Validação de um radioimunoensaio para a quantificação de metabólitos fecais de testosterona em hamster Sírio (Mesocricetus auratus)

Modelo para o estudo de diversas doenças humanas, o hamster tem sido objeto de numerosos estudos comportamentais e envolvendo estresse e/ou comportamento agonístico que supõem, muitas vezes, o monitoramento das flutuações hormonais nos indivíduos envolvidos. O objetivo do presente trabalho foi confirmar a adequação de um conjunto comercial para dosagem de testosterona em sangue humano para a quantificação de metabólitos fecais de testosterona (MFT) em hamsters Sírios machos e fêmeas. Dez machos foram submetidos a um desafio com um agonista de GnRH para estimular a atividade testicular, elevando os níveis circulantes de testosterona. Cinco fêmeas receberam uma injeção de testosterona enquanto cinco outras receberam uma injeção de solução salina. Amostras de fezes coletadas antes e depois dos procedimentos, assim como amostras fecais de 20 fêmeas gestantes coletadas ao longo da gestação foram analisadas com um conjunto comercial para radioimunoensaio. Um pico de MFT 12h após a injeção seguido de uma queda abaixo do nível basal comprovou que, nos machos, as alterações nos níveis de MFT refletem as alterações da concentração de testosterona no sangue. Nestes observou-se um ciclo circadiano das concentrações de MFT com acrofase no início do período claro correspondendo ao ciclo descrito para as concentrações sanguíneas na literatura. Nas fêmeas a administração de testosterona exógena provocou uma elevação importante dos níveis de MFT, mas as concentrações medidas ao longo da gestação não refletiram o padrão dos níveis sanguíneos do hormônio endógeno. O conjunto para radioimunoensaio para testosterona em sangue humano mostrou-se adequado para o monitoramento da função testicular no hamster macho, mas um ensaio mais específico seria necessário para as fêmeas.

Assessment of the potential genotoxic risk of medicinal Tamarindus indica fruit pulp extract using in vivo assays

Tamarindus indica has been used in folk medicine as an antidiabetic, a digestive aid, and a carminative, among other uses. Currently, there is no information in the toxicology literature concerning the safety of T. indica extract. We evaluated the clastogenic and/or genotoxic potential of fruit pulp extract of this plant in vivo in peripheral blood and liver cells of Wistar rats, using the comet assay, and in bone marrow cells of Swiss mice, using the micronucleus test. The extract was administered by gavage at doses of 1000, 1500 and 2000 mg/kg body weight. Peripheral blood and liver cells from Wistar rats were collected 24 h after treatment, for the comet assay. The micronucleus test was carried out in bone marrow cells from Swiss mice collected 24 h after treatment. The extract made with T. indica was devoid of clastogenic and genotoxic activities in the cells of the rodents, when administered orally at these three acute doses.

Fluorescence spectroscopy for the detection of tongue carcinoma - validation in an animal model

The efficacy of fluorescence spectroscopy to detect squamous cell carcinoma is evaluated in an animal model following laser excitation at 442 and 532 nm. Lesions are chemically induced with a topical DMBA application at the left lateral tongue of Golden Syrian hamsters. The animals are investigated every 2 weeks after the 4th week of induction until a total of 26 weeks. The right lateral tongue of each animal is considered as a control site (normal contralateral tissue) and the induced lesions are analyzed as a set of points covering the entire clinically detectable area. Based on fluorescence spectral differences, four indices are determined to discriminate normal and carcinoma tissues, based on intraspectral analysis. The spectral data are also analyzed using a multivariate data analysis and the results are compared with histology as the diagnostic gold standard. The best result achieved is for blue excitation using the KNN (K-nearest neighbor, a interspectral analysis) algorithm with a sensitivity of 95.7% and a specificity of 91.6%. These high indices indicate that fluorescence spectroscopy may constitute a fast noninvasive auxiliary tool for diagnostic of cancer within the oral cavity. (C) 2008 Society of Photo-Optical Instrumentation Engineers.

Tocopherols, tocotrienols, and gamma-oryzanol contents in Japonica and Indica subspecies of rice (Oryza sativa L.) cultivated in Brazil

Whole rice contains several fat-soluble phytochemicals such. as tocopherols, tocotrienols, and gamma-oryzanol which have been reported to possess beneficial health properties. This study was conducted to determine whether brown rice belonging to indica and japonica subspecies were distinguishable from each other regarding the concentration of these compounds by analyzing 32 genotypes. The fat-soluble compounds were analyzed by normal-phase HPLC in a single run. The variability of the compounds analyzed was high, but the mean content of gamma-oryzanol across all samples was significantly higher (P < 0.01) in japonica (246.3 mg/kg) than in indica rice (190.1 mg/kg). Similar differences were found for total vitamin E contents which were 24.2 mg/kg injaponica and 17.1 mg/kg in indica rice, respectively. In japonica rice, alpha-tocopherol, alpha-tocotrienol, and gamma-tocotrienol were the most abundant homologs, while in indica rice the most abundant were gamma-tocotrienol, alpha-tocopherol, and alpha-tocotrienol. A significant Pearson coefficient (0.80, P < 0.001) between alpha-tocopherol and alpha-tocotrienol levels was found, independent of the subspecies. Both compounds were positively correlated to total tocols and gamma-oryzanol contents. Although more studies are needed to evaluate the interference of growing rice in different environments and multiple years, the present study provided information on natural variations of the vitamin E isomers and the gamma-oryzanol contents in different rice genotypes.

Modulation of human neutrophil oxidative metabolism and degranulation by extract of Tamarindus indica L. fruit pulp

The tamarind (Tamarindus indica L) is indigenous to Asian countries and widely cultivated in the American continents. The tamarind fruit pulp extract (ExT), traditionally used in spices, food components and juices, is rich in polyphenols that have demonstrated anti-atherosclerotic, antioxidant and immunomodulatory activities. This study evaluated the modulator effect of a crude hydroalcoholic ExT on some peripheral human neutrophil functions. The neutrophil reactive oxygen species generation, triggered by opsonized zymosan (OZ), n-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), and assessed by luminol- and lucigenin-enhanced chemiluminescence (LumCL and LucCL, respectively), was inhibited by ExT in a concentration-dependent manner. ExT was a more effective inhibitor of the PMA-stimulated neutrophil function [IC(50) (in mu g/10(6)cells) = 115.7 +/- 9.7 (LumCL) and 174.5 +/- 25.9 (LucCL)], than the OZ- [IC(50) = 248.5 +/- 23.1 (LumCL) and 324.1 +/- 34.6 (LucCL)] or fMLP-stimulated cells [IC(50) = 178.5 +/- 12.2 (LumCL)]. The ExT also inhibited neutrophil NADPH oxidase activity (evaluated by O(2) consumption), degranulation and elastase activity (evaluated by spectrophotometric methods) at concentrations higher than 200 mu g/10(6) cells, without being toxic to the cells, under the conditions assessed. Together, these results indicate the potential of ExT as a source of compounds that can modulate the neutrophil-mediated inflammatory diseases. (C) 2008 Elsevier Ltd. All rights reserved.

Leishmania (Viannia) braziliensis transfectants overexpressing the miniexon gene lose virulence in vivo

The miniexon gene has a central role in the processing of polycistronic pre-mRNA of kinetoplastids. It is added to the 5` extremity of each mRNA, supplying the 5`-capped structure to the molecule. Previous studies in Leishmania (Leishmania) major showed that the overexpression of the miniexon array attenuates the Virulence of the parasite in in vivo assays. The results presented here extend those findings to Vionnia subgenus. Leishmania (Vionnia) braziliensis was transfected with a cosmid harboring a tandem array of one hundred miniexon gene copies and then characterized by Northern blot analysis. The overexpression of the exogenous gene was confirmed and its effect on the virulence of L (V.) braziliensis was investigated in hamsters. In BALB/c mice we could not detect parasites during the course of 15 weeks of infection. In addition, hamsters infected with transfectants overexpressing the miniexon gene exhibited only a minor footpad swelling of late onset and failed to develop progressive lesion, these attenuated parasites could be recovered from the inoculation site 1 year after infection. The persistence of parasites in the host indicates that a stable line overexpressing the miniexon may be tested as live vaccine against leishmaniasis. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Melatonin reduces the severity of experimental amoebiasis

Background: Melatonin has immunomodulatory effects but very little is known about its influence in protozoan infections, such as Entamoeba histolytica, which causes amoebiasis, a disease with significant morbidity and mortality. In this study, we evaluated the effects of exogenous melatonin interference in experimental amoebiasis and on interactions between human blood cells and E. histolytica trophozoites. Methods: The effect of melatonin was investigated in models of experimental amoebiasis in hamsters and rats by evaluating the area of necrosis induced by E. histolytica. The activity of melatonin on the interactions between leukocytes and amoebae was determined by examining leukophagocytosis. For in vitro tests, polymorphonuclear and mononuclear human blood leucocytes were incubated with E. histolytica trophozoites. Results: The areas of amoebic necrosis were significantly reduced in animals treated with melatonin. Melatonin treatment increased leukophagocytosis but was associated with a greater number of dead amoebae. Conclusions: These results suggest that melatonin may play a beneficial role in the control of amoebic lesions, raising the possibility that this drug may be used as an adjuvant in anti-amoebic therapy.

Evaluation of the Expression and Protective Potential of Leptospiral Sphingomyelinases

Leptospirosis is a zoonotic disease of global distribution, which affects both animals and humans. Pathogenic leptospires, the bacteria that cause this disease, require iron for their growth, and these spirochetes probably use their hemolysins, such as the sphingomyelinases, as a way to obtain this important nutrient from host red blood cells during infection. We expressed and purified the leptospiral sphingomyelinases Sph1, Sph2, Sph4, and SphH in a heterologous system. However, the recombinant proteins were not able to lyse sheep erythrocytes, despite having regular secondary structures. Transcripts for all sphingomyelinases tested were detected by RT-PCR analyses, but only Sph2 and SphH native proteins could be detected in Western blot assays using Leptospira whole extracts as well as in renal tubules of infected hamsters. Moreover, antibodies present in the serum of a human patient with laboratory-confirmed leptospirosis recognized Sph2, indicating that this sphingomyelinase is expressed and exposed to the immune system during infection in humans. However, in an animal challenge model, none of the sphingomyelinases tested conferred protection against leptospirosis.

LipL53, a temperature regulated protein from Leptospira interrogans that binds to extracellular matrix molecules

The regulation of gene expression by environmental signals, such as temperature and osmolarity, has been correlated with virulence. In this study, we characterize the protein LipL53 from Leptospira interrogans, previously shown to react with serum sample of individual diagnosed with leptospirosis and to be up-regulated by shift to physiological osmolarity. The recombinant protein was expressed in Escherichia coli system, in insoluble form, recovered by urea solubilization and further refolded by decreasing the denaturing agent concentration during the purification procedure. The secondary structure content of the recombinant LipL53, as assessed by circular dichroism, showed a mixture of beta-strands and alpha-helix. The presence of LipL53 transcript at 28 degrees C was only detected within the virulent strains. However, upon shifted of attenuated cultures of pathogenic strains from 28 degrees C to 37 degrees C and to 39 degrees C, this transcript could also be observed. LipL53 binds laminin, collagen IV, cellular and plasma fibronectin in dose-dependent and saturable manner. Animal challenge studies showed that LipL53, although immunogenic, elicited only partial protection in hamsters. LipL53 is probably surface exposed as seen through immunofluorescence confocal microscopy. Our results suggest that LipL53 is a novel temperature regulated adhesin of L. interrogans that may be relevant in the leptospiral pathogenesis. (C) 2009 Elsevier Masson SAS. All rights reserved.

Axonal regeneration of retinal ganglion cells after optic nerve pre-lesions and attachment of normal or pre-degenerated peripheral nerve grafts

Axonal regeneration of retinal ganglion cells (RGCs) into a normal or pre-degenerated peripheral nerve graft after an optic nerve pre-lesion was investigated. A pre-lesion performed 1-2 weeks before a second lesion has been shown to enhance axonal regeneration in peripheral nerves (PN) but not in optic nerves (ON) in mammals. The lack of such a beneficial pre-lesion effect may be due to the long delay (1-6 weeks) between the two lesions since RGCs and their axons degenerate rapidly 1-2 weeks following axotomy in adult rodents. The present study examined the effects of the proximal and distal ON pre-lesions with a shortened delay (0-8 days) on axonal regeneration of RGCs through a normal or pre-degenerated PN graft. The ON of adult hamsters was transected intraorbitallv at 2 mm. (proximal lesion) or intracranially at 7 mm (distal lesion) from the optic disc. The pre-lesioned ON was re-transected at 0.5 mm from the disc after 0, 1, 2, 4, or 8 days and a normal or a pre-degenerated PN graft was attached onto the ocular stump. The number of RGCs regenerating their injured axons into the PN graft was estimated by retrograde labeling with FluoroGold 4 weeks after grafting. The number of regenerating RGCs decreased significantly when the delay-time increased in animals with both the ON pre-lesions (proximal or distal) compared to control animals without an ON pre-lesion. The proximal ON pre-lesion significantly reduced the number of regenerating RGCs after a delay of 8 days in comparison with the distal lesion. However, this adverse effect can be overcome, to some degree, by a pre-degenerated PN graft applied 2, 4, or 8 days after the distal ON pre-lesion enhanced more RGCs to regenerate than the normal PN graft. Thus, in order to obtain the highest number of regenerating RGCs, a pre-degenerated PN should be grafted immediately after an ON lesion.

Infecções naturais de mamíferos silvestres em área endêmica de leishmaniose tegumentar do Estado de São Paulo, Brasil

Descreve-se a presença de foco de leishmaniose tegumentar no vale do rio Moji-Guaçú, em região comum aos municípios de Luiz Antonio, São Carlos, Rincão e Santa Rita do Passa Quatro, no Estado de São Paulo, Brasil. Os casos humanos apresentavam formas clínicas caracterizadas por lesões úlcero-vegetantes, de evolução lenta e pobres em parasitos. As investigações sobre infecção natural em animais silvestres levou ao isolamento de roedores, de três cepas em cultura, duas procedentes de Akodon arviculoides e uma de Oryzomys nigripes. As provas de inoculação em hamsters foram, até o momento, positivas para duas delas, mas com evolução lenta, com manifestações clínicas muito discretas e pobres em parasitos. Pelos dados disponíveis até o momento, parece tratar-se de cepas filiáveis à raça "lenta", à qual se atribui papel na etiologia da forma cutaneo-mucosa da leishmaniose.

Infecção natural de flebotomíneos em foco enzoótico de leishmaniose tegumentar no Estado de São Paulo, Brasil

Relata-se o encontro de infecções naturais de Pintomyia pessoai e Psychodopygus intermedius em foco enzoótico de leishmaniose tegumentar no Estado de São Paulo, Brasil. A natureza leishmaniótica desses encontros obteve confirmação através da inoculação experimental em hamsters.

Nota sobre infecção natural de Oryzomys capito laticeps em foco enzoótico de leishmaniose tegumentar no Estado de São Paulo, Brasil

Assinala-se o isolamento de cepa de Leishmania braziliensis braziliensis em exemplar de Oryzomys capito laticeps capturado em foco enzoótico do Estado de São Paulo, Brasil. A identificação do parasita fez-se de acordo com o seu comportamento em inoculação experimental feita em hamsters. Pela primeira vez, relata-se possível papel de reservatório natural, desempenhado por esse roedor, em relação à leishmoniose cutâneo-mucosa.

Physiological age in Lutzomyia youngi (Diptera: Psychodidae) populations from an endemic area for cutaneous leishmaniasis, Venezuela

Batches of sylvatic females of Lutzomyia youngi (Phlebotominae) captured in a Shannon trap on twelve occasions over one year in a locality where subcutaneous leishmaniasis is endemic, near the city of Trujillo, Venezuela, were used to study: 1) the percentages of parous females according to previously established criteria and 2) the average number of eggs laid spontaneously by isolated females during 7 days after feeding on hamsters. The data on the batches of females captured on nights previous to the rainy period (prepluvial) were compared with those on females captured after the rains (postpluvial) . Significant differences were detected by variation analysis for two variables and different number of N, as also were consistent groupings by Duncan's Test for pre-and postpluvial lots of females. The females captured on nights prior to the rainy periods (January-March and August-September) presented higher rates of nulliparity (86-72%) and contained or laid a greater number of eggs (71-67) than those captured after the rains (March-June and November-December) which presented lower rates of nulliparity (60-24%) and a smaller number of eggs (50-30). The rainfall peaks occurred in April and September-October, respectively. It is considered that these differences can be used by epidemiological studies as a means of estimating the physiological age of female populations of L. youngy.

Alberprosenia malheiroi Serra, Atzingen & Serra, 1987 (Hemiptera, Reduviidae): redescrição e bionomia

Sobre Alberprosenia malheiroi n. sp. deu-se a conhecer uma diagnose resumida em 1980, porém sem valor bibliográfico. Em 1987 publicou-se a mesma diagnose anexando uma foto e alguns comentários, porém sem realizar uma descrição formal. Descreve-se para esta espécie, os adultos, os estádios imaturos, determina-se a série sintípica e apresenta-se dados bionômicos e de criação em insetário. As diferenças mais evidentes com A. goyovargasi, a única espécie que se conhecia do gênero até então, são a coloração geral negra, o espaço interocular maior que o tamanho de um olho visto dorsalmente, os tubérculos do colar com o ápice agudo e o tamanho maior, na nova espécie, quase o dobro do da primeira espécie. Os ovos são pequenos, fixados ao substrato em grupos de 3 ou 4, elipsóides, não achatados lateralmente, com o opérculo proeminente, convexo, sem estruturas evidentes. As ninfas apresentam em todos os estádios caracteres típicos do gênero e da tribo, com região anteocular menos longa que a post-ocular e característica pilosidade do tegumento que vai se acentuando a cada estádio. A. malheiroi n. sp. foi capturado em ecótopos silvestres em palmeiras em floresta no Estado do Pará, associados com morcegos ou aves. Nenhum dos exemplares estava infectado com Trypanosoma cruzi. Esses triatomíneos foram mantidos em insetários a ± 25°C e ± 60% UR, são insetos ágeis e voam com relativa facilidade. Alimentaram-se bem em pombos e morcegos e não aceitaram alimentação em ratos, camundongos ou hamsters. O período de incubação dos ovos foi em média treze dias e o tempo de evolução do período ninfal foi em média cento e trinta e dois dias.