Biomedical subjects
J P Silva
Publications and source records attributed to J P Silva.
Kinetics of eosinophil and IgE-mast cell changes following infection with Angiostrongylus costaricensis in Wistar rats.
Human abdominal angiostrongyliasis is a severe eosinophilic disease caused by Angiostrongylus costaricensis. Previous studies have demonstrated that wild rodents are critically involved as definitive hosts to this nematode in nature. In this study, we have evaluated the susceptibility of Wistar rats (Rattus norvegicus) to A. costaricensis infection. Kinetics of parasitological and pathological changes, including the number of adult worms recovered from mesenteric arteries, and of IgE, mast cell and eosinophil levels in several compartments have been assessed. The oral inoculation of third-stage larvae (L3) into adult Wistar rats led to a marked accumulation of worms in the branches of the mesenteric arteries 25 and 50 days post-inoculation. Intense bone marrow eosinophilia ranging from 7 to 50 days was accompanied by marked accumulation of eosinophils in the blood, peritoneal and bronchoalveolar spaces. Eosinophilic periarteritis, oedema and granuloma in the intestinal and lung tissues were also histologically evident. Total serum IgE and specific anti-parasite IgE peaked at 25 days post-infection, as measured by ELISA and by the passive cutaneous anaphylaxis test, respectively. At that time point, there was a drastic reduction in the number of intact mast cells in the peritoneal effluent. These findings indicate that Wistar rats are permissive to A. costaricensis infection. IgE-mast cell activation and massive tissue eosinophil infiltration are marked features in the process and are likely to play a crucial role in the immune-response evoked by this parasite.
Late-onset corticohippocampal neurodepletion attributable to catastrophic failure of oxidative phosphorylation in MILON mice.
We generated mitochondrial late-onset neurodegeneration (MILON) mice with postnatal disruption of oxidative phosphorylation in forebrain neurons. They develop normally and display no overt behavioral disturbances or histological changes during the first 5 months of life. The MILON mice display reduced levels of mitochondrial DNA and mitochondrial RNA from 2 and 4 months of age, respectively, and severely respiratory chain-deficient neurons from 4 months of age. Surprisingly, these respiratory chain-deficient neurons are viable for at least 1 month without showing signs of neurodegeneration or major induction of defenses against oxidative stress. Prolonged neuronal respiratory chain deficiency is thus required for the induction of neurodegeneration. Before developing neurological symptoms, MILON mice show increased vulnerability to excitotoxic stress. We observed a markedly enhanced sensitivity to excitotoxic challenge, manifest as an abundance of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) reactive cells after kainic acid injection, in 4-month-old MILON mice, showing that respiratory chain-deficient neurons are more vulnerable to stress. At approximately 5-5.5 months of age, MILON mice start to show signs of disease, followed by death shortly thereafter. The debut of overt disease in MILON mice coincides with onset of rapidly progressive neurodegeneration and massive cell death in hippocampus and neocortex. This profound neurodegenerative process is manifested as axonal degeneration, gliosis, and abundant TUNEL-positive nuclei. The MILON mouse model provides a novel and powerful tool for additional studies of the role for respiratory chain deficiency in neurodegeneration and aging.
Increased in vivo apoptosis in cells lacking mitochondrial DNA gene expression.
We have attempted to determine whether loss of mtDNA and respiratory chain function result in apoptosis in vivo. Apoptosis was studied in embryos with homozygous disruption of the mitochondrial transcription factor A gene (Tfam) and tissue-specific Tfam knockout animals with severe respiratory chain deficiency in the heart. We found massive apoptosis in Tfam knockout embryos at embryonic day (E) 9.5 and increased apoptosis in the heart of the tissue-specific Tfam knockouts. Furthermore, mtDNA-less (rho(0)) cell lines were susceptible to apoptosis induced by different stimuli in vitro. The data presented here provide in vivo evidence that respiratory chain deficiency predisposes cells to apoptosis, contrary to previous assumptions based on in vitro studies of cultured cells. These results suggest that increased apoptosis is a pathogenic event in human mtDNA mutation disorders. The finding that respiratory chain deficiency is associated with increased in vivo apoptosis may have important therapeutic implications for human disease. Respiratory chain deficiency and cell loss and/or apoptosis have been associated with neurodegeneration, heart failure, diabetes mellitus, and aging. Furthermore, chemotherapy and radiation treatment of cancer are intended to induce apoptosis in tumor cells. It would therefore be of interest to determine whether manipulation of respiratory chain function can be used to inhibit or enhance apoptosis in these conditions.
Modulation of eotaxin formation and eosinophil migration by selective inhibitors of phosphodiesterase type 4 isoenzyme.
1. This study was undertaken to investigate the possible contribution of the blockade of eotaxin generation to the anti-eosinophilotactic effect of phosphodiesterase (PDE) type 4 inhibitors. In some experiments, the putative synergistic interaction between PDE type 4 inhibitors and the beta2-agonist salbutamol was also assessed. 2. Sensitized guinea-pigs aerosolized with antigen (5% ovalbumin, OVA) responded with a significant increase in eotaxin and eosinophil levels in the bronchoalveolar lavage fluid (BALF) at 6 h. Eosinophil recruitment was inhibited by both PDE type 4 inhibitors rolipram (5 mg kg(-1), i.p.) and RP 73401 (5 mg kg(-1), i.p.) treatments. In contrast, only rolipram inhibited eotaxin production. 3. Sensitized rats intrapleurally challenged (i.pl.) with antigen (OVA, 12 microg cavity(-1)) showed a marked eosinophil infiltration at 24 h, preceded by eotaxin generation at 6 h. Intravenous administration of a rabbit anti-mouse eotaxin antibody (0.5 mg kg(-1)) significantly reduced allergen-evoked eosinophilia in this model. 4. Local pretreatment with rolipram (40 microg cavity(-1)) or RP 73401 (40 microg cavity(-1)) 1 h before challenge reduced eosinophil accumulation evaluated in the rat pleural effluent, but only the former was active against eotaxin generation. The inhibitors of PDE type 3 (SK&F 94836) and type 5 (zaprinast) failed to alter allergen-evoked eosinophil recruitment in rats. 5. Local injection of beta2-agonist salbutamol (20 microg cavity(-1)) inhibited both eosinophil accumulation and eotaxin production following pleurisy. The former was better inhibited when salbutamol and rolipram were administered in combination. 6. Treatment with rolipram and RP 73401 dose-dependently inhibited eosinophil adhesion and migration in vitro. These effects were clearly potentiated by salbutamol at concentrations that had no effect alone. 7. Our findings indicate that although rolipram and RP 73401 are equally effective in inhibiting allergen-induced eosinophil infiltration only the former prevents eotaxin formation, indicating that PDE 4 inhibitors impair eosinophil accumulation by mechanisms independent of eotaxin production blockade.
Using kaon regeneration to probe the quark mixing parameter cos2beta in B --> psiK decays.
We suggest a novel method to determine the sign of cos2beta in the decays B --> psiK, by creating interference between K(L) and K(S) final states via "regeneration," that is propagation through a matter target region to convert some K(L) to K(S). The determination of this quantity resolves an ambiguity between beta and 90 degrees -beta that remains after the standard measurements of sin2beta and may turn out to be important in resolving whether the result is in agreement with standard model predictions or indicates the presence of new physics. We find the measurement is feasible at a B factory, but requires several years of high-luminosity running with a regeneration target affecting a significant fraction of the detector.
Impaired insulin secretion and beta-cell loss in tissue-specific knockout mice with mitochondrial diabetes.
Mitochondrial dysfunction is an important contributor to human pathology and it is estimated that mutations of mitochondrial DNA (mtDNA) cause approximately 0.5-1% of all types of diabetes mellitus. We have generated a mouse model for mitochondrial diabetes by tissue-specific disruption of the nuclear gene encoding mitochondrial transcription factor A (Tfam, previously mtTFA; ref. 7) in pancreatic beta-cells. This transcriptional activator is imported to mitochondria, where it is essential for mtDNA expression and maintenance. The Tfam-mutant mice developed diabetes from the age of approximately 5 weeks and displayed severe mtDNA depletion, deficient oxidative phosphorylation and abnormal appearing mitochondria in islets at the ages of 7-9 weeks. We performed physiological studies of beta-cell stimulus-secretion coupling in islets isolated from 7-9-week-old mutant mice and found reduced hyperpolarization of the mitochondrial membrane potential, impaired Ca(2+)-signalling and lowered insulin release in response to glucose stimulation. We observed reduced beta-cell mass in older mutants. Our findings identify two phases in the pathogenesis of mitochondrial diabetes; mutant beta-cells initially display reduced stimulus-secretion coupling, later followed by beta-cell loss. This animal model reproduces the beta-cell pathology of human mitochondrial diabetes and provides genetic evidence for a critical role of the respiratory chain in insulin secretion.
Complex genetic counselling and prenatal analysis in a woman with external ophthalmoplegia and deleted mtDNA.
Single large mitochondrial DNA deletions (DeltamtDNA) are usually spontaneously occurring and cause a wide variety of symptoms, ranging from severe infantile multisystem disorders to adult onset progressive external ophthalmoplegia (PEO). There is always heteroplasmy with a mixture of normal and mutant mtDNA and the levels usually vary widely between tissues. There is at present insufficient scientific basis for accurate genetic counselling of women with DeltamtDNA, but it is reasonable to assume that DeltamtDNA can be transmitted if it is present in the female germ cells. Here, we present the results of prenatal analysis in a woman with DeltamtDNA and PEO. No DeltamtDNA was detected by Southern blot and PCR analyses of chorionic villi from the first trimester of pregnancy, in cord blood obtained at birth or in peripheral blood from the child at six months of age. This makes it unlikely that the child will develop a severe infantile mitochondrial disorder due to transmission of high levels of DeltamtDNA. However, the complex mitochondrial genetics and the limited access to human tissues makes it impossible to exclude transmission of low levels of DeltamtDNA that possibly could cause disease later in life.
Effects of immunotherapy on symptoms, PEFR, spirometry, and airway responsiveness in patients with allergic asthma to house-dust mites (D. pteronyssinus) on inhaled steroid therapy.
The present study was designed to investigate the effects of immunotherapy (IT) with an extract of Dermatophagoides pteronyssinus (Alergo-Merck Depot) during a 27-month period in patients with allergic asthma to house-dust mites. We included 11 patients (mean age 18 years) treated with a combination of IT and inhaled beclomethasone dipropionate (BDP) in comparison to another 11 (mean age 22 years) treated with BDP alone. We evaluated symptom scores, salbutamol use, peak expiratory flow rates (PEFR), spirometry, and bronchial hyperresponsiveness (BHR) during 18 months of therapy with BDP and in the 9 months after BDP interruption. The two kinds of treatment were efficient and comparable in relation to symptom score, salbutamol use, morning PEFR, FVC, and FEV1, but patients treated with IT and BDP had a faster improvement of BHR and PEFR variability. The interruption of BDP after 18 months of therapy was linked to an impairment of all end points, which were more pronounced in patients previously treated only with BDP. These findings suggest that in selected asthmatic patients allergic to house-dust mites, the association of IT and BDP is more effective than therapy with this inhaled steroid alone due to a faster and more striking improvement during the first months of treatment and to a lower rate of relapse after the interruption of therapy with BDP.
A cytogenetic analysis of rye introgression into a Portuguese bread wheat cultivar Barbela.
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[Mammary-coronary anastomosis: a new technique of approach through the brachial route for angiographic study and angioplasty].
PURPOSE: A new technique of mammary artery catheterization, by a brachial artery approach, utilizing a pre-molded conventional Sones catheter is described. METHODS: In a series of 300 patients, 308 procedures were performed. Three hundred internal mammary-coronary anastomosis were studied. In eight cases angioplasty were performed, five in the anterior descending artery and three in the internal mammary artery itself, with recanalization of one of the three cases. The approach was through the brachial artery homolateral to the anastomosed mammary artery. After the conventional coronarographic and bypass studies were performed, the catheter was withdrawn and pre-molded, forming a closed loop of approximately 10 mm in its distal extremity. The loop was introduced through the arteriotomy reaching the origin of the vertebral artery. The internal mammary artery was then catheterized utilizing rotation and traction movements. In the percutaneous transluminal coronary angioplasty (PTCA) procedures, the Sones catheter was replaced by a Myler right coronary catheter with a 260 cm metallic wire. RESULTS: In the 308 procedures, the internal mammary artery was catheterized in 305 instances (99.03%). In the remaining three cases selective catheterization of the internal mammary artery was not possible. In these three cases there was extreme tortuosity of the subclavian artery. The only complication observed in this series was thrombosis of the brachial artery in two cases (90.65%). In the eight patients submitted to PTCA the existing lesions were successfully dilated. CONCLUSION: Catheterization of the internal mammary artery through a brachial approach utilizing a pre-molded Sones catheter was an efficient procedure, with low incidence of complications. This approach could be the elective technique in the services that habitually utilize the brachial artery approach. It could be also an alternative for those utilizing the Judkins technique, whenever the internal mammary artery catheterization is impossible due to the anatomic characteristics of the patient.
Chromoblastomycosis: a retrospective study of 325 cases on Amazonic Region (Brazil).
A retrospective study of 325 cases of chromoblastomycosis diagnosed in the last 55 years in the Amazon region was carried out by the main Mycology services of the state of Pará, Brazil (Department of Tropical Pathology--UFPA and Mycology Department of the Evandro Chagas Institute/FNS). The data obtained showed that: (a) the main age group affected by the diseases range from 41 to 70 years-old, (b) 86.1% of the patients were agricultural-workers, (c) 93.2% of them were males and (d) 80.7% showed lesions on the lower limbs (feet and legs). The diagnosis of 62% of the cases was confirmed by laboratory studies considering the tissue form in histopathological analysis. In 24% of patients (78 cases), the etiological agent was isolated and identified through culture. Fonsecaea pedrosoi was present in 77 cases and Phialophora verucosa in only one case.
Effect of drug properties on the release from CAP microspheres prepared by a solvent evaporation method.
Drugs with different water-solubility and molecular weights were microencapsulated in cellulose acetate phthalate, using an emulsion-solvent evaporation technique with a continuous oil-phase. The mean size of the particles was approximately 600 microns, and they were non-porous. The capacity of the microspheres to retain the drugs was evaluated by in vitro release studies in acidic medium. For low molecular weight compounds the release rates increased with solubility: for thiamin hydrochloride and phenacetin, a highly and a poorly soluble compound respectively, the percentages released at 60 min were 90 and 10%. Drugs with molecular weights above approximately 700 Da were retained in the microspheres. The above dependence on solubility was corroborated by release studies in ethanol, and by modelling the release of phenacetin in acidic media. Microspheres with a different polymer matrix, Eudragit RS PO, were also prepared by a similar technique, and these particles prolonged the release of thiamin for over 6 h, under simulated GI conditions.
t-Butylhydroperoxide and gliotoxin stimulate Ca2+ release from rat skeletal muscle mitochondria.
Rat liver mitochondria have a specific Ca2+ release pathway which operates when NAD+ is hydrolysed to nicotinamide and ADPribose. NAD+ hydrolysis is Ca(2+)-dependent and inhibited by cyclosporine A (CSA). Mitochondrial Ca2+ release can be activated by the prooxidant t-butylhydroperoxide (tbh) or by gliotoxin (GT), a fungal metabolite of the epipolythiodioxopiperazine group. Tbh oxidizes NADH to NAD+ through an enzyme cascade consisting of glutathione peroxidase, glutathione reductase, and the energy linked transhydrogenase, whereas GT oxidizes some vicinal thiols to the disulfide form, a prerequisite for NAD+ hydrolysis. We report now that rat skeletal muscle mitochondria also contain a specific Ca2+ release pathway activated by both tbh and GT. Ca2+ release increases with the mitochondrial Ca2+ load, is completely inhibited in the presence of CSA, and is paralleled by pyridine nucleotide oxidation. In the presence of tbh and GT, mitochondria do not lose their membrane potential and do not swell, provided continuous release and re-uptake of Ca2+ ('Ca2+ cycling') is prevented. These data support the notion that both tbh- and GT-induced Ca2+ release are not the consequence of an unspecific increase of the inner membrane permeability ('pore' formation). Tbh induces Ca2+ release from rat skeletal muscle less efficiently than from liver mitochondria indicating that the coupling between tbh and NADH oxidation is much weaker in skeletal muscle mitochondria. This conclusion is corroborated by a much lower glutathione peroxidase activity in skeletal muscle than in liver mitochondria. The prooxidant-dependent pathway promotes, under drastic conditions (high mitochondrial Ca2+ loads and high tbh concentrations), Ca2+ release to about the same extent and rate as the Na+/Ca2+ exchanger. This renders the prooxidant-dependent pathway relevant in the pathophysiology of mitochondrial myopathies where its activation by an increased generation of reactive oxygen species probably results in excessive Ca2+ cycling and damage to mitochondria.
Merkel cell carcinoma of the eyelid: a case report.
A 22-year-old woman who presented with a tumour of the upper eyelid with clinical, histological, immunohistochemical and ultrastructural features of a Merkel cell carcinoma is reported. This is a unique case of Merkel cell carcinoma in such a young patient. This neuroendocrine tumour is typically found in the elderly. It can grow rapidly, has the potential for local recurrence and early metastatic spread. The authors recommend wide resection of the primary site, which proved effective in this case.
Intestinal parasitism in the Xavánte Indians, central Brazil.
This paper reports the findings of a survey for intestinal parasites among the Xavánte Indians from Central Brazil. A. lumbricoides (25.0%) and hookworms (33.6%) were the two most common helminths; E. histolytica complex (7.8%) and G. lamblia (8.6%) the most common protozoans. The majority (58.5%) of positive individuals hosted only one species of helminth. Egg counts for helminths, and for A. lumbricoides in particular, were found to be not dispersed at random, with a few individuals, all of whom young children, showing very high counts. The prevalence rates of intestinal parasites for the Xavánte are below those reported for other Amerindian populations from Brazil.