Persistent activation of Akt or ERK prevents the toxicity induced by saturated and polyunsaturated fatty acids in RINm5F beta-cells

The aim of this study was to investigate whether the toxicity of saturated and polyunsaturated fatty acids (PUFA) on RINm5F cells is related to the phosphorylation state of Akt, ERK and PKC delta. The regulation of these kinases was compared in three experimental designs: (a) 4 h-exposure, (b) 4 h-exposure and a subsequent withdrawn of the FA for a 20 h period and (c) 24 h-exposure. Saturated and PUFA were toxic to RINm5F cells even at low concentrations. Also, evidence is provided for a late (i.e. the effect only appeared hours after the treatment) and a persistent regulation (i.e. maintenance of the effect for several hours) of Akt, ERK and PKC delta phosphorylation by the FA. Late activation of PKC delta seems important for palmitate cytotoxicity. Persistent activation of the survival proteins Akt and ERK by stearate, oleate and arachidonate might play an important role to prevent the toxic effect of posterior PKC delta activation. The results shown may explain why a short-period exposure to FA is not enough to induce cytotoxicity in pancreatic beta-cells, since survival pathways are activated. Besides, when this activation is persistent, it may overcome a posterior induction of death pathways. (c) 2008 Elsevier Ltd. All rights reserved.

Exercise training prevents beta-adrenergic hyperactivity-induced myocardial hypertrophy and lesions

Background: Sustained beta-adrenoreceptor activation promotes cardiac hypertrophy and cellular injury. Aims: To evaluate the cardioprotective effect of exercise on damage induced by beta-adrenergic hyperactivity. Methods: Male Wistar rats were randomised into four groups (n=8 per group): sedentary non-treated control (C), sedentary treated with isoproterenol 0.3 mg/kg/day administered subcutaneously for 8 days (1), exercised non-treated (E) and exercised plus isoproterenol administered during the last eight days of exercise (IE). Exercised animals ran on a treadmill for 1 h daily 6 times a week for 13 weeks. Results: Isoproterenol caused increases in left ventricle (LV) wet and dry weight/body weight ratio, LV water content and cardiomyocyte transverse diameter. Additionally, isoproterenol induced severe cellular lesions, necrosis, and apoptosis, increased collagen content and reduced capillary and fibre fractional areas. Notably, all of these abnormalities were completely prevented by exercise. Conclusion: Our data have demonstrated that complete cardioprotection is possible through exercise training; by preventing p-adrenergic hyperactivity-induced cardiac hypertrophy and structural injury. (c) 2008 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Cutaneuos cryoglobulinemic vasculitis induced by chronic hepatitis C virus infection

Cutaneous vasculitis may represent a great clinical challenge, even after careful dermatological examination and laboratory assessment. The authors present a case of cutaneous cryoglobulinemic vasculitis associated to chronic hepatitis C virus infection, pointing out the importance of the dermatological examination for diagnostic investigation. They discuss about the importance of defining the etiology and making correct classification for appropriate prognosis and treatment of cutaneous vasculitis.

Inflammatory related changes in lung tissue mechanics after bleomycin-induced lung injury

The impact of lung remodelling in respiratory mechanics has been widely studied in bleomycin-induced lung injury. However, little is known regarding the relationship between the amount of lung inflammation and pulmonary tissue mechanics. For this purpose, rats were intratracheally instilled with bleomycin (n = 29) or saline (n = 8) and sacrificed at 3, 7, or 15 days. Forced oscillatory mechanics as well as indices of remodelling (elastic fibre content and hydroxyproline) and inflammation (myeloperoxidase content, total cell count, alveolar wall thickness, and lung water content) were studied in lung tissue strips. Tissue resistance increased significantly at day 15, while hysteresivity was significantly higher in bleomycin group compared to control at all time points. Elastic fibres, hydroxyproline and myeloperoxidase, contents augmented after bleomycin at days 7 and 15. Tissue resistance and hysteresivity were significantly correlated with myeloperoxidase, elastic fibre and lung water content. In conclusion, inflammatory structural changes and elastogenesis are the main determinants for hysteretic changes in this 2-week bleomycin-induced lung injury model. (c) 2007 Elsevier B.V. All rights reserved.

Comparison of Full Versus Short Induced-Sleep Polysomnography for the Diagnosis of Sleep Apnea

Objectives/Hypothesis: Polysomnography (PSG) is the gold-standard method for diagnosing obstructive sleep apnea (OSA). However, the gap between demand and capacity in performing PSG is a major health-care problem. We sought to validate a short day-time induced sleep for the diagnosis of OSA. Study Design: Prospective diagnostic method validation. Methods: We studied 25 consecutive patients referred to the sleep laboratory and 15 healthy volunteers. All subjects were evaluated by means of full overnight PSG (Full-PSG) and short day-time induced-sleep PSG (Induced-PSG). Sleep was monitored during both procedures (Embla, 16 channels). Sleep was induced by slow intravenous drip infusion of midazolam. Results: The population studied (N = 40) was 60% male (mean age, 42 +/- 10 years; body mass index, 29 +/- 6.5 kg/m(2)). Sleep was successfully induced in all subjects, and no complications were observed (midazolam doses, 6.2 +/- 3.8 mg; time of induced sleep 41.5 +/- 18.9 minutes). The apnea-hypopnea index (AHI) and minimal oxygen saturation during Full-PSG versus Induced-PSG were similar: median AHI (with 25%-75% interquartile range) was 13 (3-35) events per hour versus 17 (4-36) events per hour, and median oxygen saturation was 84% (75-90) versus 85% (76-92); P = .89 and P = .53, respectively. The majority of the respiratory events during induced sleep were obstructive and similar to those observed during Full-PSG. AHI and lowest oxygen saturation during Induced-PSG correlated significantly with Full-PSG (r = 0.67 and r = 0.77, respectively). Sensitivity and specificity for the diagnosis of OSA (AHI > 15 events per hour) by Induced-PSG were 0.83 and 0.72, respectively. Conclusions: Induced-PSG by midazolam during the day is safe and correlates with Full-PSG; it therefore is a promising alternative method in the diagnosis of OSA.

Enhancement of diagnostic efficiency by a gamma interferon release assay for pulmonary tuberculosis

This study was designed to examine the use of the QuantiFERON-TB Gold assay as an aid in the diagnosis of active pulmonary tuberculosis (TB) in Brazilian patients. Using the receiver operating characteristic curve, the cutoff was adjusted to >= 0.20 IU/ml. The sensitivity increased to 86%, with 100% specificity. All TB patients with negative sputum smear microscopy and negative culture results were positive using this test.

Changes in postnatal skeletal muscle development induced by alternative immobilization model in female rat

The objective of this study was to adapt a model of hind limb immobilization to newly weaned female rats and to determine the morphology of shortened soleus and plantaris muscles. Female Wistar rats were divided into three groups: control zero (n = 3) and control and free (n = 8), animals aged 21 and 31 days, respectively, submitted to no intervention, and immobilized (n = 25), animals aged 21 days submitted to immobilization for 10 days and sacrificed at 31 days of age. The device used for immobilization had advantages such as easy connection, good fit, and low cost. The immobilized rats showed a reduction in muscle fiber area and in connective tissue. The adaptation of this immobilization model originally used for adult rats was an excellent alternative for newly weaned rats and was also efficient in inducing significant hind limb disuse.

Effect of academic psychological stress in post-graduate students: The modulatory role of cortisol on superoxide release by neutrophils

Experimental and clinical evidence shows that neutrophils play an important role in the mechanism of tissue injury in immune complex diseases through the generation of reactive oxygen species. In this study, we examined the influence of academic psychological stress in post-graduate students on the capacity of their blood neutrophils to release superoxide when stimulated by immune complexes bound to nonphagocytosable surfaces and investigated the modulatory effect of cortisol on this immune function. The tests were performed on the day before the final examination. The state-trait anxiety inventory questionnaire was used to examine whether this stressful event caused emotional distress. In our study, the psychological stress not only increased plasma cortisol concentration, but it also provoked a reduction in superoxide release by neutrophils. This decrease in superoxide release was accompanied by diminished mRNA expression for subunit p47(phox) of the phagocyte superoxide-generating nicotinamide adenine dinucleotide phosphate-oxidase. These inhibitory effects were also observed by in vitro exposure of neutrophils from control volunteers to 10(-7) M hydrocortisone, and could be prevented by the glucocorticoid receptor antagonist RU-486. These results show that in a situation of psychological stress, the increased levels of cortisol could inhibit superoxide release by neutrophils stimulated by IgG immune complexes bound to nonphagocytosable surfaces, which could attenuate the inflammatory state.

Expression of Mycobacterium leprae HSP65 in tobacco and its effectiveness as an oral treatment in adjuvant-induced arthritis

Transgenic plants are able to express molecules with antigenic properties. In recent years, this has led the pharmaceutical industry to use plants as alternative systems for the production of recombinant proteins. Plant-produced recominant proteins can have important applications in therapeutics, such as in the treatment of rheumatoid arthritis (RA). In this study, the mycobacterial HSP65 protein expressed in tobacco plants was found to be effective as a treatment for adjuvant-induced arthritis (AIA). We cloned the hsp65 gene from Mycobacterium leprae into plasmid pCAMBIA 2301 under the control of the double 35S promoter from cauliflower mosaic virus. Agrobacterium tumefaciens bearing the pChsp65 plasmid was used to transform tobacco plants. Incorporation of the hsp65 gene was confirmed by PCR, reverse transcription-PCR, histochemistry, and western blot analyses in several transgenic lines of tobacco plants. Oral treatment of AIA rats with the HSP65 protein allowed them to recover body weight and joint inflammation was reduced. Our results suggest a synergistic effect between the HSP65 expressed protein and metabolites presents in tobacco plants.

Rabbit Rubeosis Iridis Induced by Intravitreal Latex-derived Angiogenic Fraction

Purpose: To describe the presence of iris neovascularization in a rabbit-model of retinal neovascularization induced by the intravitreal injection of latex-derived angiogenic fraction microspheres (LAF). Materials and Methods: Eight New Zealand rabbits received one intravitreal injection of PLGA (L-lactide-coglycolide) microspheres with 50 ug of LAF in the right eye (Group A). Microspheres without the LAF (0.1 ml) were injected in controls (Group B; n = 8). Follow-up with clinical evaluation and iris fluorescein angiography was performed after 4 weeks when eyes were processed for light microscopy. Results: All eyes from Group A showed significant vascular dilation, conjunctival hyperemia and neovascularization on the iris surface, after LAF injection. No vascular changes were observed in Group B. Conclusions: The intravitreal injection of microspheres containing the LAF can induce rubeosis iridis in rabbits and could be used as a simple experimental model for iris neovascularization.

Increased Vascular Permeability, Angiogenesis and Wound Healing Induced by the Serum of Natural Latex of the Rubber Tree Hevea brasiliensis

Increases in vascular permeability and angiogenesis are crucial events to wound repair, tumoral growth and revascularization of tissues submitted to ischemia. An increased vascular permeability allows a variety of cytokines and growth factors to reach the damaged tissue. Nevertheless, the angiogenesis supply tissues with a wide variety of nutrients and is also important to metabolites clearance. It has been suggested that the natural latex from Hevea brasiliensis showed wound healing properties and angiogenic activity. Thus, the purpose of this work was to characterize its angiogenic activity and its effects on vascular permeability and wound healing. The serum fraction of the latex was separated from the rubber with reduction of the pH. The activity of the dialyzed serum fraction on the vascular permeability injected in subcutaneous tissue was assayed according Mile`s method. The angiogenic activity was determined using a chick embryo chorioallantoic membrane assay and its effects on the wound-healing process was determined by the rabbit ear dermal ulcer model. The serum fraction showed evident angiogenic effect and it was effective in enhancing vascular permeability. In dermal ulcers, this material significantly accelerated wound healing. Moreover, the serum fraction boiled and treated with proteases lost these activities. These results are in accordance with the enhancement of wound healing observed in clinical trials carried out with a biomembrane prepared with the same natural latex. Copyright (C) 2009 John Wiley & Sons, Ltd.

Modulation of cutaneous inflammation induced by ticks in contrasting phenotypes of infestation in bovines

Tick saliva contains molecules that are inoculated at the site of attachment on their hosts in order to modulate local immune responses and facilitate a successful blood meal. Bovines express heritable, contrasting phenotypes of infestations with the cattle tick, Rhipicephalus (Boophilus) microplus: breeds of Bos taurus indicus are significantly note resistant than those of Bos taurus taurus. Tick saliva may contain molecules that interfere with adhesion of leukocytes to endothelium and resistant hosts may mount an inflammatory profile that is more efficient to hamper the tick`s blood meal. We show in vitro that adhesion of peripheral blood mononuclear cells to monolayers of cytokine-activated bovine umbilical endothelial cells was significantly inhibited by tick saliva. The inflammatory response to bites of adults of R. microplus mounted by genetically resistant and susceptible bovine hosts managed in the same pasture was investigated in vivo. The inflammatory infiltrates and levels of message coding for adhesion molecules were measured in biopsies of tick-bitten and control skin taken when animals of both breeds were exposed to low and high tick infestations. Histological studies reveal that cutaneous reactions of resistant hosts to bites of adult ticks contained significantly more basophils and eosinophils compared with reactions of the susceptible breed. Expression of the adhesion molecules - intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin - was higher in adult-infested skin of susceptible hosts undergoing low infestations compared to resistant hosts; when host was exposed to high infestations expression of these adhesion molecules was down-regulated in both phenotypes of infestations. Expression of leukocyte adhesion glycoprotein-1 (LFA-1) was higher in skin from susceptible hosts undergoing low or high infestations compared to resistant hosts. Conversely, higher levels of E-selectin, which promotes adhesion of memory T cells, were expressed in skin of resistant animals. This finding may explain the resistant host`s ability to mount more rapid and efficient secondary responses that limit hematophagy and infestations. The expression profiles observed for adhesion molecules indicate that there are differences in the kinetics of the inflammatory reactions mounted by resistant and susceptible hosts and the balance between tick and host is affected by the number of tick bites a host receives. We show that the contrasting phenotypes of infestations seen in bovines infested with R. microplus are correlated with differences in the cellular and molecular composition of inflammatory infiltrates elicited by bites with adult ticks. (C) 2009 Published by Elsevier B.V.

Characterization and in vitro activities of cell-free antigens from Histoplasma capsulatum-loaded biodegradable microspheres

In the last decades, the incidence of histoplasmosis, a pulmonary fungal disease caused by Histoplasma capsulatum, has increased worldwide. In this context, vaccines for the prevention of this infection or therapies are necessary. Cell-free antigens (CFAgs) from H. capsulatum when administered for murine immunization purposes are able to confer protection and control of the infection, since they activate cellular immunity. However the most of vaccination procedures need several anti, gens administrations and immunoadjuvants, which are not approved for use in humans. The aim of this study was to develop and characterize a vaccination approach using biodegradable PLGA microspheres (MS) that could allow the controlled and/or sustained release of the encapsulated antigens from H. capsulatum. CFAgs-loaded MS presented a size less than 10 mu m, were marked engulfed by bone marrow-derived macrophages (BMDM phi) and induced the nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by these cells. Our data show that CFAgs-loaded MS induce cell activation, suggesting an immunostimulant effect to be further investigated during immunization procedures. CFAgs-loaded MS present potential to be used as vaccine in order to confer protection against H. capsulatum infection. (C) 2009 Elsevier B.V. All rights reserved.

Cytotoxic effects of crotamine are mediated through lysosomal membrane permeabilization

Crotamine, one of the main toxic components of Crotalus durissus terrificus venom, is a small non-enzymatic basic polypeptide, which causes hind limb paralysis and necrosis of muscle cells. it is well-known that several toxins penetrate into the cytosol through endocytosis, although in many cases the mechanism by which this occurs has not been fully investigated. Recently, using low concentrations of crotamine, we demonstrated the uptake of this toxin into actively proliferative cells via endocytosis, an event that ensues crotamine binding to cell membrane heparan sulfate proteoglycans. Thus, crotamine can be regarded as a cell-penetrating peptide that, additionally, has been shown to be able of delivering some biologically active molecules into various cells. Herein, we investigate one of the mechanisms by which crotamine exerts its cytotoxic effects by following its uptake into highly proliferative cells, as CHO-K1 cells. Crotamine accumulation in the acidic endosomal/lysosomal vesicles was observed within 5 min after treatment of these cells with a cytotoxic concentration of this toxin, a value determined here by classical MTT assay. This accumulation caused disruption of lysosomal vesicles accompanied by the leakage of these vesicles contents into the cytosol. This lysosomal lysis also promoted the release of cysteine cathepsin and an increase of caspase activity in the cytoplasm. This chain of events seems to trigger a cell death process. Overall, our data suggest that lysosomes are the primary targets for crotamine cytotoxicity, a proposal corroborated by the correlation between both the kinetics and concentration-dependence of crotamine accumulation in lysosome compartments and the cytotoxic effects of this protein in CHO-K1 cells. Although crotamine is usually regarded as a myotoxin, we observed that intraperitoneal injection of fluorescently labeled crotamine in living mice led to significant and rapid accumulation of this toxin in the cell cytoplasm of several tissues, suggesting that crotamine cytotoxicity might not be restricted to muscle cells. (C) 2008 Elsevier Ltd. All rights reserved.

Coxsackievirus 135 induced apoptosis of HeLa cells: Effects on p53 and SUMO

Coxsackievirus B5 (CVB5), a human enterovirus of the family Picornaviridae, is a frequent cause of acute and chronic human diseases. The pathogenesis of enteroviral infections is not completely understood, and the fate of the CVB5-infected cell has a pivotal role in this process. We have investigated the CVB5-induced apoptosis of HeLa cells and found that it happens by the intrinsic pathway by a mechanism dependent on the ubiquitin-proteasome system, associated with nuclear aggregation of p53. Striking redistribution of both SUMO and UBC9 was noted at 4 h post-infection, simultaneously with a reduction in the levels of the ubiquitin-ligase HDM2. Taken together, these results suggest that CVB5 infection of HeLa cells elicit the intrinsic pathway of apoptosis by MDM2 degradation and p53 activation, destabilizing protein sumoylation, by a mechanism that is dependent on a functional ubiquitin-proteasome system. (C) 2009 Elsevier Inc. All rights reserved.