[Susceptibility of Vero cell line to vaccine strains of the measles virus]. / 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.

Hemosomegenesis and hemoglobin biosynthesis in vertebrates.

1. Ultrastructural observations on maturing rabbit embryo erythroid cells led to the finding of hemoglobinized organelles distinguishable from mitochondria due to their highly dense matrix, two or three longitudinally arranged double lamellae, and smaller diameters. Intraorganellar 50-60 A particles identical to those contained in the hemoglobinized cytoplasm were found. 2. Their hemoglobin (Hb) content was demonstrated by electrophoresis of the concentrated supernatant from the isolated, washed, and osmotically lysed organellar fraction. We have proposed that these organelles are the sites for heme integration into the globin (G) polypeptide chains and subunits assembly. The term hemosome has been suggested for such entities. 3. This hypothesis has been sustained by several analytical and experimental works based on the postulation that hemosomes should be found at higher frequencies where the Hb biosynthesis rate is more intensive, or where the induction of this biosynthesis is always dependent on the formation of hemosomes. 4. Maturing erythroid cells of the circulating embryo blood contain hemosomes in higher frequency than in liver erythroid cells, coinciding with the higher Hb biosynthesis rate in peripheral blood than in the liver. In bleeding anemia, the decay of Hb concentration parallels the reduction of the mean number of hemosomes per reticulocyte, in comparison with normal reticulocytes. 5. In HeLa cells and epithelial cultured cells induced to synthesize Hb, it was shown that this biosynthesis is ever concomitant with the formation of hemosomes and depends on the presence of erythropoietin, as occurs in erythroid cells. 6. Studies on hemosomegenesis and Hb biosynthesis experimentally effected in epithelial cultured cells, allowed the interpretation of the sequence of events leading to hemosome formation in maturing erythroid cells. Simultaneously with iron uptake, mitochondria differentiate to lamellated bodies and, successively, expansions rise for ferruginous compounds and G polypeptides gathering, followed by prehemosome vesicles formation, which condense and change to prohemosomes that afterwards evolve to hemosomes. 7. These dynamics, and organellar Hb have been detected in immature erythrocytes of mammalians, including humans, avians, reptilians, amphibians and representative fish specimens. It appears that these events occur in the erythrocytary maturation of all vertebrate classes.

[Identification of mycoplasma by the growth inhibition of samples isolated from cell cultures]. / Identificação de microplasmas pela inibição de crescimento de amostras isoladas de culturas celulares.

Cell cultures must be continuously screened for the presence of mycoplasma because, although these microorganisms sometimes pass unnoticed, they may cause chromosomic alterations and interfere with viral replication, antibody and interferon production etc. The International Organization for Mycoplasmology (IOM) recommends the isolation and identification of mycoplasma with a view to the detection of the origin of the infection and the improvement of the quality of the cultures. In this paper, 37 samples belonging to 27 cell lines contaminated with mycoplasma were assayed by the growth inhibition test. It is known that Mycoplasma orale is the most common human mycoplasma contaminant of cell cultures, the major vehicle of contamination being mouth pippeting, while commercial bovine serum in the main source for Mycoplasma arginini and Acholeplasma laidlawii. M. arginini was found in 18 (48.65%) of the cell samples tested, A. laidlawii in 15 (40.55%), and M. orale in two (5.40%). Two other samples could not be identified by the antisera used (antisera against M. arginini, M. orale, Mycoplasma hyorhinis and A. laidlawii) their characteristics being "fried egg" colonies, digitonine sensitivity, Dienes stained, positive glucose catabolism, negative arginini hydrolysis, and negative tetrazolium reduction. No more than one type of mycoplasma was found in each cell culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemoglobin biosynthesis enhancement following mitochondrial membrane growth and differentiation.

1. A quantitative increase of organelles in early reticulocytes has been observed compared to that found in late erythroblasts of the peripheral rabbit embryo blood. 2. The increase is due to the formation of hemosomes, organelles taken as sites for final hemoglobin (Hb) biosynthesis or where the assembly of heme and globin polypeptides could occur. 3. These organelles derive indirectly from mitochondria whose internal membrane grows concomitantly to its differentiation, originating lamellated bodies that modify successively to prehemosomal vesicles, prohemosomes and hemosomes. 4. The occurrence of membrane synthesis for the formation of lamellated bodies could explain the increase of organelles per cell and, thereby, the enhancement of the Hb biosynthesis rate in peripheral embryo blood in relation to this biosynthesis rate in the liver, as had been biochemically ascertained by other authors.

[Photosensitivity and stability of freeze-dried and/or reconstituted measles vaccines (Biken CAM-70 strain)]. / 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)

[Biochemical characterization of calf sera used in the maintenance of cell cultures used in virology]. / Caracterização bioquímica de soros de bezerros utilizados na manutenção de culturas celulares usadas em virologia.

Eight lots of the calf sera employed to supplement culture media for the cultivation of animal cells, of widespread use in virology obtained from calves above and below six months of age were rated as good or as poor cell growth promoters according to their growth promoting capacity (GPC). Parameters related to macronutrients contained in these serum lots were then evaluated with the purpose of establishing their biochemical profiles. The results obtained can be considered as normal values for apparently healthy animal donors. Fluctuations found between the data of this investigation and those mentioned in the literature for certain biochemical parameters are probably due to the methodology employed, the breed and age of the animals, or even to regional diet. Student's "t" test was applied for the statistical analysis of the results and demonstrated that, as far as serum fractions were concerned, no significant differences occurred between sera rated as good and poor cell growth promoters, taking tc = 2.45. For calf sera from animals above and below six months of age, two tests relating to alfa and beta fractions were significant (t = 2.68 and 2.61 respectively). It was demonstrated that the evaluation of the biochemical parameters mentioned "per se" neither leads to the identification of calf sera presenting good or poor GPC, nor of sera harvested from calves younger or older than six months.

[Mycoplasma as a contaminant of cell cultures maintained in laboratories of private and official institutions]. / 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)

Sorbitol-gelatin and glutamic acid-lactose solutions for stabilization of reference preparations of measles virus.

The work reported here sought to assess the protection afforded by two stabilizing solutions (sorbitol-gelatin and glutamic acid-lactose) in preserving the potency of freeze-dried Schwarz strain measles virus during storage with a view to the production of reference preparations and working lots of virus suspensions. Stabilized virus suspensions and control suspensions were stored at -70 degrees C or were freeze-dried and stored at -20 degrees C, and their potency was determined over a storage period of 21 months. It was found that the sorbitol-gelatin imparted more satisfactory stability (r = +0.18) to the freeze-dried virus suspensions than did the glutamic acid-lactose. The results also indicate that sorbitol-gelatin, used under the conditions of this study, is an effective stabilizer in the preparation of freeze-dried suspensions of Schwarz strain measles virus employed as reference preparation working lots.

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.