RESUMO
BACKGROUND: In vitro study showed that NADPH oxidase (NOx), the most important enzyme producing reactive oxygen species (ROS), plays a role in the process of platelet activation. However, it is unclear if changes in its activity affect platelet activation in vivo. METHODS AND RESULTS: In vivo and ex vivo experiments assessing platelet activation were investigated in healthy subjects, obese patients, and subjects with different low rates of NOx2 activity, namely X-linked chronic granulomatous disease (X-CGD) patients and X-CGD carriers. We included 27 X-CGD patients, 31 women carriers of hereditary deficiency of NOx2, 31 obese women, and 62 healthy subjects matched for sex and age. Plasma levels of soluble sCD40 L (sCD40L) and soluble P (sP)-selectin, 2 markers of in vivo platelet activation, were reduced in X-CGD patients (sCD40L=-55%; sP-selectin=-51%, P<0.001) and in X-CGD carriers (sCD40L=-41%; sP-selectin=-57%, P<0.001) compared with respective controls. Conversely, obese women, who disclosed NOx2 upregulation, had significantly higher plasma levels of sCD40L (+47%, P<0.001) and sP-selectin (+70%, P<0.001) compared with controls. Ex vivo study showed platelet isoprostane downexpression and enhanced platelet NO generation in both X-CGD patients and X-CGD carriers compared with controls; opposite findings were observed in obese patients. Correlation analysis showed that platelet NOx2 regulation was directly associated with plasma levels of sCD40L (R=0.336, P<0.001) and sP-selectin (R=0.441; P<0.001). CONCLUSIONS: The study provides the first evidence that in vivo platelet activation is significantly and directly associated with NOx2 activity. Platelet NOx2 may be a novel target for platelet activation inhibition.
Assuntos
Doença Granulomatosa Crônica/enzimologia , Glicoproteínas de Membrana/fisiologia , NADPH Oxidases/fisiologia , Ativação Plaquetária/fisiologia , Adolescente , Adulto , Plaquetas/enzimologia , Ligante de CD40/sangue , Estudos de Casos e Controles , Criança , Feminino , Doença Granulomatosa Crônica/sangue , Doença Granulomatosa Crônica/fisiopatologia , Heterozigoto , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , Obesidade/sangue , Obesidade/enzimologia , Obesidade/fisiopatologia , Selectina-P/sangueRESUMO
NADPH oxidase is known to modulate the arterial tone, but the role of its specific subunits is still unclear. The objective of this study was to compare the role of p47 and gp91phox (NOX2) on artery dilatation. We conducted a multicenter study enrolling 30 patients with chronic granulomatous disease (CGD) (25 with NOX2 deficiency and 5 with p47(phox) deficiency) and 30 healthy subjects (HS), matched for gender and age, in whom flow-mediated dilation (FMD), serum activity of NOX2 (soluble NOX2-derived peptide [sNOX2-dp]), urinary isoprostanes (8-iso-PGF2α), and platelet production of isoprostanes and NOX2 were determined. Compared to HS, patients with CGD had significantly higher FMD and lower sNOX2-dp and 8-iso-PGF2α levels. Compared to patients with NOX2 deficiency and HS, patients with p47(phox) hereditary deficiency had intermediate FMD and oxidative stress, that is, higher and lower FMD and lower and higher isoprostanes compared to HS and patients with NOX2 deficiency, respectively. In agreement with this finding, an ex vivo study showed higher inhibition of NOX2 activity and lower isoprostane formation in platelets from patients with NOX2 deficiency compared to platelets from ones with p47(phox) deficiency. Our observations lead to the hypothesis that oxidants are implicated in artery vasoconstriction.
Assuntos
Doença Granulomatosa Crônica/fisiopatologia , Glicoproteínas de Membrana/deficiência , NADPH Oxidases/deficiência , Vasodilatação , Adolescente , Plaquetas/enzimologia , Artérias Carótidas/diagnóstico por imagem , Artérias Carótidas/patologia , Espessura Intima-Media Carotídea , Estudos de Casos e Controles , Criança , Feminino , Doença Granulomatosa Crônica/enzimologia , Doença Granulomatosa Crônica/urina , Humanos , Isoprostanos/urina , Masculino , NADPH Oxidase 2 , Adulto JovemRESUMO
OBJECT: Platelet isoprostane 8-ISO-prostaglandin F2α (8-iso-PGF2α), a proaggregating molecule, is believed to derive from nonenzymatic oxidation of arachidonic acid. We hypothesized that NADPH is implicated in isoprostane formation and platelet activation. METHODS AND RESULTS: We studied 8-iso-PGF2α in platelets from 8 male patients with hereditary deficiency of gp91(phox), the catalytic subunit of NADPH oxidase, and 8 male controls. On stimulation, platelets from controls produced 8-iso-PGF2α, which was inhibited -8% by aspirin and -58% by a specific inhibitor of gp91(phox). Platelets from patients with gp91(phox) hereditary deficiency had normal thromboxane A(2) formation but marked 8-iso-PGF2α reduction compared with controls. In normal platelets incubated with a gp91(phox) inhibitor or with SQ29548, a thromboxane A(2)/isoprostane receptor inhibitor, platelet recruitment, an in vitro model of thrombus growth, was reduced by 44% and 64%, respectively; a lower effect (-17%) was seen with aspirin. Moreover, thrombus formation under shear stress (blood perfusion at the wall shear rate of 1500 s(-1)) was reduced in samples in which isoprostane formation was inhibited by NADPH oxidase inhibitors. In gp91(phox)-deficient patients, agonist-induced platelet aggregation was within the normal range, whereas platelet recruitment was reduced compared with controls. Incubation of platelets from gp91(phox)-deficient patients with 8-iso-PGF2α dose-dependently (1 to 100 pmol/L) increased platelet recruitment by mobilizing platelet Ca(2+) and activating gpIIb/IIIa; a further increase in platelet recruitment was detected by platelet coincubation with l-NAME, an inhibitor of NO synthase. CONCLUSIONS: This study provides the first evidence that platelet 8-iso-PGF2α maximally derives from gp91(phox) activation and contributes to platelet recruitment via activation of gpIIb/IIIa.
Assuntos
Plaquetas/metabolismo , Deficiências Nutricionais/metabolismo , Isoprostanos/metabolismo , Glicoproteínas de Membrana/deficiência , NADPH Oxidases/deficiência , Adulto , Aspirina/farmacologia , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Estudos de Casos e Controles , Deficiências Nutricionais/patologia , Dinoprosta/análogos & derivados , Dinoprosta/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , NADP/metabolismo , NADPH Oxidase 2 , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismoRESUMO
BACKGROUND: NADPH oxidase is believed to modulate arterial tone, but its role in humans is still unclear. The objective of this study was to evaluate whether NADPH oxidase is involved in flow-mediated arterial dilation (FMD). METHODS AND RESULTS: Twenty-five patients with hereditary deficiency of gp91(phox), the catalytic core of NADPH oxidase, (X-CGD), 25 healthy subjects, and 25 obese patients matched for sex and age were recruited. FMD, platelet gp91(phox), serum levels of nitrite and nitrate as markers of nitric oxide generation, oxidized low-density lipoprotein, and urinary excretion of isoprostanes as markers of oxidative stress were determined. Platelet gp91(phox) expression was downregulated in X-CGD patients (1.0+/-0.8 mean fluorescence; P<0.001) and upregulated in obese patients (4.1+/-2.2 mean fluorescence; P=0.01) compared with healthy subjects (2.9+/-1.7 mean fluorescence). Urinary excretion of isoprostanes was reduced in X-CGD patients (41.7+/-33.3 pg/mg creatinine; P=0.04) and increased in obese patients (154.4+/-91 pg/mg creatinine; P<0.001) compared with healthy subjects (69.5+/-52.4 pg/mg creatinine). Obese patients had higher serum oxidized low-density lipoprotein than healthy subjects (35.3+/-6.7 versus 24.8+/-9.8 U/L; P<0.001) and X-CGD patients (28.5+/-7.2 U/L; P<0.001). X-CGD patients had significantly higher FMD (14.7+/-5.9%) compared with healthy subjects (7.9+/-2.5%; P<0.001); obese patients had lower FMD (5.3+/-3.0%; P=0.028) compared with healthy subjects. Serum nitrite and nitrate levels were significantly higher in patients with X-CGD (36.0+/-10.8 micromol/L; P=0.016) and lower in obese patients (9.3+/-11.0 micromol/L; P=0.001) compared with healthy subjects (27.1+/-19.1 micromol/L). Serum nitrite and nitrate levels significantly correlated with FMD (R(s)=0.403, P<0.001) and platelet gp91(phox) (R(s)=-0.515, P<0.001). FMD inversely correlated with platelet gp91(phox) (R(s)=-0.502, P<0.001) and isoprostanes (R(s)=-0.513, P<0.001). CONCLUSIONS: This study provides the first evidence that, in humans, gp91(phox) is implicated in the modulation of arterial tone.
Assuntos
Artérias/fisiopatologia , Glicoproteínas de Membrana/deficiência , Erros Inatos do Metabolismo/fisiopatologia , NADPH Oxidases/deficiência , Vasodilatação , Adolescente , Adulto , Plaquetas/metabolismo , Criança , Regulação para Baixo , Humanos , Isoprostanos/urina , Masculino , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/urina , NADPH Oxidase 2 , NADPH Oxidases/sangue , NADPH Oxidases/urina , Nitratos/sangue , Nitritos/sangue , Obesidade/sangue , Obesidade/fisiopatologia , Obesidade/urina , Fluxo Sanguíneo Regional , Regulação para Cima , Adulto JovemAssuntos
Agamaglobulinemia/fisiopatologia , Subpopulações de Linfócitos B/imunologia , Memória Imunológica , Agamaglobulinemia/diagnóstico , Agamaglobulinemia/imunologia , Biomarcadores/sangue , Pré-Escolar , Diagnóstico Diferencial , Humanos , Síndromes de Imunodeficiência/diagnóstico , Lactente , Valor Preditivo dos TestesRESUMO
Chronic granulomatous disease (CGD) is a primary phagocytic disorder characterized by greatly increased susceptibility to severe bacterial and fungal infections. Patients with CGD may have gastrointestinal manifestations, commonly colitis, usually mimicking Crohn disease. We report an adult case, the second in literature, of CGD with severe colitis displaying histologic features of ulcerative colitis. The patient had a prompt improvement (continuing up to more than 5 y of follow-up) of the clinical picture after ileostomy and fecal diversion.
Assuntos
Colite Ulcerativa/etiologia , Colite Ulcerativa/cirurgia , Doença Granulomatosa Crônica/complicações , Ileostomia/métodos , Adulto , Colite Ulcerativa/diagnóstico , Doença de Crohn/diagnóstico , Diagnóstico Diferencial , Humanos , MasculinoRESUMO
The molecular and antigenic properties of a Sabin-like type 2 poliovirus, isolated from the stool samples of a 2-year-old agammaglobulinaemic child who developed paralysis 1 year after receiving the third dose of oral poliovirus vaccine, were analysed. The virus revealed 0.88 % genome variation in the VP1 region compared with the standard reference strain, compatible with replication of the virus in the intestine over approximately 1 year. The typical mutations in the 5'NCR and VP1 associated with reversion to neurovirulence for Sabin type 2 poliovirus were found. Despite this, the virus was characterized by both PCR and ELISA tests as Sabin-like and showed temperature sensitivity and neurovirulence in transgenic mice typical of the Sabin type 2 vaccine strain. Gammaglobulin replacement therapy led rapidly to virus clearance, which, when combined with treatment with the antiviral drug pleconaril, stopped virus excretion; no further virus shedding occurred. This is the first case of poliomyelitis and long-term excretion from an immunodeficient patient to be reported in Italy through the active 'Acute Flaccid Paralysis' surveillance system.