Extract from Arrabidaea chica (Fridericia chica) leaves show preventive action for the mitigation of doxorubicin-induced cardiotoxicity / [Extrato das folhas de Arrabidaea chica (Fridericia chica) mostrou potencial ação no combate à cardiotoxicidade induzida pela doxorrubicina]

A doxorrubicina (dox) é um medicamento antineoplásico que induz cardiotoxicidade por estresse oxidativo. Os flavonoides são antioxidantes extraídos de plantas como Camellia sinensis e Arrabidaea chica (Fridericia chica). Esta pesquisa objetivou avaliar efeitos protetores do extrato de A. chica (AC), comparado ao de C. sinensis (CS), frente ao estresse oxidativo induzido pela dox, no coração. Cardiomiócitos e células neoplásicas MDA-MB 231 foram incubados com AC e CS. Depois, adicionou-se dox e avaliaram-se taxas de viabilidade e morte celular. A citometria de fluxo para o ensaio de iodeto de propídeo (IP) em cardiomiócitos mostrou as seguintes taxas de morte celular: controle 53%; dox 78% (maior que controle, P=0,015); AC_12,5µg/mL + dox 65% (menor que dox, P=0,031); AC_25µg/mL + dox 62% (menor que dox, P=0,028); AC_50µg/mL + dox 63% (menor que dox, P=0,030); CS_12,5µg/mL + dox 71% (menor que dox, P=0,040); CS_25µg/ml + dox 69% (menor que dox, P=0,037); CS_50µg/mL + dox 74% (menor que dox, P=0,044). Resultados das células MDA-MB 231 mostraram que nenhum extrato interferiu na atividade antitumoral da dox. Os dados de IP foram corroborados pelos de MTT. Este estudo reporta promissora utilização de A. chica na prevenção da cardiotoxicidade induzida pela dox.(AU)

Matriz porosa do BV60S associada a células osteoprogenitoras alógenas no tratamento de defeitos ósseos críticos em rádios de cães / Porous matrix of BG60S associated with allogenic osteoprogenitor cells in the treatment of critical bone defects in the radius of dogs

O objetivo do estudo foi avaliar o efeito da matriz porosa do biovidro 60S (BV60S) associada a células osteoprogenitoras (CO) alógenas no tratamento de defeitos ósseos críticos de cães. Foram utilizados 20 cães, machos, sem raça definida, com dois anos de idade e massa corporal média de 25kg. Com os cães sob anestesia geral, foram criados defeitos ósseos críticos no terço médio dos ossos rádios. Procedeu-se à fixação óssea com uma placa em ponte, e os defeitos foram tratados de acordo com cada grupo experimental. Constituíram-se três grupos experimentais, em que os defeitos ósseos foram preenchidos com: BV60S associado a CO alógenas (grupo BV60S+CO), osso autógeno (grupo C+), ou não preenchidos (grupo C-). A regeneração óssea foi avaliada por meio de exames radiográficos, densitométricos e histomorfométricos ao longo de 90 dias. Os grupos C- e BV60S+CO mostraram preenchimento ósseo parcial do defeito de, no máximo, 56,68% e 35,23%, respectivamente, sem a formação de ponte óssea entre as extremidades, e o controle positivo (C+) mostrou regeneração óssea completa. Conclui-se que a matriz porosa do BV60S associada às células osteoprogenitoras não é eficiente no tratamento de defeitos ósseos críticos em rádios de cães.(AU)

The objective of this study was to evaluate the effect of the porous matrix of bioglass 60S (BV60S) associated with allogenic osteoprogenitor cells (CO) in the treatment of critical bone defects of dogs. 20 male mongrel dogs at two years old and mean weight of 25kg were used. Dogs were anesthetized and critical bone defects were created in the middle third of the radios bones. With dogs under general anesthesia, critical bone defects were created in the middle third of bone radios. Bone fixation was done with a bridge plate and defects treated according to each experimental group. Three experimental groups were formed according to the treatment. The defects filled with BV60S associated with allogenic CO (Group-BV60S+CO), autogenous bone (Group-C+) or unfilled (Group-C-). Bone regeneration was evaluated by radiography, bone densitometry and histomorphometry over 90 days. The BV60S+CO and C- groups showed partial bone filling of the defect of at most 56.68% and 35.23%, respectively. No bone bridge formation was observed between the extremities in the BV60S+CO and C- groups. Positive control showed complete bone regeneration at 90 days. It was concluded that the porous matrix of BV60S associated with osteoprogenitor cells was not effective in the treatment of critical bone defects in the radius of dogs.(AU)

Benefícios da condroitinase abc associada a células-tronco mesenquimais na lesão espinhal aguda em ratos / Benefits of chondroitinase abc associated with mesenchymal stem cells in acute spinal cord injury in rats

Com o objetivo de estudar o efeito da condroitinase associada às células-tronco mesenquimais na lesão aguda da medula espinhal, utilizaram-se 50 ratos Lewis, distribuídos igualmente nos grupos: controle negativo (CN), tratamento com placebo (PLA), condroitinase (CDN), células-tronco mesenquimais (CTM) e condroitinase mais células-tronco mesenquimais (CDN+CTM). Todos os animais tiveram a medula espinhal exposta por laminectomia, e os grupos PLA, CDT, CTM e CDT+CTM sofreram também trauma medular compressivo. Após sete dias, procedeu-se à reexposição da medula espinhal, quando os grupos PLA e CTM receberam 4µL de líquido cefalorraquidiano artificial via intralesional, e os grupos CDT e CDT+CTM receberam o mesmo líquido contendo 2,2U de condroitinase. Após 14 dias da cirurgia inicial, todos os animais receberam 0,2mL de PBS via endovenosa, contudo, nos grupos CTM e CDT+CTM, esse líquido continha 1x106 CTM. Avaliou-se a capacidade motora até o 28o dia pós-trauma e, posteriormente, as medulas espinhais foram analisadas por RT-PCR, para quantificação da expressão gênica para BDNF, NT-3, VEGF, KDR e PECAM-1, e por imunoistoquímica, para detecção das células-tronco GFP injetadas (anti-GFP), quantificação dos neurônios (anti-NeuN) e da GFAP e vimentina, para avaliação da cicatriz glial. As análises estatísticas foram realizadas com o auxílio do Prism 5 for Windows, com o nível de significância de 5%. Não houve diferença entre os grupos quanto à capacidade motora. O grupo CDT+CTM apresentou maior imunoexpressão de neurônios viáveis do que o placebo. No CTM, houve maior expressão dos fatores neurotróficos BDNF e VEGF. E no CDT, houve menor imunoexpressão de vimentina. Concluiu-se que a associação CDT+CTM favorece a viabilidade neuronal após o trauma, que o tratamento com CTM promove aumento na expressão dos fatores tróficos BDNF e VEGF e que o tratamento com condroitinase é efetivo na redução da cicatriz glial.(AU)

The aim of this work was to study the effect of chondroitinase associated with mesenchymal stem cells in acute spinal cord injury. Therefore, 50 Lewis rats were distributed in the following groups: negative control (NC), treatment with placebo (PLA), chondroitinase (CDT), mesenchymal stem cells (MSC), and chondroitinase associated with mesenchymal stem cells (CDT + MSC). All animals had their spinal cord exposed by laminectomy, and the groups named PLA, CDT, MSC and CDT + MSC also suffered compressive spinal cord trauma. After seven days, the spinal cord was re-exposed, when the PLA and MSCs groups received 4uL of artificial cerebrospinal fluid through the lesion, and the CDT group and CDT + MSC received the same fluid containing 2,2U of chondroitinase. 14 days after the first surgery, all animals received 0.2ml of PBS intravenously; however, the MSC and CDT + MSC groups received the same liquid also containing 1x106 MSCs. The motor skills were evaluated up to 28 days post-injury and, subsequently, the spinal cords were analyzed by RT-PCR for BDNF, NT-3, VEGF, PECAM-1 and KDR gene expression quantification, immunohistochemistry to detect injected stem cells GFP (anti-GFP), to quantify neurons (anti-NeuN), GFAP and detect vimentin in order to evaluate the glial scar. Statistical analyzes were performed by Prism 5 for Windows using a 5% level of significance. There was no difference between groups with regarding motor capacity. The CDT + MSC group showed increased immunoreactivity of viable neurons than placebo. In MSC, there was a greater expression of neurotrophic factors BDNF and VEGF. Also, there was less vimentin immunostaining in group CDT. It was concluded that CDT + MSC association promotes neuronal viability after trauma, in which treatment with MSC promotes increased expression of BDNF and VEGF trophic factors, and also that treatment with chondroitinase is effective in reducing the glial scar.(AU)

Osteogenic differentiation of adipose tissue-derived mesenchymal stem cells cultured with different concentrations of prolactin / Diferenciação osteogênica de células tronco mesenquimais do tecido adiposo cultivadas com diferentes concentrações de prolactina

The objective was to evaluate the in vitro effect of prolactin in osteogenic potential of adipose tissue-derived mesenchymal stem cells (ADSCs) in female rats. ADSCs were cultured in osteogenic medium with and without the addition of prolactin and distributed into three groups: 1) ADSCs (control), 2) ADSCs with addition of 100ng/mL of prolactin and 3) ADSCs with addition of 300ng/mL of prolactin. At 21 days of differentiation, the tests of MTT conversion into formazan crystals, percentage of mineralized nodules and cells per field and quantification of genic transcript for alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I by real-time RT-PCR were made. The addition of prolactin reduced the conversion of MTT in group 3 and increased the percentage of cells per field in the groups 2 and 3, however without significantly increasing the percentage of mineralized nodules and the expression of alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I. In conclusion, the addition of prolactin in concentrations of 100ng/mL and 300ng/mL does not change the osteogenic differentiation to the ADSCs of female rats despite increase in the cellularity of the culture.(AU)

O objetivo do presente trabalho foi avaliar o efeito in vitro da prolactina sobre o potencial osteogênico de células-tronco mesenquimais do tecido adiposo (CTM-TA) em ratas. CTM-TA foram cultivadas em meio osteogênico com e sem adição de prolactina e distribuídas em três grupos: 1) CTM-TA (controle), 2) CM-TA com adição de 100ng/mL de prolactina e 3) CTM-TA com adição de 300ng/mL de prolactina. Aos 21 dias de diferenciação, foram realizados os testes de conversão do MTT em cristais de formazan, porcentagem de nódulos mineralizados e células por campo e quantificação dos transcritos gênicos para fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. A adição de prolactina reduziu a conversão do MTT no grupo 3 e aumentou a porcentagem de células por campo nos grupos 2 e 3, sem alterar significativamente a porcentagem de nódulos mineralizados e a expressão de fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. Conclui-se que a adição de prolactina nas concentrações de 100ng/mL e 300ng/mL não altera a diferenciação osteogênica das CTM-TA de ratas, apesar do aumento de celularidade da cultura.(AU)

Efeito do produto iônico do biovidro 60S na diferenciação osteogênica de células-tronco mesenquimais do tecido adiposo de cães / Effect of ionic product of bioglass 60S in osteogenic differentiation of mesenchymal stem cells from the adipose tissue of dogs

O objetivo do presente trabalho foi avaliar o produto iônico do biovidro 60S (BV60S) na diferenciação osteogênica de células-tronco mesenquimais de origem adiposa (CTM-AD) de cães. As CTM-AD foram diferenciadas sem OSTe com o produto iônico (PI OST) por sete, 14 e 21 dias. Avaliou-se o MTT, a fosfatase alcalina (FA), o colágeno, mineralização e as expressões de osterix (OSX), sialoproteína óssea (BSP), osteonectina (ON) e osteocalcina (OC). O grupo PI OSTmostrou menor conversão de MTT aos sete dias e maior conversão aos 21 dias. A atividade de FA foi maior no grupo OST, aos 14 e 21 dias. A síntese de colágeno foi maior no grupo OST aos sete e 21 dias. Verificou-se maior área mineralizada no grupo PI OSTem todos os tempos. Não houve diferenças nas expressões de OSX e OC entre os grupos. Observou-se maior expressão de BSP no grupo PI OST, aos 14 e 21 dias. A expressão de ON foi maior no grupo OST aos 14 dias. Concluiu-se que o produto iônico do BV60S favorece a osteogênese in vitro de CTM-AD de cães.

The aim was to evaluate the ionic product of 60S bioglass (BV60S) in osteogenic differentiation of mesenchymal stem cells from adipose tissue (ADMSCs) in dogs. ADMSCs were differentiated without the ionic product (OST) and with the ionic product (PI-OST) for 7, 14 and 21 days. We evaluated the MTT, alkaline phosphatase (ALP), collagen mineralization and expressions of osterix (OSX), bone sialoprotein (BSP), osteonectin (ON) and osteocalcin (OC). The PI-OST group had a lower MTT conversion to 7days and higher conversion at 21 days. The ALP activity was higher in the OST group at 14 and 21 days. Collagen synthesis was higher in the OST group at 7 and 21 days. A higher mineralized area in the PI-OST group was observed at all times. There were no differences in expressions of OSX and OC between groups. We observed increased expression of BSP in the PI-OST group at 14 and 21 days. The expression of ON was higher in the OST group at 14 days. It was concluded that the ionic product of BV60S promotes in vitro osteogenesis of MSC-AD from dogs.

Primary intraocular chondrosarcoma in a dog / Condrossarcoma intraocular primário em cão

A five-year-old male Cocker Spaniel was presented for evaluation of the right eye due to discomfort, abundant purulent discharge and progressive enlargement of the eyeball. The owner revealed that the right eye has appeared to be inflamed and smaller then the left eye for years. Ophthalmic examination revealed corneal perforation, buphthalmia and conjuctival hyperemia. Enucleating was performed due to signs of endophthalmitis and ocular discomfort. Histopathology revealed a multilobulated proliferation of chondrocytes producing hyaline cartilage with occasional pleomorphism and binucleate cells. A diagnosis of primary intraocular chondrosarcoma was done.

Foi atendido um cão, da raça Cocker Spaniel, de cinco anos de idade, com desconforto ocular, secreção purulenta abundante e aumento progressivo do bulbo ocular. Ao exame oftálmico, evidenciaram-se perfuração corneana, buftalmia e hiperemia conjuntival. Foi realizada enucleação em decorrência do desconforto ocular intenso e dos sinais de endoftalmite. Exame histopatológico revelou proliferação multilobulada de condrócitos produzindo cartilagem hialina com pleomorfismo ocasional e células binucleadas. Foi diagnosticado condrossarcoma intraocular primário.

Eficácia dos tratamentos estabelecidos pelo Manual da IETS, em oócitos, expostos à Leptospira interrogans / Effectiveness of treatments established by the Manual of IETS, in oocytes, exposed to Leptospira interrogans

Avaliou-se a eficácia dos tratamentos, definidos pela International Embryo Transfer Society (IETS), de oócitos bovinos, maturados in vitro e expostos experimentalmente à Leptospira interrogans sorovar Grippotyphosa. Os oócitos foram obtidos por meio de punção folicular, selecionados e distribuídos em quatro grupos, expostos ao patógeno e submetidos aos diferentes tipos de tratamentos. Foram expostos à cepa na concentração de 4,7.10(5)/µL, virulenta e não adaptada ao meio de manutenção EMJH, e, de 6,3.10(5)/µL, avirulenta e adaptada ao meio, por 24 horas. Os grupos tratados com tripsina ou antibióticos apresentaram eficácia de 21,7 por cento, e o grupo lavado sequencialmente 33,4 por cento. Os tratamentos não foram eficazes para os contaminados com a cepa avirulenta. Concluiu-se que as normas de controle de qualidade estabelecidas pela IETS poderiam ser revisadas e, possivelmente, redefinidas, uma vez que a eficácia dos tratamentos, provavelmente, não depende somente da espécie do patógeno, pois há interferência da virulência e de ação dos tratamentos sobre o tipo de patógeno.

The research purpose was to evaluate the effectiveness of treatments established by the IETS, in bovine oocytes maturated in vitro, exposed experimentally to Leptospira interrogans serovar Grippotyphosa. The oocytes were obtained through follicular puncture, selected and randomly allotted in four groups, exposed to the pathogen and subjected to different types of treatments. They were exposed to the strain in the concentration of 4.7x10(5)/µL virulent and not adapted to the EMJH, and to 6,3x10(5)/µL, virulent and adapted to the medium, for 24 hours. The treatments presented for the groups with trypsin or antibiotics, 21.7 percent efficiency, and the group washed sequentially presented 33.4 percent efficiency. The treatments were not effective for those infected with avirulent strain. In the statistical analysis, by χ², was found significance in the results obtained. The standards of quality control established by IETS could be reviewed and possibly redefined, since the effectiveness of treatment probably depends not only on the species of the pathogen, but is also affected by its virulence and treatment effectiveness.

Detecção direta de Actinobacillus pleuropneumoniae em órgãos de suídeos do estado de São Paulo pela técnica de reação em cadeia pela polimerase (nested-PCR) / Direct detection of Actinobacillus pleuropneumoniae in suidae organs in São Paulo state, Brazil, by polymerase chain reaction (nested-PCR)

A pleuropneumonia suína, causada pelo Actinobacillus pleuropneumoniae, é uma importante doença respiratória, responsável por prejuízos e queda de produtividade nas criações. Este trabalho teve como objetivo determinar a ocorrência de A. pleuropneumoniae em amostras de campo, mediante a adaptação e emprego de uma técnica de nested-PCR dirigida ao gene Apx IV. Definiu-se a sensibilidade analítica das técnicas de PCR e nested-PCR utilizando a amostra padrão A. pleuropneumoniae sorotipo III, em concentrações de DNA variando entre 30 µg/mL a 0,01 ng/ mL. Um total de trinta e sete amostras de campo encaminhadas ao Instituto Biológico entre 1995 a 2007 foram analisadas pelas técnicas de PCR e nested-PCR. A avaliação da sensibilidade analítica revelou que a PCR possui capacidade de gerar sinal a partir de 2 ng/mL de DNA extraído e a nested-PCR a partir de 0,4 ng/mL. Uma vez que a nested-PCR apresentou sensibilidade analítica cinco vezes maior se comparada à PCR para detecção de A. pleuropneumoniae em amostra padrão, o seu emprego pode minimizar a ocorrência de resultados tipo "falso-negativo". Dentre as amostras testadas, dez foram positivas à nested-PCR, sendo observada a ocorrência de A. pleuropneumoniae em nove diferentes animais, um deles javali. A presente técnica de nested-PCR pode ser utilizada para detecção direta de A. pleuropneumoniae em amostras de campo, mesmo após congelamento da amostra por longos períodos e sem necessidade de isolamento bacteriano prévio.

Porcine pleuropneumonia, caused by Actinobacillus pleuropneumoniae, is an important respiratory disease, responsible for economic losses and reduced productivity. The aim of this study was to determine occurrence of A. pleuropneumoniae in field samples, using an adapted nested-PCR reaction targeting the Apx IV gene. Different DNA concentrations (from 30 µg/mL to 0.01 ng/mL) of A. pleuropneumoniae serotype III reference strain were used to determine the level of sensitivity of first generation and nested-PCR reactions. Thirty-seven field samples sent to Instituto Biológico from 1995 to 2007 were tested by PCR and nested-PCR. Determination of the level of sensitivity showed that PCR could amplify to 2 ng/mL of extracted DNA and nested-PCR to 0.4 ng/mL. Since the nested reaction exhibited a level of sensitivity 5 times greater than the PCR reaction to detect a reference strain, using nested-PCR could minimize the occurrence of false-negative results. Among tested samples, 10 of them were nested-PCR positive, showing occurrence of A. pleuropneumoniae in 9 different animals (including one wild boar). This nested-PCR reaction can be used for direct detection of A. pleuropneumoniae in field samples, even after frozen storage for long periods, without the need for previous bacterial isolation.

Anti-parasitic action and elimination of intracellular Toxoplasma gondii in the presence of novel thiosemicarbazone and its 4-thiazolidinone derivatives

Toxoplasma, which infects all eukaryotic cells, is considered to be a good system for the study of drug action and of the behavior of infected host cells. In the present study, we asked if thiosemicarbazone derivatives can be effective against tachyzoites and which morphological and ultrastructural features of host cells and parasites are associated with the destruction of Toxoplasma. The compounds were tested in infected Vero cell culture using concentration screens (0.1 to 20 mM). The final concentration of 1 mM was chosen for biological assay. The following results were obtained: 1) These new derivatives decreased T. gondii infection with an in vitro parasite IC50 percent of 0.2-0.7 mM, without a significant effect on host cells and the more efficient compounds were 2, 3 (thiosemicarbazone derivatives) and 4 (thiazolidinone derivative); 2) The main feature observed during parasite elimination was continuous morphological disorganization of the tachyzoite secretory system, progressive organelle vesiculation, and then complete disruption; 3) Ultrastructural assays also revealed that progressive vesiculation in the cytoplasm of treated parasites did not occur in the host cell; 4) Vesiculation inside the parasite resulted in death, but this feature occurred asynchronously in different intracellular tachyzoites; 5) The death and elimination of T. gondii was associated with features such as apoptosis-like stage, acidification and digestion of parasites into parasitophorous vacuoles. Our results suggest that these new chemical compounds are promising for the elimination of intracellular parasites by mainly affecting tachyzoite development at 1 mM concentration for 24 h of treatment.

Behavior of mesenchymal stem cells stained with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) in osteogenic and non osteogenic cultures

4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) is a DNA dye widely used to mark and trace stem cells in therapy. We here studied the effect of DAPI staining on the behavior of mesenchymal stem cells cultured in either a control, non-osteogenic medium or in an osteogenic differentiation medium. In the control medium, the number of stem cells/field, as well as the number of fluorescent cells/field increased up to the sixth day in both control and DAPI-treated cultures. Afterwards, both the number of fluorescent cells and their fluorescence intensity decreased. Control cells were fusiform and with some long extensions that apparently linked them to neighboring cells, while DAPI-treated cells were mostly round cells with fine and short extensions. The trypan-blue exclusion method showed 99% cell viability in both groups, however, both alkaline phosphatase activity and the thiazolyl blue formazan assay (indicative of mitochondrial metabolism) gave significantly lower values in DAPI-marked cells The mitochondrial mass, as indicated by specific staining and flow cytometry, showed no differences between groups. Mesenchymal stem cells gave origin to mineralized nodules in the osteogenic differentiation medium and there were not DAPI-marked cells on the ninth day of culture. Alkaline phosphatase activity, viability assay and number of cells/field and of mineralized nodules/field were similar in both groups. So, DAPI treatment did not change cell viability and proliferation during osteogenic differentiation of mesenchymal stem cells. However, since these cells loose DAPI marking after 9 days in osteogenic cultures suggests that DAPI may not be an effective marker for mesenchymal stem cells implanted in bone tissue for long periods.

In vitro response of the human breast cancer cell line MDAMB-231 and human peripheral blood mononuclear cells exposed to 60Co at single fraction

Radiotherapy using gamma rays is a common modality of breast cancer treatment. The aim of this research is to investigate the biological response of the human breast cancer cell line MDAMB-231 and human peripheral blood mononuclear cells (PBMC) exposed in vitro to 60 Co irradiation at a single fraction of 10 Gy, 25 Gy and 50 Gy doses at 136,4 cGy.min-1 rate. Cells were irradiated at room temperature by the Theratron 80 radiotherapy system. Biological response was evaluated through cellular viability using MTT assay and nucleus damages visualized by Propidium Iodide assay and electrophoresis agarose gel after gamma irradiation. Nucleus damages induced by 60Co irradiation were compared to damage caused by cell exposure to 10 percent methanol. The 50 Gy dose of irradiation did not stimulate nuclus damages at the same level as that affected by 10 percent methanol induction in the MDAMB-231. Further studies are necessary to understand these mechanisms in the MDAMB-231 human breast carcinoma cell line.

Radioterapia utilizando radiação gama é uma modalidade comum no tratamento do câncer de mama. A proposta deste estudo é investigar a resposta biológica in vitro da linhagem celular MDAMB-231 de câncer de mama humano e células do sangue periférico humano (PBMC) expostas à irradiação pelo Co60 em frações simples de 10Gy, 25Gy e 50Gy e 136,4cGy min-1 rate. As células foram irradiadas a temperatura ambiente usando o equipamento de radioterapia Theratron 80 radiotherapy system. A resposta biológica, após irradiação gama, foi avaliada através do ensaio do MTT para viabilidade celular e o do ensaio com Iodeto de Propídio para visualização do dano nuclear, além da eletroforese em gel de agarose. Os danos nucleares induzidos pelo Co60 foram comparados aos danos causados pela exposição das células à solução de metanol a 10 por cento. Nós observamos que a dose de 50Gy não estimulou a mesma quantidade de danos nucleares que a solução de metanol a 10 por cento nas células MDAMB-231. Maiores estudos são necessários para a compreensão destes mecanismos nas células de câncer de mama humano MDAMB-231.

Antigenic stimulation is more efficient than LPS in inducing nitric oxide production by human mononuclear cells on the in vitro granuloma reaction in schistosomiasis

Nitric oxide (NO) is an extremely important and versatile messenger in biological systems. It has been identified as a cytotoxic factor in the immune system, presenting anti- or pro-inflammatory properties under different circumstances. In murine monocytes and macrophages, stimuli by cytokines or lipopolysaccharide (LPS) are necessary for inducing the immunologic isoform of the enzyme responsible for the high-output production of NO, nitric oxide synthase (iNOS). With respect to human cells, however, LPS seems not to stimulate NO production in the same way. Addressing this issue, we demonstrate here that peripheral blood mononuclear cells (PBMC) obtained from schistosomiasis-infected patients and cultivated with parasite antigens in the in vitro granuloma (IVG) reaction produced more nitrite in the absence of LPS. Thus, LPS-induced nitrite levels are easily detectable, although lower than those detected only with antigenic stimulation. Concomitant addition of LPS and L-N-arginine methyl ester (L-NAME) restored the ability to produce detectable levels of nitrite, which had been lost with L-NAME treatment. In addition, LPS caused a mild decrease of the IVG reaction and its association with L-NAME was responsible for reversal of the L-NAME-exacerbating effect on the IVG reaction. These results show that LPS alone is not as good an NO inducer in human cells as it is in rodent cells or cell lines. Moreover, they provide evidence for interactions between LPS and NO inhibitors that require further investigation.

Main features of DNA-based immunization vectors

DNA-based immunization has initiated a new era of vaccine research. One of the main goals of gene vaccine development is the control of the levels of expression in vivo for efficient immunization. Modifying the vector to modulate expression or immunogenicity is of critical importance for the improvement of DNA vaccines. The most frequently used vectors for genetic immunization are plasmids. In this article, we review some of the main elements relevant to their design such as strong promoter/enhancer region, introns, genes encoding antigens of interest from the pathogen (how to choose and modify them), polyadenylation termination sequence, origin of replication for plasmid production in Escherichia coli, antibiotic resistance gene as selectable marker, convenient cloning sites, and the presence of immunostimulatory sequences (ISS) that can be added to the plasmid to enhance adjuvanticity and to activate the immune system. In this review, the specific modifications that can increase overall expression as well as the potential of DNA-based vaccination are also discussed

Immunological properties of gene vaccines delivered by different routes

Gene vaccines represent a new and promising approach to control infectious diseases, inducing a protective immune response in the appropriate host. Several routes and methods of genetic immunization have been shown to induce antibody production as well as T helper (Th) cell and cytotoxic T lymphocyte activation. However, few studies have compared the nature of the immune responses generated by different gene vaccination delivery systems. In the present study we reviewed some aspects of immunity induced by gene immunization and compared the immune responses produced by intramuscular (im) DNA injection to gene gun-mediated DNA transfer into the skin of BALB/c mice. Using a reporter gene coding for ß-galactosidase, we have demonstrated that im injection raised a predominantly Th1 response with mostly IgG2a anti-ßgal produced, while gene gun immunization induced a mixed Th1/Th2 profile with a balanced production of IgG2a and IgG1 subclasses. Distinct types of immune responses were generated by different methods of gene delivery. These findings have important implications for genetic vaccine design. Firstly, a combination between these two systems may create optimal conditions for the induction of a broad-based immune response. Alternatively, a particular gene vaccine delivery method might be used according to the immune response required for host protection. Here, we describe the characteristics of the immune response induced by gene vaccination and the properties of DNA involved in this process

The role of T cell subsets and cytokines in the regulation of intracellular bacterial infection

Cellular immune responses are a critical part of the host's defense against intracellular bacterial infections. Immunity to Brucella abortus crucially depends on antigen-specific T cell-mediated activation of macrophages, which are the major effectors of cell-mediated killing of this organism. T lymphocytes that proliferate in response to B. abortus were characterized for phenotype and cytokine activity. Human, murine, and bovine T lymphocytes exhibited a type 1 cytokine profile, suggesting an analogous immune response in these different hosts. In vivo protection afforded by a particular cell type is dependent on the antigen presented and the mechanism of antigen presentation. Studies using MHC class I and class II knockout mice infected with B. abortus have demonstrated that protective immunity to brucellosis is especially dependent on CD8+ T cells. To target MHC class I presentation we transfected ex vivo a murine macrophage cell line with B. abortus genes and adoptively transferred them to BALB/c mice. These transgenic macrophage clones induced partial protection in mice against experimental brucellosis. Knowing the cells required for protection, vaccines can be designed to activate the protective T cell subset. Lastly, as a new strategy for priming a specific class I-restricted T cell response in vivo, we used genetic immunization by particle bombardment-mediated gene transfer.

Human giant cell formation induced in vitro by Schistosoma mansoni antigens

Although multinucleated giant cells have been described for many years in association with different chronic inflammatory responses, their participation in immunoregulatory mechanisms within the schistosome egg granulomas remains to be clarified. In this study we determined if soluble egg antigen (SEA) or adult worm antigen preparations (SWAP) from S. mansoni induce giant cell formation in vitro and their relationship with the intensity of granulomatous reactivity. Antigenic stimulation of peripheral blood mononuclear cells (PBMC) from patients (N = 9) with active schistosomiasis infection increased giant cell formation per field after the 12th day in culture when treated with S. mansoni SEA conjugated to polyacrylamide beads (PB-SEA) (17 +/- 1.2) and SWAP (PB-SWAP) (18.5 +/- 1.5). The increase in the number of giant cells was statistically significant when compared to the control polyacrylamide beads (PB) (9 +/- 1.1) and purified protein derivative conjugated to beads (PB-PPD) (11.6 +/- 1.7). We also observed a correlation between an increase in the number of giant cells and a decrease in in vitro granuloma index (GI) to PB-SEA (GI decreased from 4.3 +/- 0.2 on the 6th day to 3.2 +/- 0.2 on the 12th day) and PB-SWAP (GI decreased from 4.8 +/- 0.3 on the 6th day to 3.5 +/- 0.05 on the 12th day). These data suggest that giant cell formation may be one of the immunoregulatory mechanisms involved in the down-regulation of the granuloma reaction against S. mansoni eggs

In vitro granuloma modulation induced by immune complexes in human Schistosomiasis mansoni

Immune complexes (IC) were isolated from sera of six chronic schistosomiasis patients in order to study the regulatory mechanisms of granulomatous hypersensitivity to Schistosoma mansoni egg antigens in vitro. Purified blood mononuclear cells (PBMC) from patients (N = 14) with active schistosome infection when treated with a pool of isolated IC were able to inhibit granulomatous hypersensitivity as determined in an in vitro model of granuloma formation. The suppressive effect of IC on granuloma index varied from 33 per cent to 73 per cent . Analysis of the in vitro proliferation of PBMC from individuals infected with S. mansoni on blastogenesis assay (N = 7) showed that isolated IC were able to induce a suppression degree on cell proliferation from 31 per cent to 93 per cent . Significant inhibition of the in vitro granuloma reaction continued to be present after treatment of PBMC with supernatant from IC treated chronic patient cells. These results demonstrate that circulating IC may down-regulate granulomatous hypersensitivity to S. mansoni eggs in patients with chronic intestinal schistosomiasis

Immunoregulation in human Schistosomiasis by idiotypic interactions and lymphokine-mediated mechanisms

Anti-idiotypic (anti-Id) T cells from schistosomiasis patients or former patients proliferate upon exposure to polyclonal or monoclonal anti-soluble egg antigen (SEA) antibodies. Chloroquine does not inhibit, the response, which is induced by F(ab')2 (but not soluble Fab) fragments of these antibodies. Purified T cells from former patients require macrophages or exogenous IL-1 to respond to anti-SEA Ids and can respond to matrix-bound Fab fragments in the presence of IL-1. These anti-Id T cells recognize the Ids directly. Chronic schistosomiasis patients immunoregulate the production of a non-IL-2 lymphokine that stimulates IL-2 receptor expression on resting T cells. This regulation is reversed upon chemotherapeutic cure.

Nivel sorologico para rubeola em uma populacao de gestantes da cidade do Salvador. / Serum level for rubella in a population of pregnant women in the city of Salvador

Os autores testaram o nivel sorologico para rubeola em 416 soros de gestantes de duas maternidades que atendem pacientes de diferentes condicoes socio-economicas, na cidade do Salvador (utilizou-se a tecnica de Inibicao de Hemaglutinacao). A positividade nas gestantes foi de 74,5%, obtendo-se um percentual de 25,5% de suscetiveis. A susceptibilidade foi significantemente maior entre as gestantes da maternidade de nivel socio-economico mais baixo.Estudaram-se tambem as faixas etarias, estando os grupos mais jovens com maior chance de infeccao