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During the last 10 years an increasing number of plants for re-use of refuse have been constructed in Europe and the U.S.A. During the same period several cases of occupational respiratory diseases among workers have been reported in the recycling industry. The aim of this project was to show, in vitro, if there is any change in the cytotoxic potential of garbage dust during the process of converting household waste to compost. Two cell lines have been exposed to extracts of waste fuel pellets and compost, taken from three different time periods in the composting process. Sig nificant differences were found in the cytotoxic potential of extracts of household waste (P-1 raw compost, fresh compost and matured compost show a cytotoxic effect at 97, 41 and 44%, respectively of unexposed cells. In conclusion, these results show the greatest cytotoxic potential when the microbial activity seems to be at its height in the composting process. Earlier, studies on the effect of endotoxin (lipopolysacchande, LPS) on the cells, and with pure endotoxin did not find any cytotoxic effect in the assay. Further mvestigations are needed in order to find which micro-organisms or components from these are responsible for the cytotoxic potential. © 1997 ISWA

Objectives: Drugs used for toxoplasmosis have limited efficacy and also severe side effects. A new drug with good efficacy and limited side effects is need of the hour. We studied the effects of artemether on Toxoplasma gondii in vitro conditions. Materials and Methods: Artemether (methyl-ether-qinghaosu) was tested for tachyzoites, J774, and Vero cell lines infected by T. gondii. For evaluating the effect of drugs on Vero cells infected with T. gondii, we designed two separate experiments; in the first experiment, the Vero cells were infected with tachyzoites and then treated with artemether; while in the second one, the tachyzoites were exposed to artemether and then Vero cells were infected with treated tachyzoites. For evaluating the apoptotic effect of artemether on tachyzoites and infected J774 macrophages cell line with T. gondii , we used flow cytometry method. Inhibitory concentration (IC 50 ) was evaluated by intracellular replication of tachyzoites in Vero cells. Results: IC 50 for infected Vero cells with tachyzoites was determined as 49.13 μg/ml. In pretreated tachyzoites with artemether before entering into Vero cells, IC 50 was calculated as 13.15 μg/ml. In both experiments, artemether showed a higher inhibitory effect than sulfadiazine (positive control). Artemether even at the highest concentrations only showed low cytotoxicity on Vero and J774 cell lines. Apoptosis in tachyzoites rise with an increasing concentration of artemether. Conclusions: Our findings indicate that artemether is effective to control the tachyzoites of T. gondii in vitro and maybe a good alternative drug for toxoplasmosis.

Influenza viruses unable to express NS1 protein (delNS1) replicate poorly and induce large amounts of interferon (IFN). They are therefore considered candidate viruses for live-attenuated influenza vaccines. Their attenuated replication is generally assumed to result from the inability to counter the antiviral host response, as delNS1 viruses replicate efficiently in Vero cells, which lack IFN expression. In this study, delNS1 virus was parallel passaged on IFN-competent MDCK cells, which resulted in two strains that were able to replicate to high virus titers in MDCK cells due to adaptive mutations especially in the M-gene segment but also in the NP and NS gene segments. Most notable were clustered U-to-C mutations in the M segment of both strains and clustered A-to-G mutations in the NS segment of one strain, which presumably resulted from host cell-mediated RNA editing. The M segment mutations in both strains changed the ratio of M1 to M2 expression, probably by affecting splicing efficiency. In one virus, 2 amino acid substitutions in M1 additionally enhanced virus replication, possibly through changes in the M1 distribution between the nucleus and the cytoplasm. Both adapted viruses induced levels of IFN equal to that of the original delNS1 virus. These results show that the increased replication of the adapted viruses is not primarily due to altered IFN induction but rather is related to changes in M1 expression or localization. The mutations identified in this paper may be used to enhance delNS1 virus replication for vaccine production.

The filovirus plaque assay serves as the assay of choice to measure infectious virus in a cell culture, blood, or homogenized tissue sample. It has been in use for more than 30 years and is the generally accepted assay used to titrate virus in samples from animals treated with a potential antiviral therapeutic or vaccine. As these animal studies are required for the development of vaccines and therapeutics under the FDA Animal Rule, it is essential to have a standardized assay to compare their efficacies against the various filoviruses. Here, we present an evaluation of the conditions under which the filovirus plaque assay performs best for the Ebola virus Kikwit variant and the Angola variant of Marburg virus. The indicator cell type and source, inoculum volumes, length of incubation and general features of filovirus biology as visualized in the assay are addressed in terms of the impact on the sample viral titer calculations. These optimization studies have resulted in a plaque assay protocol which can be used for preclinical studies, and as a standardized protocol for use across institutions, to aid in data comparison. This protocol will be validated for use in GLP studies supporting advanced development of filovirus therapeutics and vaccines.

The present work was carried out to evaluate the in vitro development of bovine embryo co-cultured in granulosa, oviduct, BRL or VERO cells co-cultures, supplemented with 5% or 10% of Fetal Calf Serum (FCS). Cummulus oocyte complexes were aspirated, matured and fertilized in vitro. Embryonic structures were divided into eight treatments. They were placed in culture media TCM 199 containing granulosa, oviduct, BRL or VERO cells, each of them added with 5% or 10% FCS. The conditions for the co-culture were 38.5 ºC, 5% CO2 in air and high humidity for ten consecutive days. Cleavage, blastocyst and hatching rates did not differ (p > 0.05) in co-culture with primary cells (granulosa and oviduct) when FCS concentration increased from 5 to 10%. However, in continuous cells co-culture (BRL and VERO), when FCS concentration increased from 5% to 10%, the blastocyst development rate decreased significantly (p < 0.05) from 33.6 to 16.3% and from 40 to 16.5% in embryo co-culture with BRL and VERO cells, respectively. O presente trabalho foi realizado com o objetivo de avaliar o desenvolvimento in vitro de embriões bovinos co-cultivados em células da granulosa, do oviduto, BRL e VERO, suplementados com 5% ou 10% de Soro Fetal Bovino (SFB). Os complexos cummulus oócitos foram aspirados, maturados e fecundados in vitro. As estruturas embrionárias foram divididas em oito tratamentos: co-cultivo em TCM 199 contendo células da granulosa, do oviduto, BRL ou VERO adicionadas com 5% ou 10% de SFB. As condições de cultivo foram 38.5 ºC, 5% CO2 em ar e alta humidade por dez dias consecutivos. Os índices de clivagem, blastocisto e eclosão não diferiram (p > 0,05) no co-cultivo com células primárias (granulosa e oviduto) quando a concentração de SFB aumentou de 5 para 10%. Entretanto, no co-cultivo com células de linhagens contínuas (BRL e VERO), quando a concentração de SFB aumentou de 5% para 10%, os índices de blastocistos diminuíram significativamente (p < 0,05) de 33,6 para 16,3 % e de 40 para 16,5% nos embriões bovinos co-cultivados com células VERO e BRL, respectivamente.

The Laboratory diagnosis of Bartonella henselae infection is mainly based on serological testing by indirect immunofluorescence assay (IFA). Cell line co-cultivation with B.henselae and agar derivated antigens are the two major procedures used for evaluation of anti-Bartonella antibodies. Vero and Hep-2 cell lines are the most commonly used media for co-cultivation both in-house and commercial diagnostic kits production. However, HeLa cells which are easily supplied and grown, also can easily be infected by B.henselae. The aim of this study was to compare the performances of antigens obtained by co-cultivation of B.henselae ATCC 49882 (Houston-1) in Vero and HeLa Cells in IFA serology. Out of 381 sera samples, 127 (33.3%) were found positive and 195 (51.2%) were found negative by IFA performed by both cell line co-cultivations. The total agreement between the methods were found as 84.5% (322/381), and Cohen kappa value was calculated as 0.68 (strong, coherent). As a result, HeLa cells were found to be useful for the preparation of B.henselae antigens to be used in IFA for the serologic diagnosis of B.henselae infections. However different genotype strains and cross reactions with other infectious agents should be investigated by further studies before routine applications of HeLa cell co-cultivations procedure is established.

The presentation of a recent book by Andrea Nicolotti provides a valuable opportunity to examine the procedures of so-called sindonology. In turn, this is the chance to reflect upon a recent, worrying trend by authors, publishers and mass media, who popularise documentary forgeries and arbitrary reconstructions of fictitious historical events. Nicolotti's book develops his useful analyses to examine recent attempts to corroborate two theses, which turn out to be completely unfounded on close examination. The first claims the identification of the Shroud of Turin ‒ whose existence cannot be historically documented before the second half of the XIVth century – and the famous Mandylion, preserved in Edessa and transferred to Constantinople in 944. The second, building upon the first one, aims to demonstrate the acquisition of Jesus' Shroud by the Templars in the second half of the XIIIth century, after it had reached the West as crusaders's spoil stolen during the pillage of the Byzantine capital in 1204. Consequent to this absurd build-up of hypotheses, the mysterious idol, which some Templars under torture during their trial in 1307 confessed to worship in their initiation ceremonies, would be nothing but the burial sheet of Jesus of Nazareth, which has been brought to Turin in 1578 and is still preserved there today.

Introducción: Lippia origanoides H.B.K. (Verbenacea), es una planta aromática conocida comúnmente como "orégano". Los aceites esenciales de 8 muestras de L. origanoides y algunos de sus componentes mayoritarios fueron evaluados in vitro sobre la línea tumoral HeLa y la línea no tumoral Vero para identificar su potencial citotóxico. Materiales y métodos: la concentración inhibitoria cincuenta (IC50) se determinó mediante la técnica fotocolorimétrica del MTT (3-(4,5-Dimetiltiazol-2-il)-2,5-bromuro difeniltetrazolio) y los valores de IC50 se obtuvieron por análisis estadístico mediante regresión lineal simple. El índice de selectividad (IS), definido como la IC50 en células Vero sobre IC50 en células HeLa, fue calculado con el fin de encontrar aceites o componentes con potencial citotóxico selectivo hacia líneas celulares tumorales. Resultados y conclusiones: se determinó por cromatografía de gases y espectrometría de masas GC/MS la composición química de los aceites más citotóxicos. El aceite de L. origanoides que presentó la mayor actividad citotóxica sobre células HeLa con un valor de IC50 de 9,1 ± 1 μg/mL e índice de selectividad de 7,1, fue identificado como quimiotipo trans-β-cariofileno/ρ-cimeno. Los componentes mayoritarios del aceite quimiotipo trans-β-cariofileno/ρ-cimeno fueron: trans-β-cariofileno (11,3%), ρ-cimeno (11,2%), α-felandreno (9,9%), limoneno (7,2%), 1,8-cineol (6,5%) y α-humuleno (6,0%). Los componentes mayoritarios evaluados no mostraron actividad citotóxica relevante sobre células HeLa, sólo el limoneno y β-mirceno presentaron valores de IS, respectivamente, de 6,97 y 3,01. Sin embargo, los valores de IC50 fueron más altos que el del aceite activo. Estos resultados sugieren que la actividad citotóxica de los aceites no se debe sólo a sus componentes mayoritarios, sino a un sinergismo entre sus componentes. Salud UIS 2009; 41: 215-222 Introduction: Lippia origanoides H.B.K. (Verbenaceae) is an aromatic plant commonly called as "oregano". Eight essential oils of L. origanoides and some of their main components were evaluated in vitro on tumor cell line HeLa and non-tumor cell line Vero to identify tumoural cytotoxic potential. Materials and methods: Inhibition 50% of cell population (IC50) was determined using the photo-colorimeter technique MTT (3 - (4.5-dimethylthiazol-2-yl)-2,5-difeniltetrazolium bromide). IC50 values were obtained by linear regression analysis. The selectivity index (SI), defined as Vero IC50 on HeLa IC50, it was calculated in order to find oil or major components with selective tumor cytotoxic potential. Results and conclusions: the chemical composition of the oil most cytotoxic was determined by gas chromatography and mass spectrometry GC/MS. The L. origanoides oil identified as chemotype trans-β-caryophyllene/p-cymene showed the highest cytotoxic activity on HeLa cells with IC50 value of 9.1 ± 1 μg/mL and selectivity index of 7.1. The main components were: trans-β-caryophyllene (11.3 ρ-cymene (11.2%), α-phellandrene (9.9%), limonene (7.2%), 1.8-cineol (6.5%) and α-humulene (6.0%). The most of the major components did not show cytotoxic activity on HeLa cells, only limonene and β-myrcene showed IS values of 6.97 and 3.01, respectively. However, the IC50 values were higher than active oil. These results suggest that cytotoxic activities of the oils are not only due to their main components, but to a synergism among its components. Salud UIS 2009; 41: 215-222.

Conselho Nacional de Desenvolvimento Científico e Tecnológico Neosporosis is a parasitic disease of wide distribution and great importance to the cattle industry, mainly due to its associated reproductive losses. The life cycle of Neospora caninum typified by the tree know infectious stages: tachyzoites, tissue cysts with bradyzoites, and oocysts. Transmission routes can be horizontal and/or vertical. The vertical transmission or transplacentally is the most frequent form of infection, and an important form of maintain the agent in herds. With aim of to determine the occurrence of anti-Neospora caninum antibodies in serum samples of 260 bovine fetuses, collected in a slaughter in the municipality of Santa Maria, in Rio Grande do Sul, Brazil. For detection antibodies anti-N. caninum indirect fluorescent antibody test was used and immunoglobulin G and M were detected, using a cut-off 1:25. Of the 260 serum samples tested, 15% (39/260) were positive for the presence of anti-N. caninum. Of these, in 38 the presence of IgG where detected (97.4%) and in six IgM were present (10.3%). Five samples (15.4%) tested were positive for both IgG and IgM. The results reaffirming the ability of N. caninum in determine fetal infection. The results presented on the first chapter indicated that the search of IgM anti-N. caninum is of very limited help in the detection of the transplacental infection in cattle. In second chapter, was evaluated the susceptibility to infection by N. caninum in different cell cultures, for the purpose of observe the ability in vitro multiplication this agent. For this, eight cell cultures were tested, among the cell cultures tested, four presented good susceptibility to agent: cell lines VERO (yield of 21.2 tachyzoites/cell) and MA-104 (17.1); primary bovine testicle (16.3) and lung cells (13.6). Primary bovine kidney (8.2 tachyzoites/cell), MDBK (5.1) and RK-13 cell lines (0.4) presented moderate to low sensitivity. No viable tachyzoites were detected in the culture of MDCK cells. These results demonstrate that MA-104 cells present adequate susceptibility to N. caninum compared to VERO cells, which have been largely used to multiply the parasite in vitro. Due to the easy manipulation, quick multiplication and relatively low nutritional equirements, these results indicate that MA-104 cells are adequate for multiplication of N. caninum in vitro. A neosporose é uma doença parasitária de ampla distribuição e com grande importância para a bovinocultura, principalmente pelas perdas reprodutivas que determina. O ciclo do Neospora caninum caracteriza-se por apresentar três estágios infecciosos: os taquizoítos, os cistos teciduais contendo bradizoítos e os oocistos. Suas rotas de transmissão podem ser horizontal e/ou vertical. A infecção vertical ou transplacentária é a forma mais freqüente de infecção, sendo uma importante forma de manutenção do agente nos rebanhos. Com o objetivo de determinar a ocorrência de anticorpos anti-N. caninum em fetos bovinos, foram coletadas 260 amostras de soro em abatedouro localizado no município de Santa Maria, Rio Grande do Sul, Brasil. Para detecção de anticorpos anti-N. caninum, utilizou-se a técnica de imunofluorescência indireta com a presença de imunoglobulinas G e M (IgG e IgM), sendo analisada com ponto de corte de 1:25. Do número total de amostras testadas, 15% (39/260) foram positivas para anticorpos anti-N. caninum. Destas, em 38 (97,4%) foi detectada a presença de IgG anti-N. caninum e em seis (15,4%) de IgM. Em cinco amostras (12,8%) detectaram-se ambos, IgG e IgM. Os resultados reafirmam a capacidade do N. caninum determinar infecção fetal. Os resultados obtidos no primeiro capítulo desta dissertação permitiram demonstrar que a pesquisa de IgM foi de limitada importância na detecção da infecção via transplacentária em soro fetal bovino. No segundo capítulo, foi avaliada a susceptibilidade à infecção pelo N. caninum em diferentes cultivos celulares, com a finalidade de observar a capacidade de multiplicação deste agente in vitro. Para isto, foram testados oito cultivos, sendo que quatro apresentaram boa susceptibilidade a multiplicação pelo N. caninum: células VERO (produção de 21,2 taquizoítos/célula), MA-104 (17,1), cultivo primário de testículo (16,3) e pulmão bovino (13,6). O cultivo primário de rim bovino (8,2), células MDBK (5,1) e RK-13 (0,4) apresentaram baixa sensibilidade, enquanto células MDCK não produziram taquizoítos viáveis. Os resultados obtidos demonstram que as células MA-104 apresentaram susceptibilidade semelhante a das células VERO linhagem tradicionalmente utilizada para o cultivo deste protozoário. Pela facilidade de cultivo e rápida multiplicação, menor exigência nutricional e produção de taquizoítos em níveis semelhantes às células VERO, as células MA-104 demonstraram ser adequadas para a manutenção e multiplicação do N. caninum in vitro.

Mepacrine is an antiproliferative agent, characterised by an aliphatic chain similar to that of natural polyamines whose activation is closely associated with cell proliferation and may lead to malignant transformation and neurodegenerative diseases. This study aims to investigate a possible antagonism between mepacrine and polyamines in tumour proliferation.MCF-7 and Vero cells were cultured in Eagle's minimum essential medium and then subjected to graded concentrations of putrescine, spermine and spermidine alone and in combination with mepacrine. Methyl thiazole tetrazolium test and Western-blotting were performed.Putrescine and spermidine at 0.5 mg/l significantly stimulated cell growth, whereas mepacrine treatment confirmed the enhanced p21 expression previously reported by a recent study and growth inhibition. When used in combination, mepacrine antagonized MCF-7 growth induced by polyamines.Our results suggest that mepacrine may represent a choice in the treatment of tumours induced by the modified concentration of polyamines.

Previous in vivo studies performed in our laboratories demonstrated that anti-malarial drugs may or enhance or slow down Ehrlich's ascites tumour progression in infected mice. In the light of these observations, an in vitro study was undertaken to assess the response of human tumour cells to various anti-malarial drugs and consequently the safety of the anti-malarial therapy.MCF-7 cells and Vero cells (control line) were cultured in Eagle's minimum Essential medium (EMEM) and then subjected to graded concentrations of different anti-malarial drugs. Trypan-blue exclusion, MTT and Western blotting tests were performed.The findings showed that pyrimethamine (12.5 mg/L), chloroquine (12.5 mg/L) and primaquine (1.56 mg/L) stimulated MCF-7 cell growth. The proliferative effect was inhibited by doxorubicin only in cultures treated with chloroquine and primaquine. These results might indicate that some anti-malarial drugs have a worrying tumour-promoting effect which should not be underestimated when undertaking anti-malarial prophylactic measures.

During HSV-1 infection, IE gene expression triggers apoptosis, but subsequent synthesis of infected cell proteins blocks apoptotic death from ensuing. This “HSV-1-dependent” apoptosis was identified in HEp-2/HeLa cells infected with wild-type HSV-1 in the presence of an inhibitor of protein synthesis or a virus lacking ICP27 {HSV-1(vBSΔ27)}. Unlike HEp-2/HeLa cells, vBSΔ27-infected Vero cells fail to exhibit dramatic apoptotic morphologies at times prior to 24 hpi. Here, we examined the basis of these different apoptotic responses to HSV-1. We found that infected Vero cells take substantially longer than HEp-2/HeLa cells to display membrane blebbing, chromatin condensation, DNA laddering, and PARP cleavage. Vero, but not HEp-2/HeLa, cells required de novo protein synthesis to exhibit efficient HSV-1-dependent apoptosis, which included changes in mitochondrial membrane potential, and these factors were produced prior to 3 hpi. Vero cells infected with recombinant viruses devoid of the ICP27 and ICP4 proteins alone or both the ICP27 and ICP22 proteins were apoptotic. These results indicate a requirement for cellular or other viral protein synthesis in Vero cells and provide insight into cell type differences in HSV-1-dependent apoptosis.

A comparative study was performed in the production of different viral antigens by using microcarrier systems and traditional systems. Vero, BHK and MA 104 cells were cultivated in microcarriers (2mg/ml) using a bioreactor with a working capacity of 3.7 liters, in parallel with conventional Roux bottles. After four days (BHK cells), and seven days of culture (Vero and MA-104 cells), the cells were infected with 0.1 MOI (multiplicity of infection) of rabies virus, measles virus, poliovirus and rotavirus. The yields of the cells and virus in microcarriers and in the conventional system were determined. It was observed that in the microcarrier system, an average increase of twenty-fold more cells/ml was obtained in relation to the conventional monolayer culture, using Roux bottle. On the other hand, cells grown in Roux bottles presented 1.3 to 6.7 more viruses/ml culture than those in the microcarrier systems. However, the overall data showed that yieldings, in terms of viruses per batch, were statistically similar for both systems (p > 0.05). The amount of viral antigen production seems to depend not only on cell concentration, but also on other culture factors such as the characteristic of the cell-growth surface. Thus, the present findings provide a baseline for further improvements and strategies to be established for a scaling-up virus production since depending on the type of virus the optimal conditions found for a small-scale virus production seem unsuitable for large-scale production, requiring new standardization and evaluation. Foi realizado estudo comparativo na produção de diferentes antígenos virais usando sistema de microcarregador e sistema tradicional. Células Vero, BHK e MA-104 foram cultivadas em microcarregadores (2mg/ml) utilizando-se biorreatores com capacidade de 3,7 litros e, em paralelo, no sistema convencional com garrafas Roux. Após quatro dias de cultura para as células BHK e sete dias para as células Vero e MA-104, as células foram infectadas com 0,1 MOI (multiplicidade de infecção) de vírus da raiva, vírus do sarampo, poliovírus e rotavírus. Foi determinado o rendimento das células e dos vírus em microcarregadores e sistema convencional. Foi observado no sistema de microcarregador um aumento médio obtido de vinte vezes mais células/ml em relação à cultura convencional em monocamadas, usando garrafas Roux. Por outro lado, as células que cresceram em garrafas Roux apresentaram 1,6 a 6,7 mais vírus/ml em culturas do que no sistema de microcarregador. Contudo o total das amostras em termos de vírus por grupo foi estatisticamente similar para ambos os sistemas (p > 0,05). O rendimento na produção de antígeno viral pode depender não somente da concentração das células, mas também de outros fatores da cultura, como características do suporte no crescimento. Assim, o estudo deste parâmetro pode proporcionar uma linha de base para um futuro melhoramento e estratégias para se estabelecer um aumento em escala na produção de vírus, já que, dependendo do tipo de vírus, a ótima condição encontrada para uma produção de vírus em pequena escala pode não ser adequada para a produção em grande escala, requerendo novas padronização e avaliação.

L' omaggio di un allievo al metodo critico del proprio Maestro

Resumen basado en el de la publicación En el llamado proceso de Copenhague se ha puesto de manifiesto la insuficiencia de los modelos tradicionales para la medida de la calidad –ISO (Organización Internacional de Normalización), FQM (Fundación Europea para la Gestión de la Calidad)- lo que ha motivado que grupos diferentes de trabajo busquen otras respuestas más adecuadas a la solución del problema de la citada medida, desde la preparación y aproximación definidora de las competencias de los formadores. En la búsqueda los modelos de autoevaluación contrastados por expertos de los 25 países de la Unión Europea han dado respuestas altamente positiva. En la búsqueda ha resultado de estimable claridad el modelo VERO (Verificación de Escenarios Organizacionales). ESP

The response to lead nitrate has been assessed in two cell lines of renal origin. The range of toxic concentrations was determined by Neutral Red assay after 24-h of exposure. Morphological changes in the Buffalo Green Monkey (BGM) and VERO cell lines after exposure to subcytotoxic doses (1.38 mM and 1.04 mM, respectively) equivalent to EC10 (effective concentrations 10%) of lead nitrate were evaluated at the ultrastructural level by transmission microscopy. The most notable finding in treated cells was the presence of inclusion bodies in the form of irregular granules of varying size in both cytoplasm and lysosomes. Cell membrane integrity was not affected. The number of phagolysosomes and myeline figures associated to the inclusion bodies was higher than in the control cultures. We conclude that the phagolysosomic mechanism fails to digest this metal ion and the BGM and VERO renal cell lines can be considered as useful tools for toxicological studies involving lead nitrate.

An acceptable live-attenuated dengue virus vaccine candidate should have low potential for transmission by mosquitoes. We have identified and characterized a mutation in dengue virus type 4 (DEN4) that decreases the ability of the virus to infect mosquitoes. A panel of 1248 mutagenized virus clones generated previously by chemical mutagenesis was screened for decreased replication in mosquito C6/36 cells but efficient replication in simian Vero cells. One virus met these criteria and contained a single coding mutation: a C-to-U mutation at nucleotide 7129 resulting in a Pro-to-Leu change in amino acid 101 of the nonstructural 4B gene (NS4B P101L). This mutation results in decreased replication in C6/36 cells relative to wild-type DEN4, decreased infectivity for mosquitoes, enhanced replication in Vero and human HuH-7 cells, and enhanced replication in SCID mice implanted with HuH-7 cells (SCID-HuH-7 mice). A recombinant DEN4 virus (rDEN4) bearing this mutation exhibited the same set of phenotypes. Addition of the NS4B P101L mutation to rDEN4 bearing a 30 nucleotide deletion (Δ30) decreased the ability of the double-mutant virus to infect mosquitoes but increased its ability to replicate in SCID-HuH-7 mice. Although the NS4B P101L mutation decreases infectivity of DEN4 for mosquitoes, its ability to enhance replication in SCID-HuH-7 mice suggests that it might not be advantageous to include this specific mutation in an rDEN4 vaccine. The opposing effects of the NS4B P101L mutation in mosquito and vertebrate systems suggest that the NS4B protein is involved in maintaining the balance between efficient replication in the mosquito vector and the human host.

Dengue virus type 2 (DENV-2) infection induced apoptotic cellular DNA fragmentation in Vero cells within 8 days of infection. The addition of high concentrations of extracellular Zn(2+) but not Ca(2+), Mg(2+) or Mn(2+) to the cell culture medium hastened the detection of apoptosis to within 4 h after infection. No apoptotic cellular DNA fragmentation was detected in the cell culture treated with Zn(2+) alone or infected with heat- or ultraviolet light-inactivated DENV-2 in the presence of Zn(2+). These results suggest that (i) apoptosis is induced in African green monkey kidney cells infected with live DENV-2 and (ii) the addition of high extracellular Zn(2+) accelerates detection of apoptosis in the DENV-2-infected cells.

O presente trabalho foi realizado com o objetivo de avaliar o desenvolvimento in vitro de embriões bovinos co-cultivados em células da granulosa, do oviduto, BRL e VERO, suplementados com 5% ou 10% de Soro Fetal Bovino (SFB). Os complexos cummulus oócitos foram aspirados, maturados e fecundados in vitro. As estruturas embrionárias foram divididas em oito tratamentos: co-cultivo em TCM 199 contendo células da granulosa, do oviduto, BRL ou VERO adicionadas com 5% ou 10% de SFB. As condições de cultivo foram 38.5 ºC, 5% CO2 em ar e alta humidade por dez dias consecutivos. Os índices de clivagem, blastocisto e eclosão não diferiram (p > 0,05) no co-cultivo com células primárias (granulosa e oviduto) quando a concentração de SFB aumentou de 5 para 10%. Entretanto, no co-cultivo com células de linhagens contínuas (BRL e VERO), quando a concentração de SFB aumentou de 5% para 10%, os índices de blastocistos diminuíram significativamente (p < 0,05) de 33,6 para 16,3 % e de 40 para 16,5% nos embriões bovinos co-cultivados com células VERO e BRL, respectivamente.

Previous papers have reported that the syncytial mutant HSV-1(13)S11 carries three segregable syn mutations and exhibits its altered phenotype in four different cell lines, i.e. HEp-2, VERO, BHK and HEL both at 34 degrees C and 39 degrees C. Those studies have shown that one of three syncytial loci, designated Syn 5, is located in the Bam HI Q fragment spanning map units 0.296-0.317 of the prototype arrangement. Recombinants obtained from marker transfer experiments with donor BamHI Q fragment, have shown that locus Syn 5 is able to induce cell-to-cell fusion in VERO, BHK and HEL but not in HEp-2 cells. In this paper we have characterized the syn mutant HSV-1(13)S11 with regard to plaque morphology, synthesis of viral polypeptides and glycoproteins, thymidine kinase activity and physical map position of locus Syn 5 on the genome. Pertinent to the syn phenotype, earlier papers claimed that two different polypeptides, thymidine kinase (TK) and glycoprotein H (gH), whose genes map in BamHI Q, may be responsible for the fusion activity. Functional studies on the TK of the syn mutant HSV-1(13)S11 indicate that this polypeptide accumulates normally in infected cells and is a fully active enzyme. The other gene product, gH, has been studied with SDS-PAGE and in radioimmunoprecipitation (RIP) experiments using specific monoclonal antibodies. The results indicate that the amount of gH accumulation in the syn mutant-infected cells is greater than its parental strain. However, new marker transfer experiments described here located locus Syn 5 in 663 base pairs between SstI and EcoRI restriction endonuclease sites at the right end of the BamHI Q fragment, where TK gene overlaps in opposite orientation with UL 24 gene. Altogether these results indicate that the Syn 5 locus segregates from the gene specifying gH, to a region encompassing portions of the TK and UL 24 genes, and that the syn mutation does not affect the expression or activity of TK.

The evidence supporting organic dust as a cause of occupationally-related lung disease has substantially increased in the post two decades. The aim of this project was to test an assay which could be used to detect cytotoxic potential of organic dusts found in working environments. A cytotoxic assay with two cell lines was used: VERO and human A 549 carcinoma cells. Cells were exposed to extracts of dust samples collected from five different occupational environments with known adverse respiratory effects, using two different extraction protocols. Compared to water, alkaline soluble extract exerted the greater cytotoxic effect on the cells. With this assay it was possible to distinguish differences in the cytotoxic potential of the different types of organic dusts. The most aggressive dusts proved to be those with a high microbial content, such as compost and grain dust, which exerted an effect already after 2 hours of incubation at low concentrations.

The laboratory diagnosis of Bartonella henselae infection is mainly based on serological testing by indirect immunofluorescence assay (IFA). Cell line co-cultivation with B.henselae and agar derivated antigens are the two major procedures used for evaluation of arnti-Bartonella antibodies. Vero and Hep-2 cell lines are the most commonly used media for co-cultivation both in-house and commercial diagnostic kits production. However, HeLa cells which are easily supplied and grown, also can easily be infected by B.henselae. The aim of this study was to compare the performances of antigens obtained by co-cultivation of B.henselae ATCC 49882 (Houston-1) in Vero and HeLa Cells in IFA serology. Out of 381 sera samples, 127 (33.3%) were found positive and 195 (51.2%) were found negative by IFA performed by both cell line co-cultivations. The total agreement between the methods were found as 84.5% (322/381), and Cohen kappa value was calculated as 0.68 (strong, coherent). As a result, HeLa cells were found to be useful for the preparation of B.henselae antigens to be used in IFA for the serologic diagnosis of B.henselae infections. However different genotype strains and cross reactions with other infectious agents should be investigated by further studies before routine applications of HeLa cell co-cultivations procedure is established.

Background: Mentha species are widely used in traditional medicine mostly as anti-flatulence. Nowadays, their usage as flavor and preservative in food, cosmetic and pharmaceutical industries has been developed. Moreover, cytotoxic effects of some Mentha species have been reported. Objective: In this study, cytotoxic properties of Mentha piperita, M. spicata, M. aquatica, M. crispa, M. pulegium and M. longifolia have been investigated. Methods: Different concentrations of essential oils and total extracts of six Mentha species were tested by MTT assay against Vero, Hep2 and Hela cell lines. Results: The results showed that all samples were toxic against Vero, Hela and Hep2 cell lines (IC50 28.1-166.2 µg/ml). Conclusion: All examined Mentha species extracts and essential oils have cytotoxic effects but some of them could be considered as potent toxic agents.