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Lisbon, Portugal

Dogi C.A.,CONICET | Weill F.,University of Buenos Aires | Perdigon G.,CONICET | Perdigon G.,Institute Microbiologia
Immunobiology | Year: 2010

The gut associated lymphoid tissue (GALT) is anatomical and functionally divided in inductive and effectors sites. In previous works we demonstrated that non-pathogenic bacteria with probiotic characteristics can improve the gut mucosal immune system, with an increase in the number of IgA and cytokines producing cells in the effector site of the intestine. In the present work we studied the effect of non-pathogenic Gram(+), Gram(-) bacteria and a Gram(+) probiotic strain on the inductor site (PP) after the oral administration to BALB/c mice. We also studied some signals induced by the assayed strain in the effectors site, such as the enzyme calcineurin and TLR-9 as a way to understand the mechanisms induced in such bacterial stimulation. The implicance of the lipoteichoic acid (LTA) in the immunostimulation was analyzed. All strains increased the number of IFN-γ and TNF-α(+) cells, but not of IL-10(+) cells in the total population of PP. The release of IFN-γ and TNF-α was only induced by LPS stimulation. All assayed strains increased the number of calcineurin(+) cells, while only Gram(+) strains increased the number of TLR-9(+) cells. The immunostimulatory properties of the purified LTA from Gram(+) strains was evaluated on a monocyte-macrophage U937 cell line. These cells showed capacity to release TNF-α and IL-10 in response to all LTA assayed in a dose-dependent way. Gram(+) strains induced signals through the calcineurin enzyme able to activate the transcriptional factor NFAT and through TLR-9. The LTA molecule from Gram(+) strains would not be the only structure involved in the immunostimulatory properties observed, specially for the probiotic strain. © 2009 Elsevier GmbH. All rights reserved. Source


Ale C.E.,CONICET | Bru E.,CONICET | Strasser de Saad A.M.,Institute Microbiologia | Pasteris S.E.,CONICET
Journal of Applied Microbiology | Year: 2014

Aim: To evaluate the effect of temperature, pH and SO2 on growth and glycerol production improvement by Saccharomyces cerevisiae mc2, Kloeckera apiculata mF and Oenococcus oeni X2L using the response surface method (RSM). Methods and Results: Multifactorial design of cultures with physicochemical factors variations was performed. The micro-organisms grew in all cultures conditions. Overall, after 6 days yeasts prevailed, especially S. cerevisiae (109 CFU ml-1), while O. oeni reached 107 CFU ml-1. At initial fixed pH 5·5, metabolic behaviour of cultures showed a temperature-dependent response. Total malate consumption occurred at 26°C, 50 mg l-1 SO2. Glucose and pentoses utilization was highly modified when varying SO2. Ethanol showed negative interaction with temperature-SO2 relationship. At low SO2, glycerol and acetate production increased when temperature enhanced. Predictive results of RSM indicate that 26°C, 60·24 mg l-1 SO2 and pH 5·5 were the optimal conditions for glycerol and organic acids synthesis compatible with wine quality. Conclusions: We propose a predictive condition to improve the performance of mixed cultures for must fermentations. Significance and Impact of the Study: To optimize the culture conditions to design mixed starters containing autochthonous yeasts and O. oeni strains for winemaking and to obtain products with high glycerol content, low acidity and maintenance of regional characteristics. © 2014 The Society for Applied Microbiology. Source


Rebelo M.,Institute Medicina Molecular | Tempera C.,Institute Medicina Molecular | Fernandes J.F.,University of Tubingen | Fernandes J.F.,University of Amsterdam | And 4 more authors.
Malaria Journal | Year: 2015

Background: In vitro sensitivity assays are crucial to detect and monitor drug resistance. Plasmodium falciparum has developed resistance to almost all anti-malarial drugs. Although different in vitro drug assays are available, some of their inherent characteristics limit their application, especially in the field. A recently developed approach based on the flow cytometric detection of haemozoin (Hz) allowed reagent-free monitoring of parasite maturation and detection of drug effects in culture-adapted parasites. In this study, the set-up, performance and usefulness of this novel assay were investigated under field conditions in Gabon. Methods: An existing flow cytometer (Cyflow Blue) was modified on site to detect light depolarization caused by Hz. Blood from malaria patients was incubated for 72 hrs with increasing concentrations of chloroquine, artesunate and artemisinin. The percentage of depolarizing red blood cells (RBC) was used as maturation indicator and measured at 24, 48 and 72 hrs of incubation to determine parasite growth and drug effects. Results: The flow cytometer was easily adapted on site to detect light depolarization caused by Hz. Analysis of ex vivo cultures of parasites, obtained from blood samples of malaria patients, showed four different growth profiles. In 39/46 samples, 50% inhibitory concentrations (IC50) were successfully determined. IC50 values for chloroquine were higher than 200 nM in 70% of the samples, indicating the presence of chloroquine-resistant parasites. For artesunate and artemisinin, IC50 values ranged from 0.9 to 60 nM and from 2.2 nM to 124 nM, respectively, indicating fully sensitive parasites. Conclusion: Flow cytometric detection of Hz allowed the detection of drug effects in blood samples from malaria patients, without using additional reagents or complex protocols. Adjustment of the initial parasitaemia was not required, which greatly simplifies the protocol, although it may lead to different IC50 values. Further investigation of set-up conditions of the Hz assay, as well as future studies in various settings should be performed to further determine the usefulness of this assay as a tool for rapid resistance testing in malaria-endemic countries. © 2015 Rebelo et al.; licensee BioMed Central. Source


Neumann A.,University of Veterinary Medicine Hannover | Vollger L.,University of Veterinary Medicine Hannover | Berends E.T.M.,University Utrecht | Molhoek E.M.,TNO | And 10 more authors.
Journal of Innate Immunity | Year: 2014

Neutrophil extracellular traps (NETs) have been described as a fundamental innate immune defence mechanism. They consist of a nuclear DNA backbone associated with different antimicrobial peptides (AMPs) which are able to engulf and kill pathogens. The AMP LL-37, a member of the cathelicidin family, is highly present in NETs. However, the function of LL-37 within NETs is still unknown because it loses its antimicrobial activity when bound to DNA in the NETs. Using im-munofluorescence microscopy, we demonstrate that NETs treated with LL-37 are distinctly more resistant to S. aureus nuclease degradation than nontreated NETs. Biochemical assays utilising a random LL-37-fragment library indicated that the blocking effect of LL-37 on nuclease activity is based on the cationic character of the AMP, which facilitates the binding to neutrophil DNA, thus protecting it from degradation by the nuclease. In good correlation to these data, the cationic AMPs human beta defensin-3 and human neutro-phil peptide-1 showed similar protection of neutrophil-de-rived DNA against nuclease degradation. In conclusion, this study demonstrates a novel role of AMPs in host immune defence: beside its direct antimicrobial activity against various pathogens, cationic AMPs can stabilise neutrophil-de-rived DNA or NETs against bacterial nuclease degradation. © 2014 S. Karger AG, Basel. Source


De Barbosa C.B.,Federal University of Rio de Janeiro | Lazzarini L.C.O.,Federal University of Rio de Janeiro | Elias A.R.,Oswaldo Cruz Institute | Leung J.A.M.,Clementino Fraga Filho University Hospital | And 9 more authors.
International Journal of Tuberculosis and Lung Disease | Year: 2012

BACKGROUND: We recently described the Mycobacterium tuberculosis RD Rio genotype, a clonally derived sublineage within the Latin American-Mediterranean (LAM) family. Genetic diversity of M. tuberculosis likely affects the clinical aspects of tuberculosis (TB). Prospective studies that address this issue are scarce and remain controversial. OBJECTIVE: To determine the association of differential clinical features of pulmonary TB with the RD Rio M. tuberculosis etiology. METHODS: Culture-proven pulmonary TB patients (n = 272) were clinically evaluated, including history, physical examination, chest X-ray and anti-human immunodeficiency virus serology. Isolates were classified as RD Rio or non-RD Rio M. tuberculosis by multiplex polymerase chain reaction and further spoligotyped. Clinical and M. tuberculosis genotype data were analyzed. RESULTS: RD Rio M. tuberculosis caused disease in 26.5% (72/270) of all TB cases. The LAM genotype, of which RD Rio strains are members, was responsible for 46.0% of the TB cases. Demographic data, major signs and symptoms, radiographic presentation, microbiological features and clinical outcomes were not significantly different among patients with TB caused by RD Rio and non-RD Rio strains. CONCLUSIONS: Disease caused by M. tuberculosis RD Rio strains was not clinically distinctive or more severe than disease caused by non-RD Rio strains in this series of TB patients. Larger prospective studies specifically designed to disclose differential clinical characteristics of TB caused by specific M. tuberculosis lineages are needed. © 2012 The Union. Source

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