Effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs on the production of reactive oxygen species by activated rat neutrophils

The release of reactive oxygen specie (ROS) by activated neutrophil is involved in both the antimicrobial and deleterious effects in chronic inflammation. The objective of the present investigation was to determine the effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs (NSAIDs) on the production of ROS by stimulated rat neutrophils. Diclofenac (3.6 µM), indomethacin (12 µM), naproxen (160 µM), piroxicam (13 µM), and tenoxicam (30 µM) were incubated at 37ºC in PBS (10 mM), pH 7.4, for 30 min with rat neutrophils (1 x 10(6) cells/ml) stimulated by phorbol-12-myristate-13-acetate (100 nM). The ROS production was measured by luminol and lucigenin-dependent chemiluminescence. Except for naproxen, NSAIDs reduced ROS production: 58 ± 2% diclofenac, 90 ± 2% indomethacin, 33 ± 3% piroxicam, and 45 ± 6% tenoxicam (N = 6). For the lucigenin assay, naproxen, piroxicam and tenoxicam were ineffective. For indomethacin the inhibition was 52 ± 5% and diclofenac showed amplification in the light emission of 181 ± 60% (N = 6). Using the myeloperoxidase (MPO)/H2O2/luminol system, the effects of NSAIDs on MPO activity were also screened. We found that NSAIDs inhibited both the peroxidation and chlorinating activity of MPO as follows: diclofenac (36 ± 10, 45 ± 3%), indomethacin (97 ± 2, 100 ± 1%), naproxen (56 ± 8, 76 ± 3%), piroxicam (77 ± 5, 99 ± 1%), and tenoxicam (90 ± 2, 100 ± 1%), respectively (N = 3). These results show that therapeutic levels of NSAIDs are able to suppress the oxygen-dependent antimicrobial or oxidative functions of neutrophils by inhibiting the generation of hypochlorous acid.

Lack of antidiabetic effect of a Eugenia jambolana leaf decoction on rat streptozotocin diabetes

Streptozotocin-diabetic rats were treated for 17 days with a decoction of Eugenia jambolana (Myrtaceae) leaves (15%, w/v) as a substitute for water. Body weight, food and fluid intake, urine volume, glycemia, urinary glucose and urea were evaluated every 5 days. The animals were sacrificed by decapitation and blood samples collected for the determination of glycemia, serum cholesterol, HDL-cholesterol, triglycerides and angiotensin-converting enzyme. The weight of adipose and muscle tissues was also determined. There were no statistically significant differences between treated and untreated rats for any of the biochemical or physiological parameters. We conclude that, at least in this experimental model, Eugenia jambolana leaf decoction has no antidiabetic activity.

Diagnosis of Smith-Lemli-Opitz syndrome by ultraviolet spectrophotometry

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder due to an inborn error of cholesterol metabolism, characterized by congenital malformations, dysmorphism of multiple organs, mental retardation and delayed neuropsychomotor development resulting from cholesterol biosynthesis deficiency. A defect in 3ß-hydroxysteroid-delta7-reductase (delta7-sterol-reductase), responsible for the conversion of 7-dehydrocholesterol (7-DHC) to cholesterol, causes an increase in 7-DHC and frequently reduces plasma cholesterol levels. The clinical diagnosis of SLOS cannot always be conclusive because of the remarkable variability of clinical expression of the disorder. Thus, confirmation by the measurement of plasma 7-DHC levels is needed. In the present study, we used a simple, fast, and selective method based on ultraviolet spectrophotometry to measure 7-DHC in order to diagnose SLOS. 7-DHC was extracted serially from 200 µl plasma with ethanol and n-hexane and the absorbance at 234 and 282 nm was determined. The method was applied to negative control plasma samples from 23 normal individuals and from 6 cases of suspected SLOS. The method was adequate and reliable and 2 SLOS cases were diagnosed.

Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CCR5 genotype and plasma ß-chemokine concentration of Brazilian HIV-infected individuals

The 32-bp deletion in the HIV-1 co-receptor CCR5 confers a high degree of resistance to HIV-1 infection in homozygous individuals for the deleted allele and partial protection against HIV-1 during disease progression in heterozygotes. Natural ligands for CCR5, MIP-1alpha, MIP-1ß and RANTES, have been shown to inhibit HIV replication in CD4+ T cells. In the present study, we examined the CCR5 genotype by PCR and the plasma levels of RANTES and MIP-1alpha by ELISA among blood donors (N = 26) and among HIV-1-infected individuals (N = 129). The control group consisted of healthy adult volunteers and HIV-1-infected subjects were an asymptomatic and heterogeneous group of individuals with regard to immunologic and virologic markers of HIV-1 disease. The frequency of the CCR5 mutant allele (delta32ccr5) in this population was 0.032; however, no delta32ccr5 homozygote was detected. These results could be related to the intense ethnic admixture of the Brazilian population. There was no correlation between circulating ß-chemokines (MIP-1alpha, RANTES) and viral load in HIV-infected individuals. RANTES concentrations in plasma samples from HIV+ patients carrying the homozygous CCR5 allele (CCR5/CCR5) (28.23 ng/ml) were higher than in the control samples (16.07 ng/ml; P<0.05); however, this HIV+ patient group (mean 26.23 pg/ml) had significantly lower concentrations of MIP-1alpha than those observed in control samples (mean 31.20 pg/ml; P<0.05). Both HIV-1-infected and uninfected individuals heterozygous for the delta32ccr5 allele had significantly lower concentrations of circulating RANTES (mean 16.07 and 6.11 ng/ml, respectively) than CCR5/CCR5 individuals (mean 28.23 and 16.07 ng/ml, respectively; P<0.05). These findings suggest that the CCR5 allele and ß-chemokine production may affect the immunopathogenesis of HIV-1.

Cytotoxicity of chlorhexidine digluconate to murine macrophages and its effect on hydrogen peroxide and nitric oxide induction

Chlorhexidine, even at low concentrations, is toxic for a variety of eukaryotic cells; however, its effects on host immune cells are not well known. We evaluated in vitro chlorhexidine-induced cytotoxicity and its effects on reactive oxygen/nitrogen intermediate induction by murine peritoneal macrophages. Thioglycollate-induced cells were obtained from Swiss mice by peritoneal lavage with 5 ml of 10 mM phosphate-buffered saline, washed twice and resuspended (10(6) cells/ml) in appropriate medium for each test. Cell preparations contained more than 95% macrophages. The cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the presence of hydrogen peroxide (H2O2) and nitric oxide (NO) by the horseradish peroxidase-dependent oxidation of phenol red and Griess reaction, respectively. The midpoint cytotoxicity values for 1- and 24-h exposures were 61.12 ± 2.46 and 21.22 ± 2.44 µg/ml, respectively. Chlorhexidine did not induce synthesis or liberation of reactive oxygen/nitrogen intermediates. When macrophages were treated with various sub-toxic doses for 1 h (1, 5, 10, and 20 µg/ml) and 24 h (0.5, 1, and 5 µg/ml) and stimulated with 200 nM phorbol myristate acetate (PMA) solution, the H2O2 production was not altered; however, the NO production induced by 10 µg/ml lipopolysaccharide (LPS) solution varied from 14.47 ± 1.46 to 22.35 ± 1.94 µmol/l and 13.50 ± 1.42 to 20.44 ± 1.40 µmol/l (N = 5). The results showed that chlorhexidine has no immunostimulating activity and sub-toxic concentrations did not affect the response of macrophages to the soluble stimulus PMA but can interfere with the receptor-dependent stimulus LPS.

Repeated exposure of adolescent rats to oral methylphenidate does not induce behavioral sensitization or cross-sensitization to nicotine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of simultaneous exposure to stress and nicotine on nicotine-induced locomotor activation in adolescent and adult rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effect of cocaine on periadolescent rats with or without early maternal separation

Cocaine-induced behavioral sensitization and weight loss were investigated in periadolescent Wistar rats kept with their mothers or subjected to repeated maternal separation. Litters allocated to the separation procedure were placed in a temperature-controlled (33ºC) chamber for 3 h per day from postnatal day 6 (P6) to P20. Non-handled rats were left undisturbed until weaning. Treatments were started on P30-31 and the test was performed on P36-37. Animals received injections of saline or cocaine (10 mg/kg, sc) twice daily for 5 days. on day 6 all animals received saline. on day 7 animals were challenged with 10 mg/kg cocaine and their locomotion was evaluated in activity cages. A third group received saline throughout the 7-day period. Body weights were recorded on P30-31 and P36-37. Two-way ANOVA on body weights showed a main effect of treatment group (F(1,35) = 10.446, P = 0.003; N = 10-12). Non-handled rats treated with cocaine for 5 days gained significantly less weight, while no significant effect was observed in maternally separated rats. Two-way ANOVA revealed a main effect of drug treatment on locomotor activity (F(2,32) = 15.209, P<0.001; N = 6-8), but not on rearing condition (F(1,32)<0.001, P = 0.998). Animals pretreated with cocaine showed a clear behavioral sensitization relative to the saline group. No difference in the magnitude of sensitization was found between separated and non-handled animals. Only the effect of cocaine on weight gain was significantly affected by repeated episodes of early maternal separation during the pre-weaning period.

Presence of autoantibodies against HeLa small nuclear ribonucleoproteins in chagasic and non-chagasic cardiac patients

We detected anti-human small nuclear ribonucleoprotein (snRNP) autoantibodies in chagasic patients by different immunological methods using HeLa snRNPs. ELISA with Trypanosoma cruzi total lysate antigen or HeLa human U small nuclear ribonucleoproteins (UsnRNPs) followed by incubation with sera from chronic chagasic and non-chagasic cardiac patients was used to screen and compare serum reactivity. Western blot analysis using a T. cruzi total cell extract was also performed in order to select some sera for Western blot and immunoprecipitation assays with HeLa nuclear extract. ELISA showed that 73 and 95% of chronic chagasic sera reacted with HeLa UsnRNPs and T. cruzi antigens, respectively. The Western blot assay demonstrated that non-chagasic cardiac sera reacted with high molecular weight proteins present in T. cruzi total extract, probably explaining the 31% reactivity found by ELISA. However, these sera reacted weakly with HeLa UsnRNPs, in contrast to the chagasic sera, which showed autoantibodies with human Sm (from Stefanie Smith, the first patient in whom this activity was identified) proteins (B/B', D1, D2, D3, E, F, and G UsnRNP). Immunoprecipitation reactions using HeLa nuclear extracts confirmed the reactivity of chagasic sera and human UsnRNA/RNPs, while the other sera reacted weakly only with U1snRNP. These findings agree with previously reported data, thus supporting the idea of the presence of autoimmune antibodies in chagasic patients. Interestingly, non-chagasic cardiac sera also showed reactivity with T. cruzi antigen and HeLa UsnRNPs, which suggests that individuals with heart disease of unknown etiology may develop autoimmune antibodies at any time. The detection of UsnRNP autoantibodies in chagasic patients might contribute to our understanding of how they develop upon initial T. cruzi infection.

Rapid test for the evaluation of the activity of the prodrug hydroxymethylnitrofurazone in the processing of Trypanosoma cruzi messenger RNAs

No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 µM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.

Involvement of dopamine receptors in diethylpropion-induced conditioning place preference

Diethylpropion (DEP) is an amphetamine-like agent used as an anorectic drug. Abuse of DEP has been reported and some restrictions of its use have been recently imposed. The conditioning place preference (CPP) paradigm was used to evaluate the reinforcing properties of DEP in adult male Wistar rats. After initial preferences were determined, animals weighing 250-300 g (N = 7 per group) were conditioned with DEP (10, 15 or 20 mg/kg). Only the dose of 15 mg/kg produced a significant place preference (358 ± 39 vs 565 ± 48 s). Pretreatment with the D1 antagonist SCH 23390 (0.05 mg/kg, sc) 10 min before DEP (15 mg/kg, ip) blocked DEP-induced CPP (418 ± 37 vs 389 ± 31 s) while haloperidol (0.5 mg/kg, ip), a D2 antagonist, 15 min before DEP was ineffective in modifying place conditioning produced by DEP (385 ± 36 vs 536 ± 41 s). These results suggest that dopamine D1 receptors mediate the reinforcing effect of DEP

Experimental murine mycobacteriosis: evaluation of the functional activity of alveolar macrophages in thalidomide- treated mice

Thalidomide is a selective inhibitor of tumor necrosis factor-alpha (TNF-alpha), a cytokine involved in mycobacterial death mechanisms. We investigated the role of this drug in the functional activity of alveolar macrophages in the presence of infection induced by intranasal inoculation of Mycobacterium avium in thalidomide-treated and untreated adult Swiss mice. Sixty animals were inoculated with 5 x 10(6) M. avium by the respiratory route. Thirty animals received daily thalidomide (30 mg/kg mouse) and 30 received water by gavage up to sacrifice. Ten non-inoculated mice were used as a control group. Lots of animals from each group were evaluated until 6 weeks after inoculation. Infection resulted in an increased total number of inflammatory cells as well as increased activity of pulmonary macrophages. Histologically, intranasal inoculation of bacilli resulted in small mononuclear infiltrates located at the periphery of the organ. Culture of lung fragments revealed the presence of bacilli only at the beginning and at the end of the experimental period. Thalidomide administration did not affect the microbiological or histological features of the infection. Thalidomide-treated and untreated animals showed the same amount of M. avium colonies 3 weeks after infection. Although it did not affect bacillary clearance, thalidomide administration resulted in a decreased percent of spread cells and release of hydrogen peroxide, suggesting that factors other than TNF-alpha play a role in the killing of mycobacteria by alveolar macrophages. Thalidomide administration also reduced the number of spread cells among resident macrophages, suggesting a direct effect of the drug on this phenomenon.

Reduced pancreatic β-cell mass is associated with decreased FoxO1 and Erk1/2 protein phosphorylation in low-protein malnourished rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of gender on training-induced vascular remodeling in SHR

There is accumulating evidence that physical inactivity, associated with the modern sedentary lifestyle, is a major determinant of hypertension. It represents the most important modifiable risk factor for cardiovascular diseases, which are the leading cause of morbidity and mortality for both men and women. In addition to involving sympathetic overactivity that alters hemodynamic parameters, hypertension is accompanied by several abnormalities in the skeletal muscle circulation including vessel rarefaction and increased arteriole wall-to-lumen ratio, which contribute to increased total peripheral resistance. Low-intensity aerobic training is a promising tool for the prevention, treatment and control of high blood pressure, but its efficacy may differ between men and women and between male and female animals. This review focuses on peripheral training-induced adaptations that contribute to a blood pressure-lowering effect, with special attention to differential responses in male and female spontaneously hypertensive rats (SHR). Heart, diaphragm and skeletal muscle arterioles (but not kidney arterioles) undergo eutrophic outward remodeling in trained male SHR, which contributed to a reduction of peripheral resistance and to a pressure fall. In contrast, trained female SHR showed no change in arteriole wall-to-lumen ratio and no pressure fall. on the other hand, training-induced adaptive changes in capillaries and venules (increased density) were similar in male and female SHR, supporting a similar hyperemic response to exercise.