GABA(A) receptor blockade in dorsomedial and ventromedial nuclei of the hypothalamus evokes panic-like elaborated defensive behaviour followed by innate fear-induced antinociception

Dysfunction in the hypothalamic GABAergic system has been implicated in panic syndrome in humans. Furthermore, several studies have implicated the hypothalamus in the elaboration of pain modulation. Panic-prone states are able to be experimentally induced in laboratory animals to study this phenomenon. The aim of the present work was to investigate the involvement of medial hypothalamic nuclei in the organization of panic-like behaviour and the innate fear-induced oscillations of nociceptive thresholds. The blockade of GABA(A) receptors in the neuronal substrates of the ventromedial. or dorsomedial hypothalamus was followed by elaborated defensive panic-like reactions. Moreover, innate fear-induced antinociception was consistently elicited after the escape behaviour. The escape responses organized by the dorsomedial and ventromedial hypothalamic nuclei were characteristically more elaborated, and a remarkable exploratory behaviour was recorded during GABA(A) receptor blockade in the medial hypothalamus. The motor characteristic of the elaborated defensive escape behaviour and the patterns of defensive alertness and defensive immobility induced by microinjection of the bicuculline either into the dorsomedial. or into the ventromedial hypothalamus were very similar. This was followed by the same pattern of innate fear-induced antinociceptive response that lasted approximately 40 min after the elaborated defensive escape reaction in both cases. These findings suggest that dysfunction of the GABA-mediated neuronal system in the medial hypothalamus causes panic-like responses in laboratory animals, and that the elaborated escape behaviour organized in both dorsomedial and ventromedial hypothalamic nuclei are followed by significant innate-fear-induced antinociception. Our findings indicate that the GABA(A) receptor of dorsomedial and ventromedial hypothalamic nuclei are critically involved in the modulation of panic-like behaviour. (C) 2009 Elsevier B.V. All rights reserved.

Effects of type II pyrethroid cyhalothrin on rat innate immunity: A flow cytometric study

Synthetic type II pyrethroids induce anxiety, immunosuppresion or, alternatively, immunostimulatory effects in laboratory animals. Macrophages and neutrophils are known to be key elements in cellular immune responses. The present study was designed to investigate the in vivo effects of cyhalothrin (1.0 and 3.0 mg/kg/once daily for 7 days) on macrophage and neutrophil activities, using a flow cytometry method. Results showed that cyhalothrin treatment decreased the percentage and intensity of phagocytosis performed by macrophages, but did not alter these parameters in neutrophils: and also decreased basal neutrophil oxidative burst and increased S. aureus-induced neutrophil oxidative burst, but did not alter these responses in macrophages. The present results are discussed in the light of a possible indirect action of cyhalothrin on macrophage and neutrophil activities via hypothalamic pituitary adrenal (HPA) axis activation. A possible direct effect of cyhalothrin on macrophage and neutrophil activities is also considered. (C) 2008 Elsevier B.V. All rights reserved.

Cyhalothrin increased c-fos immunoreactivity at the paraventricular nucleus of the hypothalamus in rats, and suppressed macrophage activity in an adrenal-dependent fashion

Synthetic type II pyrethroid insecticides, such as cyhalothrin at certain dosage levels, simultaneously induce stress-like symptoms and innate immunosuppressive effects in laboratory animals. The present study was designed to further analyze the stress-like effects induced by cyhalotrin and also investigate the role of Hypothalamus-Hypophysis-Adrenal (HHA) axis and Sympathetic Nervous Systems (SNS) and their effects on macrophage activity of rats. Results showed that cyhalothrin treatment (3.0 mg/kg/day. for 7 days) increased corticosterone serum levels and c-fos immunoreactivity at the paraventricular nucleus of the hypothalamus (PVN) but induced no changes in c-fos expression at the basolateral amygdala (BLA). Both areas were related to HHA axis and SNS activations by stress. Further analysis showed that adrenalectomy partially abrogated the suppression effects of cyhalothrin on macrophage activity and that 6-OHDA-induced peripheral symphatectyomy had no effects on this innate immune cell activity. The present observed data support and reinforce the notion that cyhalotrin at this treatment schedule induces stress-like symptoms and suggest that other factors, beyond indirect neuroadaptative responses, are necessary for the suppression effects of insecticide on innate immune response. (C) 2008 Elsevier B.V. All rights reserved.

Exposição profissional ao chumbo : análise da indução de efeitos genotóxicos

RESUMO - A exposição contínua a substâncias químicas tem consequências para a saúde humana, algumas das quais não estão ainda totalmente estabelecidas. A toxicologia ocupacional é uma área interdisciplinar que envolve conhecimentos de higiene e de medicina ocupacional, de epidemiologia e de toxicologia e que tem por principal objectivo prevenir a ocorrência de efeitos adversos decorrentes do ambiente ocupacional sendo um dos seus principais papéis fornecer o máximo de dados que possam contribuir para o conhecimento dos potenciais efeitos na saúde. O chumbo é um tóxico de características cumulativas que provoca na saúde efeitos principalmente sistémicos, ou seja, o efeito tóxico manifesta-se em locais afastados do contacto inicial que resultam essencialmente de exposições crónicas, resultantes de períodos de exposição mais ou menos longos ao metal (entre meses e anos). Pode interagir com diferentes órgãos e tecidos, ligando-se a moléculas e constituintes celulares. Uma vez que não possui qualquer função fisiológica, a presença do chumbo no organismo humano resulta numa série de efeitos prejudiciais que afectam diversos órgãos e sistemas. A toxicidade do chumbo manifesta-se em diversos órgãos e tecidos, nomeadamente no sistema hematopoiético, no sistema nervoso, no rim, no aparelho reprodutor, no sistema cardiovascular, no sistema endócrino e no sistema imunitário. Da interferência do chumbo com o funcionamento de alguns sistemas biológicos resultam um conjunto de alterações fundamentais ao nível dos processos de transporte através das membranas, da integridade estrutural e funcional das enzimas e de várias vias metabólicas, em especial da fosforilação oxidativa e da síntese do heme sendo os primeiros efeitos bioquímicos do chumbo detectados a partir de valores de plumbémia inferiores a 10 μg/dL. As medidas de higiene e segurança actualmente em vigor nos países desenvolvidos asseguram que os casos de intoxicação grave são cada vez menos frequentes. No entanto, o risco de exposição a nível ocupacional existe em todas as actividades que envolvem materiais que o contenham como as explorações mineiras, as fundições primária e secundária, a produção de baterias de chumbo ácido, a produção de vidro com pigmentos de chumbo, as soldaduras de reparação automóvel e a instrução de tiro. Desde 2006 o chumbo é considerado pela International Agency for Research on Cancer (IARC) uma substância carcinogénica do grupo 2A (provável carcinogénio para o ser humano). Considera-se, assim, que o chumbo tem, inequivocamente, capacidade de induzir cancro em animais experimentais mas que, embora haja fortes indícios de que os mecanismos que medeiam a carcinogénese desses compostos ocorrem no ser humano, os dados disponíveis ainda não podem assegurar essa relação. Com este estudo pretendeu-se contribuir para o conhecimento da toxicidade do chumbo através do estudo da exposição ao chumbo e da influência da susceptibilidade individual (em industrias sem co-exposição significativa a outros agentes conhecidos ou suspeitos de serem carcinogénicos). Pretendeu-se estudar o caso através de uma abordagem múltipla que permitisse relacionar diferentes tipos de marcadores biológicos uma vez que a monitorização biológica integra todas as possíveis vias de entrada no organismo (para além da via respiratória), eventuais exposições fora do contexto estritamente profissional assim como uma série de factores intrínsecos individuais (relacionados com modos de via, de natureza fisiológica e comportamentais). Sendo a co-exposição a outros compostos com propriedades genotóxicas e carcinogénicas uma questão difícil de tornear quando se quer avaliar o potencial genotóxico do chumbo em populações expostas, ocupacional ou ambientalmente este estudo tem a vantagem de ter sido efectuado em populações sem co-exposição conhecida a outras substâncias deste tipo, permitindo concluir sobre os efeitos resultantes apenas da exposição a chumbo na população humana, contribuindo para explicar algumas das aparentes inconsistências e contradições entre diferentes estudos sobre este tema. Os indicadores de exposição usados foram: indicadores de dose interna (doseamento de chumbo e de PPZ no sangue), indicadores de efeitos adversos no heme e genotóxicos (actividade da ALAD, teste do cometa e mutação em TCR) e indicadores de susceptibilidade (polimorfismos genéticos de ALAD e VDR) através de uma abordagem estatística de comparação directa de sub-grupos previamente definidos na população e da aplicação de um modelo de regressão múltipla. Este estudo revelou que os níveis de plumbémia na população portuguesa baixaram significativamente nos últimos 10 anos, tanto na população ocupacionalmente exposta como na população em geral e que a presença do genótipo B-B (do gene VDR) é preditiva das variações de plumbémia, quando comparada com o genótipo mais frequente na população, B-b; ao contrário, o genótipo b-b não aparenta ter influência em nenhum dos marcadores estudados. No que diz respeito a efeitos genotóxicos concluiu-se que estes não se manifestaram na população estudada, levando a concluir que nos níveis de exposição estudados, o chumbo não tem capacidade de induzir este tipo de efeitos per si levando ao reforço da hipótese, já levantada por outros autores, de que o mecanismo de genotoxicidade do chumbo seja essencialmente de promoção de processos de genotoxicidade desencadeados por outros agentes. A realização de estudos de efeitos genotóxicos e de stress oxidativo desenhados de forma a comparar grupos de trabalhadores expostos apenas a chumbo com grupos de trabalhadores com o mesmo nível de exposição a chumbo, mas com co-exposição a outros agentes reconhecidamente carcinogénicos poderá ajudar a aumentar o conhecimento deste efeito do chumbo na saúde humana.

American tripanosomiasis: a study on the prevalence of Trypanosoma cruzi and Trypanosoma cruzi-like organisms in wild rodents in San Luis province, Argentina

INTRODUCTION: Chagas disease is caused by Trypanosoma cruzi. Wild and perianthropic mammals maintain the infection/transmission cycle, both in their natural habitat and in the peridomestic area. The aim of this paper was to present the results from a study on wild rodents in the central and northern regions of San Luis province, Argentina, in order to evaluate the prevalence of this infection. METHODS: Sherman traps were set up in capture areas located between latitudes 32º and 33º S, and longitudes 65º and 66º W. The captured rodents were taxonomically identified and hemoflagellates were isolated. Morphological, biometric and molecular studies and in vitro cultures were performed. Infection of laboratory animals and histological examination of the cardiac muscle and inoculation area were also carried out. Parasites were detected in circulating blood in Calomys musculinus, Graomys griseoflavus, Phyllotis darwini and Akodon molinae. The parasites were identified using biological criteria. Molecular PCR studies were performed on some isolates, which confirmed the characterization of these hemoflagellates as Trypanosoma cruzi. RESULTS AND CONCLUSIONS: Forty-four percent of the 25 isolates were identified as Trypanosoma cruzi, and the remaining 56% as Trypanosoma cruzi-like. These findings provide evidence that wild rats infected with Trypanosoma cruzi and Trypanosoma cruzi-like organisms are important in areas of low endemicity.

Evaluation of antihypertensive drugs in patients with obstructive sleep apnea and in rats submitted to chronic intermittent hypoxia

RESUMO: A hipertensão arterial (HA) é uma patologia altamente prevalente, embora claramente subdiagnosticada, em doentes com síndrome de apneia obstrutiva do sono (SAOS). Estas duas patologias apresentam uma estreita relação e a monitorização ambulatória da pressão arterial (MAPA), por um período de 24 horas, parece ser o método mais preciso para o diagnóstico de hipertensão em doentes com SAOS. No entanto, esta ferramenta de diagnóstico para além de ser dispendiosa e envolver um número acrescido de meios técnicos e humanos, é mais morosa e, por conseguinte, não é utilizada por rotina no contexto do diagnóstico da SAOS. Por outro lado, apesar da aplicação de pressão positiva contínua nas vias aéreas (CPAP – Continous Positive Airway Pressure) ser considerada a terapêutica de eleição para os doentes com SAOS, o seu efeito no abaixamento da pressão arterial (PA) parece ser modesto, exigindo, por conseguinte, a implementação concomitante de terapêutica anti-hipertensora. Acontece que são escassos os dados relativos aos regimes de fármacos anti-hipertensores utilizados em doentes com SAOS e, acresce ainda que, as guidelines terapêuticas para o tratamento farmacológico da HA, neste grupo particular de doentes, permanecem, até ao momento, inexistentes. A utilização de modelos animais de hipóxia crónica intermitente (CIH), que mimetizam a HA observada em doentes com SAOS, revela-se extremamente importante, uma vez que se torna imperativo identificar fármacos que promovam um controle adequado da PA neste grupo de doentes. No entanto, estudos concebidos com o intuito de investigar o efeito anti-hipertensor dos fármacos neste modelo animal revelam-se insuficientes e, por outro lado, os escassos estudos que testaram fármacos anti-hipertensores neste modelo não foram desenhados para responder a questões de natureza farmacológica. Acresce ainda que se torna imprescindível garantir a escolha de um método para administração destes fármacos que seja não invasivo e que minimize o stress do animal. Embora a gavagem seja uma técnica indiscutivelmente eficaz e amplamente utilizada para a administração diária de fármacos a animais de laboratório, ela compreende uma sequência de procedimentos geradores de stress para os animais e, que podem por conseguinte, constituir um viés na interpretação dos resultados obtidos. O objectivo global da presente investigação translacional foi contribuir para a identificação de fármacos anti-hipertensores mais efectivos para o tratamento da HT nos indivíduos com SAOS e investigar mecanismos subjacentes aos efeitos sistémicos associadas à SAOS bem como a sua modulação por fármacos anti-hipertensores. Os objectivos específicos foram: em primeiro lugar,encontrar novos critérios, baseados nas medidas antropométricas, que permitam a identificação de doentes com suspeita de SAOS, que erroneamente se auto-classifiquem como nãohipertensos, e desta forma promover um uso mais criterioso do MAPA; em segundo lugar, investigar a existência de uma hipotética associação entre os esquemas de fármacos antihipertensores e o controle da PA (antes e após a adaptação de CPAP) em doentes com SAOS em terceiro lugar, avaliar a eficácia do carvedilol (CVD), um fármaco bloqueador β-adrenérgico não selectivo com actividade antagonista α1 intrínseca e propriedades anti-oxidantes num modelo animal de hipertensão induzida pela CIH; em quarto lugar, explorar os efeitos da CIH sobre o perfil farmacocinético do CVD; e, em quinto lugar, investigar um método alternativo à gavagem para a administração crónica de fármacos anti-hipertensores a animais de laboratório. Com este intuito, na primeira fase deste projecto, fizemos uso de uma amostra com um número apreciável de doentes com SAOS (n=369), que acorreram, pela primeira vez, à consulta de Patologia do Sono do CHLN e que foram submetidos a um estudo polissonográfico do sono, à MAPA e que preencheram um questionário que contemplava a obtenção de informação relativa ao perfil da medicação anti-hipertensora em curso. Numa segunda fase, utilizámos um modelo experimental de HT no rato induzida por um paradigma de CIH. Do nosso trabalho resultaram os seguintes resultados principais: em primeiro lugar, o índice de massa corporal (IMC) e o perímetro do pescoço (PP) foram identificados como preditores independentes de “auto-classificação errónea” da HA em doentes com suspeita de SAOS; em segundo lugar, não encontramos qualquer associação com significado estatístico entre os vários esquemas de fármacos anti-hipertensores bem como o número de fármacos incluídos nesse esquemas, e o controle da PA (antes e depois da adaptação do CPAP); em terceiro lugar, apesar das doses de 10, 30 e 50 mg/kg de carvedilol terem promovido uma redução significativa da frequência cardíaca, não foi observado qualquer decréscimo na PA no nosso modelo animal; em quarto lugar, as razões S/(R+S) dos enantiómeros do CVD nos animais expostos à CIH e a condições de normóxia revelaram-se diferentes; e, em quinto lugar, a administração oral voluntária mostrou ser um método eficaz para a administração diária controlada de fármacos anti-hipertensores e que é independente da manipulação e contenção do animal. Em conclusão, os resultados obtidos através do estudo clínico revelaram que o controle da PA, antes e após a adaptação do CPAP, em doentes com SAOS é independente, quer do esquema de fármacos anti-hipertensores, quer do número de fármacos incluídos num determinado esquema. Os nossos resultados salientam ainda a falta de validade da chamada self-reported hypertension e sugerem que em todos os doentes com suspeita de SAOS, com HA não diagnosticada e com um IMC e um PP acima de 27 kg/m2 e 39 cm, respectivamente, a confirmação do diagnóstico de HA deverá ser realizada através da MAPA, ao invés de outros métodos que com maior frequência são utilizados com este propósito. Os resultados obtidos no modelo animal de HA induzida pela CIH sugerem que o bloqueio do sistema nervoso simpático, juntamente com os supostos efeitos pleiotrópicos do CVD, não parece ser a estratégia mais adequada para reverter este tipo particular de hipertensão e indicam que as alterações farmacocinéticas induzidas pela CIH no ratio S/(R+S) não justificam a falta de eficácia anti-hipertensora do CVD observada neste modelo animal. Por último, os resultados do presente trabalho suportam ainda a viabilidade da utilização da administração oral voluntária, em alternativa à gavagem, para a administração crónica de uma dose fixa de fármacos anti-hipertensores.---------------------------- ABSTRACT: Hypertension (HT) is a highly prevalent condition, although under diagnosed, in patients with obstructive sleep apnea (OSA). These conditions are closely related and 24-hour ambulatory blood pressure monitoring (ABPM) seems to be the most accurate measurement for diagnosing hypertension in OSA. However, this diagnostic tool is expensive and time-consuming and, therefore, not routinely used. On the other hand, although continuous positive airway pressure (CPAP) is considered the gold standard treatment for symptomatic OSA, its lowering effect on blood pressure (BP) seems to be modest and, therefore, concomitant antihypertensive therapy is still required. Data on antihypertensive drug regimens in patients with OSA are scarce and specific therapeutic guidelines for the pharmacological treatment of hypertension in these patients remain absent. The use of animal models of CIH, which mimic the HT observed in patients with OSA, is extremely important since it is imperative to identify preferred compounds for an adequate BP control in this group of patients. However, studies aimed at investigating the antihypertensive effect of antihypertensive drugs in this animal model are insufficient, and most reports on CIH animal models in which drugs have been tested were not designed to respond to pharmacological issues. Moreover, when testing antihypertensive drugs (AHDs) it becomes crucial to ensure the selection of a non-invasive and stress-free method for drug delivery. Although gavage is effective and a widely performed technique for daily dosing in laboratory rodents, it comprises a sequence of potentially stressful procedures for laboratory animals that may constitute bias for the experimental results. The overall goal of the present translational research was to contribute to identify more effective AHDs for the treatment of hypertension in patients with OSA and investigate underlying mechanisms of systemic effects associated with OSA, as well as its modulation by AHDs. The specific aims were: first, to find new predictors based on anthropometric measures to identify patients that misclassify themselves as non-hypertensive, and thereby promote the selective use of ABPM; second, to investigate a hypothetical association between ongoing antihypertensive regimens and BP control rates in patients with OSA, before and after CPAP adaptation; third, to determine, in a rat model of CIH-induced hypertension, the efficacy of carvedilol (CVD), a nonselective beta-blocker with intrinsic anti-α1-adrenergic activity and antioxidant properties; fourth, to explore the effects of CIH on the pharmacokinetics profile of CVD and fifth, to investigate an alternative method to gavage, for chronic administration of AHDs to laboratory rats. For that, in the first phase of this project, we used a sizeable sample of patients with OSA (n=369), that attended a first visit at Centro Hospitalar Lisboa Norte, EPE Sleep Unit, and underwent overnight polysomnography, 24-h ABPM and filled a questionnaire that included ongoing antihypertensive medication profile registration. In the second phase, a rat experimental model of HT induced by a paradigm of CIH that simulates OSA was used. The main findings of this work were: first, body mass index (BMI) and neck circumference (NC) were identified as independent predictors of hypertension misclassification in patients suspected of OSA; second, in patients with OSA, BP control is independent of both the antihypertensive regimen and the number of antihypertensive drugs, either before or after CPAP adaptation; third, although the doses of 10, 30 and 50 mg/Kg of CVD promoted a significant reduction in heart rate, no decrease in mean arterial pressure was observed; fourth, the S/(R+S) ratios of CVD enantiomers, between rats exposed to CIH and normoxic conditions, were different and fifth, voluntary ingestion proved to be an effective method for a controlled daily dose administration, with a define timetable, that is independent of handling and restraint procedures. In conclusion, the clinical study showed that BP control in OSA patients is independent of both the antihypertensive regimen and the number of antihypertensive drugs. Additionally, our results highlight the lack of validity of self-reported hypertension and suggest that all patients suspected of OSA with undiagnosed hypertension and with a BMI and NC above 27 Kg/m2 and 39 cm should be screened for hypertension, through ABPM. The results attained in the rat model of HT related to CIH suggest that the blockade of the sympathetic nervous system together with the putative pleiotropic effects of carvedilol is not able to revert hypertension induced by CIH and point out that the pharmacokinetic changes induced by CIH on S/(R+S) ratio are not apparently responsible for the lack of efficacy of carvedilol in reversing this particular type of hypertension. Finally, the results here presented support the use of voluntary oral administration as a viable alternative to gavage for chronic administration of a fixed dose of AHDs.

Comparison of recombinant A2-ELISA with rKE16 dipstick and direct agglutination tests for diagnosis of visceral leishmaniasis in dogs in Northwestern Iran

INTRODUCTION: Various methods are used for the diagnosis of visceral leishmaniasis (VL), such as microscopic examination, culture and inoculation of laboratory animals; however, serological assays are commonly used for the detection of antibodies in serum samples with a wide range of specificity and sensitivity. METHODS: The purpose of this study was to compare three serological methods, including rA2-ELISA, the recombinant KE16 (rKE16) dipstick test and the direct agglutination test (DAT), for the detection of antibodies against VL antigens. The assays utilized 350 statistically based random serum samples from domestic dogs with clinical symptoms as well as samples from asymptomatic and healthy dogs from rural and urban areas of the Meshkinshahr district, northwestern Iran. RESULTS: Samples were assessed, and the following positive rates were obtained: 11.5% by rKE16, 26.9% by DAT and 49.8% by ELISA. The sensitivity among symptomatic dogs was 32.4% with rKE16, 100% with DAT and 52.9% with ELISA. Conversely, rA2-ELISA was less specific for asymptomatic dogs, at 46.5%, compared with DAT, at 88.9%. CONCLUSIONS : This study recommends rA2-ELISA as a parallel assay combined with DAT to detect VL infection among dogs. Further evaluations should be performed to develop an inexpensive and reliable serologic test for the detection of Leishmania infantum among infected dogs.

Hind limb ischemia in type 1 diabetic mice as useful tool to evaluate the neovascularization of tissue engineering constructs

Hind-limb ischemia has been used in type 1 diabetic mice to evaluate treatments for peripheral arterial disease or mechanisms of vascular impairment in diabetes [1]. Vascular deficiency is not only a pathophysiological condition, but also an obvious circumstance in tissue regeneration and in tissue engineering and regenerative medicine (TERM) strategies. We performed a pilot experiment of hind-limb ischemia in streptozotocin(STZ)-induced type 1 diabetic mice to hypothesise whether diabetes influences neovascularization induced by biomaterials. The dependent variables included blood flow and markers of arteriogenesis and angiogenesis. Type 1 diabetes was induced in 8-week-old C57BL/6 mice by an i.p. injection of STZ (50 mg/kg daily for 5 days). Hind-limb ischemia was created under deep anaesthesia and the left femoral artery and vein were isolated, ligated, and excised. The contralateral hind limb served as an internal control within each mouse. Non-diabetic ischaemic mice were used as experiment controls. At the hind-limb ischemia surgical procedure, different types of biomaterials were placed in the blood vessels gap. Blood flow was estimated by Laser Doppler perfusion imager, right after surgery and then weekly. After 28 days of implantation, surrounding muscle was excised and evaluated by histological analysis for arteriogenesis and angiogenesis. The results showed that implanted biomaterials were promote faster restoration of blood flow in the ischemic limbs and improved neovascularization in the diabetic mice. Therefore, we herein demonstrate that the combined model of hind-limb ischemia in type 1 diabetes mice is suitable to evaluate the neovascularization potential of biomaterials and eventually tissue engineering constructs.  

Producción in vitro de Embriones Bovinos Transgénicos mediante Inyección Intracitoplasmática de Espermatozoides (ICSI) con Enzimas de Restricción. In vitro Production of Transgenic Bovine Embryos using Intracitoplasmic Sperm Injection (ICSI) and Restriction Enzymes

La ingeniería genética y la reprogramación de organismos vivos representan las nuevas fronteras biotecnológicas que permitirán generar animales con modificaciones precisas en sus genomas para un sinnúmero de aplicaciones biomédicas y agropecuarias. Las técnicas para inducir modificaciones génicas intencionales en animales, especialmente en especies mayores de interés agropecuario, se encuentran rezagadas si se compara con los avances significativos que se han producido en el área de la transgénesis de roedores de laboratorio, especialmente el ratón. Es así que, el presente proyecto persigue desarrollar y optimizar protocolos para generar embriones bovinos transgénicos para aplicaciones biotecnológicas. La estrategia propuesta, se basa en conseguir la presencia simultánea en el interior celular de una enzima de restricción (I-SceI) más un transgén (formado por casetes de expresión de una proteína fluorescente -ZsGreen1- y neomicina fosfotransferasa). Específicamente, proyectamos estudiar una vía alternativa para generar embriones bovinos transgénicos mediante la incorporación del transgén (casetes ZsGreen1 y neo) flanqueado por sitios I-SceI más la enzima I-SceI al interior del ovocito junto con el espermatozoide durante la técnica conocida como inyección intracitoplasmática de espermatozoides (ICSI). Los embriones así generados se cultivarán in vitro, inspeccionándolos diariamente para detectar la emisión de fluorescencia, indicativa de la expresión de la proteína ZsGreen1. Los embriones que alcancen el estado de blastocisto y expresen el transgén se transferirán quirúrgicamente al útero de ovejas sincronizadas y se mantendrán durante 7 días. Al cabo de este período, los embriones se recolectarán quirúrgicamente del útero ovino y se transportarán al laboratorio para determinar el número de sitios de integración y número de copias del transgén mediante el análisis de su ADN por Southern blot. Se prevé que los resultados de esta investigación permitirán sentar las bases para el desarrollo de métodos eficientes para obtener modificaciones precisas en el genoma de los animales domésticos para futuras aplicaciones biotecnológicas. Genetic engineering and reprogrammed organisms represent the new biotechnological frontiers which will make possible to generate animals with precise genetic modifications for agricultural and biomedical applications. Current methods used to generate genetically modified large animals, lay behind those used in laboratory animals, specially the mouse. Therefore, we seek to develop and optimize protocols to produce transgenic bovine embryos through the use of a non-viral vector. The strategy involves the simultaneous presence inside the cell of a restriction enzyme (I-SceI) and a transgene (carrying cassettes for a fluorescent protein -ZsGreen1- and neomycin phosphotransferase) flanked by restriction sites for the endonuclease. We plan to develop an alternative approach to generate transgenic bovine embryos by coinjecting the transgene flanked by I-SceI restriction sites plus the enzyme I-SceI along with the spermatozoon during the technique known as intracytoplasmic sperm injection (ICSI). Embryos will be cultured in vitro and inspected daily with a fluorescence microscope to characterize transgene expression. Embryos that reach the blastocyst stage and express the transgene will be surgically transfer to the uterus of a synchronized ewe. After 7 days, the embryos will be flushed out the ovine uterus and transported to the laboratory to determine the number of integration sites and transgene copies by Southern blot. We anticipate that results from this research will set the stage for the development of efficient strategies to achieve precise genetic modifications in large domestic animals for future biotechnological applications.

Toxicidade de algumas crotolárias

This paper deals with some testes of Crotalaria as forage, using rabbits as laboratory animals to detect toxicity, at Piracicaba, São Paulo, Brazil. 1. C. intermedia and C. juncea were palatable, non toxic, the last more productive. Tests on cattle are recommended and selection to improve characteristics as forage too. 2. C. incana and C. verrucosa were little palatable, probably non toxic, poor productive. 3. C. paulina, C. lanceolata (four strains), C. mysorensis, C. gratiana, C. spectabilis, C. breviflora and C. campista were toxic. This results are not according those of others AA. Others species are beeing tested since Crotalaria is a drought resistent legume on this area.

Análise comparativa entre tipos de alimentação de Micrurus corallinus (Serpentes, Elapidae) em cativeiro

Micrurus corallinus (Merrem, 1820) kept in laboratory animal rooms swallow voluntarily various colubrid species. The most accepted species belong to the genera Sibynomorphus Fitzinger, 1843, Oxyrhopus Wagler, 1830 and Liophis Wagler, 1830. The voluntary feeding guaranteed resistance against pathogenic agents. The forced feeding was unsatisfactory due to stress resulting from handling, feeding frequency (30 days), susceptibility to pathogenic agents and high occurrence of pathologies.

Cultura cromogênica dum bacilo ácido-álcool resistente isolado de pus de lesão fechada de lepra humana

Last October 2nd the Author smeared nine tubes of Loewenstein medium with material obtained from closed pustulae of a seven years old boy, L2 case of leprosy. This material was very rich in Hansen bacilli in its different forms, inclusive globus, as is seen in the figures 2 and 3 of Plate 1. Part of this material obtained from pustulae opened by galvanocautery, was inoculated, at the same day, into white rats and guinea-pigs. November 26th a new biopsy gave more rich material, which was smeared again into Loewenstein fresh medium. December 15th three of the first and two of the second series of tubes of cultures showed germination of a yellow, dry and rough culture, covering almost the total surface of the medium. Microscopic examination of the culture showed that it was a pure culture of an acid-fast organism. Passages into glycerinated potatoes germinated well covering the surface of the same with a clear yellow granulated culture remaining the fluid (glycerinated water) quite limpid. The germination in glycerinated broth produced a yellow velum in the surface of the medium, as is seen in fig. 3 of Plate 2, without becoming turbid. The microorganism isolated twice from the same source of material was cocciforme (as Mycobacterium pulviforme of Marchoux), in the original culture, becoming more bacilliforme, always acid-fast, after passage into glycerinated media. The A. sent his culture to foreigner colleagues to study it and will inoculate it soon into laboratory animals.

Poderá o carrapato transmitir a lepra?: isolamento e cultura dum bacilo acido-álcool resistente de sedimento de "Amblyomma cajennense" capturado em leproso: 2ª nota

In this 2nd note upon the possibility of transmission of human leprosy by ticks, the A. relates his stepps to obtain the collaboration of his colleagues working in leprosaria in various States of Brazil, Argentina and Paraguay in such studies. Firstly the A. describes the positive results of examination of sediment of ticks, the cattle tick Boophilus microplus (Canestrini, 1888), received from Paraná (Leprosário São Roque) , which were put on active lepers, two of them sucking during 9 days and one during 7 days. Two out of three were killed for examination and were very strongly positive for acid-fast bacilli. A series of tubes of Loewenstein medium was smeared with the sediment of such ticks. Secondly the A. relates his personnal experiment, carried out in Rio de Janeiro, trying to infect normal ticks in lepers. The experiment with Boophilus microplus was negative and was twicely positive the experiment with Amblyomma cajennense Fabricius, 1794. The experiment is being in progress and will be continued in other places of Brazil. Finally, after being given the general characteristics of Boophilus microplus, the A. describes the non-chromogenic culture of a acid-fast bacillus isolated by him from sediment of ticks (Amblyomma cajennense) captured in lepers from Colônia Santa Isabel (Minas gerais), which parasitism was spontaneous. The first isolation was obtained in Loewenstein medium after 62 days incubation at 37°C. The culture is pure and the bacillus is permanent acid-fast. The plate1, in full color, represents this culture in its four generations. The colonies are pearl-white in color, dry, elevated and rough, developing slowly and beginning as white pinhead points scattered upon the surface of the medium. The culture is not yet rich enough to be inoculated into laboratory animals, which will be done when possible.

Poderá o carrapato transmitir a lepra?: mais quatro amostras de culturas de bacilos acido-alcool resistentes obtidas de carrapatos (2 de "Amblyomma cajennense"e 2 de "Boophilus microplus") infectados em leprosos do Paraná: 3ª nota

The AA. carried out experiments in the leprosarium São Roque, State of Paraná, South Brazil, to verify if the cattle tick Boophilus microplus could be experimentally infected in lepers, which was true. The AA. Tried also to be ascertained if Boophilus microplus and Amblyomma cajennense could change of hosts during their feedings which was true, both ticks continue feeding, the last species for many days, after being transferred from one to another leper. The junior A. describes in full their experiments and also a dermatites caused by tick bites. The senior A. brought to Rio de Janeiro most of the infected ticks for examination, which revealed a very high positivity. He smeared the sediments of lots of both species of ticks in Loewenstein medium and after a variable periode of incubation at 37° C. he obtained four new samples of cultures of acid-fast organisms, two from Amblyomma cajennense and two from Boophilus microplus. These cultures are being studied and will be inoculated into laboratory animals. The senior A. inoculated new batches of white rats with sediments of many ticks infected in lepers. Various hypotheses of both previous notes upon the subject now are verified facts. The A. is accumulating facts to draw the conclusions in the future. He also suggested the leprosy workers in the interior of the country to cooperate with him in such important studies, specially in the habitat of lepers in the rural zones of various States.

Parasite Genotypically Related to a Monoxenous Trypanosomatid of Dog's Flea Causing Opportunistic Infection in an HIV Positive Patient

An HIV positive patient presenting a clinical picture of visceral leishmaniasis co-infection was submitted to a bone marrow aspiration after admission to hospital. Amastigotes forms were seen in the bone marrow aspirate and the parasite grew in culture as promastigotes. Molecular analyses showed that the flagellates isolated did not belong to the genera Leishmania, Trypanosoma or Sauroleishmania. It was not possible to establish infection in laboratory animals. In vitro culture of mouse peritoneal macrophages revealed the invasion of the host cells by the flagellates and their killing 48 hr after infection. Opportunistic infection with an insect trypanosomatid was suspected. Further hybridization analyses against a pannel of different monoxenous and heteroxenous trypanosomatids showed kDNA cross-homology with Leptomonas pulexsimulantis a trypanosomatid found in the dog's flea