Platelet adhesion and intracellular calcium levels in antigen-challenged rats

There is considerable evidence that platelet activation occurs in allergic airways diseases. In this study we aimed to investigate platelet adhesion to immobilized fibrinogen and intracellular calcium levels in a rat model of allergic inflammation. Male Wistar rats were challenged with ovalbumin (OVA). At 30 min to 24 h after OVA-challenge, assays of platelet adhesion to immobilized fibrinogen and intracellular calcium levels using fura 2-AM loaded platelets were performed. The serum levels of IgE were approximately 5-fold greater in OVA-sensitized rats. A marked eosinophil influx in bronchoalveolar lavage (BAL) fluid of OVA-challenged rats at 24 h after OVA-challenge was also seen. OVA-challenge resulted in a marked thrombocytopenia, as observed within 12 h after OVA-challenge. The agonists ADP (0.5–50 μM) and thrombin (30–100 mU/ml) concentration-dependently increased platelet adhesion to immobilized fibrinogen. At an early time after OVA-challenge (30 min), platelets exhibited greater platelet adhesion compared with the non-sensitized group, whereas at a late time (24 h) they exhibited lower platelet adhesion to both agonists. Moreover, at 30 min after OVA-challenge, intracellular calcium levels to ADP (20 μM) and thrombin (100 mU/ml)-activated platelets were greater compared with non-challenged rats. As opposed, at 24 h after OVA challenge, a lower intracellular calcium level to ADP- and thrombin-activated platelets was observed. In conclusion, OVA-challenge in rats promotes a biphasic response in platelet adhesion consisting of an increased adhesion and intracellular calcium levels at an early phase (30 min), which progress to a reduction in adhesion and intracellular calcium levels at a late time (24 h) after antigen challenge.     

Drug use during in-hospital birth delivery stay

OBJECTIVE: Drug use in birth delivery has not been enough explored in the literature. It is a significant issue to be discussed on the theme of rational drug use. The objective was to study drug use during birth delivery stay in maternity hospitals. METHODS: A cross-sectional study was carried out using medical records of two private and public maternity hospitals of Belo Horizonte, Brazil. Data were collected on pregnant women's identification, pregnancy, delivery and drug prescribed from medical records of public hospitals and medical records and billing invoice of private maternity hospitals. Statistical analysis was conducted using odds ratio by the Chi-square test and means by t-Student test. RESULTS: Mean in-hospital stay was 2.2 days and it was lower in the private maternity hospital. Cesarean sections were performed in 52.7% of all births, 31.3% in the public hospital and 64.5% in the private hospital. Peridural anesthesia was used in 72.8% of births and local anesthesia in 22.4% (25.3% and 63.7% of births in the public and 98.2% and 0.4% in the private hospital). All women received drugs (minimum of 3 and maximum of 19 different drugs) during their hospital stay. Eighty-three medications (97 active ingredients) were utilized at a total frequency of 3,429. The higher average drug use was 8.5 drugs per woman, in the private maternity hospital. CONCLUSIONS: There was a significant difference in drug use between the two maternity hospitals, being it higher in pre- and during birth delivery procedures. The results suggest a preeminent drug use compared to those of other few studies available in the literature. The disproportionate number of cesarean sections and anesthesia explain the differences observed.     

Phoneutria nigriventer spider venom activates 5-HT4 receptors in rat-isolated vagus nerve

1. The venom of Phoneutria nigriventer spider (PNV) causes intense pain and inflammation following an attack. We have investigated the involvement of capsaicin-sensitive nerve fibres by utilizing an in vitro nerve preparation. Extracellular DC potential recordings were made from the rat-isolated vagus nerve, a preparation that is rich in capsaicin-sensitive, that is, nociceptive, C-fibres. 2. PNV (1-10 microg ml(-1)), capsaicin (0.03-0.3 microM) or 5-hydroxytriptamine (5-HT) (0.3-3 microM) induced dose-dependent depolarizations of vagus nerve fibres. Depolarizing responses to capsaicin were blocked by ruthenium red (RR, 10 microM), but responses to PNV were not. Depolarizing responses to PNV or veratridine (50 microM) were inhibited by tetrodotoxin (TTX, 10 microM), but those to capsaicin were not. This suggests that capsaicin and PNV depolarize the nerve fibres by distinct mechanisms. 3. Depolarization in response to 5-HT (3 microM) was reduced by the 5-HT(3) receptor antagonists Y25130 (0.5 micro M) and tropisetron (10 nM) or, to a lesser extent, by the 5-HT(4) receptor antagonist RS39604 (1 or 10 microM). Depolarizing responses to PNV were not affected significantly by Y25130 or tropisetron, but were blocked by RS39604. 4. These data show that 5-HT(4) receptors play a significant role in the activation of nociceptive sensory nerve fibres by PNV and suggest that this is of importance in the development of the pain and inflammation associated with bites from the P. nigriventer spider.     

Vascular effects of long-term propranolol administration after chronic nitric oxide blockade

The ability of agmatine, formed from L-arginine by the enzyme arginine decarboxylase (ADC), to modulate vasomotor function in rat aorta was investigated in the present study. Agmatine-mediated modulation of vasomotor tone was studied in organ chambers, protein expression quantified by Western blot analysis and cyclic guanosine 5'-monophosphate (cGMP) levels measured by radioimmunoassay. Agmatine (10(-10) to 10(-3) M) produced concentration-dependent relaxations (82+/-5%) in phenylephrine-contracted endothelium intact rat aorta. Relaxations to agmatine were diminished on denudation of endothelium and nitric oxide synthase (NOS) inhibition by L-Nomega-nitro arginine or soluble guanylate cyclase inhibition by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (P<0.001) abolished agmatine-mediated relaxations, while relaxations were insensitive to inducible NOS inhibition by 1400W. Agmatine-treated aorta demonstrated increased protein expression of phosphorylated S473-Akt and phosphorylated S1177-endothelial nitric oxide synthase (eNOS), and elevated the levels of cyclic GMP (P<0.01). Agmatine-mediated potentiation of relaxations and elevation of cGMP levels was sensitive to phosphatidylinositol 3'-kinase inhibitor, wortmannin. Relaxations to agmatine were also affected by pre-treatment with tetraethylammonium (P<0.01) or apamin (P<0.05), and were not affected by charybdotoxin. Relaxations to agmatine were partially affected by pre-treatment of aortic rings with barium chloride (P<0.05), and glybenclamide (P<0.05). Results obtained suggest that agmatine activates protein kinase B/Akt to phosphorylate eNOS and elevate cyclic GMP levels to produce vasodilatation of aorta. Agmatine-mediated relaxations in rat aorta seems to be mediated mainly by endothelial NO-mediated activation of small conductance Ca2+-activated K+ channels, and partly by ATP-sensitive and inward rectifying K+ channels.     

Enhancement of the pulmonary allergic granulocyte recruitment in rats exposed to DMTI-II, a Kunitz-type inhibitor isolated from Dimorphandra mollis seeds

DMTI-II (23-kDa trypsin inhibitor purified from Dimorphandra mollis seeds) promotes acute inflammation accompanied by an early infiltration of eosinophils, a critical cell type involved in allergic diseases. We have evaluated here the capacity of DMTI-II to enhance the allergic pulmonary inflammation, looking over time to the leukocyte trafficking from bone marrow to peripheral blood, and their recruitment into the allergic airways. Male Wistar rats were sensitized and challenged with ovalbumin (OVA). At 2 to 16h prior to OVA challenge, animals were exposed to DMTI-II (10μg). Bronchoalveolar lavage fluid (BAL), circulating blood and bone marrow were examined at 24h post-OVA challenge. Challenge with OVA significantly increased the influx of total inflammatory cells, neutrophils and eosinophils in BAL and lung tissue. Pre-exposure to DMTI-II potentiated total inflammatory cell and neutrophil recruitment (p<0.05). Neutropoiesis and neutrophilia accompanied pulmonary cell influx. Pre-exposure to DMTI-II also significantly increased eosinophil recruitment to BAL, an effect starting at 4h, remaining markedly elevated at 16h (p<0.05). Eosinopoiesis and eosinophilia (seen within 2 to 4h) were also observed. Exposure to DMTI-II alone increased the IL-4 levels, and further increased the IL-4 levels in OVA-challenged rats. The levels of IgE, LTB(4) and eotaxin in OVA-challenged rats were greater compared with non-sensitized rats, but DMTI-II exposure failed to further enhance such levels. In summary, our study shows that DMTI-II itself presents granulocytopoietic activity, and enhances allergen-induced neutrophil and eosinophil mobilization from bone marrow to lung tissues that is accompanied by enhanced IL-4 production.     

Genotoxicity assessment of the antimalarial compound artesunate in somatic cells of mice

Artesunate is a derivate of artemisinin that is both an antimalarial agent and acts cytotoxically on tumor cells. Despite its therapeutic use, its in vivo genotoxic potential has still not been evaluated. This study, therefore, was an investigation into the effects of a single oral administration of artesunate with an in vivo comet assay that analyzed leukocytes from peripheral blood and liver cells, and a micronucleus (MN) assay of bone marrow cells from male Swiss mice. The artesunate was administered by oral gavage at doses of 5, 50 and 100 mg/kg. Cytotoxicity was assessed by scoring 200 consecutive polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). The results demonstrate that artesunate induced significant DNA damage only in liver cells and that high doses of artesunate caused an increase in the mean number of micronucleated polychromatic erythrocytes (MNPCE). Under our experimental conditions, artesunate showed weak genotoxic effects at low doses and clastogenic effects at high doses. The PCE/NCE ratio indicated no cytotoxicity. The data obtained suggest caution about either continuous or high-dose use of artesunate by humans.     

Experimental pathogenicity of Candida albicans and Candida dubliniensis with continuous and discontinuous fringes morphotypes

A possible correlation between the presence of discontinuous fringes and high virulence has been previously suggested. The aim of this study was to compare the pathogenicity of Candida albicans and Candida dubliniensis with continuous and discontinuous fringes morphotypes on mice. For C. albicans, two discontinuous fringe morphotype isolates (PN 69, PN 74), two continuous fringe morphotype isolates (N 60, N 33) and one reference strain were used. For C. dubliniensis, three discontinuous fringe morphotype isolates (97487, 97464, 97519), two continuous fringe morphotype isolates (97040, 98026) and one reference strain were used. Swiss male mice were inoculated with a standardised suspension of the microorganisms and observed for 35 days. The pathogenicity of the isolates was analysed according to parameters proposed previously. Three isolates were considered pathogenic: PN 74, N 60 and 98026. Strain N 60 killed the highest amount of mice (80%). Animals inoculated with C. albicans did not show differences on survival estimate. Candida dubliniensis 98026 was more pathogenic than samples 97464 and 97519. On the other hand, the sample 97487 showed a higher pathogenicity when compared with 97040 (Kaplan–Meier test, P = 0.008). Strains with continuous fringe morphotypes were also associated with Candida sp. virulence in vivo.     

Effect of the phosphodiesterase 5 inhibitors sildenafil, tadalafil and vardenafil on rat anococcygeus muscle: functional and biochemical aspects

1. The anococcygeus muscle is part of the erectile machinery in male rodents. Phosphodiesterase (PDE) 5 inhibitors enhance and prolong the effects of cGMP, which has a key role in penile erection. The aim of the present study was to provide a functional and biochemical comparison of the three PDE5 inhibitors, namely sildenafil, tadalafil and vardenafil, in the rat anococcygeus muscle. 2. Muscle strips were mounted in 4 mL organ baths and isometric force recorded. Levels of cGMP were measured using an enzyme immunoassay kit. Western blots were used to determine PDE5 protein expression. 3. The PDE5 inhibitors concentration-dependently relaxed carbachol-precontracted anococcygeus muscle; however, vardenafil was more potent (pEC(50) = 8.11 +/- 0.05) than sildenafil (7.72 +/- 0.06) or tadalafil (7.69 +/- 0.05). Addition of N(G)-nitro-l-arginine methyl ester (100 micromol/L) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 micromol/L) to the organ baths caused significant rightward shifts in concentration-response curves for all PDE5 inhibitors. 4. Sildenafil, tadalafil and vardenafil (all at 0.1 micromol/L) caused leftward shifts in the glyceryl trinitrate (GTN) concentration-response curves (by 4.0-, 3.7- and 5.5-fold, respectively). In addition, all three PDE5 inhibitors significantly potentiated relaxation responses to both GTN (0.01-10 micromol/L) and electrical field stimulation (EFS; 1-32 Hz), with vardenafil having more pronounced effects. 5. All three PDE5 inhibitors reduced EFS-evoked contractions in a concentration-dependent manner over the concentration range 0.001-1 micromol/L. There were no significant differences between the effects of the three PDE5 inhibitors. 6. Vardenafil (0.01-0.1 micromol/L) was more potent in preventing cGMP degradation in vitro than sildenafil (0.01-0.1 micromol/L) and tadalafil (0.01-0.1 micromol/L). 7. Under control conditions, the expression of PDE5 was higher in the anococcygeus muscle than in the corpus cavernosum. 8. In conclusion, PDE5 inhibitors enhance exogenous and endogenous nitric oxide-mediated relaxation in the rat anococcygeus muscle. The potency of vardenafil was greater than that of either sildenafil or tadalafil.