Newer Aspects Of The Pathophysiology Of Sickle Cell Disease Vaso-occlusion

Sickle cell disease is an inherited disorder of hemoglobin (Hb) synthesis, caused by a single nucleotide substitution (GTG>GAG) at the sixth codon of the β-globin gene, leading to the production of a defective form of Hb, Hb S. When deoxygenated, Hb S polymerizes, damaging the sickle erythrocyte and it is this polymerization that is the primary indispensable event in the molecular pathogenesis of sickle cell disease. Hb S polymerization results in a series of cellular alterations in red cell morphology and function that shorten the red cell life span and leads to vascular occlusion. Sickle cell disease vaso-occlusion is now known to constitute a complex multifactorial process characterized by recurrent vaso-occlusion, ischemia-reperfusion injury, and oxidative stress with consequent vascular endothelial cell activation that induces a chronic inflammatory state in sickle cell disease individual and is propagated by elevated levels of circulating inflammatory cytokines. Activation of the endothelium results in the induction of endothelial adhesion molecule expression that mediates red and white cell adhesion to the vessel wall and the formation of heterocellular aggregates, followed by secondary red cell trapping, all of which contribute to reduced blood flow and eventually obstruction of the micro-circulation. Reduced nitric oxide bioavailability, caused principally by its consumption by cell-free Hb, liberated during intravascular hemolysis, contributes to this process by facilitating vasoconstriction and adhesion molecule activity. Copyright © Informa Healthcare USA, Inc.331116Steinberg, M.H., Management of sickle cell disease (1999) N Engl J Med, 340 (13), pp. 1021-1030Madigan, C., Malik, P., Pathophysiology and therapy for haemoglobinopathies. 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Phosphodiesterase-9 (PDE9) inhibition with BAY 73-6691 increases corpus cavernosum relaxations mediated by nitric oxide-cyclic GMP pathway in mice

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES – COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORPhosphodiesterase-9 (PDE9) specifically hydrolyzes cyclic GMP, and was detected in human corpus cavernosum. However, no previous studies explored the selective PDE9 inhibition with BAY 73-6691 in corpus cavernosum relaxations. Therefore, this study aimed to characterize the PDE9 mRNA expression in mice corpus cavernosum, and investigate the effects of BAY 73-6691 in endothelium-dependent and -independent relaxations, along with the nitrergic corpus cavernosum relaxations. Male mice received daily gavage of BAY 73-6691 (or dimethylsulfoxide) at 3 mg kg(-1) per day for 21 days. Relaxant responses to acetylcholine (ACh), nitric oxide (NO) (as acidified sodium nitrite; NaNO2 solution), sildenafil and electrical-field stimulation (EFS) were obtained in corpus cavernosum in control and BAY 73-6691-treated mice. BAY 73-6691 was also added in vitro 30 min before construction of concentration-responses and frequency curves. PDE9A and PDE5 mRNA expression was detected in the mice corpus cavernosum in a similar manner. In vitro addition of BAY 73-6691 neither itself relaxed mice corpus cavernosum nor changed the NaNO2, sildenafil and EFS-induced relaxations. However, in mice treated chronically with BAY 73-6691, the potency (pEC(50)) values for ACh, NaNO2 and sildenafil were significantly greater compared with control group. The maximal responses (E-max) to NaNO2 and sildenafil were also significantly greater in BAY 73-6691-treated mice. BAY 73-6691 treatment also significantly increased the magnitude and duration of the nitrergic corpus cavernosum relaxations (8-32 Hz). In conclusion, murine corpus cavernosum expresses PDE9 mRNA. Prolonged PDE9 inhibition with BAY 73-6691 amplifies the NO-cGMP-mediated cavernosal responses, and may be of therapeutic value for erectile dysfunction2526973FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES – COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES – COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORsem informaçãosem informaçã

Hydroxycarbamide Reduces Eosinophil Adhesion And Degranulation In Sickle Cell Anaemia Patients

Inflammation, leucocyte and red cell adhesion to the endothelium contribute to the pathogenesis of sickle cell anaemia. Neutrophils appear to be important for vaso-occlusion, however, eosinophils may also participate in this phenomenon. The role of eosinophils in the pathophysiology of sickle cell anaemia (SCA) and the effect of hydroxycarbamide (HC) therapy on the functional properties of these cells are not understood. Patients with SCA and those on HC therapy (SCAHC) were included in the study. SCAHC individuals presented significantly lower absolute numbers of eosinophils than SCA. Furthermore, SCAHC eosinophils demonstrated significantly lower adhesive properties, compared to SCA eosinophils. SCA and SCAHC eosinophils presented greater spontaneous migration when compared with control eosinophils. Baseline eosinophil peroxidase and reactive oxygen species release was higher for SCA individuals than for control individuals, as were plasma levels of eosinophil derived neurotoxin. SCAHC eosinophil degranulation was lower than that of SCA eosinophil degranulation. Eotaxin-1 and RANTES levels were higher in the plasma of SCA and SCAHC individuals, when compared with controls. These data suggest that eosinophils exist in an activated state in SCA and indicate that these cells play a role in the vaso-occlusive process. The exact mechanism by which HC may alter SCA eosinophil properties is not clear. © 2013 John Wiley & Sons Ltd.1642286295Adamko, D.J., Wu, Y., Gleich, G.J., Lacy, P., Moqbel, R., The induction of eosinophil peroxidase release: improved methods of measurement and stimulation (2004) Journal of Immunological Methods, 291, pp. 101-108Almeida, C.B., Favero, M.E., Pereira-Cunha, F.G., Lorand-Metze, I., Saad, S.T., Costa, F.F., Conran, N., Alterations in cell maturity and serum survival factors may modulate neutrophil numbers in sickle cell disease (2011) Experimental Biology and Medicine, 236, pp. 1239-1246Amer, J., Ghoti, H., Rachmilewitz, E., Koren, A., Levin, C., Fibach, E., Red blood cells, platelets and polymorphonuclear neutrophils of patients with sickle cell disease exhibit oxidative stress that can be ameliorated by antioxidants (2006) British Journal of Haematology, 132, pp. 108-113Assis, A., Conran, N., Canalli, A.A., Lorand-Metze, I., Saad, S.T., Costa, F.F., Effect of cytokines and chemokines on sickle neutrophil adhesion to fibronectin (2005) Acta Haematologica, 113, pp. 130-136Blanchard, C., Rothenberg, M.E., Biology of the eosinophil (2009) Advances in Immunology, 101, pp. 81-121Boyd, J.H., Macklin, E.A., Strunk, R.C., De-Baun, M.R., Asthma is associated with acute chest syndrome and pain in children with sickle cell anemia (2006) Blood, 108, pp. 2923-2927Bruijnzeel, P., Storz, E., van der Donk, E., Bruijnzeel-Koomen, C., Skin eosinophilia in patients with allergic asthma, patients with nonallergic asthma, and healthy controls. 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Exercise training ameliorates the impairment of endothelial and nitrergic corpus cavernosum responses in diabetic rats

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOThe effect of exercise training (ET) on vascular responsiveness in diabetes mellitus has been largely well studied. However, limited studies have investigated the effects of ET on functional responses of the corpus cavernosum (CC) in diabetic animals. Therefore, the aim of this study was to investigate whether prior ET prevents the impairment of erectile function in streptozotocin-induced diabetic rats. Main methods: Rats were exercised for four weeks prior to the induction of diabetes, and then again for another 4 weeks thereafter. Concentration-response curves to acetylcholine, sodium nitroprusside, Y-27632. BAY 412272 and phenylephrine (PE) were obtained in CC. The excitatory and inhibitory effects of electrical-field stimulation were also evaluated. Key findings: Plasma SOD levels were markedly decreased in the sedentary diabetic group (D-SD) as compared to control sedentary animals (C-SD), approximately 53% (P<0.05) and this reduction was restored in trained diabetic animals. Physical training restored the impairment of endothelium-dependent and -independent relaxation responses seen in the D-SD group. The potency values for Y-27632 in the CC were significantly reduced in the D-SD group, which was reversed by physical training. The impairment of electrical-field stimulation (EFS)-induced relaxation seen in the D-SD group was restored by physical training. On the other hand, both EFS-induced contractions and concentration-response curves to PE in cavernosal strips were not modified by either diabetes or physical training. Significance: Practice of regular physical exercise may be an important approach in preventing erectile dysfunction associated with diabetes mellitus by re-establishment of the balance between NO production and its inactivation885-6272277FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOsem informaçã

Mechanisms involved in the enhancement of allergic airways neutrophil influx by permanent C-fiber degeneration in rats

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOThis study was undertaken to clarify the mechanisms by which C-fiber degeneration at neonatal stages exacerbates the inflammatory responses of rat airways. Rats were treated with capsaicin at neonatal stages and immunized with ovalbumin (OVA) at adult ages. Challenge of capsaicin-pretreated rats with OVA promoted a higher influx of neutrophils in bronchoalveolar lavage (BAL) fluid compared with the vehicle group. No significant differences were found for the other cell types. The increased adhesion of N-formyl-methionyl-leucyl-phenylalanine (fMLP; 0.1 mu M)- and phorbol myristate acetate (PMA; 1 mu M) treated neutrophils to fibronectin-coated wells did not differ among vehicle- and capsaicin-pretreated rats. Additionally, fMLP (10 mu M), platelet-activating factor (0.1 mu M), and substance P (50 mu M) induced a significant neutrophil chemotaxis, but no differences were found among vehicle and capsaicin groups. Increased levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-10, and leukotriene B-4 in BAL fluid as well as higher expression of cytokine-induced neutrophil chemoattractant (CINC)- 3 in lung homogenates were detected in the capsaicin group compared with vehicle group. In the capsaicin group, chronic treatment with compound 48/80 restored the TNF-alpha levels to control values and prevented the neutrophil influx in BAL fluid. The enhanced production of TNF-alpha, superoxide anion, and nitrite by isolated alveolar macrophages in response to lipopolysaccharide (3 mu g/ml), PMA (10 nM), and/or zymosan (100 particles/cell) did not differ between vehicle- and capsaicin-pretreated rats. In conclusion, chronic neuropeptide depletion promoted by neonatal capsaicin treatment up-regulates airways mast cells, which upon activation by antigen at adult ages, release large amounts of cytokines such as TNF-alpha and CINC-3 that accounts for the massive airways neutrophil infiltration3131440448FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOsem informaçã

Role of nitric oxide on the increased vascular permeability and neutrophil accumulation induced by staphylococcal enterotoxin B into the mouse paw

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOThe role of nitric oxide (NO) on the increase in vascular permeability and neutrophil migration induced by staphylococcal enterotoxin B (SEB: 25 mug/paw) in the mouse was investigated in this study. The NO synthase inhibitor N-omega-nitro-L-arginine methyl ester (L-NAME) [but not its inactive enantiomer N-omega-nitro-D-arginine methyl ester (D-NAME)I, given intravenously (25-100 mu moL/kg) or subplantarly (0.25-1.0 mu mol/paw). reduced SEE-induced paw oedema significantly. A similar response was observed with aminoguanidine, given either intravenously (200-600 mu mol/kg) or subplantarly (2 mu mol/paw). In contrast to paw oedema. the plasma exudation in response to SEE was not affected by the subplantar injection of L-NAME or aminoguanidine. The inhibition of oedema and plasma exudation by systemic treatment with L-NAME or aminoguanidine was reversed by co-injection of the vasodilator iloprost (0.3 nmol/paw). Subplantar injection of SEE (25 mug/paw) increased by 69% the myeloperoxidase (MPO) activity of SEE-treated paws, indicating the presence of neutrophils. Intravenous (12.5-50 mu mol/kg) or subplantar (0.125-0.5 mu mol/paw) administration of L-NAME (but not of its inactive enantiomer, D-NAME) largely reduced the MPO activity in SEE-treated paws. Similarly, intravenous (200-600 mu mol/kg) or subplantar (2 mu mol/paw) administration of aminoguanidine significantly reduced the MPO values of the SEE-injected paws. The vasodilator iloprost (0.3 nmol/paw) completely reversed the inhibition by L-NAME or aminoguanidine of the MPO activity in SEE-injected paws. Our results show that the increased vascular permeability and neutrophil accumulation in response to subplantar injection of SEE in the mouse are inhibited by L-NAME and aminoguanidine by mechanisms probably involving reduction of local microvascular blood RowPergamon611013051311FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOsem informaçãoOxfor

Expression of the gamma-globin gene is sustained by the cAMP-dependent pathway in beta-thalassaemia

The present study found that the cyclic adenosine monophosphate (cAMP)-dependent pathway efficiently induced \u3b3-globin expression in adult erythroblasts, and this pathway plays a role in \u3b3-globin gene (HBG) expression in \u3b2-thalassaemia. Expression of HBG mRNA increased to about 46% of non-HBA mRNA in adult erythroblasts treated with forskolin, while a cyclic guanosine monophosphate (cGMP) analogue induced HBG mRNA to levels <20% of non-HBA mRNA. In patients with \u3b2-thalassaemia intermedia, cAMP levels were elevated in both red blood cells and nucleated erythroblasts but no consistent elevation was found with cGMP levels. The transcription factor cAMP response element binding protein (CREB) was phosphorylated in nucleated erythroblasts and its phosphorylation levels correlated with HBG mRNA levels of the patients. Other signalling molecules, such as mitogen-activated protein kinases and signal transducers and activators of transcription proteins, were phosphorylated at variable levels and showed no correlations with the HBG mRNA levels. Plasma levels of cytokines, such as erythropoietin, stem cell factor and transforming growth factor-\u3b2 were increased in patients, and these cytokines induced both HBG mRNA expression and CREB phosphorylation. These results demonstrate that the cAMP-dependent pathway, the activity of which is augmented by multiple cytokines, plays a role in regulating HBG expression in \u3b2-thalassaemia

Effects of neonatal capsaicin treatment in the neutrophil production, and expression of preprotachykinin-I and tachykinin receptors in the rat bone marrow

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOBone marrow is richly innervated with both myelinated and non-myelinated nerve fibers, but the role of this innervation on hemopoiesis is poorly understood. Therefore, the aim of this study was to investigate the role of C-fibers on hernatopoiesis. Wistar rats were neonatally injected with either capsaicin or its vehicle, and used at adult ages (8-10 weeks). In capsaicin-pretreated rats, the levels of substance P (SP) in bone marrow fluid were markedly reduced in comparison with the vehicle group (13.1 +/- 4.5 pg/ml versus 47.3 +/- 5.5 pg/ml, p < 0.05). In bone marrow, the number of total leukocytes was 28% higher (p < 0.05) in capsaicin-pretreated group, and this accompanied by a higher number of neutrophils, particularly of the immature forms. The mononuclear cell and eosinophils counts did not differ significantly among vehicle and capsaicin groups. In peripheral blood, the number of circulating neutrophils in the capsaicin group increased by 53.8% (p < 0.05), whereas the number of mononuclear cells did not change significantly among groups. Eosinophils were virtually absent in the circulating blood in both groups. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed that both preprotachykinin (PPT)-I mRNA and the tachykinin neurokinin (NK)-1 mRNA expression in bone marrow cells significantly increased in capsaicin group, whereas the NK-2 mRNA expression was unchanged after capsaicin pretreatment. In conclusion, our data show that chronic neuropeptide depletion enhance the neutrophil proliferation and differentiation in the rat bone marrow by mechanisms involving upregulation of PPT-I gene and NK-1 receptors40717073FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOsem informaçã