains obscure. Hence, we aimed to differentially identify potential serum immunologic diagnostic markers of pKOA and of sKOA. This case control study used 46 KOA patients (pKOA, n = 30; sKOA, n = 16), and 60 age, gender matched controls (normal healthy, n = 30; systemic lupus erythematosus [SLE] disease controls, n = 30) where serum was assayed for cytokines (TNF-α, IL-1ß, IL-6, IL-10) and nitric oxide derivatives (NOx). Sandwich ELISA assessed cytokine levels, while the 'Griess assay' quantified NOx levels. The diagnostic accuracy of optimal marker combinations was evaluated by the CombiROC web tool. Compared with pKOA, sKOA serum displayed significantly elevated levels of pro inflammatory cytokines (TNF-α, IL-1ß, IL-6) with a concurrent decrease in the anti-inflammatory cytokine, IL-10 (P<0.05). This was reiterated by significantly higher Th1:Th2 (TNF-α: IL-10) serum cytokine ratio observed in sKOA compared to that of pKOA. The CombiROC curves identified TNF-α, IL-1ß, IL-6 and NOx as the best performing panel of potential diagnostic markers to discriminate pKOA from control groups (~97% accuracy, 90% Sensitivity [SE] and 98% specificity [SP]), while TNF-α, IL-1ß and IL-6 discriminated sKOA from control groups (~100% accuracy, 100% SE, and 98% SP). The study identified discrete serum immune biomarker panels to differentiate between pKOA (TNF-α, IL-1ß, IL-6 and NOx) and sKOA (TNF-α, IL-1ß and IL-6). These findings may assist in developing distinct therapeutic agents for the two types of KOA.

Dicloridrato de sapropterina para o tratamento de pacientes com fenilcetonúria a partir de cinco anos de idade / Sapropterin dihydrochloride for the treatment of patients with phenylketonuria from five years of age

CONTEXTO: Fenilcetonúria (FNC) é uma doença genética, autossômica recessiva, de erro inato do metabolismo no aminoácido fenilalanina. A mutação do gene o qual codifica a enzima hepática fenilalanina-hidroxilase (FAH) altera a conversão de fenilalanina em tirosina, causando um acúmulo de fenilalanina no sangue. O nível elevado de fenilalanina (FAL) no sangue é neurotóxico e leva aos defeitos no desenvolvimento neuromotor e neurocognitivo. A doença é rara, mas pode variar sua prevalência ao redor do mundo, de 1: 10.000 até 1:200.000. No Brasil, estima-se prevalência de 1:30.402 recém-nascidos, identificados pela triagem neonatal por meio do teste do pezinho, no âmbito do PNTN do MS. O controle da concentração de FAL no sangue com orientação de uma dieta restritiva de consumo de FAL e suplemento alimentar é o tratamento preconizado em todo o mundo, e deve ser utilizada pela vida toda com início precoce, até o 10º dia de vida. Devido à falta de aderência à dieta pelas crianças e pelos adultos, o uso de dicloridrato de sapropterina poderia aumentar a tolerância ao consumo de FAL natural, principalmente em indivíduos acima de 5 anos de idade. TECNOLOGIA: Dicloridrato de sapropterina (KUVAN®). PERGUNTA: Dicloridrato de sapropterina é eficaz e seguro para o tratamento da fenilcetonúria (FNC) a partir de 5 anos de idade? EVIDÊNCIAS CIENTÍFICAS: Há 2 ensaios clínicos randomizados, com baixo risco de viés, que avaliaram a eficácia do dicloridrato de sapropterina na diminuição de FAL no sangue, que mostraram serem eficazes no curto prazo (6 a 10 semanas). Há 1 ensaio clínico randomizado que avaliou os efeitos clínicos, na melhora dos sintomas da TDAH, em pacientes com FNC, e demonstrou melhora de atenção, identificado em uma subescala do escore quando comparado ao grupo controle de dieta isolada. Há estudos observacionais com pacientes que fizeram uso da sapropterina por até 7 anos, e que avaliaram a segurança e o aumento na tolerância ao consumo de FAL natural. Os efeitos adversos são leves e de fácil resolução e a melhora na tolerabilidade ao consumo de FAL natural foi mantida ao longo do tempo. AVALIAÇÃO ECONÔMICA: Um modelo analítico de decisão foi desenvolvido para uma análise de custo-efetividade incremental do dicloridrato de sapropterina em conjunto com fórmula metabólica versus apenas uso de fórmula metabólica. O modelo de Markov foi utilizado e modelado com ciclos anuais. O modelo assume que uma coorte de 1.000 pacientes entra no modelo e são designados para os grupos de tratamento. O valor incremental da razão de custoutilidade (RCUI) foi de R$ 1.131.036/QALY. No entanto, as probabilidades de transição utilizadas no modelo de Markov não estão claras e podem afetar o resultado de forma significativa. AVALIAÇÃO DO IMPACTO ORÇAMENTÁRIO: Para o cenário base, estima-se que no primeiro ano o impacto orçamentário incrementalseja de R$ 30.096.210,00 e ao longo dos 5 anos o valor acumulado de R$ 241.464.528,00. A principal limitação da estimativa do impacto orçamentário é o market share e a quantidade utilizada no tratamento atual, que assume uso de 15mg/kg/dia e consumo máximo de proteína do suplemento alimentar em todos os pacientes, podendo subestimar o impacto orçamentário incremental. CONSIDERAÇÕES FINAIS: O pequeno número de indivíduos incluídos nos ensaios clínicos randomizados, e os desfechos intermediários analisados em seguimento curto, gera incerteza na efetividade clínica, principalmente no uso de longo prazo. Por outro lado, as coortes de registro com até 7 anos ajudam a entender que é uma tecnologia com perfil de segurança aceitável. Na análise econômica, ainda há muitos dados que são baseados em opinião de especialistas e não está claro se as probabilidades utilizadas são robustas. Desta forma, há elevado grau de incerteza na eficiência desta tecnologia. RECOMENDAÇÃO PRELIMINAR DA CONITEC: Considerou-se que após apreciação inicial do parecer técnico-científico, as evidências deixam dúvidas quanto ao benefício na melhora na qualidade de vida e em aspectos neuropsicológicos. Será solicitado para a próxima reunião um especialista no assunto para melhor entendimento dos benefícios da tecnologia. Além disso, no modelo econômico apresentado pelo demandante, há quantidade considerável de incertezas nos parâmetros utilizados na modelagem. Assim, a Conitec em 03/03/2021, recomendou a não incorporação no SUS do dicloridrato de sapropterina para o tratamento da fenilcetonúria em crianças acima de 5 anos de idade. CONSULTA PÚBLICA: O relatório de recomendação inicial da Conitec foi disponibilizado para contribuições por meio da consulta pública nº 22 entre os dias 22/03/2021 e 12/04/2021. Foram recebidas 2098 contribuições, sendo 243 contribuições de cunho técnico-científico e 1855 contribuições de experiência pessoal ou opinião, destas 96% discordavam da recomendação preliminar da Conitec. RECOMENDAÇÃO FINAL DA CONITEC: Os membros da Conitec presentes na 97ª Reunião Ordinária, no dia 05/05/2021, deliberaram, por unanimidade, recomendar a não incorporação do dicloridrato de sapropterina para o tratamento da fenilcetonúria acima de 5 anos de idade. Os membros da Conitec consideraram que não houve adição de evidências e mesmo com as novas considerações da avaliação econômica, o custo do tratamento em relação a efetividade alcançada mostrou-se com uma razão de custo-utilidade incremental expressiva, aquém das possibilidades de ser uma tecnologia eficiente para o SUS. Assim, foi assinado o Registro de Deliberação nº 614/2021. DECISÃO: Não incorporar o dicloridrato de sapropterina para o tratamento da fenilcetonúria em crianças acima de 5 anosde idade, no âmbito do Sistema Único de Saúde ­ SUS, conforme a Portaria nº 29, publicada no Diário Oficial da União nº 129, seção 1, página 80, em 12 de julho de 2021.

Post-Translational Modifications of Nitrate Reductases Autoregulates Nitric Oxide Biosynthesis in Arabidopsis.

Nitric oxide (NO) is a regulator of growth, development, and stress responses in living organisms. Plant nitrate reductases (NR) catalyze the reduction of nitrate to nitrite or, alternatively, to NO. In plants, NO action and its targets remain incompletely understood, and the way NO regulates its own homeostasis remains to be elucidated. A significant transcriptome overlapping between NO-deficient mutant and NO-treated wild type plants suggests that NO could negatively regulate its biosynthesis. A significant increase in NO content was detected in transgenic plants overexpressing NR1 and NR2 proteins. In turn, NR protein and activity as well as NO content, decreased in wild-type plants exposed to a pulse of NO gas. Tag-aided immunopurification procedures followed by tandem mass spectrometry allowed identifying NO-triggered post-translational modifications (PTMs) and ubiquitylation sites in NRs. Nitration of tyrosine residues and S-nitrosation of cysteine residues affected key amino acids involved in binding the essential FAD and molybdenum cofactors. NO-related PTMs were accompanied by ubiquitylation of lysine residues flanking the nitration and S-nitrosation sites. NO-induced PTMs of NRs potentially inhibit their activities and promote their proteasome-mediated degradation. This auto-regulatory feedback loop may control nitrate assimilation to ammonium and nitrite-derived production of NO under complex environmental conditions.

Recent Developments in Drug Design of NO-donor Hybrid Compounds.

The free radical nitric oxide (NO) is considered one of the most versatile endogenous molecules and is a crucial signalling molecule in numerous biochemistry pathways of the human body. NO is directly related to pathological processes and plays an important role in many different and interrelated physiological processes. In some cases, a depletion of NO or an attenuation of its effector system could exist as in hypertension, angina and impotence; in others, an overproduction of NO may be a major cause of damage, as in circulatory shock, sepsis, neurodegenerative disorders and inflammatory responses. By using certain functional groups present in molecules that already have potential therapeutic value, hybrid compounds, by means of inclusion of NO-donors (e.g., ester nitrates and nitrites, S-nitrosothiols, metal complexes, furoxans, oxadiazoles, diazeniumdiolates and NO nanoparticles), can be developed that have a NO release benefit along with maintaining the activity of the native drug. The objective of the design of NO-donor hybrid compounds is to achieve a balance between the release of therapeutic amounts of NO, especially in the site of action, and maintaining the native drug activity. This review explores some of the most promising recent advances in NO-donor drug development and addresses the challenges associated with NO as a therapeutic agent.

Effects of NO-Hybridization on the Immunomodulatory Properties of the HIV Protease Inhibitors Lopinavir and Ritonavir.

HIV protease inhibitors (PIs) are antiretroviral agents, which have been found to also affect several cellular processes, such as inflammation and cell progression. In studies on non-steroidal, anti-inflammatory drugs, the addition of a nitric oxide (NO) moiety has been shown to both reduce their toxicity and enhance their pharmacological efficacy. Along this line of research, several derivatives of PIs have been synthesized by covalent attachment of NO moiety to the parental molecules. Previous work has indicated that NO-hybridization of the prototypical PI, Saquinavir leads to a derivative named Saquinavir-NO that while retaining the antiretroviral effect, acquires antitumoural and immunomodulatory properties along with reduced toxicity in vitro and in vivo. These data prompted us to evaluate the effects of NO-hybridization on two other PIs, Lopinavir and Ritonavir. The two NO-derivatives were compared head to head with their parental compounds on human primary peripheral blood mononuclear cells as well as on human primary macrophages. Lopinavir-NO and Lopinavir were also screened in an in vivo model of autoimmune hepatitis. Our results prove that Lopinavir-NO exerts markedly superior effects as compared to the parental compound both in vitro and in vivo. On the contrary, Ritonavir-NO effects overlapped those of Ritonavir. These data demonstrate that NO-hybridization of Lopinavir generates a derivative with significantly stronger immunomodulatory effects that are apparently related to an action of the compound on T-cell secretory capacity. Lopinavir-NO deserves additional studies for its possible use in T-cell-mediated autoimmune diseases including, but not limited to autoimmune hepatitis.

Pharmacological characterization of the relaxant effect induced by adrenomedullin in rat cavernosal smooth muscle

The aim of the present study was to determine the mechanisms underlying the relaxant effect of adrenomedullin (AM) in rat cavernosal smooth muscle (CSM) and the expression of AM system components in this tissue. Functional assays using standard muscle bath procedures were performed in CSM isolated from male Wistar rats. Protein and mRNA levels of pre-pro-AM, calcitonin receptor-like receptor (CRLR), and Subtypes 1, 2 and 3 of the receptor activity-modifying protein (RAMP) family were assessed by Western immunoblotting and quantitative real-time polymerase chain reaction, respectively. Nitrate and 6-keto-prostaglandin F1α (6-keto-PGF1α; a stable product of prostacyclin) levels were determined using commercially available kits. Protein and mRNA of AM, CRLR, and RAMP 1, -2, and -3 were detected in rat CSM. Immunohistochemical assays demonstrated that AM and CRLR were expressed in rat CSM. AM relaxed CSM strips in a concentration-dependent manner. AM22-52, a selective antagonist for AM receptors, reduced the relaxation induced by AM. Conversely, CGRP8-37, a selective antagonist for calcitonin gene-related peptide receptors, did not affect AM-induced relaxation. Preincubation of CSM strips with NG-nitro-L-arginine-methyl-ester (L-NAME, nitric oxide synthase inhibitor), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, quanylyl cyclase inhibitor), Rp-8-Br-PET-cGMPS (cGMP-dependent protein kinase inhibitor), SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole, selective cyclooxygenase-1 inhibitor], and 4-aminopyridine (voltage-dependent K+ channel blocker) reduced AM-induced relaxation. On the other hand, 7-nitroindazole (selective neuronal nitric oxide synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), H89 (protein kinase A inhibitor), SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, adenylate cyclase inhibitor], glibenclamide (selective blocker of ATP-sensitive K+ channels), and apamin (Ca2+-activated channel blocker) did not affect AM-induced relaxation. AM increased nitrate levels and 6-keto-PGF1α in rat CSM. The major new contribution of this research is that it demonstrated expression of AM and its receptor in rat CSM. Moreover, we provided evidence that AM-induced relaxation in this tissue is mediated by AM receptors by a mechanism that involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, and the opening of voltage-dependent K+ channels.

Pharmacological characterization of the relaxant effect induced by adrenomedullin in rat cavernosal smooth muscle.

The aim of the present study was to determine the mechanisms underlying the relaxant effect of adrenomedullin (AM) in rat cavernosal smooth muscle (CSM) and the expression of AM system components in this tissue. Functional assays using standard muscle bath procedures were performed in CSM isolated from male Wistar rats. Protein and mRNA levels of pre-pro-AM, calcitonin receptor-like receptor (CRLR), and Subtypes 1, 2 and 3 of the receptor activity-modifying protein (RAMP) family were assessed by Western immunoblotting and quantitative real-time polymerase chain reaction, respectively. Nitrate and 6-keto-prostaglandin F(1α) (6-keto-PGF(1α); a stable product of prostacyclin) levels were determined using commercially available kits. Protein and mRNA of AM, CRLR, and RAMP 1, -2, and -3 were detected in rat CSM. Immunohistochemical assays demonstrated that AM and CRLR were expressed in rat CSM. AM relaxed CSM strips in a concentration-dependent manner. AM(22-52), a selective antagonist for AM receptors, reduced the relaxation induced by AM. Conversely, CGRP(8-37), a selective antagonist for calcitonin gene-related peptide receptors, did not affect AM-induced relaxation. Preincubation of CSM strips with N(G)-nitro-L-arginine-methyl-ester (L-NAME, nitric oxide synthase inhibitor), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, quanylyl cyclase inhibitor), Rp-8-Br-PET-cGMPS (cGMP-dependent protein kinase inhibitor), SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole, selective cyclooxygenase-1 inhibitor], and 4-aminopyridine (voltage-dependent K(+) channel blocker) reduced AM-induced relaxation. On the other hand, 7-nitroindazole (selective neuronal nitric oxide synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), H89 (protein kinase A inhibitor), SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, adenylate cyclase inhibitor], glibenclamide (selective blocker of ATP-sensitive K(+) channels), and apamin (Ca(2+)-activated channel blocker) did not affect AM-induced relaxation. AM increased nitrate levels and 6-keto-PGF1α in rat CSM. The major new contribution of this research is that it demonstrated expression of AM and its receptor in rat CSM. Moreover, we provided evidence that AM-induced relaxation in this tissue is mediated by AM receptors by a mechanism that involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, and the opening of voltage-dependent K(+) channels.

Impact of chronic congestive heart failure on pharmacokinetics and vasomotor effects of infused nitrite.

BACKGROUND AND PURPOSE: Nitrite (NO2⁻) has recently been shown to represent a potential source of NO, in particular under hypoxic conditions. The aim of the current study was to compare the haemodynamic effects of NO2⁻ in healthy volunteers and patients with stable congestive heart failure (CHF). EXPERIMENTAL APPROACH: The acute haemodynamic effects of brachial artery infusion of NO2⁻ (0.31 to 7.8 µmol·min⁻¹) was assessed in normal subjects (n = 20) and CHF patients (n = 21). KEY RESULTS: NO2⁻ infusion was well tolerated in all subjects. Forearm blood flow (FBF) increased markedly in CHF patients at NO2⁻ infusion rates which induced no changes in normal subjects (ANOVA: F = 5.5; P = 0.02). Unstressed venous volume (UVV) increased even with the lowest NO2⁻ infusion rate in all subjects (indicating venodilation), with CHF patients being relatively hyporesponsive compared with normal subjects (ANOVA: F = 6.2; P = 0.01). There were no differences in venous blood pH or oxygen concentration between groups or during NO2⁻ infusion. Venous plasma NO2⁻ concentrations were lower in CHF patients at baseline, and rose substantially less with NO2⁻ infusion, without incremental oxidative generation of nitrate, consistent with accelerated clearance in these patients. Plasma protein-bound NO concentrations were lower in CHF patients than normal subjects at baseline. This difference was attenuated during NO2⁻ infusion. Prolonged NO2⁻ exposure in vivo did not induce oxidative stress, nor did it induce tolerance in vitro. CONCLUSIONS AND IMPLICATIONS: The findings of arterial hyper-responsiveness to infused NO2⁻ in CHF patients, with evidence of accelerated transvascular NO2⁻ clearance (presumably with concomitant NO release) suggests that NO2⁻ effects may be accentuated in such patients. These findings provide a stimulus for the clinical exploration of NO2⁻ as a therapeutic modality in CHF.

[Which future in cardiovascular therapy for nitric oxide and its derivatives?]. / Quel avenir en thérapeutique cardiovasculaire pour le monoxyde d'azote et ses dérivés?

Nitric oxide (NO) is involved in the regulation of several physiological processes such as vascular homeostasis. Exogenous NO supply offers major therapeutic interest, especially in the treatment of coronary artery disease, ischemic syndromes and other cardiovascular pathologies. Nevertheless, the administration of NO itself is limited by its short half-life. NO prodrugs have been marketed for decades, e.g. organic nitrates for angina pectoris. These prodrugs display undeniable advantages such as angina crisis relief and preconditioning effect. Nevertheless, they suffer from several drawbacks: toxicity, tolerance, endothelial dysfunction exacerbation. These negative effects are related to massive production of reactive species derived from oxygen or nitrogen, which trigger oxidative and nitrosative stress. New NO donors are under development to overcome those disadvantages, among which the S-nitrosothiols family seems especially promising.

Production of nitrogen oxide derivatives under the influence of NO-synthase inhibitors and natural compounds in mice with transplanted tumors.

AIM: The aim of the present study was to investigate the dynamics of nitric oxide derivative (NOD) formation in mice with transplanted tumors and to analize whether synthetic NO-synthase inhibitors, NO-donors and natural compounds could modulate NOD synthesis. MATERIALS AND METHODS: In the study F(1)(C(57)BlxCBA), CBA/Lac, BDF and Balb/c mice were used. Endogenous NOD synthesis in mice with transplanted Ehrlich carcinoma (EC) and Lewis lung carcinoma (LLC) was estimated by measuring urine nitrates (NA) and nitrites (NI) excretion and their concentration in tumor tissue determined by cadmium-reduction method. RESULTS: It is shown that EC development is accompanied by increased endogenous NOD formation whereas LLC growth - by its decrease. Total NOD excretion with urine in EC-bearing mice during tumor development was in the range of 1.1x10(-7)-7.0x10(-6) mol/kg body weight that was 1.7-6.8 times higher than that in LLC-bearing mice. Treatment of EC-bearing animals with N(ώ)-nitro-L-arginine and aminoguanidine resulted in decreased NOD formation causing moderate tumor growth retardation. Effect of treatment with nitroprusside was shown to be dependent on the rout of its administration and dosage. Treatment of EC-bearing mice with picnogenol, tannic acid, spirulina and paprika enriched with selenium resulted in tumor growth inhibition at the early stage of EC growth accompanied by stimulation of endogenous NOD formation. CONCLUSION: Regulation of endogenous NOD formation towards normal physiological levels or hyperproduction of these compounds may result in tumor growth suppression.

An integrated approach to assessing nitroso-redox balance in systemic inflammation.

Most studies examining the metabolic fate of NO during systemic inflammation have focused on measuring the quantitatively predominating, stable anions nitrite and nitrate within the circulation. However, these are not necessarily the NO-related products that govern NO metabolism and signaling in tissues. We assessed all major NO derivatives temporally in blood and vital organs during inflammation and explored their relationship to insult severity and redox status. Male rats receiving intraperitoneal endotoxin or vehicle were sacrificed for organ and blood sampling between 0 and 24 h. Endotoxin induced transient and organ-specific changes in a variety of NO metabolites. Nitrite and nitrate increased, peaking at 8 and 12 h, respectively. S- and N-nitrosation and heme-nitrosylation products also peaked at 8 h; these posttranslational protein modifications were associated with decreased myocardial function (echocardiography). Evidence of oxidative stress and systemic inflammation was also obtained. The rise in most NO derivatives was proportional to insult severity. All metabolite levels normalized within 24 h, despite evidence of persisting myocardial dysfunction and clinical unwellness. Our findings point to a complex interplay between NO production, antioxidant defense, and redox status. Although the precise (patho)physiologic roles of specific NO derivatives and their diagnostic/prognostic utility await further investigation, nitroso species in erythrocytes are the most sensitive markers of NO in systemic inflammation, detectable before clinical symptoms manifest.

A novel nitroxide is an effective brain redox imaging contrast agent and in vivo radioprotector.

Individuals are exposed to ionizing radiation during medical procedures and nuclear disasters, and this exposure can be carcinogenic, toxic, and sometimes fatal. Drugs that protect individuals from the adverse effects of radiation may therefore be valuable countermeasures against the health risks of exposure. In the current study, the LD(50/30) (the dose resulting in 50% of exposed mice surviving 30 days after exposure) was determined in control C3H mice and mice treated with the nitroxide radioprotectors Tempol, 3-CP, 16c, 22c, and 23c. The pharmacokinetics of 22c and 23c were measured with magnetic resonance imaging (MRI) in the brain, blood, submandibular salivary gland, liver, muscle, tongue, and myocardium. It was found that 23c was the most effective radioprotector of the five studied: 23c increased the LD(50/30) in mice from 7.9±0.15Gy (treated with saline) to 11.47±0.13Gy (an increase of 45%). Additionally, MRI-based pharmacokinetic studies revealed that 23c is an effective redox imaging agent in the mouse brain, and that 23c may allow functional imaging of the myocardium. The data in this report suggest that 23c is currently the most potent known nitroxide radioprotector, and that it may also be useful as a contrast agent for functional imaging.

Study of nitric oxide in patients with chronic hepatitis C genotype 4: relationship to viremia and response to antiviral therapy.

Hepatitis C virus (HCV) infection is a global medical problem. The role of Nitric oxide (NO) in chronic viral hepatitis is still unknown. It may play a prominent role as an antiviral agent that reduces its replication or as a mediator that causes accumulation of oxidative DNA damage and oncogenesis. The present study was carried out to study effect of combined peginterferon and ribavirin therapy for hepatitis C on NO in both responders and in non responder patients. The study included seventy three patients with positive serological markers for HCV. They were classified according to presence or absence of HCV viremia and the response to therapy. In addition sixteen control subjects were included. NO levels were determined as the stable end product nitrate and nitrite. Serum nitrite and nitrate concentrations in the patients with viral hepatitis were significantly higher than normal subjects and patients with serological evidence of hepatitis C infection in absence of viral load. The levels of nitrite >or= 31 microM, nitrate >or= 15 microM and NO(2)/NO(3) ratio < 1.5 microM were associated with increased risk of resistance to therapy. The multivariate logistic regression analysis showed that NO(2)/NO(3) ratio at levels < 1.5 microM was associated with HCV eradication independently. This study provides new insight into the pathogenesis of hepatitis C and highlights the effect of combined peginterferon and ribavirin on nitrite and nitrate as markers of endogenous NO system. There is a limitation level of NO that if it is increased above it may lead to non response to antiviral therapy. Therefore, it may be an important factor for chronic hepatitis C, which suggests an additional therapeutic pathway of anti-oxidants in combination with the standard regimen for further study.

Nitric oxide derivatives and soluble plasma selectins in patients with myeloproliferative neoplasms.

Essential thrombocythaemia (ET) and polycythaemia vera (PV) are characterised by a high incidence of thrombotic complications due to high-shear stress of the vessel wall, blood hyperviscosity and hypoxaemia, all factors responsible for chronic endothelial dysfunction and platelet and leukocyte activation. We evaluated the activation status of vascular cells in 18 consecutive ET and 14 PV patients by measuring the plasma levels of the nitric oxide derivatives (NOX) (i.e. nitrites and nitrates) and of soluble selectins of platelet (P-selectin), endothelial cell (P-selectin and E-selectin) and leukocyte (L-selectin) origin. The effect of hydroxyurea (HU) therapy on these parameters was also investigated. NOX were significantly (p<0.01) increased in ET patients treated with HU (11.5 +/- 2.6 nM) compared to non-HU treated ET (1.41 +/- 0.3 nM) and to controls (4.78 +/- 2.49 nM). Multivariate analysis confirmed HU therapy as an independent predictor of higher NOX levels in ET. In addition, NOX significantly correlated with haematocrit. Plasma P-selectin was significantly elevated in ET (350 +/- 40 ng/10(6) platelets) and PV (482 +/- 53 ng/10(6) platelets) patients compared to controls (120 +/- 8 ng/10(6) platelets). In PV, also E-selectin (23.8 +/- 4.2 ng/ml) was significantly increased compared to controls (11.2 +/- 1.1 ng/ml; p<0.01). P-selectin was significantly correlated to platelet (R=0.33; p=0.01) and leukocyte count (R=0.6; p=0.000), while E-selectin (R=0.34; p=.014) and sL-selectin (R=0.3; p=0.03) were correlated with leukocyte count only. In the multivariate analysis, NOX predicted increased levels of E-selectin in ET, but not in PV patients. Our data demonstrate that ET and PV are characterised by an altered pattern of soluble selectins and NOX. HU-mediated increase of NOX levels could represent an additional antithrombotic mechanism of this drug.

Nitric oxide and downstream second messenger cGMP and cAMP enhance adipogenesis in primary human preadipocytes.

BACKGROUND AIMS: Obesity is correlated with chronic low-grade inflammation. Thus the induction of inflammation could be used to stimulate adipose tissue formation in tissue-engineering approaches. As nitric oxide (NO) is a key regulator of inflammation, we investigated the effect of NO and its downstream signaling molecule guanosine 3',5'-cyclic monophosphate (cGMP) as well as adenosine 3',5'-cyclic monophosphate (cAMP) on preadipocytes in vitro. METHODS: Preadipocytes were isolated from human subcutaneous adipose tissue, cultured until confluence, and differentiated. The NO donor diethylenetriamine (DETA)/NO (30-150 microm) was added during proliferation and differentiation. Additionally, cGMP/cAMP analogs 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP) and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), and the adenylyl cyclase activator forskolin, specific guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and adenylyl cyclase inhibitor 2'-5'-dideoxyadenosine (ddA), were applied. Proliferation and differentiation were evaluated. RESULTS: DETA/NO in combination with the standard differentiation procedure significantly enhanced maturation of precursor cells to adipocytes. Proliferation, in contrast, was inhibited in the presence of NO. The application of cGMP and cAMP, respectively, increased pre-adipocyte differentiation to an even higher extent than NO. Inhibitors of the underlying pathways caused a significant decrease in adipogenic conversion. CONCLUSIONS: Our results support the application of NO donors during transplantation of preadipocytes in a 3-dimensional setting to accelerate and optimize differentiation. The results suggest that, instead of the rather instable and reactive molecule NO, the application of cGMP and cAMP would be even more effective because these substances have a stronger adipogenic effect on preadipocytes and a longer half-life than NO. Also, by applying inhibitors of the underlying pathways, the induced inflammatory condition could be regulated to the desired level.

Urinary nitric oxide excretion in infants with eczema.

Eczema is characterized by inflammation of the skin and is commonly associated with food allergy. It has been suggested that nitric oxide (NO) is an important player in eczema, food allergy and intestinal inflammation. The aim of this study was to assess the levels of urinary NO breakdown products in infants with eczema and the effect of eczema treatment on NO levels. Ninety-four infants with eczema, 58 boys and 36 girls, with a mean age of 7.5 ± 5.2 months (mean ± s.d.) at inclusion were examined twice with an interval of 6 wk. The sum of nitrite and nitrate was measured colorimetrically in urinary samples from both visits and compared with clinical data concerning eczema severity, nutrition, gastrointestinal symptoms, asthma and skin prick positivity. The levels of NO products increased significantly from the first to the second visit: 289; 374 µm (median; IQR) vs. 457; 678 µm (median; IQR) (p < 0.001) in parallel with a significant improvement of the eczema. After eczema treatment consisting of skin care and elimination diet during the 6-wk interval between evaluations, the NO levels approached the values previously found in healthy children. The results support previous studies indicating that the homeostasis of nitrogen radicals is disturbed in childhood eczema.

pO(2)-dependent NO production determines OPPC activity in macrophages.

Stimulated macrophages produce nitric oxide (NO) via inducible nitric oxide synthase (iNOS) using molecular O(2), L-arginine, and NADPH. Exposure of macrophages to hypoxia decreases NO production within seconds, suggesting substrate limitation as the mechanism. Conflicting data exist regarding the effect of pO(2) on NADPH production via the oxidative pentose phosphate cycle (OPPC). Therefore, the present studies were developed to determine whether NADPH could be limiting for NO production under hypoxia. Production of NO metabolites (NOx) and OPPC activity by RAW 264.7 cells was significantly increased by stimulation with lipopolysaccharide (LPS) and interferon gamma (IFNgamma) at pO(2) ranging from 0.07 to 50%. OPPC activity correlated linearly with NOx production at pO(2)>0.13%. Increased OPPC activity by stimulated RAW 264.7 cells was significantly reduced by 1400 W, an iNOS inhibitor. OPPC activity was significantly increased by concomitant treatment of stimulated RAW 264.7 cells with chemical oxidants such as hydroxyethyldisulfide or pimonidazole, at 0.07 and 50% O(2), without decreasing NOx production. These results are the first to investigate the effect of pO(2) on the relationship between NO production and OPPC activity, and to rule out limitations in OPPC activity as a mechanism by which NO production is decreased under hypoxia.

Nitric oxide metabolites as biomarkers for follow-up after chronic rhinosinusitis surgery.

BACKGROUND: Nitric oxide (NO) has a variety of effects on the pathophysiology of the nasal cavity and seems to play an important role in inflammation. It is increased in the common cold but decreased in acute and chronic rhinosinusitis (CRS). Exhaled NO increases after endoscopic sinus surgery in CRS. In our previous study we showed that NO metabolite (nitrate and nitrite) levels are increased in the sinus cavity of CRS patients. We hypothesized that NO metabolite levels are increased to normal in the nasal lavage of CRS patients after endoscopic sinus surgery and NO metabolites in the nasal lavage can be used as indicators of the disease status after surgery. METHODS: This study was performed on 52 patients with CRS who did not respond to medical therapy and who underwent surgery. NO metabolite levels were measured in nasal lavages of the patients before surgery and 2 months after surgery. RESULTS: NO metabolite levels (mean +/- SEM) were 18.11 +/- 3.08 micromol/L and 35.97 +/- 4.64 micromol/L in nasal lavages of patients before and after surgery, respectively. The levels of NO metabolites were increased significantly (p < 0.01) after surgery in nasal lavages and patients reported significant improvement based on the visual analog scoring after the operation. CONCLUSION: NO metabolite levels were decreased in nasal lavages of CRS patients and were increased to normal levels after surgery along with improvement of the disease. NO metabolite levels may be used as an indicator for the follow-up of patients after endoscopic sinus surgery.

Effect of uvulopalatopharyngoplasty on endothelial function in obstructive sleep apnea.

OBJECTIVES: This study evaluates the effects of uvulopalatopharyngoplasty (UPPP) on serum levels of nitric oxide derivatives (NO(x)) and endothelial function by endothelium dependent flow-mediated dilation (FMD) in obstructive sleep apnea syndrome (OSAS). STUDY DESIGN: Prospective study. SUBJECTS AND METHODS: Fifteen healthy subjects and 30 subjects with moderately severe to severe OSAS who desired UPPP were prospectively enrolled. FMD was measured by high-resolution B-mode ultrasonography; serum level of NO(x) from peripheral blood samples was also measured. All subjects participated in sleep studies. These studies were repeated 3 months after UPPP in OSAS patients. RESULTS: For healthy patients, there was no difference in serum level of NO(x) and FMD between baseline and 3 months later. The serum levels of NO(x) in 14 of 30 patients with OSAS - designated surgical responders - increased from 13.9 +/- 5.5 microM preoperation to 28.9 +/- 8.2 microM postoperatively. FMD increased from 5.2 +/- 5.0 preoperatively to 10.0 +/- 4.7 postoperatively. For the 16 unresponsive patients, serum NOx and FMD remained impaired after UPPP. CONCLUSION: Successful treatment of OSAS with UPPP leads to restoration of FMD and normal serum levels of NO(x).