Viral antigen production in cell cultures on microcarriers Bovine parainfluenza 3 virus and MDBK cells.

Viral antigens can be obtained from infected mammalian cells cultivated on microcarriers. We have worked out parameters for the production of bovine parainfluenza 3 (PI-3) virus by Mandin-Darby Bovine Kidney (MDBK) cells cultivated on Cytodex 1 microcarriers (MCs) in spinners flasks and bioreactor using fetal bovine serum (FBS) supplemented Eagle minimal essential medium (Eagle-MEM). Medium renewal during the cell culture was shown to be crucial for optimal MCs loading (>90% MCs with confluent cell monolayers) and cell growth (2.5 x 10(6)cells/mL and a micro(x) (h(-1)) 0.05). Since cell cultures performed with lower amount of MCs (1g/L), showed good performances in terms of cell loading, we designed batch experiments with a lower concentration of MCs in view of optimizing the cell growth and virus production. Studies of cell growth with lower concentrations of MCs (0.85 g/L) showed that an increase in the initial cell seeding (from 7 to 40 cells/MC) led to a different kinetic of initial cell growth but to comparable final cell concentrations ((8-10)x10(5)cells/mL at 120 h) and cell loading (210-270 cells/MC). Upon infection with PI-3 virus, cultures showed a decrease in cell growth and MC loading directly related to the multiplicity of infection (moi) used for virus infection. Infected cultures showed also a higher consumption of glucose and production of lactate. The PI-3 virus and PI-3 antigen production among the cultures was not significantly different and attained values ranging from, respectively, 7-9 log(10) TCID(50)/mL and 1.5-2.2 OD. The kinetics of PI-3 virus production showed a sharp increase during the first 24h and those of PI-3 antigen increased after 24h. The differential kinetics of PI-3 virus and PI-3 antigen can be explained by the virus sensitivity to temperature. In view of establishing a protocol of virus production and based on the previous experiments, MDBK cell cultures performed under medium perfusion in a bioreactor of 1.2L were infected and the PI-3 virus production in 12L attained 12 log(10) TCID(50). Other than establishing a protocol for PI-3 production in MDBK cell cultures on Cytodex 1, the experiments are proposed as a basis for approaching the development of a virus production protocol in mammalian cells cultivated on microcarriers in bioreactors.

Familial dwarfism due to a novel mutation of the growth hormone-releasing hormone receptor gene.

Isolated growth hormone (GH) deficiency (IGHD) is a rare cause of short stature. The same mutation of the gene encoding the growth hormone-releasing hormone receptor (GHRHR) has been identified as the basis for IGHD in three families from the Indian subcontinent. The prevalence and heterogeneity of defects in the GHRHR gene are not known. Twenty-two dwarf members of a large, extended kindred containing at least 105 affected members with autosomal recessive short stature underwent extensive endocrine evaluation, which confirmed markedly reduced or undetectable serum concentrations of GH that did not increase in response to different stimuli. DNA sequences of the 13 exons and intron-exon boundaries of the GHRHR gene were determined in an index patient. A novel homozygous 5' splice site mutation (G-->A at position +1) in IVS1 was found. Thirty of the affected subjects tested were homozygous for this mutation, and 64 clinically unaffected patients were either heterozygous for the mutation (n = 41, including 9 obligate carriers) or homozygous for the wild-type sequence (n = 23). We describe a novel mutation in the GHRHR gene as cause of dwarfism in the largest kindred with familial IGHD described to date.

22 new data on the association between the glyoxalase I and haptoglobin loci.

No association between GLO and Hp was found in three Brazilian samples (153 Whites, 216 Blacks from Porto Alegre and 564 mixed individuals from Aracaju). In a sample of 174 Blacks (settled along the Trombetas river) a moderate (p less than 0.02) association was found, but not of the same kind as that observed by other authors. Population stratification instead of interactions in fitness may explain our findings.

Spread and diversity of human populations in Bahia, Brazil.

PIP: Data from surnames and racial subgroups were obtained in 60 localities to reconstruct the history of population spread and mixing over the state of Bahia, Brazil. Using the historically significant location of Cachoeira and Sao Felix as central points, the sampled localities were distributed along 7 main paved roads. 20% of the elementary school children were selected, but only the surnames of 12,872 boys were used in the analysis. The following parameters were estimated for each of the localities: black phenotype index (BPI), which is the proportion of 3 racial classifications determined by gene frequency analysis; black cultural index (BCI) determined by the frequency of devotional surnames; and indian cultural index (ICI) determined by the frequency of animal-plant surnames; and the isolated frequency of the surname Santos. The results of regression analysis indicate a negative association between BPI and BCI and the distance from Cachoeira-Sao Felix. Although not significant, ICI increases slightly with increased distance from Cachoeira-Sao Felix. The isolated frequency of the surname Santos is associated with the BCI but does not decrease significantly with distance from Cachoeira-Sao Felix. Construction of a map characterizing each of the 60 localities by its most representative racial admixture confirms the analysis and reveals the spread of blacks toward 4 cardinal points. A review of the historical background of the state explains the migration of blacks as related to the needs for slave labor during the development of gold mining and cocoa and sugar cultivation. The higher concentrations of indian admixtures farther from Cachoeira-Sao Felix reflects their retreat to survive the incoming foreign settlers. Given the strong link between the observed diversity in the population and major historical events, it is suggested that historical reconstruction of a population be an initial step before undertaking sampling for gene frequency analysis.^ieng

Association between HBsAg and race in a mixed population of Northeastern Brazil.

The frequency of Australian antigen carriers (HBsAg) was studied in three samples from northeastern Brazil. In sample A there were 1000 women from Tsylla Balbino Maternity in Salvador; in sample B there were 1,512 blood donors from Salvador, and in sample C there were 930 blood donors from Aracaju. In sample A, B and C the observed frequency of carriers (HBsAg) was 1.20%, 1.58% and 2.04% respectively. These differences were not significant (x22 = 2.2; p greater than 0.25). There was no association between the carrier state and sex, age, ABO and Rh blood groups, Chagas' disease and rural origin. However, the frequency of carriers (HBsAg) increases from White to Black, as follows: Whites = 0.5%; Mulattoes = 1.91%, and Black = 1.96% (x22 = 7.91; p less than 0.02).

Human aconitase polymorphism in three samples from northeastern Brazil.

Human aconitase (ACONS) polymorphism was studied in three samples from northeastern Brazil. Two of the samples were collected in the State of Bahia and one in the State of Sergipe. The main characteristic of the samples was given by different degrees of Black admixture. The results showed that the more negroid the samples the higher the frequencies of the alleles ACONS4, ACONS2 and ACONS6. These findings fit well with the known ACONS gene frequencies in present-day Nigerians and with the past history of Yoruba slaves in Bahia.