Comparative effects of macro-sized aluminum oxide and aluminum oxide nanoparticles on erythrocyte hemolysis: influence of cell source, temperature and size

Al2O3 is the most abundantly produced nanomaterial and has been used in diverse fields, including the medical, military and industrial sectors. As there are concerns about the health effects of nanoparticles, it is important to understand how they interact with cells, and specifically with red blood cells. The hemolysis induced by three commercial nano-sized aluminum oxide particles (nanopowder 13 nm, nanopowder <50 nm and nanowire 2-6 nm × 200-400 nm) was compared to aluminum oxide and has been studied on erythrocytes from humans, rats and rabbits, in order to elucidate the mechanism of action and the influence of size and shape on hemolytic behavior. The concentrations inducing 50% hemolysis (HC50) were calculated for each compound studied. The most hemolytic aluminum oxide particles were of nanopowder 13, followed by nanowire and nanopowder 50. The addition of albumin to PBS induced a protective effect on hemolysis in all the nano-forms of Al2O3, but not on Al2O3. The drop in HC50 correlated to a decrease in nanomaterial size, which was induced by a reduction of aggregation Aluminum oxide nanoparticles are less hemolytic than other oxide nanoparticles, and behave differently depending on the size and shape of the nanoparticles. The hemolytic behavior of aluminum oxide nanoparticles differs from that of aluminum oxide.

Nanoparticle-coated organic-inorganic microparticles: experimental design and gastrointestinal tolerance evaluation

The influences of the spray-drying parameters and the type of nanoparticles (nanocapsules or nanospheres) on the characteristics of nanoparticle-coated diclofenac-loaded microparticles were investigated by using a factorial design 3². Gastrointestinal tolerance following oral administration in rats was evaluated. Formulations were selected considering the best yields, the best encapsulation efficiencies and the lowest water contents, presenting surfaces completely coated by nanostructures and a decrease in the surface areas in relation to the uncoated core. In vitro drug release demonstrated the influence of the nanoparticle-coating on the dissolution profiles of diclofenac. Nanocapsule-coated microparticles presented a protective effect on the gastrointestinal mucosa.

Multigram synthesis and in vivo efficacy studies of a novel multitarget anti-Alzheimer's compound

We describe the multigram synthesis and in vivo efficacy studies of a donepezil‒huprine hybrid that has been found to display a promising in vitro multitarget profile of interest for the treatment of Alzheimer's disease (AD). Its synthesis features as the key step a novel multigram preparative chromatographic resolution of intermediate racemic huprine Y by chiral HPLC. Administration of this compound to transgenic CL4176 and CL2006 Caenorhabditis elegans strains expressing human Aβ42, here used as simplified animal models of AD, led to a significant protection from the toxicity induced by Aβ42. However, this protective effect was not accompanied, in CL2006 worms, by a reduction of amyloid deposits. Oral administration for 3 months to transgenic APPSL mice, a well-established animal model of AD, improved short-term memory, but did not alter brain levels of Aβ peptides nor cortical and hippocampal amyloid plaque load. Despite the clear protective and cognitive effects of AVCRI104P4, the lack of Aβ lowering effect in vivo might be related to its lower in vitro potency toward Aβ aggregation and formation as compared with its higher anticholinesterase activities. Further lead optimization in this series should thus focus on improving the anti-amyloid/anticholinesterase activity ratio.

Photoinhibition of Photosystem II. Kinetics, Photoprotection and Mechanism

Photosystem II (PSII) is susceptible to light-induced damage defined as photoinhibition. In natural conditions, plants are capable of repairing the photoinhibited PSII by on-going degradation and re-synthesis of the D1 reaction centre protein of PSII. Photoinhibition is induced by both visible and ultraviolet light and photoinhibition occurs under all light intensities with the same efficiency per photon. In my thesis work, I studied the reaction kinetics and mechanism of photoinhibition of PSII, as well as photoprotection in leaves of higher plants. Action spectroscopy was used to identify photoreceptors of photoinhibition. I found that the action spectrum of photoinhibition in vivo shows resemblance to the absorption spectra of manganese model compounds of the oxygen evolving complex (OEC) suggesting a role for manganese as a photoreceptor of photoinhibition under UV and visible light. In order to study the protective effect of non-photochemical quenching, the action spectrum was measured from leaves of wild type Arabidopsis thaliana and two mutants impaired in nonphotochemical quenching of chlorophyll a excitations. The findings of action spectroscopy and simulations of chlorophyll-based photoinhibition mechanisms suggested that quenching of antenna excitations protects less efficiently than would be expected if antenna chlorophylls were the only photoreceptors of photoinhibition. The reaction kinetics of prolonged photoinhibition was studied in leaves of Cucurbita maxima and Capsicum annuum. The results indicated that photoinhibitory decrease in both the oxygen evolution activity and ratio of variable to maximum fluorescence follows firstorder kinetics in vivo. The persistence of first-order kinetics suggests that already photoinhibited reaction centres do not protect against photoinhibition and that the mechanism of photoinhibition does not have a reversible intermediate. When Cucurbita maxima leaves were photoinhibited with saturating single-turnover flashes and continuous light, the light response curve of photoinhibition was found to be essentially a straight line with both types of illumination, suggesting that similar photoinhibition mechanisms might function during illumination with continuous light and during illumination with short flashes.

A Brazilian glycoprotein E-negative bovine herpesvirus type 1.2a (BHV-1.2a) mutant is attenuated for cattle and induces protection against wild-type virus challenge

The authors previously reported the construction of a glycoprotein E-deleted (gE-) mutant of bovine herpesvirus type 1.2a (BHV-1.2a). This mutant, 265gE-, was designed as a vaccinal strain for differential vaccines, allowing the distinction between vaccinated and naturally infected cattle. In order to determine the safety and efficacy of this candidate vaccine virus, a group of calves was inoculated with 265gE-. The virus was detected in secretions of inoculated calves to lower titres and for a shorter period than the parental virus inoculated in control calves. Twenty one days after inoculation, the calves were challenged with the wild type parental virus. Only mild signs of infection were detected on vaccinated calves, whereas non-vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Six months after vaccination, both vaccinated and control groups were subjected to reactivation of potentially latent virus. The mutant 265gE- could not be reactivated from vaccinated calves. The clinical signs observed, following the reactivation of the parental virus, were again much milder on vaccinated than on non-vaccinated calves. Moreover, parental virus shedding was considerably reduced on vaccinated calves at reactivation. In view of its attenuation, immunogenicity and protective effect upon challenge and reactivation with a virulent BHV-1, the mutant 265gE- was shown to be suitable for use as a BHV-1 differential vaccine virus.

Efficacy of a gE-deleted, bovine herpesvirus 1 (BoHV-1) inactivated vaccine

Bovine herpesvirus type 1 (BoHV-1) is recognized as a major cause of economic losses in cattle. Vaccination has been widely applied to minimize losses induced by BoHV-1 infections. We have previously reported the development of a differential BoHV-1 vaccine, based on a recombinant glycoprotein E (gE)-deleted virus (265gE-). In present paper the efficacy of such recombinant was evaluated as an inactivated vaccine. Five BoHV-1 seronegative calves were vaccinated intramuscularly on day 0 and boostered 30 days later with an inactivated, oil adjuvanted vaccine containing an antigenic mass equivalent to 10(7.0) fifty per cent cell culture infectious doses (CCID50) of 265gE-. Three calves were kept as non vaccinated controls. On day 60 post vaccination both vaccinated and controls were challenged with the virulent parental strain. No clinical signs or adverse effects were seen after or during vaccination. After challenge, 2/5 vaccinated calves showed mild clinical signs of infection, whereas all non vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Serological responses were detected in all vaccinated animals after the second dose of vaccine, but not on control calves. Following corticosteroid administration in attempting to induce reactivation of the latent infection, no clinical signs were observed in vaccinated calves, whereas non vaccinated controls showed clinical signs of respiratory disease. In view of its immunogenicity and protective effect upon challenge with a virulent BoHV-1, the oil adjuvanted preparation with the inactivated 265gE- recombinant was shown to be suitable for use as a vaccine.

AT1-receptor mediated vascular damage in myocardium, kidneys and liver in rats

The systemic aspect of vascular damage induced by angiotensin II (ANG II) has been poorly explored in the literature. Considering the presence of ANG II and its specific receptor AT1, in several organs, all tissues might be potentially affected by its effects. The aims of this study were: To evaluate the early histological changes in the heart, liver and kidneys, produced by ANG II infusion, to evaluate the protective effect of losartan. Wistar rats were distributed into three groups: control (no treatment), treated with ANG II, and treated with ANG II + losartan. ANG II was continuously infused over 72 hours by subcutaneous osmotic pumps. Histological sections of the myocardium, kidneys and liver were stained and observed for the presence of necrosis. There were ANG II-induced perivascular inflammation and necrosis of the arteriolar wall in the myocardium, kidney, and liver by, which were partially prevented by losartan. There was no significant correlation between heart and kidney damage. Tissue lesion severity was lower than that of vascular lesions, without statistical difference between groups. ANG II causes vascular injury in the heart, kidneys and liver, indicating a systemic vasculotoxic effect; the mechanisms of damage/protection vary depending on the target organ; perivascular lesions may occur even when anti-hypertensive doses of losartan are used.

Factors predicting mortality in type 2 diabetes. With special reference to physical exercise, blood pressure, proteinuria, inflammation and P wave duration

Background: Type 2 diabetes patients have a 2-4 fold risk of cardiovascular disease (CVD) compared to the general population. In type 2 diabetes, several CVD risk factors have been identified, including obesity, hypertension, hyperglycemia, proteinuria, sedentary lifestyle and dyslipidemia. Although much of the excess CVD risk can be attributed to these risk factors, a significant proportion is still unknown. Aims: To assess in middle-aged type 2 diabetic subjects the joint relations of several conventional and non-conventional CVD risk factors with respect to cardiovascular and total mortality. Subjects and methods: This thesis is part of a large prospective, population based East-West type 2 diabetes study that was launched in 1982-1984. It includes 1,059 middle-aged (45-64 years old) participants. At baseline, a thorough clinical examination and laboratory measurements were performed and an ECG was recorded. The latest follow-up study was performed 18 years later in January 2001 (when the subjects were 63-81 years old). The study endpoints were total mortality and mortality due to CVD, coronary heart disease (CHD) and stroke. Results: Physically more active patients had significantly reduced total, CVD and CHD mortality independent of high-sensitivity C-reactive protein (hs-CRP) levels unless proteinuria was present. Among physically active patients with a hs-CRP level >3 mg/L, the prognosis of CVD mortality was similar to patients with hs-CRP levels ≤3 mg/L. The worst prognosis was among physically inactive patients with hs-CRP levels >3 mg/L. Physically active patients with proteinuria had significantly increased total and CVD mortality by multivariate analyses. After adjustment for confounding factors, patients with proteinuria and a systolic BP <130 mmHg had a significant increase in total and CVD mortality compared to those with a systolic BP between 130 and 160 mmHg. The prognosis was similar in patients with a systolic BP <130 mmHg and ≥160 mmHg. Among patients without proteinuria, a systolic BP <130 mmHg was associated with a non-significant reduction in mortality. A P wave duration ≥114 ms was associated with a 2.5-fold increase in stroke mortality among patients with prevalent CHD or claudication. This finding persisted in multivariable analyses. Among patients with no comorbidities, there was no relationship between P wave duration and stroke mortality. Conclusions: Physical activity reduces total and CVD mortality in patients with type 2 diabetes without proteinuria or with elevated levels of hs-CRP, suggesting that the anti-inflammatory effect of physical activity can counteract increased CVD morbidity and mortality associated with a high CRP level. In patients with proteinuria the protective effect was not, however, present. Among patients with proteinuria, systolic BP <130 mmHg may increase mortality due to CVD. These results demonstrate the importance of early intervention to prevent CVD and to control all-cause mortality among patients with type 2 diabetes. The presence of proteinuria should be taken into account when defining the target systolic BP level for prevention of CVD deaths. A prolongation of the duration of the P wave was associated with increased stroke mortality among high-risk patients with type 2 diabetes. P wave duration is easy to measure and merits further examination to evaluate its importance for estimation of the risk of stroke among patients with type 2 diabetes.

Morphological and physiological alterations induced by lactofen in soybean leaves are reduced with nitric oxide

Lactofen is a diphenylether herbicide recommended to control broad-leaved weeds in soybean (Glycine max) fields and its mechanism of action is the inhibition of protoporphyrinogen-IX oxidase (Protox), which acts in the chlorophyll biosynthesis. This inhibition results in an accumulation of protoporphyrin-IX, which leads to the production of reactive oxygen species (ROS) that cause oxidative stress. Consequently, spots, wrinkling and leaf burn may occur, resulting in a transitory crop growth interruption. However, nitric oxide (NO) acts as an antioxidant in direct ROS scavenging. Thus, the aim of this work was to verify, through phytometric and biochemical evaluations, the protective effect of NO in soybean plants treated with the herbicide lactofen. Soybean plants were pre-treated with different levels of sodium nitroprusside (SNP), a NO-donor substance, and then sprayed with 168 g a.i. ha-1 lactofen. Pre-treatment with SNP was beneficial because NO decreased the injury symptoms caused by lactofen in young leaflets and kept low the soluble sugar levels. Nevertheless, NO caused slower plant growth, which indicates that further studies are needed in order to elucidate the action mechanisms of NO in signaling the stress caused by lactofen in soybean crop.

The role of IL-12 in experimental Trypanosoma cruzi infection

Host resistance to Trypanosoma cruzi infection is dependent on both natural and acquired immune responses. During the early acute phase of infection in mice, natural killer (NK) cell-derived IFN-g is involved in controlling intracellular parasite replication, mainly through the induction of nitric oxide biosynthesis by activated macrophages. We have shown that IL-12, a powerful inducer of IFN-g production by NK cells, is synthesized soon after trypomastigote-macrophage interaction. The role of IL-12 in the control of T. cruzi infection in vivo was determined by treating infected mice with anti-IL-12 monoclonal antibody (mAb) and analyzing both parasitemia and mortality during the acute phase of infection. The anti-IL-12 mAb-treated mice had higher levels of parasitemia and mortality compared to control mice. Also, treatment of infected mice with mAb specific for IFN-g or TNF-a inhibited the protective effect of exogenous IL-12. On the other hand, TGF-ß and IL-10 produced by infected macrophages inhibited the induction and effects of IL-12. Therefore, while IL-12, TNF-a and IFN-g correlate with resistance to T. cruzi infection, TGF-ß and IL-10 promote susceptibility. These results provide support for a role of innate immunity in the control of T. cruzi infection. In addition to its protective role, IL-12 may also be involved in the modulation of T. cruzi-induced myocarditis, since treatment of infected mice with IL-12 or anti-IL-12 mAb leads to an enhanced or decreased inflammatory infiltrate in the heart, respectively. Understanding the role of the cytokines produced during the acute phase of T. cruzi infection and their involvement in protection and pathogenesis would be essential to devise new vaccines or therapies.

Neuronal and endothelial nitric oxide synthase gene knockout mice

Targeted disruption of the neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) genes has led to knockout mice that lack these isoforms. These animal models have been useful to study the roles of nitric oxide (NO) in physiologic processes. nNOS knockout mice have enlarged stomachs and defects in the inhibitory junction potential involved in gastrointestinal motility. eNOS knockout mice are hypertensive and lack endothelium-derived relaxing factor activity. When these animals are subjected to models of focal ischemia, the nNOS mutant mice develop smaller infarcts, consistent with a role for nNOS in neurotoxicity following cerebral ischemia. In contrast, eNOS mutant mice develop larger infarcts, and show a more pronounced hemodynamic effect of vascular occlusion. The knockout mice also show that nNOS and eNOS isoforms differentially modulate the release of neurotransmitters in various regions of the brain. eNOS knockout mice respond to vessel injury with greater neointimal proliferation, confirming that reduced NO levels seen in endothelial dysfunction change the vessel response to injury. Furthermore, eNOS mutant mice still show a protective effect of female gender, indicating that the mechanism of this protection cannot be limited to upregulation of eNOS expression. The eNOS mutant mice also prove that eNOS modulates the cardiac contractile response to ß-adrenergic agonists and baseline diastolic relaxation. Atrial natriuretic peptide, upregulated in the hearts of eNOS mutant mice, normalizes cGMP levels and restores normal diastolic relaxation.

Ghost protein damage by peroxynitrite and its protection by melatonin

We have studied the effect of peroxynitrite (ONOO-) on the membrane cytoskeleton of red blood cells and its protection by melatonin. Analysis of the protein fraction of the preparation by SDS-PAGE revealed a dose-dependent (0-600 µM ONOO-) disappearance at pH 7.4 of the main proteins: spectrin, band 3, and actin, with the concomitant formation of high-molecular weight aggregates resistant to reduction by ß-mercaptoethanol (2%) at room temperature for 20 min. These aggregates were not solubilized by 8 M urea. Incubation of the membrane cytoskeleton with ONOO- was characterized by a marked depletion of free sulfhydryl groups (50% at 250 µM ONOO-). However, a lack of effect of ß-mercaptoethanol suggests that, under our conditions, aggregate formation is not mediated only by sulfhydryl oxidation. The lack of a protective effect of the metal chelator diethylenetriaminepentaacetic acid confirmed that ONOO--induced oxidative damage does not occur only by a transition metal-dependent mechanism. However, we demonstrated a strong protection against cytoskeletal alterations by desferrioxamine, which has been described as a direct scavenger of the protonated form of peroxynitrite. Desferrioxamine (0.5 mM) also inhibited the loss of tryptophan fluorescence observed when the ghosts were treated with ONOO-. Glutathione, cysteine, and Trolox® (1 mM), but not mannitol (100 mM), were able to protect the proteins against the effect of ONOO- in a dose-dependent manner. Melatonin (0-1 mM) was especially efficient in reducing the loss of spectrin proteins when treated with ONOO- (90% at 500 µM melatonin). Our findings show that the cytoskeleton, and in particular spectrin, is a sensitive target for ONOO-. Specific antioxidants can protect against such alterations, which could seriously impair cell dynamics and generate morphological changes.

Inhibition of rat microsomal lipid peroxidation by the oral administration of D002

The effect of D002, a defined mixture of higher primary alcohols purified from bee wax, on in vivo and in vitro lipid peroxidation was studied. The extent of lipid peroxidation was measured on the basis of the levels of thiobarbituric acid reactive substances (TBARS). When D002 (5-100 mg/kg body weight) was administered orally to rats for two weeks, a partial inhibition of the in vitro enzymatic and non-enzymatic lipid peroxidation was observed in liver and brain microsomes. Maximal protection (46%) occurred at a dose of 25 mg/kg. D002 behaved differently depending on both the presence of NADPH and the integrity of liver microsomes, which suggests that under conditions where microsomal metabolism was favored the protective effect of D002 was increased. D002 (25 mg/kg) also completely inhibited carbon tetrachloride- and toluene-induced in vivo lipid peroxidation in liver and brain. Also, D002 significantly lowered in a dose-dependent manner the basal level of TBARS in liver (19-40%) and brain (28-44%) microsomes. We conclude that the oral administration of D002 (5, 25 and 100 mg/kg) for two weeks protected rat liver and brain microsomes against microsomal lipid peroxidation in vitro and in vivo. Thus, D002 could be useful as a dietary natural antioxidant supplement. More studies are required before these data can be extrapolated to the recommendation for the use of D002 as a dietary antioxidant supplement for humans.

Antioxidant properties of natural compounds used in popular medicine for gastric ulcers

There is evidence concerning the participation of reactive oxygen species in the etiology and physiopathology of human diseases, such as neurodegenerative disorders, inflammation, viral infections, autoimmune pathologies, and digestive system disorders such as gastrointestinal inflammation and gastric ulcer. The role of these reactive oxygen species in several diseases and the potential antioxidant protective effect of natural compounds on affected tissues are topics of high current interest. To consider a natural compound or a drug as an antioxidant substance it is necessary to investigate its antioxidant properties in vitro and then to evaluate its antioxidant functions in biological systems. In this review article, we shall consider the role of natural antioxidants derived from popular plants to reduce or prevent the oxidative stress in gastric ulcer induced by ethanol.

Temperature, air pollution, and mortality from myocardial infarction in São Paulo, Brazil

An increase in daily mortality from myocardial infarction has been observed in association with meteorological factors and air pollution in several cities in the world, mainly in the northern hemisphere. The objective of the present study was to analyze the independent effects of environmental variables on daily counts of death from myocardial infarction in a subtropical region in South America. We used the robust Poisson regression to investigate associations between weather (temperature, humidity and barometric pressure), air pollution (sulfur dioxide, carbon monoxide, and inhalable particulate), and the daily death counts attributed to myocardial infarction in the city of São Paulo in Brazil, where 12,007 fatal events were observed from 1996 to 1998. The model was adjusted in a linear fashion for relative humidity and day-of-week, while nonparametric smoothing factors were used for seasonal trend and temperature. We found a significant association of daily temperature with deaths due to myocardial infarction (P < 0.001), with the lowest mortality being observed at temperatures between 21.6 and 22.6ºC. Relative humidity appeared to exert a protective effect. Sulfur dioxide concentrations correlated linearly with myocardial infarction deaths, increasing the number of fatal events by 3.4% (relative risk of 1.03; 95% confidence interval = 1.02-1.05) for each 10 µg/m³ increase. In conclusion, this study provides evidence of important associations between daily temperature and air pollution and mortality from myocardial infarction in a subtropical region, even after a comprehensive control for confounding factors.