BackgroundStreptococcus pneumoniae (S. pneumoniae) causes several serious diseases including pneumonia, septicemia and meningitis. The World Health Organization estimates that streptococcal pneumonia is the cause of approximately 1.9 million deaths of children under five years of age each year. The large number of serotypes underlying the disease spectrum, which would be reflected in the high production cost of a commercial vaccine effective to protect against all of them and the higher level of amino acid sequence conservation as compared to polysaccharide structure, has prompted us to attempt to use conserved proteins for the development of a simpler vaccine. One of the most prominent proteins is pneumolysin (Ply), present in almost all the serotypes known at the moment, which shows an effective protection against S. pneumoniae infections.ResultsWe have cloned the pneumolysin gene from S. pneumoniae serotype 14 and studied the effects of eight variables related to medium composition and induction conditions on the soluble expression of rPly in Escherichia coli (E. coli) and a 28-4 factorial design was applied. Statistical analysis was carried out to compare the conditions used to evaluate the expression of soluble pneumolysin; rPly activity was evaluated by hemolytic activity assay and served as the main response to evaluate the proper protein expression and folding. The optimized conditions, validated by the use of triplicates, include growth until an absorbance of 0.8 (measured at 600 nm) with 0.1 mM IPTG during 4 h at 25°C in a 5 g/L yeast extract, 5 g/L tryptone, 10 g/L NaCl, 1 g/L glucose medium, with addition of 30 μg/mL kanamycin.ConclusionsThis experimental design methodology allowed the development of an adequate process condition to attain high levels (250 mg/L) of soluble expression of functional rPly in E. coli, which should contribute to reduce operational costs. It was possible to recover the protein in its active form with 75% homogeneity.

BackgroundSeveral countries have used pegylation technology to improve the pharmacokinetic properties of essential drugs. Recently, a novel interferon alfa-2b protein conjugated to four-branched 12 kDa polyethylene glycol molecules was developed jointly between Cuba and Brazil. The aim of this study was to compare the pharmacokinetic properties of BIP48 (pegylated interferon alfa-2b from Bio-Manguinhos/Fiocruz, Brazil) to those of PEGASYS® (commercially available pegylated interferon alfa-2a from Roche Pharmaceutical).MethodsThis phase I, single-centre, randomized, double-blind crossover trial enrolled 31 healthy male volunteers aged 19 to 35 who were allocated to two stages, either side of a 5-week wash-out period, with each arm lasting 14 consecutive days after subcutaneous administration of 180 μg of one formulation or the other (study or comparator). The main outcome variable was serum pegylated interferon concentrations in 15 samples collected during the course of the study and tested using an enzyme immunoassay.ResultsThere were no differences between formulations in terms of magnitude or absorption parameters. Analysis of time parameters revealed that BIP48 remained in the body significantly longer than PEGASYS® (Tmax: 73 vs. 54 h [p = 0.0010]; MRT: 133 vs. 115 h [p = 0.0324]; ke: 0.011 vs. 0.013 h(−1) [p = 0.0153]; t1/2: 192 vs. 108 h [p = 0.0218]).ConclusionBIP48 showed the expected pharmacokinetic profile for a pegylated product with a branched molecular structure. Compared to PEGASYS®, the magnitude absorption was similar, but time parameters were consistent with slower elimination. Further studies should be conducted to evaluate the clinical implications of these findings. A phase II-III repeated-dose clinical trial is ongoing to study these findings in patients with chronic hepatitis C virus infection.Trial registrationThis study is registered on the ClinicalTrials.gov platform (accession number NCT01889849). This trial was retrospectively registered in June 2013.

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RESUMO.-[Avaliação do antígeno recombinante p24 para a detecção de anticorpos específicos do Vírus da Imunodeficiência Felina.] O vírus da imunodeficiência felina tem distribuição mundial e é considerado um patógeno significativo. No Brasil, a prática diagnóstica é baseada principalmente em teste rápidos, importados e de custo elevado, disponíveis comercialmente. Devido ao seu custo proibitivo em nosso país, o diagnóstico da infecção pelo FIV é raramente realizado. Ademais, os lentivírus se multiplicam lenta e pobremente em cultura de células, o que torna a produção de antígeno por meios clássicos e o estabelecimento do imunodiagnóstico impraticável. Com o objetivo de lidar com esta questão, técnicas de DNA recombinante foram utilizadas para produção de um antígeno do capsídeo viral, a proteína p24. A avaliação da reatividade realizada por Western blot indicou que este antígeno recombinante pode ser útil para o imunodiagnóstico de infecções pelo FIV. TERMOS DE INDEXAÇÃO: FIV, vírus da imunodeficiência felina, gatos. INTRODUCTIONFeline Immunodeficiency Virus (FIV) is a lentivirus that causes a chronic and progressive immunodeficiency and is considered one of the main pathogens of domestic felines. Infected cats show a progressive depletion of CD4+ Tlymphocytes and immunological deficits which, after a long asymptomatic period, may lead to severe opportunistic infections followed by death, characterizing the clinical phase known as Feline Acquired Immunodeficiency Syndrome (FAIDS). Due to the similarity to HIV, FIV-infected cats have been used as experimental models for HIV/AIDS (De Rozieres et al. 2004).FIV is structured like other lentiviruses. Virions measure 80-100 nm in diameter and consist of an envelope, a nucleocapsid, a nucleoid and a matrix protein.

Techniques of production and transfer of embryos is safe as long as it follows the control regulations defined by the manual of the International Embryo Transfer Society (IETS) for treating oocytes with trypsin, antibiotics, and TCM-199 medium. The aim of this work was to evaluate the effectiveness of treatments, established by IETS, in bovine oocytes experimentally exposed to Leptospira interrogans serovar Grippotyphosa and to assist implementation of quality control standards on in vitro embryo production. The oocytes were obtained through follicular puncture of ovaries derived from the slaughterhouse. They were selected and divided into 4 groups: the control group and groups exposed to 5, 10, and 30 μL of an L. interrogans strain at 4.7 × 105 μL-1; and 4 additional groups exposed to the same concentrations of another L. interrogans strain at 6.3 × 105 μL-1, in which the gene for virulence is not expressed. The groups were kept in maturation medium (TCM-199 medium, 0.5 (iLof FSH, 50IU mL-1 hCG, 1μL mL-1 17-βiestradiol) and incubated at 38°C, 5% CO2, and 95% humidity for 24 h. All the groups were separately subjected to the treatments with antibiotic, trypsin, and TCM-199 medium after maturation. The treatment involved 10 drops (each 200 μL), with 8 drops of TCM-199 medium and 2 drops of antibiotic (penicillin 10000 IU mL-1 and streptomycin 10 mg mL-1) or trypsin 0.25%; exposed to trypsin and antibiotic for 120 s. For the sequential washes, all drops contained TCM-199 medium. The analysis for presence of the pathogen by dark-field optical microscopy showed that in the groups exposed to L. interrogans and subjected to antibiotic washes, the effectiveness was 50% (100/200) for the group exposed to 5 μL, 40% (80/200) for that exposed to 10 μL, and 22.5% (45/200) for that exposed to 30 μL. We found the same results after the trypsin washes. After the washings with TCM-199 medium, the groups infected with 5 and 10 μL presented 100% of effectiveness; however, for the group infected with 30 μL, the washings were not effective. For the groups exposed to L. interrogans that did not express virulence, after the washings with antibiotics as well as with trypsin, the results showed no effectiveness in all of them (n = 200). Yet, after washings with TCM-199, the group exposed to 5 μL showed 28.5% (57/200) of effectiveness, whereas in those exposed to 10 and 30 μL, the medium washes were not effective. Complementary studies are being made with ultramicrotome cut and polymerase chain reaction for more reliable conclusions, to confirm the results. With such results, we conclude that the quality control regulations established by IETS for IVP could be reviewed and possibly redefined, because the effectiveness of the treatment may depend not only on the pathogen species, but also its virulence as well as its concentration and the action of the treatments on the type of pathogen. We thank Vitrocel/Embriolife for supporting the laboratory.