In vitro cytotoxic activity of Baccharis dracunculifolia and propolis against HEp-2 cells.

Baccharis dracunculifolia is the most important vegetal source of propolis in southeast Brazil, and researchers have been investigating its biological properties. Propolis is a complex resinous hive product collected by bees from several plants, showing a very complex chemical composition. It has been employed since ancient times due to its therapeutic properties, such as antimicrobial, anti-inflammatory, antioxidant, immunomodulatory and antitumour activities, among others. The goal of this work was to compare the cytotoxic action of B. dracunculifolia, propolis and two isolated compounds (caffeic and cinnamic acids) on human laryngeal epidermoid carcinoma (HEp-2) cells in vitro. These cells were incubated with different concentrations of each variable, and cell viability was assessed by the crystal violet method. Lower concentrations of B. dracunculifolia (extract and essential oil), propolis, as well as caffeic and cinnamic acids, showed no cytotoxic activity against HEp-2 cells. On the other hand, elevated concentrations (50 and 100 µg per 100 µL) exerted a cytotoxic action, and propolis showed a more efficient action than its vegetal source and isolated compounds. Further investigation is still needed in order to explore the potential of these variables as antitumour agents and to understand their mechanisms of action.

Anti-poliovirus activity of Baccharis dracunculifolia and propolis by cell viability determination and real-time PCR.

AIMS: The aim of this work was to evaluate the antiviral activities of Baccharis dracunculifolia (extract and essential oil), propolis and some isolated compounds (caffeic and cinnamic acids) against poliovirus type 1 (PV1) replication in HEp-2 cells. METHOD: Three different protocols (pre-, simultaneous and post-treatments) were used to verify the effect of addition time of the variables on PV1 replication by crystal violet method and relative viral RNA quantification by real-time PCR for analysing in which step of virus replication the variables could interfere. CONCLUSIONS: Data revealed that the B. dracunculifolia showed the best antiviral activity percentage in the simultaneous treatment, as well as lower relative viral quantification by real-time PCR. Variables might block partially the viral entry within cells, affect the steps of viral cycle replication into cells, or lead to RNA degradation before the virus entry into cells or after their release to the supernatant. SIGNIFICANCE AND IMPACT OF THE STUDY: Baccharis dracunculifolia is the most important botanical source of the south-eastern Brazilian propolis, and its potential for the development of new phytotherapeutic medicines has been investigated. Propolis is commonly used for its antimicrobial and immunomodulatory activities. Nevertheless, B. dracunculifolia and propolis effects on PV1 have not been investigated yet.

Inflammatory cytokine mRNA detection by real time PCR in chorioamniotic membranes from pregnant women with preterm premature rupture of membranes.

OBJECTIVE: To quantify the expression of interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor alpha (TNF-alpha) in chorioamniotic membranes of PPROM pregnant women with chorioamnionits. STUDY DESIGN: The study included 25 PPROM women in labor, 15 PPROM without labor, and 25 pregnant women in preterm labor (PTL). Chorioamniotic membranes were collected for histopathological analyses and cytokine mRNA expression quantification by real time PCR. Comparisons were performed using the Mann-Whitney, Kruskal-Wallis, Fisher's exact test or z test with significance set at p<0.05. The software employed was the SigmaStat version 3.1. RESULTS: During the study PPROM incidence was 4.6% and chorioamnionits was present in 75% of the samples. IL-1beta, IL-6, and IL-8 mRNA expression did not statistically differ among study groups. TNF-alpha mRNA expression was statistically higher in PTL. No difference in the mRNA concentration of the cytokines studied in the presence of chorioamnionitis was observed. CONCLUSION: Chorioamniotic membranes are sources of IL-1beta, IL-6, IL-8, and TNF-alpha and their mRNA concentrations in PPROM are not related to the presence of chorioamnionitis.

PCR detection of staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk.

Milk is considered a nutritious food because it contains several important nutrients including proteins and vitamins. Conversely, it can be a vehicle for several pathogenic bacteria such as Staphylococcus aureus. This study aimed to analyze the frequency of genes encoding the staphylococcal enterotoxins (SEs) SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI and SEJ in S. aureus strains isolated from raw or pasteurized bovine milk. S. aureus was found in 38 (70.4%) out of 54 raw milk samples at concentrations of up to 8.9 x 10(5) CFU/ml. This microorganism was present in eight samples of pasteurized milk before the expiration date and in 11 samples analyzed on the expiration date. Of the 57 strains studied, 68.4% were positive for one or more genes encoding the enterotoxins, and 12 different genotypes were identified. The gene coding for enterotoxin A, sea, was the most frequent (16 strains, 41%), followed by sec (8 strains, 20.5%), sed (5 strains, 12.8%), seb (3 strains, 7.7%) and see (2 strains, 5.1%). Among the genes encoding the other enterotoxins, seg was the most frequently observed (11 strains, 28.2%), followed by sei (10 strains) and seh and sej (3 strains each). With the recent identification of new SEs, the perceived frequency of enterotoxigenic strains has increased, suggesting that the pathogenic potential of staphylococci may be higher than previously thought; however, further studies are required to assess the expression of these new SEs by S. aureus, and their impact in foodborne disease. The quality of Brazilian milk is still low, and efforts from the government and the entire productive chain are required to attain consumer safety.

Videoscopic assessment of the maintenance status of gamma radiography exposure containers employed in Brazil.

Industrial radiography is the most frequent method of non-destructive testing (NDT) used by Brazilian industrial facilities for investigating the material integrity of a test object. In Brazil, industrial radiography employs around 220 x-ray and 290 gamma radiography machines. About 90% of the latter uses iridium ((192)Ir) sources. The large majority of (192)Ir projectors in operation in Brazil have been in continuous usage for more than 25 years, which means that they are old and worn-out. Usually the majority of accidents concerning gamma radiography occur during the return of the source into the exposure container. Poor maintenance or imperfections of the internal channel of the exposure container can lead to accidental source exposure. In the present work the internal tube of 65 gamma machines from nine Brazilian companies that render gamma radiography services were analysed using an industrial videoscope. The internal images from the projectors were compared with the internal image of an apparatus that had never been used, i.e. has never received a radioactive source. From the 65 machines evaluated, nine showed irregularities of the internal tube. It was also observed that each company follows a different methodology for the maintenance and lubrication of the exposure containers and drive cables.

Prevalence of diarrheogenic Escherichia coli and rotavirus among children from Botucatu, São Paulo State, Brazil.

In a one-year prospective study carried out to define the role of rotavirus and Escherichia coli in local childhood diarrhea, we determined the prevalence of both agents in 54 diarrheic children attending a health center in Botucatu. Diarrheogenic E. coli (DEC) strains were characterized by O:H serotyping, a search for virulence genetic markers, and assays of adherence to HEp-2 cells. Except for enteroaggregative E. coli (EAEC), no other DEC category was detected in the children's stools. Both EAEC and rotavirus were isolated from 22 of the 54 (41.0%) diarrheic children as single agents or in combination with other enteropathogens. However, when considering the presence of a single agent, EAEC was dominant and isolated from 20.4% of the patients, whereas rotavirus was detected in 14.8%. These results indicate that rotavirus and EAEC play a significant role as agents of childhood diarrhea in the local population.

Prevalence of diarrheogenic Escherichia coli and rotavirus among children from Botucatu, Säo Paulo State, Brazil

In a one-year prospective study carried out to define the role of rotavirus and Escherichia coli in local childhood diarrhea, we determined the prevalence of both agents in 54 diarrheic children attending a health center in Botucatu. Diarrheogenic E. coli (DEC) strains were characterized by O:H serotyping, a search for virulence genetic markers, and assays of adherence to HEp-2 cells. Except for enteroaggregative E. coli (EAEC), no other DEC category was detected in the children's stools. Both EAEC and rotavirus were isolated from 22 of the 54 (41.0 percent) diarrheic children as single agents or in combination with other enteropathogens. However, when considering the presence of a single agent, EAEC was dominant and isolated from 20.4 percent of the patients, whereas rotavirus was detected in 14.8 percent. These results indicate that rotavirus and EAEC play a significant role as agents of childhood diarrhea in the local population

Cd8(+)/V beta 5.1(+) large granular lymphocyte leukemia associated with autoimmune cytopenias, rheumatoid arthritis and vascular mammary skin lesions: successful response to 2-deoxycoformycin.

We report a case of CD8(+)/V beta 5.1(+) T-cell large granular lymphocyte leukemia (T-LGL leukemia) presenting with mild lymphocytosis, severe autoimmune neutropenia, thrombocytopenia, polyarthritis and recurrent infections with a chronic disease course. Immunophenotyping showed an expansion of CD3(+)/TCR alpha beta(+)/CD8(+bright)/CD11c(+)/CD57(-)/CD56(-) large granular lymphocytes with expression of the TCR-V beta 5.1 family. Southern blot analysis revealed a clonal rearrangement of the TCR beta-chain gene. Hematopoietic growth factors, high dose intravenous immunoglobulin and corticosteroids were of limited therapeutic benefit to correct the cytopenias. During the disease course, the patient developed a severe cutaneous leg ulcer and bilateral vascular mammary skin lesions. Treatment with 2-deoxycoformycin resulted in both clinical and hematological complete responses, including the resolution of vascular skin lesions. Combined immuno-staining with relevant T-cell associated and anti-TCR-V beta monoclonal antibodies proved to be a sensitive method to assess the therapeutic effect of 2-deoxycoformicin and to evaluate the residual disease.

Differences in phenotype, growth factor requirements, pattern of expression of adhesion molecules and rate of apoptosis displayed by three new myeloid sister leukemic cell lines.

We established three new human myeloid cell lines from one patient, in the presence of granulocyte-macrophage colony-stimulating factor (UPM1-GM), interleukin-3 (UMP1-IL-3) or without exogenous growth factors (UPM1). The 3 lines were characterized by phenotypic, genotypic and functional studies. These cell lines may provide useful tools to study different aspects of leukemic cell biology

Factors that can interfere with virus concentration from wastewater when using zeta plus 60S filter membranes

Zeta plus filter membranes (ZP60S) have been shown to be efficient for rotavirus concentration from wastewater and for the reduction of cytotoxicity for cell cultures. Recently a variability in both properties was observed. In view of the low costs and the high virus recovery rates obtained in the past, we re-evaluated the application of ZP60S filter membranes for virus concentration from environmental samples. Some factors that could interfere with the concentration strategy using ZP60S were also considered and assessed including the type of water to be filtered and the possible release of toxic substances from the membrane matrix during filtration.

Factors that can interfere with virus concentration from wastewater when using Zeta Plus 60S filter membranes.

Zeta plus filter membranes (ZP60S) have been shown to be efficient for rotavirus concentration from wastewater and for the reduction of cytotoxicity for cell cultures. Recently a variability in both properties was observed. In view of the low costs and the high virus recovery rates obtained in the past, we re-evaluated the application of ZP60S filter membranes for virus concentration from environmental samples. Some factors that could interfere with the concentration strategy using ZP60S were also considered and assessed including the type of water to be filtered and the possible release of toxic substances from the membrane matrix during filtration.

Enumeration of CD4(+) T-cells in the peripheral blood of HIV-infected patients: an interlaboratory study of the FACSCount system.

The aim of the present study was to assess the interlaboratory reproducibility of the FACSCount system for the enumeration of peripheral blood (PB) CD4(+) T-cells. In each of the seven participating centers, both previously stained and unstained PB samples (n = 49) were received and either analyzed or stained and then analyzed. Interlaboratory reproducibility was checked in two different groups of centers (n = 3 and n = 4) where the study was performed in parallel. In addition, both the intralaboratory precision and accuracy of this system were analyzed in comparison with results obtained with conventional flow cytometry. Accordingly, upon comparing both methods, a high degree of correlation was observed in the total number of CD3(+) T-cells (coefficient of correlation of 0.9750 +/- 0.0184, slope of the best linear fit: 0. 9214 +/- 0.0311, y-intercept of 12 +/- 47) as well as in the number of CD3(+)/CD4(+) (coefficient of correlation of 0.9794 +/- 0.1457, slope of the best linear fit: 0.9463 +/- 0.0753, y-intercept of -11 +/- 36) and CD3(+)/CD8(+) (coefficient of correlation of 0.9728 +/- 0.0192, slope of the best linear fit: 0.9682 +/- 0.0735, y-intercept of 7 +/- 95) major subsets. In addition, low coefficients of variation (CV) were obtained for replicates, indicating the method's high degree of accuracy. The present study shows that with respect to the interlaboratory reproducibility reported for most techniques used for the enumeration of PB CD4(+) T-cells, the FACSCount system results in data with much lower coefficients of variance (CVs) (mean CV of less than 10%). Upon measuring the impact on results of different variables associated with either sample preparation or data acquisition and analysis, our study clearly shows that data acquisition and analysis does not influence the results by increasing variability since the coefficients of variation obtained for samples prepared in the same laboratory under the same conditions and read in different laboratories with different instruments were identical to those obtained for the replicates of the same samples read in each individual center. In contrast, interlaboratory variability, although low, significantly increased when sample preparation was carried out in different laboratories, suggesting that pipetting still represents the major source of variability in the FACSCount system.

The impact of soccer training on the immune system.

OBJECTIVE: The aim of this study was to evaluate whether the level of some immune markers changed in soccer players during pre-season and during playing-season. DESIGN: An observational study for one soccer season (one year). SETTING: Professional soccer players. PARTICIPANTS: Thirteen professional soccer players (age: 26.3 +/- 3.7 years). MEASURES: The effect of 11 months of soccer training sessions and matches on circulating leukocyte and lymphocyte subpopulations was investigated. Blood samples were taken before the season and also 6 weeks, 6 months and 11 months later. All samples were taken at 08:00 h. RESULTS: Total lymphocyte counts did not change in all samples. During the pre-season, however, the concentrations of some inflammatory lymphocyte subpopulations were changed: CD4CD45RA+ was elevated and CD57 and CD8CD57 showed a significant decrease (p < 0.05), At the competitive period, CD4 and CD8 were increased. At the end of the season there were significant changes in some immune cells: total leukocyte and neutrophil number and CD8+ cell concentration was increased compared to pre-season values and the CD4/CD8 ratio was decreased. CONCLUSIONS: The results suggest that athletes exposed to a long-term training periods can exhibit variations in some immune cells. The clinical significance of these variations requires more detailed investigation.

Detection of rotavirus in sewage and creek water: efficiency of the concentration method

Simian rotavirus SA-11, experimentally seede, was recovered from raw domestic sewage by a two-step concentration procedure, using filtration through a positively charged microporous filter (Zeta Plus 60 S) followed by ultracentrifugation, effecting an 8000-fold concentration. By this method, a mean recovery of 81 per centñ7.5 of the SA-11 virus was achieved.

Detection of rotavirus in sewage and creek water: efficiency of the concentration method.

Simian rotavirus SA-11 experimentally seeded, was recovered from raw domestic sewage by a two-step concentration procedure, using filtration through a positively charged microporous filter (Zeta Plus 60 S) followed by ultracentrifugation, effecting an 8,000-fold concentration. By this method, a mean recovery of 81% +/- 7.5 of the SA-11 virus, was achieved.