Functional tests associated with sarcopenia in moderate chronic obstructive pulmonary disease.

Objectives: To evaluate the association of physical and functional measures with sarcopenia in moderate chronic obstructive pulmonary disease (COPD) and to establish cutoff points for sarcopenia screening.Methods: The study included COPD with and without sarcopenia, of both sexes who were over 50 years old. Participants were assessed for lung function, body composition, grip strength, Short Physical Performance Battery (SPPB), 5-repetition, 10-repetition and 30-s sit-to-stand tests (5STS, 10STS, and 30STS, respectively). In addition, 6-min walking test, respiratory muscular strength, and physical activity level were tested.Results: The study had 35 participants, 24 men (68.6%) and moderate COPD (51.4%). COPD-sarcopenia showed lower values in lean mass, body fat and body mass alongside lower performance in 10 and 30 STS tests, SPPB and gait speed compared to non-sarcopenic group. The cutoff points with better sensitivity and specificity to identify sarcopenia were 10.88 and 34.14 s, 15 repetitions, and 10 points in the 5STS, 10STS, 30STS, and SPPB, respectively. The comparison of the receiver operating curves evidenced no differences between the functional tests. Only 30STS and SPPB showed acceptable discriminatory power.Conclusion: Functional tests, especially 30STS and SPPB, are simple and affordable tools for screening sarcopenia in COPD with moderate obstruction.

Does whole body vibration exercise improve oxidative stress markers in women with fibromyalgia?

The objective of this study was to investigate the effect of whole body vibration (WBV) exercise on oxidative stress markers in a group of women with fibromyalgia (FM) compared to a group of healthy women (CT). Twenty-one women diagnosed with FM and 21 age- and weight-matched healthy women were enrolled the study. Plasma oxidative stress markers (primary outcomes) were evaluated at rest and after WBV, and included thiobarbituric acid reactive substances (TBARS), iron reduction capacity (FRAP), superoxide dismutase antioxidant enzymes activity (SOD), and catalase (CAT). At rest, the FM group had higher TBARS (P<0.001) and FRAP (P<0.001), and lower CAT (P=0.005) compared to the CT. In the CT group, the WBV had no effect on TBARS (P=0.559) and FRAP (P=0.926), whereas it increased both SOD (P<0.001) and CAT (P<0.001). In the FM group, the WBV reduced TBARS (p <0.001), FRAP (P<0.001), and CAT (P=0.005), while it increased SOD (P=0.019). There was an interaction effect (moments vs groups) in the TBARS (effect size=1.34), FRAP (effect size=0.93), CAT (effect size=1.45), and SOD (effect size=1.44) (P<0.001). A single trial of WBV exercise improved all oxidant and antioxidant parameters towards a greater adaptation to the stress response in FM women.

Does whole body vibration exercise improve oxidative stress markers in women with fibromyalgia?

The objective of this study was to investigate the effect of whole body vibration (WBV) exercise on oxidative stress markers in a group of women with fibromyalgia (FM) compared to a group of healthy women (CT). Twenty-one women diagnosed with FM and 21 age- and weight-matched healthy women were enrolled the study. Plasma oxidative stress markers (primary outcomes) were evaluated at rest and after WBV, and included thiobarbituric acid reactive substances (TBARS), iron reduction capacity (FRAP), superoxide dismutase antioxidant enzymes activity (SOD), and catalase (CAT). At rest, the FM group had higher TBARS (P<0.001) and FRAP (P<0.001), and lower CAT (P=0.005) compared to the CT. In the CT group, the WBV had no effect on TBARS (P=0.559) and FRAP (P=0.926), whereas it increased both SOD (P<0.001) and CAT (P<0.001). In the FM group, the WBV reduced TBARS (p <0.001), FRAP (P<0.001), and CAT (P=0.005), while it increased SOD (P=0.019). There was an interaction effect (moments vs groups) in the TBARS (effect size=1.34), FRAP (effect size=0.93), CAT (effect size=1.45), and SOD (effect size=1.44) (P<0.001). A single trial of WBV exercise improved all oxidant and antioxidant parameters towards a greater adaptation to the stress response in FM women.

Detection and Molecular Characterization of Yellow Fever Virus, 2017, Brazil.

At the end of 2016, Brazil experienced an unprecedented yellow fever (YF) outbreak. Clinical, molecular and ecological aspects of human and non-human primate (NHP) samples collected at the beginning of the outbreak are described in this study. Spatial distribution analyses demonstrated a strong overlap between human and NHP cases. Through molecular analyses, we showed that the outbreak had a sylvatic origin, caused by the South American genotype 1 YFV, which has already been shown to circulate in Brazil. As expected, the clusters of cases were identified in regions with a low vaccination coverage. Our findings highlight the importance of the synchronization of animal surveillance and health services to identify emerging YF cases, thereby promoting a better response to the vulnerable population.

Genomic and epidemiological monitoring of yellow fever virus transmission potential.

The yellow fever virus (YFV) epidemic in Brazil is the largest in decades. The recent discovery of YFV in Brazilian Aedes species mosquitos highlights a need to monitor the risk of reestablishment of urban YFV transmission in the Americas. We use a suite of epidemiological, spatial, and genomic approaches to characterize YFV transmission. We show that the age and sex distribution of human cases is characteristic of sylvatic transmission. Analysis of YFV cases combined with genomes generated locally reveals an early phase of sylvatic YFV transmission and spatial expansion toward previously YFV-free areas, followed by a rise in viral spillover to humans in late 2016. Our results establish a framework for monitoring YFV transmission in real time that will contribute to a global strategy to eliminate future YFV epidemics.

Analyzing similarities in genome sequences.

This article investigates aspects of similarity between complete sequences of mitochondrial DNA by determining the distribution of the relative frequencies of words with different lengths and the characteristics of their relevance throughout the sequences. The degree of similarity is obtained by comparing the distances between words contained within these sequences. Our results indicate that the best groupings among different species depend on the lengths of words and their respective relative frequencies. We also observed that the longer the word the more consistent the grouping between the sequences becomes. The application of our results, together with the perspective of analyzing DNA sequences belonging to a single biological species, may be important for the construction of phylogenetic trees, which are appropriate structures for understanding the evolutionary history of the species.

Effect of phosphating on soil chemical attributes and cultivation of sugarcane for forage / Efeitos da fosfatagem nos atributos químicos do solo e produção de cana-de-açúcar para forragem

Phosphorus (P) is an essential element for the cultivation of sugarcane, which plays an important role in rooting, tillering and forage production. With the objective to evaluate the effects of pre-planting phosphating on soil chemical attributes and sugarcane growth for forage production, an experiment was conducted at the Santa Mercedes plant, Tupi Paulista, SP, using the RB 86 7515 variety. The experimental design consisted of random blocks with 8 treatments and 4 replicates, totaling 32 experimental units, in a 2 x 4 factorial scheme including two P sources (decanted phosphate and monoammonium phosphate) and four P doses (0, 80, 120, and 160 kg/ ha P2O5). The P sources and doses tested increase the availability of P in soil, with the dose of 120 kg/ha P2O5 providing the highest production of green forage mass. Monoammonium phosphate as P source reduces soil pH and increases the availability of micronutrients such as iron, boron and zinc.

O fósforo (P) é considerado um elemento essencial para o cultivo da cana-de-açúcar, assumindo grande importância no enraizamento, no perfilhamento e na produção de forragem. Com o objetivo de avaliar os efeitos da fosfatagem em pré-plantio nos atributos químicos do solo e no desenvolvimento da cultura da cana-de-açúcar para produção de forragem foi realizado um experimento com a variedade RB 86 7515, junto à unidade produtora da Usina Santa Mercedes, Tupi Paulista, SP. O delineamento experimental foi em blocos ao acaso com 8 tratamentos e 4 repetições, totalizando 32 unidades experimentais, em esquema fatorial 2 x 4, duas fontes de fósforo (fosfato decantado e fosfato de monoamônio) e quatro doses de fósforo (0; 80; 120; 160 kg/ha P2O5). As fontes e doses testadas de fósforo (P) aumenta a disponibilidade de P no solo, sendo a dose de 120 kg/ha P2O5 que apresenta maior produção de massa verde de forragem. A fonte fosfato monoamônico reduz o pH do solo e aumenta a disponibilidade dos micronutrientes como ferro, boro e zinco.

Effect of phosphating on soil chemical attributes and cultivation of sugarcane for forage / Efeitos da fosfatagem nos atributos químicos do solo e produção de cana-de-açúcar para forragem

Phosphorus (P) is an essential element for the cultivation of sugarcane, which plays an important role in rooting, tillering and forage production. With the objective to evaluate the effects of pre-planting phosphating on soil chemical attributes and sugarcane growth for forage production, an experiment was conducted at the Santa Mercedes plant, Tupi Paulista, SP, using the RB 86 7515 variety. The experimental design consisted of random blocks with 8 treatments and 4 replicates, totaling 32 experimental units, in a 2 x 4 factorial scheme including two P sources (decanted phosphate and monoammonium phosphate) and four P doses (0, 80, 120, and 160 kg/ ha P2O5). The P sources and doses tested increase the availability of P in soil, with the dose of 120 kg/ha P2O5 providing the highest production of green forage mass. Monoammonium phosphate as P source reduces soil pH and increases the availability of micronutrients such as iron, boron and zinc.(AU)

O fósforo (P) é considerado um elemento essencial para o cultivo da cana-de-açúcar, assumindo grande importância no enraizamento, no perfilhamento e na produção de forragem. Com o objetivo de avaliar os efeitos da fosfatagem em pré-plantio nos atributos químicos do solo e no desenvolvimento da cultura da cana-de-açúcar para produção de forragem foi realizado um experimento com a variedade RB 86 7515, junto à unidade produtora da Usina Santa Mercedes, Tupi Paulista, SP. O delineamento experimental foi em blocos ao acaso com 8 tratamentos e 4 repetições, totalizando 32 unidades experimentais, em esquema fatorial 2 x 4, duas fontes de fósforo (fosfato decantado e fosfato de monoamônio) e quatro doses de fósforo (0; 80; 120; 160 kg/ha P2O5). As fontes e doses testadas de fósforo (P) aumenta a disponibilidade de P no solo, sendo a dose de 120 kg/ha P2O5 que apresenta maior produção de massa verde de forragem. A fonte fosfato monoamônico reduz o pH do solo e aumenta a disponibilidade dos micronutrientes como ferro, boro e zinco.(AU)

Detection of Vaccinia virus during an outbreak of exanthemous oral lesions in Brazilian equids.

REASONS FOR PERFORMING STUDY: In August 2014, an outbreak of oral exanthematous disease in equids was reported in Brazil, affecting 11 donkeys and 3 mules. OBJECTIVES: To investigate if Vaccinia virus (VACV) was the aetiological agent in this outbreak. STUDY DESIGN: Investigation of clinical cases using serological, molecular and phylogenetic approaches. METHODS: To analyse the presence of neutralising antibodies against VACV, samples were submitted in triplicate to a plaque-reduction neutralisation test (PRNT50% ). On the basis of previous studies which detected VACV DNA in sera, we submitted extracted DNA samples to different polymerase chain reaction (PCR) platforms targeting Orthopoxvirus (OPV) genes (C11R, A56R and A26L). The PCR products were directly sequenced in both orientations using specific primers and capillary electrophoresis. The alignment and phylogenetic analysis of the A26L and A56R nucleotide sequences (maximum likelihood) were prepared with the obtained nucleotide fragments. RESULTS: Serological and molecular data suggested VACV as the aetiological agent. The neutralising antibodies against OPV were detected in 5 (55.5%) of the equids, with titres ≥40 neutralising u/ml. Based on the results obtained from all PCR platforms, all samples were positive for OPV: 9 (100%) for A56R, 4 (44.4%) for C11R and 3 (33.3%) for A26L. The alignment of the nucleotide sequences of the A26L and A56R fragments revealed that the samples were highly similar to the homologous genes from other Brazilian VACV Group 1 isolates (98.8% identity on average). Furthermore, both the A26L and A56R sequences showed signature deletions also present in the sequences of Group 1 VACV isolates from Brazil. CONCLUSIONS: Our data raises questions about the role of equids in the chain of VACV epidemiology. The surveillance of equids in VACV-affected areas worldwide is relevant.

A Correlational Analysis of Tethered Swimming, Swim Sprint Performance and Dry-land Power Assessments.

Swimmers are often tested on both dry-land and in swimming exercises. The aim of this study was to test the relationships between dry-land, tethered force-time curve parameters and swimming performances in distances up to 200 m. 10 young male high-level swimmers were assessed using the maximal isometric bench-press and quarter-squat, mean propulsive power in jump-squat, squat and countermovement jumps (dry-land assessments), peak force, average force, rate of force development (RFD) and impulse (tethered swimming) and swimming times. Pearson product-moment correlations were calculated among the variables. Peak force and average force were very largely correlated with the 50- and 100-m swimming performances (r=- 0.82 and -0.74, respectively). Average force was very-largely/largely correlated with the 50- and 100-m performances (r=- 0.85 and -0.67, respectively). RFD and impulse were very-largely correlated with the 50-m time (r=- 0.72 and -0.76, respectively). Tethered swimming parameters were largely correlated (r=0.65 to 0.72) with mean propulsive power in jump-squat, squat-jump and countermovement jumps. Finally, mean propulsive power in jump-squat was largely correlated (r=- 0.70) with 50-m performance. Due to the significant correlations between dry-land assessments and tethered/actual swimming, coaches are encouraged to implement strategies able to increase leg power in sprint swimmers.

Pamidronate affects the mandibular cortex of children with osteogenesis imperfecta.

We hypothesized that mandibular cortical width (MCW) is smaller in children with osteogenesis imperfecta (OI) than in healthy children and that pamidronate can improve the cortical mandibular thickness. The aim of this study was to assess changes in the MCW on dental panoramic radiographs (DPRs) of children with normal bone mineral density (BMD) and with OI. We also compared the MCW of children with different types of OI regarding the number of pamidronate cycles and age at the beginning of treatment. MCW measurements were retrospectively obtained from 197 DPRs of 66 children with OI types I, III, and IV who were in treatment with a comparable dosage of cyclical intravenous pamidronate between 2007 and 2013. The control group had 92 DPRs from normal BMD children. Factorial analysis of variance was used to compare MCW measurements among different age groups and between sexes and also to compare MCW measurements of children with different types of OI among different pamidronate cycles and age at the beginning of treatment. No significant differences in results were found between male and female subjects in both OI and healthy children, so they were evaluated altogether (P > 0.05). There was an increase of MCW values related to aging in all normal BMD and OI children but on a smaller scale in children with OI types I and III. Children with OI presented lower mean MCW values than did children with normal BMD at the beginning of treatment (P < 0.05). A linear model estimated the number of pamidronate cycles necessary to achieve mean MCW values equivalent to those of healthy children. The thinning of the mandibular cortex depended on the number of pamidronate cycles, the type of OI, and the age at the beginning of treatment. DPRs could thus provide a way to identify cyclic pamidronate treatment outcomes in patients with OI.

Assessment of the in vitro and in vivo genotoxicity of extracts and indole monoterpene alkaloid from the roots of Galianthe thalictroides (Rubiaceae).

Roots of Galianthe thalictroides K. Schum. (Rubiaceae) are used in folk medicine in the State of Mato Grosso do Sul, Brazil, for treating and preventing cancer. To gain information about the genotoxicity of extracts (aqueous and EtOH), the CHCl3 phase resulting from partition of the EtOH extract and the indole monoterpene alkaloid 1 obtained from this plant. The genotoxicity of 1 and extracts was evaluated in vivo through the Drosophila melanogaster wing Somatic Mutation and Recombination Test - SMART, while in vitro cytotoxic (MTT) and Comet assays were performed only with alkaloid 1. The results obtained with the SMART test indicated that the aqueous extract had no genotoxic activity. The EtOH extract was not genotoxic to ST descendants but genotoxic to HB ones. The CHCl3 phase was genotoxic and cytotoxic. Alkaloid 1 showed significant mutational events with SMART, in the cytotoxicity assay (MTT), it showed a high cytotoxicity for human hepatoma cells (HepG2), whereas for the Comet assay, not showing genotoxic activity. The ethanol extract was shown to be genotoxic to HB descendants in the SMART assay, while the results obtained in this test for the monoterpene indole alkaloid 1 isolated from this extract.

Contrast-enhanced CT and MRI for detecting neck metastasis of oral cancer: comparison between analyses performed by oral and medical radiologists.

OBJECTIVES: The aim of the study was to verify the concordance of contrast-enhanced CT (CECT) and MRI evaluation among four radiologists in detecting metastatic cervical lymph nodes of oral cancer patients. METHODS: Ten patients underwent clinical and imaging examinations (CECT and MRI). Four radiologists, two oral and maxillofacial radiologists (OMRs) and two medical radiologists (MRs), independently analysed the images twice. Cohen's kappa index and Wilcoxon signed-rank test were used to verify the concordance between all analyses. RESULTS: Regarding the interobserver agreement, the OMRs presented excellent kappa values for determining the regional lymph nodes (N-stage) in both CECT and MRI. The MRs presented moderate agreement for CECT evaluation at the first reading, but no concordance was found for the other analyses. When each imaging modality was analysed separately, kappa values were higher between all examiners. Greater variability was demonstrated between N-stage evaluation using different examinations. All radiologists were able to identify a greater number of metastatic lymph nodes in CECT than in MRI, except one MR, but no significant difference was found for all readers. The differences between the number of metastatic lymph nodes among all radiologists were not statistically significant. Moderate intraobserver agreement was observed for CECT and MRI evaluation, except for one MR. CONCLUSIONS: The differences found between the N-stage performed by OMRs and MRs support the necessity of a multidisciplinary approach in the imaging evaluation of metastatic nodes. Further studies are necessary to confirm which imaging modality should be employed when evaluating neck areas.

PP139. The elective prematurity birth and its perinatal effects of syndromes in hypertensive pregnancy.

INTRODUCTION: The hypertensive pregnancy disorders affect 7.5% of Brazilian women, being a major cause of maternal and perinatal morbidity and mortality, affecting 6-10% of primigravidae. They represent a significant cause of prematurity birth, with elective responsible for 20-30% of premature births, with higher incidence of neonatal morbidity due to high rates of complications in short and long term. OBJECTIVES: Identify the repercussions in a short time, on caused by elective obstetric parity decisions in women with hypertensive neonatals pregnancy syndromes. METHODS: Retrospective analysis of medical records of pregnant women admitted to Hospital Guilherme Álvaro, in the city of Santos/SP - Brazil, between Jan/2005 to Jan/2012. Calculations were performed for dichotomous variables and an epidemiological analysis with data obtained on pregnant women with hypertension with a single fetus, subjected to delivery before 37 weeks. Classification of Hypertensive Syndromes followed the criteria of the NHBPEP - 2000, after blood pressure measurements were calibrated following standard technique: Chronic hypertension (CH), Pre-eclampsia, superimposed pre-eclampsia and late gestational hypertension and multiple births were excluded, premature labor and fetal malformations, totaling 102 cases. RESULTS: Throughout the study, it was observed that 67.6% are appropriate for gestational age, birth weight 1500-2500grams (43.1%). Support in the neonatal were excluded ICU was necessary in the vast majority (75.7%), and length of stay exceeding 28 days in 29.3% of cases. The main indication was respiratory distress syndrome (63.6%) requiring intubation orotraquel and jaundice in 18.2% of premature infants was a common comorbidity (39.4%). Even with all the support in-hospital, the neonatal mortality was present in 8.1% of cases. The main indication of the resolution was for obstetric maternal complications (61.8%) where pre-eclampsia and preeclampsia superimposed were expressions which caused more frequent (42.2 and 40.2% respectively), showing the level of proteinuria between two -4.9g (32.1%). Gestational age at delivery was 32-34 weeks (43.1%), corticosteroid therapy was not performed (54.9%) and cesarean delivery was (94.1%). The most frequent age group was 20-34 years (53.9%), Caucasian (54.9%), primigravida (62.7%) and less than five prenatal visits (36.3%) were performed. CONCLUSION: The hypertensive pregnancy are risk factors for preterm elective delivery and is more frequent before 34 weeks, resulting in high perinatal morbidity and mortality, especially in cases of preeclampsia with severe clinical presentation and long hospital stays, with consequent costs and conducting excessive procedures in the neonate. Enable early referral to specialist services and consequent prenatal follow up, enables the adoption of early action, such as clinical support in a timely manner to facilitate the handling of cases which are still stable, optimizing the opportunity for administration of antenatal corticosteroids. It is necessary to review decisions about the mode of delivery in prematurity related to hypertension cases, with the aim of increasing the occurrence of vaginal deliveries.

EGFR is involved in control of gastric cell proliferation through activation of MAPK and Src signalling pathways in early-weaned rats.

OBJECTIVES: Early weaning (EW) increases proliferation of the gastric epithelium in parallel with higher expression of transforming growth factor alpha and its receptor epidermal growth factor receptor (EGFR). The primary objective of the present study was to examine involvement of EGFR signalling in regulating mucosal cell proliferation during the early weaning period. MATERIALS AND METHODS: Fifteen-day-old rats were split into two groups: suckling (control) and EW, in which pups were separated from the dam. Animals were killed daily until the 18th day, 3 days after onset of treatment. To investigate the role of EGFR in proliferation control, EW pups were injected with AG1478, an EGFR inhibitor; signalling molecules, proliferative indices and cell cycle-related proteins were evaluated. RESULTS: EW increased ERK1/2 and Src phosphorylation at 17 days, but p-Akt levels were unchanged. Moreover, at 17 days, AG1478 administration impaired ERK phosphorylation, whereas p-Src and p-Akt were not altered. AG1478 treatment reduced mitotic and DNA synthesis indices, which were determined on HE-stained and BrdU-labelled sections. Finally, AG1478 injection decreased p21 levels in the gastric mucosa at 17 days, while no changes were detected in p27, cyclin E, CDK2, cyclin D1 and CDK4 concentrations. CONCLUSIONS: EGFR is part of the mechanism that regulates cell proliferation in rat gastric mucosa during early weaning. We suggest that such responses might depend on activation of MAPK and/or Src signalling pathways and regulation of p21 levels.

The role of stochasticity on compactness of the native state of protein peptide backbone.

A restricted angular random-walk model to build up polypeptide structures, which encompasses properties of the dihedral-angle Ramachandran map of folded proteins, is proposed to study the role of stochasticity on the compactness of the native state of proteins. Sample structures will be built with lengths ranging from 125 up to 400 amino acids for the different fractions of secondary structure motifs, from which dihedral angles were randomly chosen according to narrow Gaussian probability distributions. Physical properties of these polypeptide protein backbones such as the radius of gyration, the compactness parameter, the number of contacts, and the associated energy were computed and analyzed from an ensemble of thousands of realizations of protein peptide chains built with different rates of alpha-helix or beta-strand motifs. Such geometric and physical parameters are compared to data from several globular proteins extracted from the Protein Data Bank indicating that a certain (small fraction) randomness is an essential ingredient for achieving the folded state of proteins, suggesting that they are neither driven by deterministic nor random-walk processes.

Comparison between computed tomography and clinical evaluation in tumour/node stage and follow-up of oral cavity and oropharyngeal cancer.

OBJECTIVES: The aim was to verify the concordance of CT evaluation among four radiologists (two oral and maxillofacial and two medical radiologists) at the TN (tumour/node) stage and in the follow-up of oral cavity and oropharyngeal cancer patients. The study also compared differences between clinical and CT examinations in determining the TN stage. METHODS: The following clinical and tomographic findings of 15 non-treated oral cavity and oropharyngeal cancer patients were compared: tumour size, bone invasion and lymph node metastases. In another 15 patients, who had previously been treated, a clinical and tomographic analysis comparison for the presence of tumoural recurrence, post-therapeutic changes in muscles and lymph node metastases was performed. The concordances of tomographic evaluation between the radiologists were analysed using the kappa index. RESULTS: Significant agreement was verified between all radiologists for the T stage, but not for the N stage. In the group of treated patients, CT disclosed post-therapeutic changes in muscles, tumour recurrence and lymph node metastases, but no concordance for the detection of lymph node metastases was found between radiologists. In the first group, for all radiologists, no concordance was demonstrated between clinical and tomographic staging. CT was effective for delimitating advanced lesions and for detecting lymph node involvement in N0 stage patients. CT revealed two cases of bone invasion not clinically detected. CONCLUSIONS: Interprofessional relationships must be stimulated to improve diagnoses, and to promote a multidisciplinary approach to oral cavity and oropharyngeal cancer. Although CT was important in the diagnosis and follow-up of cancer patients, differences between medical and dental analyses should be acknowledged.

Induction of apoptosis in Caco-2 and HT-29 human intestinal epithelial cells by enterohemolysin produced by classic enteropathogenic Escherichia coli.

AIMS: Detect the cytotoxic effects of the Enterohemolysin from enteropathogenic Escherichia coli C3888 (O 26: H-) on Caco 2 and HT-29-human epithelial intestinal cells. METHODS AND RESULTS: The Caco 2 and HT-29 cells, which were treated with Enterohemolysin (EHly) within 10-15 min, became round, lost attachment to substrate, showed extensive surface blebbing, nucleus shrank, and the chromatin became more compact. After 10 min of exposure to the EHly, the cells showed lactate dehydrogenase (LDH) leakage and reduction of mitochondrial activity. The cells showed disorganization of the actin fibers at 15 min. The death of these human epithelial intestinal cells by apoptosis was confirmed by annexin V. CONCLUSIONS: Enterohemolysin induced apoptosis on human epithelial intestinal cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding of EHly cytotoxic activity suggests the involvement of this hemolysin in the (Enteropathogenic Escherichia coli) EPEC infection mechanism and may facilitate the understanding of the diarrhea caused by EPEC.

Cytotoxic activity of clinical Stenotrophomonas maltophilia.

AIMS: To determine the potential virulence factors produced by culture supernatants of clinical isolates of Stenotrophomonas maltophilia. METHODS AND RESULTS: Culture supernatants of clinical isolates of S. maltophilia were assayed for haemolytic, enzymatic (lipase, protease and phospholipase) and cytotoxic activity. Cytotoxic activity was assayed in Vero (African green monkey), HeLa (human cervix) and HEp-2 (human larynx epidermoid carcinoma) cells. Microscopic analyses revealed intensive rounding, loss of intercellular junctions and membrane alterations (blebbing) followed by death of HEp-2 cells. In Vero and HeLa cells, the cytotoxic effects were characterized by vigorous endocytosis and cell aggregation. The viability of cultured mammalian cells was determined with neutral red and demonstrated that the sensitivity among the cells was different. This activity was inactivated by heating at 56 degrees C for 15 min and protease inhibitors did not inhibit cytotoxic activity. The clinical S. maltophilia presented a cell-free haemolytic activity similar to the 'hot-cold' haemolysins. CONCLUSIONS: S. maltophilia culture supernatants caused vigorous endocytosis and cell aggregation in HeLa and Vero cells, produced haemolytic and enzymatic activities. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed the presence of putative virulence factors that could be associated with human infections involving Stenotrophomonas maltophilia strains.

Thiol-independent activity of a cholesterol-binding enterohemolysin produced by enteropathogenic Escherichia coli

Enterohemolysin produced by Escherichia coli associated with infant diarrhea showed characteristics similar to those of thiol-activated hemolysins produced by Gram-positive bacteria, including inactivation by cholesterol, lytic activity towards eukaryotic cells and thermoinstability. However, enterohemolysin activity was not inactivated by oxidation or by SH group-blocking agents (1 mM HgCl2, 1 mM iodoacetic acid) and the hemolysin (100 æg/ml) was not lethal to mice, in contrast to the lethality of the thiol-activated hemolysin family to animals. Earlier reports showed that intravenous injection of partially purified streptolysin O preparations (0.2 æg) was rapidly lethal to mice. These results suggest that E. coli enterohemolysin is not a thiol-activated hemolysin, despite its ability to bind cholesterol, probably due to the absence of free thiol-group(s) that characterize the active form of the thiol-activated hemolysin molecule.