Edda de Rizzo

Induction of hemosome formation and hemoglobin biosynthesis in HeLa cells.

SummaryTissue cultures of HeLa cells in Hanks medium suspension, previously mixed with a solution containing reticulocyte polysomes and/or free globin, hemoglobin and blood plasma from bled rabbits, were homogenized, and an electrophoretic pattern of hemoglobin band was obtained from the supernatant; hemoglobin denatures under the tissue culture conditions. HeLa cells show typical hemosomes, identical to those found in immature erythrocytes of embryos, and adult mammals with hemolytic anemias. When HeLa cell suspensions are mixed with solutions void of anemic blood plasma, or containing normal rabbit blood plasma, anomalous organelles are formed in the cells, and no hemoglobin biosynthesis occurs.

Micoplasma como contaminante de culturas celulares mantidas em laboratórios de instituições particulares e oficiais

Mycoplasma is one of the most serious contaminants of cell cultures. Its detection is very important in virology, as well as its eradication. The aim of this study was to verify the incidence of mycoplasma in cell lines maintained in seven laboratories of private, government and college institutions of the State of São Paulo, Brazil, for the purposes of research, production of reagents for diagnosis and production of biologicals for human and animal use. Of the 29 cell lines, eight were derived from human tissues and 21 from other animal species (dog, rabbit, mouse, hamster, monkey, pig, chicken and ox). Using the direct method with specific liquid and solid media for detection of mycoplasma, 48 out of the 106 cell samples tested were positive, corresponding to a contamination index of 45.28%. The incidence of contamination among the 35 cell samples of human origin was 51.43% (18 positive). Of the 71 samples originated from other species, 30 were positive (42.25%). The high incidence of contamination found calls for the adoption of measures for the prevention of this hazard: the elimination of mouth pipetting, the use of aseptic techniques and a rigid control of trypsin, serum and other components of cell culture media. The substitution of mycoplasma-free cultures for all contaminated ones and the performance of periodical tests for mycoplasma detection must also be carried out to prevent and avoid the dissemination of these organisms. Data obtained showed that contamination appeared in the 2nd (72.92%), in the 3rd (20.83%) and in the 4th passage (6.25%).(ABSTRACT TRUNCATED AT 250 WORDS)Foi realizado estudo sobre a incidencia de contaminacao por micoplasma em 29 tipos de linhagens celulares pertencentes a sete laboratorios de instituicoes particulares, oficiais e de ensino superior. Utilizando o metodo de cultivo direto e oito passagens seriadas em meios especificos, liquido e solido, verificou-se que, do total de 106 amostras, 48 apresentaram-se contaminadas por micoplasma (45,28%), o que constitui elevado indice de contaminacao. O fato indica que testes periodicos para a determinacao da presenca de micoplasma nas culturas em utilizacao e recomendavel e que as culturas contaminadas devem ser eliminadas para evitar a disseminacao do microrganismo. Outras medidas preventivas devem ser adotadas, como a eliminacao da pipetagem bucal, execucao de tecnicas assepticas mais estritas no manuseio das celulas, controle dos soros de origem animal, da tripsina e de outros componentes dos meios de cultura utilizados em cultura celular. O estudo mostrou que, ao inves das oito passagens seriadas propostas inicialmente, cinco foram suficientes para a deteccao dos micoplasmas, o que representa economia de tempo e de materiais de custo elevado, reduzindo de 848 para 530 o numero de passagens e a duracao do teste, de oito para cinco semanas.

The mitochondrial function in hemosome formation and hemoglobin biosynthesis

1. Rabbit-kidney epithelial cell cultures were induced to synthesize hemoglobin, by previously mixing cell suspensions with solutions containing reticulocyte free globin, hemoglobin and anemic rabbit blood plasma. As control, a solution without globin was used. 2. After a 24 hr culture growth period, hemoglobin was absent, as stated through electrophoresis, suggesting hemoglobin denaturation: mitochondria interacted with the incorporated material and particles resembling ferritin molecules were found within 48 hr. 3. Mitochondria modified remarkably giving rise to lamellated bodies which recomposed to form prohemosomes, presumably containing globin and newly synthesized heme: hemoglobin was still absent up to 72 hr. 4. After 96 hr hemosomes developed and hemoglobin, apparently constituted by reticulocyte globin, was detected.

Avaliação das condições de estocagem de vacinas vivas, atenuadas contra o sarampo, em postos de vacinação credenciados e em centros de saúde do Estado de São Paulo (Brasil)

In the State of S. Paulo, Brazil, health centers sponsored by the State, as well as private health services, located in throughout large districts, are in charge of the vaccination against the various diseases affecting children, including measles. In the present study three of the above mentioned districts, covering 385 State Health Centers (SHC) and 200 Private Health Services (PHS) were surveyed. From these totals 117 SHC and 71 PHS were chosen for the evaluation of: a) quality of the existing cold chain for measles vaccine storage (temperatura); b) vaccine titer; c) validity; d) protection from light. Of the SHC inspected, 85.33% kept vaccines at the recommended temperature, 100% presented vaccines with titers above the minimum required and within expiration date. Deficiency relating to protection from light, was found in 83.06%. Inspection of the PHS revealed that whereas 66.20% kept vaccine within the right temperature range, 76.06% did not protect them against light, only 96.47% had vaccines with potency above the level required and only 85.51%, observed the expiration date. The study led to the conclusion that in the State of S. Paulo, measles vaccines are better stored in the State Health Centers than in the Private Health Services surveyed indicating that the slight preference that parents show towards having their children vaccinated in PHS is not justified. It was suggested that some conditions of measles vaccine storage montly, temperature and protection from light should be improved and that instruction leaflets must always accompany immunobiological materials distributed to all health centers, to be consulted when doubts about the proper storage and administration of these vaccines arise.Para avaliar as condicoes de estocagem de vacinas vivas, atenuadas contra o sarampo, da rede de vacinacao do Estado de Sao Paulo (Brasil), foram visitados 71 Postos de Vacinacao Credenciados particulares (PVC), assim como 117 Centros de Saude oficiais (CS), sobre os quais interessava saber a respeito da qualidade da estocagem a frio. Os parâmetros adotados foram: a) temperatura das geladeiras de uso (+2 a +8°C) e de estoque (+ 8°C); b) validade do produto; c) titulo das vacinas conservadas nestas geladeiras, avaliado pela inoculacao de diluicoes das amostras de vacinas em celulas Vero; d) protecao a luz. Dos CS pesquisados, 85,33% apresentaram geladeiras com temperatura de acordo com a recomendada e 100% das vacinas neles estocadas com titulo e validade satisfatorios. Nos PVC foram encontrados, com maior frequencia, lotes de vacina fora do prazo de validade (14,49%), com titulos abaixo do minimo requerido (3,53%) e geladeiras de uso e de estoque com temperaturas inadequadas (33,80%). Necessario se faz que as condicoes de estocagem das vacinas contra o sarampo (temperatura e protecao a luz), prevalentes no momento, sejam melhoradas e que as bulas passem a acompanhar o produto a eles entregue, para que os responsaveis pela vacinacao obedecam as recomendacoes do laboratorio produtor com relacao as condicoes de estocagem, validade e administracao do imunobiologico, uma vez que a pesquisa revelou que estas nao sao observadas com o rigor necessario.

Fotossensibilidade e termoestabilidade de vacinas contra o sarampo (cepa Biken CAM-70) liofilizadas e/ou reconstituídas para administração

Three different lots of measles vaccines produced with the Biken CAM-70 virus strain were requested from the central cold store of the Public Health Department of the State of S. Paulo, Brazil, and assays on photosensitivity at 2-8 degrees C, and on stability at 28, 36.5 and 45 degrees C were carried out to find out for how long these vaccines would maintain their minimum potency, established as being 3.70 log10 or 5000 TCID50 (50% tissue culture infective dose) per human dose. The analysis of the adjusted straight regression lines indicated that, with the passage of time, the potency of lyophilized or reconstituted vaccines, as well as of vaccines exposed to or protected from light decreased. Light-exposed vaccines, however, became less potent than vaccines protected from the light. None of the vaccine lots studied, reconstituted and stored at 2-8 degrees C, exhibited homogeneity as to sensitivity to light. When freeze-dried vaccines had their photosensitivity studied at 2-8 degrees C, lots 1 and 2 presented greater thermal degradation when exposed to light than when protected from it. However, in both instances, it was found that potency fell below that taken as minimum for the Biken CAM-70 virus strain. At all other temperatures considered, even when protected from light, lots 1 and 2 did not retain the minimum potency. Lot 3 kept the expected stability for 60 days at 2-8 degrees C when protected from light and for 40 days when unprotected, but its thermal degradation at other temperatures was more intense (28 degrees C: 5 days; 36.5 degrees C: 2 days; 45 degrees C: 0.5 day).(ABSTRACT TRUNCATED AT 250 WORDS)Tres lotes de vacinas contra o sarampo, produzidos com a cepa de virus Biken CAM-70, sob as formas liofilizada e reconstituida, pertencentes ao estoque da rede de vacinacao da Secretaria de Estado da Saude de Sao Paulo, Brasil, foram submetidos a testes de sensibilidade a luz, a temperatura de 2 a 8°C, e de termoestabilidade (protegidos da luz) as temperaturas de 28, 36,5 e 45°C, objetivando verificar por quanto tempo retinham sua potencia, isto e, a concentracao ideal recomendada para a cepa de virus presente (3,70 log 10 DICT50 ou 5.000 doses infectantes de cultura de tecidos 50% por dose). A analise de retas de regressao ajustadas demonstrou que, de modo geral, tanto os lotes de vacinas liofilizados como os reconstituidos, mantidos as referidas temperaturas, expostos ou protegidos da luz, apresentaram queda de potencia no decorrer do experimento, a qual foi mais acentuada para vacinas expostas a luz. Reconstituidos e mantidos a 2 a 8°C, os lotes nao apresentaram homogeneidade no referente a sensibilidade a luz. Quando a fotossensibilidade de lotes de vacinas liofilizadas foi testada a 2 a 8°C eles mostraram-se mais sensiveis a degradacao termica quando expostos a luz do que quando protegidos dela. Entretanto, expostos ou protegidos, a potencia foi inferior a minima aceita para a cepa Biken CAM-70. As demais temperaturas, mesmo ao abrigo da luz, os dois lotes nao retiveram potencia minima. Quanto as vacinas do lote 3, conservadas a 2 a 8°C, mantiveram-se de acordo com os requerimentos minimos de potencia durante 60 dias quando protegidas da luz, e durante 40 dias quando expostas a ela. A degradacao termica as demais temperaturas foi mais acentuada (28°C: 5 dias; 36,5°C: 2 dias; 45°C: 0,5 dia). Considerando a concentracao viral minima que vacinas produzidas com a cepa Biken CAM-70 devem conter para induzir efetiva resposta imunologica, os lotes de vacinas pesquisados (sob a forma liofilizada ou reconstituidos para administracao) apresentaram, alem de baixa estabilidade ao calor, pouca homogeneidade com relacao a este parâmetro.

Identificação de micoplasmas pela inibição de crescimento de amostras isoladas de culturas celulares

As culturas celulares devem ser continuamente monitoradas quanto a presenca de micoplasmas, pois, embora as vezes eles passem despercebidos, podem causar alteracoes cromossomicas, interferir na replicacao viral, na producao de anticorpos e interferon. A Organizacao Internacional em Micoplasmologia (IOM) recomenda o isolamento e a identificacao de micoplasmas, visando detectar as provaveis origens da infeccao e melhorar a qualidade das culturas. Assim, foram analisadas pela inibicao de crescimento, 37 amostras pertencentes a 27 linhagens celulares contaminadas por micoplasmas. Em nenhuma amostra foi observada a ocorrencia de duas especies. Foram identificados 18 (48,65%) Mycoplasma arginini, 15 (40,55%) Acholeplasma laidlawii, dois (5,40%) Mycoplasma orale, sendo que duas amostras (5,40%) nao foram identificadas. Considerando as especies caracterizadas na pesquisa, os autores sugerem: a) a adocao do teste de isolamento de micoplasmas em carater de rotina; b) o aprimoramento das tecnicas de assepsia e desinfeccao; c) a eliminacao da pipetagem bucal; d) a utilizacao de soros e de outros componentes de meios de cultura de qualidade certificada; e) o questionamento da presenca de micoplasmas quando linhagens celulares sao permutadas pelas instituicoes; f) a avaliacao cautelosa de resultados obtidos quando se utilizam culturas infectadas por esse microrganismo.

Identificação da microbiota fúngica de ambientes considerados assépticos

The isolation and identification of the fungic microbiota present in areas considered aseptic (open and/or restricted, with or without air filtration (HEPA filters) where production of immunobiologicals is carried out)were investigated Three exposures were made with appropriate culture media, in different seasons of the year, before and immediately after the performance of the technical work. Agar-Sabouraud and agar-soy media supplemented with 0.2% yeast extract and without chloranphenicol had their efficacy tests in isolating the filamentous fungia e (molds) and yeasts in the light of the number of cultures grown and the frequency exhibited by the various specimens isolated. Sixty-seven specimens (64 of filamentous fungiae (molds) and three yeasts) have been identified. Of the filamentous fungiae (molds), 54 belonged to 22 genera of the division Deuteromycota, families Moniliaceae and Dematiaceae, five were included in the order Agonomycetales (Mycelia Sterilia), and one was classified in the order Sphaeropsidales, class Coelomycetes. Of the division Zygomycota, order Mucorales, family Mucoraceae, only one specimen could be identified as to genus. The three yeasts, belonging also to the division Deuteromycota (Fungi Imperfecti), family Cryptococcaceae, were identified as two Rhodotorula rubra and one Torulopsis candida. It was demonstrated that the number of colonies isolated in the areas studied increased after the performance of technical maneuvers and that air filtration by means of HEPA filters, reducing to number of colonies isolated in restricted areas, increased safety and is consequently recommended for working areas where successful or unsuccessful results depend on a low incidence of contamination.Objetivou-se isolar e identificar a microbiota fungica em ambientes considerados assepticos, atraves de exposicoes com meios de cultivo adequados, em tres epocas distintas do ano, antes e imediatamente apos as manobras tecnicas realizadas em tres areas de trabalho: ambiente aberto, ambiente fechado sem filtracao de ar e ambiente fechado com filtracao de ar, utilizadas em producao de imunobiologicos. Os meios agar-Sabouraud e agar-soja, enriquecidos com 0,2% de extrato de levedura e sem cloranfenicol, foram estudados quanto a sua eficacia no isolamento de bolores e leveduras, considerando-se o numero de colonias desenvolvidas e a frequencia dos diversos fungos isolados. Isolaram-se 67 especimens, sendo 64 fungos filamentosos (bolores) e tres leveduras. Dos bolores, 54 pertenciam a 22 generos da divisao Deuteromycota, familias Moniliaceae e Dematiaceae, cinco amostras filamentosas foram incluidas na ordem Agonomycetales (Mycelia Sterilia), e uma amostra foi classificada na divisao Deuteromycota, ordem Sphaeropsidales, classe Coelomycetes. Da divisao Zygomycota, ordem Mucorales, familia Mucoraceae, um unico mucoraceo foi identificado ate genero. As tres leveduras pertenciam tambem a divisao Deuteromycota (Fungi Imperfecti), familia Cryptococcaceae, e foram identificadas como sendo duas Rhodotorula rubra e uma Torulopsis candida. Comprovou-se que o numero de colonias isoladas aumentou apos a realizacao das monobras tecnicas e que a filtracao de ar atraves de filtros tipo HEPA, reduzindo o numero de colonias isoladas nos ambientes fechados, aumenta a seguranca do trabalho; comumente e recomendada para areas de atividade tecnica cujos resultados satisfatorios estao diretamente relacionados com uma baixa incidencia de contaminantes.

Suscetibilidade da linhagem de células Vero a cepas vacinais do vírus do sarampo

Vero cells used by distinct measles vaccine control laboratories had their susceptibility to Moraten, Schwarz and Biken CAM-70 vaccine strains assayed. Of a total of 72 lots of measles vaccine whose potency was titrated by microtechnique in two Vero cell samples (Vero IB and Vero INCQS), 25 had been produced with Moraten strain, 24 with Schwarz and 23 with Biken CAM-70. The statistical analysis of the results demonstrated that both Vero cells assayed presented comparable susceptibility to Moraten and Biken CAM-70 strains. As to the Schwarz strain, Vero IB cells were more susceptible than the other cell sample tested, thus confirming the existence of different sensitivities of Vero cells to some measles vaccine strains, or even to viruses derived from the same strain but with different passage histories. An altered cell susceptibility to virus replication may significantly alter the results in potency testing. Such alteration may be caused not only by the adoption of distinct protocols for the maintenance of cell cultures by different control laboratories but also by the methodology followed in the vaccine titration. In order to minimize the differences existing among the results obtained in the potency testing, it is suggested that all control laboratories should use the same protocols for cell culture maintenance as well as for vaccine potency testing.

Seringas hipodérmicas descartáveis versus reutilizáveis: estudo de possíveis efeitos sobre o vírus da vacina viva, atenuada contra o sarampo

Objetivou-se verificar entre seringas hipodermicas descartaveis e reutilizaveis qual interfere mais com o virus vivo presente na vacina contra o sarampo. Vacinas pertencentes a dois lotes foram reconstituidas com os dois tipos de seringas, de modo a formarem dois pools distintos, mantidos a temperatura de +2 a+8°C e protegidos da luz. De cada lote foram realizadas, no minimo, seis titulacoes em paralelo, com amostragem a cada hora, de zero a seis horas apos reconstituicao. A analise estatistica dos resultados obtidos nas titulacoes, feita pelo sistema de retas de regressao demonstrou que embora as vacinas manipuladas com ambos os tipos de seringas apresentassem um decrescimo de titulo estatisticamente significativo com o decorrer das horas, ele foi bem mais acentuado para as vacinas reconstituidas com seringas reutilizaveis. A menor interferencia das descartaveis no titulo da vacina viva, atenuada contra o sarampo, demonstrou que a preconizacao e uso desse tipo de seringas pela Secretaria de Estado da Saude de Sao Paulo e o ideal e recomendavel, por preservar mais a vacina desde a reconstituicao ate sua administracao e, consequentemente, a sua eficacia na prevencao dessa infeccao.The study was performed in the State of S.Paulo, SP, Brazil, with the purpose of finding out whether reusable (glass) and disposable hypodermic syringes used for administration of live attenuated measles vaccines would interfere with their virus. At least six different experiments using two distinct lots of vaccines were carried out. Each time, a lot was reconstituted with reusable and disposable syringes in parallel to form separate pools from which samples were collected hourly from zero to the sixtieth hour after reconstitution for virus titration in monolayers of Vero cells. The straight line regression system chosen for the analysis of the results demonstrated that although vaccines reconstituted with both types of syringes presented a statistically significative titer decrease as time went by, there was a more pronounced decrease for vaccines manipulated with the glass syringes. The fact that the disposable syringes affected the titer of the virus present in the live, attenuated measles vaccine less confirmed that the preconization and routine usage of this type of syringe by the Health Department of the State of S.Paulo, Brazil, is ideal and highly recommended because it preserves the vaccine from reconstitution to administration better, and thus, its efficacy in preventing the infection.

Reações locais e níveis de antitoxina circulante decorrentes de administração do toxóide tetânico: estudo comparativo entre Ped-o-Jet e seringa hipodérmica

Com o objetivo de comparar reacoes locais e conversao sorologica apresentadas por adultos que receberam o toxoide tetânico atraves de Ped-o-Jet (via subcutânea) ou de seringa hipodermica (via intramuscular), o toxoide foi administrado a 472 recrutas do Exercito. Em observacoes realizadas 4 e 24 horas apos a vacinacao verificou-se que as reacoes locais dos individuos vacinados com Ped-o-Jet eram significativamente mais frequentes e mais intensas do que aquelas dos vacinados com seringa hipodermica, nao tendo ocorrido, entretanto, reacoes graves. A conversao sorologica dos nao imunes vacinados com Ped-o-Jet ocorreu numa frequencia maior do que nos individuos vacinados com seringa hipodermica. Conclui-se portanto, que o Ped-o-Jet pode ser utilizado em campanhas de vacinacao em massa contra o tetano, embora a via de administracao preferencial, ate o momento, seja a intramuscular.