Trypan blue exclusion assay by flow cytometry

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Perfis clínico e hematológico de camundongos submetidos ao envenenamento escorpiônico experimental por Tityus fasciolatus / Hematological and clinical profiles of mice submitted to experimental poisoning with Tityus fasciolatus venom

Estudou-se o quadro clínico do veneno decorrente do envenenamento por Tityus fasciolatus e sua ação refletida nos componentes hematológicos no modelo murino. Para o estudo do perfil hematológico, foram utilizados 54 camundongos Swiss CF1, machos, com 30g, distribuídos em três grupos (G) (n=18) inoculados via subcutânea com: 50µL de PBS (G1); 24µg de veneno de T. fasciolatus (G2) e 8µg de veneno de T. serrulatus (G3). Cada grupo foi subdividido em três subgrupos (n=6) de acordo com o momento da coleta de sangue que foi estipulada em uma, oito e 24h após a inoculação do veneno, e foram realizados o hemograma e a dosagem de proteínas totais e fracionadas. O veneno de T. fasciolatus na dose de 24µg causou piloereção, comportamento nociceptivo, secreção nasal e oral acentuada, dispneia, prurido na face e reflexos exacerbados. No exame hematológico, foram observadas policitemia relativa e leucocitose com linfocitose.

The aim of this study was to evaluate the clinical picture caused by Tityus fasciolatus and its action reflected in mouse blood profile. To study the blood profile, 54 Swiss CF1 mice, male, 30g, distributed into three groups (G) (n= 18) and inoculated subcutaneously with: 50µl PBS (G1), 24µg of venom of T. fasciolatus (G2) and 8µg venom T. serrulatus (G3) were used. Each group was subdivided into three subgroups (n= 6) according to the time of blood collection that was set at 1h, 8h and 24h after inoculation of the venom, and blood profile, total protein and fractionated were evaluated. The venom of T. fasciolatus at a dose of 24µg caused piloerection, behavior pain, nasal and oral sharp, dyspnea, rash on the face and exaggerated reflexes. In blood profile were observed relative polycythemia, leukocytosis with lymphocytosis.

Hematological and biochemical profiles of rats submitted to experimental poisoning with Tityus serrulatus venom / Perfis hematológicos de ratos submetidos ao envenenamento escorpiônico experimental por Tityus serrulatus

The hematological and biochemical profiles of newly weaned rats submitted to experimental poisoning with T. serrulatus venom were evaluated. Fifteen recently weaned male Wistar rats (mean weight 130g) were distributed into three equal groups (n = 5). Animals in the control group (group A) received a subcutaneous injection of 400μL of ultra-pure water, while those in the experimental groups received, by identical route, 400μL of a solution containing 100μg (group B) or 450μg (group C) of scorpion venom dissolved in ultra-pure water. Red blood cells indexes, and differential leukocyte and total platelet counts were determined, together with levels of serum glucose, urea, creatinine, lactic dehydrogenase, aspartate aminotransferase, amylase, insulin, and cortisol. No significant differences between the control and experimental groups regarding red blood cells indexes were found. In contrast, significant increases (P<0.05) in neutrophils, lymphocytes, and monocytes were observed in animals from groups B and C compared with the control group, while the number of platelets decreased. Serum glucose concentration remained unchanged in all groups, but important alterations were observed in the values of urea and creatinine. The results show that scorpion venom was detrimental to renal function as demonstrated by the altered urea and creatinine levels. Pancreatic function was also impaired, as revealed by the increase in amylase activity and the reduction in insulin levels.

Avaliaram-se os perfis hematológico e bioquímico de ratos recém-desmamados submetidos ao envenenamento experimental com veneno de Tityus serrulatus. Quinze ratos Wistar machos, média de peso de 130g, foram distribuídos aleatoriamente em três grupos (n = 5). Os animais do grupo-controle A foram inoculados com 400μL de água ultrapura, os do grupo experimental B receberam 400μL de uma solução contendo 100μg de veneno e os do grupo experimental C receberam 400μL de uma solução contendo 450μg de veneno. Foram determinados os índices da série vermelha, a contagem total e diferencial dos leucócitos e a contagem total de plaquetas, bem como os níveis da desidrogenase lática, aspartato aminotransferase, amilase, glicose, ureia, creatinina, cortisol e insulina. Não houve diferenças significativas entre o grupo-controle e os experimentais com relação aos índices da série vermelha. Foram observados aumentos significativos (P<0,05) no número de neutrófilos, linfócitos e monócitos nos ratos dos grupos B e C, enquanto o número de plaquetas diminuiu. A concentração de glicose permaneceu inalterada em todos os grupos, mas foram observadas importantes alterações nos valores séricos de ureia e creatinina. Esses resultados mostraram que o veneno de escorpião comprometeu o funcionamento dos rins. Como demonstrado pelo aumento da atividade da amilase sérica e a redução dos níveis de insulina, a função pancreática também foi afetada.

Cardiorespiratory evaluation of juvenile rats experimentally envenomed with Tityus serrulatus venom

Accidental envenomation caused by Tityus serrulatus scorpions is very common in Brazil and may result in serious cardiorespiratory alterations that are frequently fatal to children. In the present study, the effects of T. serrulatus venom on the cardiorespiratory system of recently weaned male Wistar rats were evaluated. Fifteen animals were distributed into three groups (n = 5). The control group A received 400 miuL ultrapure water by subcutaneous injection, while the experimental groups B and C were injected with scorpion venom (100 and 450 miug, respectively, in 400 miuL water). Electrocardiogram (ECG) traces were obtained prior to the experiment, at five-minute intervals up to 30 minutes after treatment. At 40 minutes after envenomation, the animals had severe acute symptoms and were subsequently anesthetized for blood collection by means of intracardiac puncture. Biochemical profiles for the cardiac muscle were established by colorimetric analysis of creatine kinase (CK) and CK-MB isoenzyme. Semiquantitative analysis of troponin was performed using the immunochromatographic assay. Following euthanasia, the lungs and hearts were removed and subjected to histopathological examination. All experimental animals had ECG alterations compatible with electrolytic imbalance, myocarditis and alterations of the cardiac conduction system. Envenomed animals had accentuated bradycardia at 25 and 30 minutes after venom inoculation. All experimental animals had myocardial lesions, which were confirmed by increased serum levels of CK and CK-MB, although there were no alterations in the serum concentration of troponin. Pulmonary hemorrhage was detected in whole lungs and microscopically confirmed by the presence of congested capillaries and erythrocytes in the alveolar parenchyma. In conclusion, T. serrulatus venom caused great cardiorespiratory damage to weaned rats.

Hemograma de cães submetidos ao envenenamento experimental por Tityus serrulatus / Canine blood profile after experimental envenomation by Tityus serrulatus

Avaliou-se o hemograma de 12 cães adultos, saudáveis (14,2±5,4kg) após a inoculação de veneno do escorpião amarelo (Tityus serrulatus). Os animais foram distribuídos em dois grupos (G), com seis em cada: os do GI foram usados como controle e receberam 0,5mL de salina tamponada com fosfato (PBS) por via subcutânea (SC) na face medial da coxa esquerda (FMCE), e os do GII receberam veneno liofilizado do T. serrulatus (250µg/kg) diluído em PBS por via SC na FMCE. Foram realizadas colheitas de sangue com anticoagulante EDTA a 10 por cento antes da inoculação do veneno (T0) e após 2h, (T1), 6h (T2), 12h (T3), 24h (T4), 48h (T5) e 72h (T6), para contagem de eritrócitos, leucócitos e plaquetas em aparelho contador eletrônico e esfregaços sanguíneos para contagem diferencial de leucócitos. Houve aumento significativo (P<0,05) dos valores de eritrócitos, volume globular e hemoglobina 2h e 6h após o envenenamento, devido à contração esplênica decorrente da dor local causada pelo veneno e pela liberação de catecolaminas. Foi observada leucocitose por aumento significativo (P<0,05) de neutrófilos e linfócitos 2h e 6h após o envenenamento. Concluiu-se que o veneno de Tityus serrulatus na dose de 250µg/kg, é capaz de aumentar os valores do eritrograma e do leucograma dos cães, provavelmente devido à dor local, com liberação de catecolaminas.

The canine blood profile after scorpion envenomation was evaluated using 12 healthy mongrel male dogs (14.2±5.4kg) distributed in two groups, with six animals in each: group I (control group) and group II (venom group). The lyophilized yellow scorpion (Tityus serrulatus) venom (250µg/kg) diluted in 0.5mL phosphate buffered saline (PBS) was given to group II animals by subcutaneous injection, in the medial face of the left thigh. Group I animals received only 0.5mL of PBS, by subcutaneous injection, in the medial face of the left thigh. Blood samples were collected with EDTA before (T0) and 2 (T1), 6 (T2), 12 (T3), 24 (T4), 48 (T5), and 72h (T6) after envenomation. Significant increases (P<0.05) in erythrocytes counting, hematocrit and hemoglobin concentration, 2 and 6h after envenomation were observed. Leukocytosis with significant increases (P<0.05) of neutrophils and lymphocytes 2 and 6h after envenomation was found. Then, T. serrulatus venom may induce alterations in blood profile in dogs, probably due to spleen contraction evoked by pain and catecholamines releasing.

Intoxicação experimental com extratos de Mascagnia rigida (Malpighiaceae) em camundongos / Experimental intoxication in mice with extracts of Mascagnia rigida (Malpighiaceae)

Foi realizado o fracionamento químico do extrato aquoso da Mascagnia rigida Griseb., uma importante planta tóxica no Brasil, para se obter cinco substratos ricos em diferentes grupos químicos - alcalóides, flavanóides, taninos, saponinas e açúcares, ácidos orgânicos e aminoácidos -, e investigar a toxicidade dessas frações, exceto a última, em 75 camundongos. Os animais, distribuídos aleatoriamente em cinco grupos, receberam: grupo I - alcalóides; grupo II - flavanóides; grupo III - taninos; grupo IV - saponinas e grupo V - placebo, este último funcionando como controle negativo. Todos os grupos, com exceção do grupo-controle, redistribuídos em três subgrupos, A, B e C, com cinco animais cada, receberam, respectivamente, 9g/kg, 18g/kg e 27g/kg de cada substrato. As frações foram fornecidas via oral, diariamente, por sete dias; no sétimo dia, foi coletado sangue para o estudo do perfil sangüíneo e dosagem de enzimas musculares. As frações de alcalóides e taninos foram capazes de causar alteração no perfil enzimático-muscular, com aumento significativo da enzima miocárdica. Observou-se, também, aumento significativo na porcentagem da CK-MB após a administração das frações de saponinas e taninos, comprovando a ação tóxica da M. rigida sobre a fibra muscular cardíaca.

A study was carried out to examine the toxic effects of Mascagnia rigida Griseb. Four classes of compounds were extracted from the plant by phytochemistry study and the individual effect of each one on mice was examined as follows: group I - alkaloids; group II- flavones; group III - tannins; group IV - saponins; and group V - water (control). Each group was further divided in three subgroups, A (9g/kg), B (18g/kg), and C (27g/kg), with five mice orally receiving a particular dose, once per day for one week. On the 7th day, blood was collected and hematological exams and levels of muscle enzymes were analyzed. The results showed that both alkaloids and tannins caused a significant increase in myocardial enzyme. Administration of either saponins or tannins fractions caused an increase of CK-MB enzyme. This study showed that Mascagnia rigida Griseb has the ability to cause damage to myocardial fibers.

Efeito do praziquantel incorporado a lipossomas nos diferentes estágios de desenvolvimento dos ovos de Schistosoma mansoni / Effect of praziquantel in liposomes on Schistosoma mansoni eggs at different development stages

A esquistossomose mansônica é causada pelo trematódeo digenético intravascular Schistosoma mansoni. Para o tratamento dessa enfermidade o praziquantel (PZQ) e a oxamniquina (OXA) são os fármacos escolhidos. Noentanto, esses fármacos apresentam limitações quanto à ação e casos de resistência ou tolerância já foram relatados. Por esse motivo, são necessários os estudos de novas alternativas que visam melhorar os fármacos já existentes, como a incorporação desses em lipossomas. Este estudo verificou a ação do praziquantel incorporado a lipossomas (lip.PZQ) sobre os ovos de S. mansoni, linhagem BH em camundongos Mus musculus (Swiss-SPF). Para tanto, foram testadas quatro doses de PZQ elip.PZQ (47; 60; 250 e 300mg/kg) sendo que parte dos camundongos foi tratada após 30 dias de infecção e outra após 45 dias. A análise do o ograma mostrou que a dose lip.PZQ 300mg/kg administrada no 45º dia de infecção foi mais eficaz, pois reduziu a oviposição pelas fêmeas de S. mansoni.

Isolation and isoenzyme characterization of Leishmania (Viannia) braziliensis from a case of human cutaneous leishmaniasis in northeast centre of the state of São Paulo

The diagnosis of human cutaneous leishmaniasis in small towns is sometimes made without the species identification of the Leishmania, even in areas without previous epidemiological surveys. Here we report the isolation of a Leishmania strain from a patient of Rincão, state of São Paulo, that was identified by isoenzyme characterization as L. (Viannia) braziliensis. Sand fly collections were made in the area where the patient live in order to investigate the likely vector species.

Evaluation of some organic compounds on bloodstream forms of Trypanosoma cruzi

Accidental transmission of Chagas' disease to man by blood transfusion is a serious problem in Latin-America. This paper describes the testing of several synthetic, semi-synthetic, and natural compounds for their activity against blood trypomastigotes in vitro at 4-C. The compounds embody several types of chemical structures: benzoquinone, naphthoquinone, anthracenequinone, phenanthrenequinone, imidazole, piperazine, quinoline, xanthene, and simple benzenic and naphthalenic derivates. Some of them are for the first time tested against Trypanosoma cruzi. The toxic effect these compounds on this parasite was done by two quite distinct sets of experiments. In one set, the compounds were added to infected blood as ethanolic solution. In this situation the most active one was a furan-1, 2-naphthoquinone, in the same range as gentian violet, a new fact to be considered in the assessment of structure-activity relationships in this class of compounds. In other set, we tentatively evaluated the biological activity of water insoluble compounds by adding them in a pure form without solvent into infected blood. In this way some appear to be very active and it was postulated that the effectiveness of such compounds must result from interactions between them and specific blood components

Influência da fluoretaçäo da água de abastecimento nos valores densitométricos e na área da imagem radiográfica do osso semilunar. Estudo comparativo / Influence of water supply fluoridation on the densitometric values and radiographic image area of the lunate bone. A comparative study

Foram obtidas radiografias do carpo de 200 criancas brancas de ambos os sexos, com idade de 7 a 8 anos, nascidas e residentes em Araraquara (0,7 ppm F) e Américo Brasiliense (0,1 ppm F). Foi analisada a transmissäo da luz e medida a área da imagem radiográfica do osso semilunar. Os resultados foram analisados estatisticamente e permitiram concluir pela diferença näo significante entre os valores de transmissäo da luz e os valores médios da área da imagem radiográfica do osso semilunar, quando comparadas ambas as comunidades

O autismo infantil - analise de uma hipotese etiopatogenetica. Consideracoes sobre a neurobiologia da senso-percepcao. / Infantile autism - analysis of an etiopathogenetic hypothesis. Considerations on neurobiology of senso-perception

Este artigo explora a hipotese de que no autismo infantil de Kanner, fetos com um elevado potencial e superioridade intelectual herdados estao com sua maturidade cerebral completa, pronta para receber estimulos de socializacao primaria, muito antes do nascimento. Ao inves de serem "marcados" no periodo critico pos-natal, como a maioria das criancas, estes fetos, neurologicamente superiores e precocemente maduros, sao sujeitos a privacao sensorial no ambiente restrito do utero materno. Os conceitos tradicionais de percepcao,aprendizagem e memoria nao tem sido uteis na descricao de contribuicoes da experiencia precoce para o desenvolvimento comportamental. Portanto, torna-se necessaria uma atualizacao do assunto e a analise das influencias dos estimulos ambientais em estadios iniciais da embriogenese