Dynamics and kinetics of natural killer cell cytotoxicity in human malaria as evaluated by a novel stepwise cytotoxicity assay

Malaria causes important functional alterations of the immune system, but several of them are poorly defined. To evaluate thoroughly the natural killer cell cytotoxicity in patients with malaria, we developed a technique capable to assess both the dynamics and the kinetics of the process. For the kinetics assay, human peripheral blood mononuclear cells were previously incubated with K562 cells and kept in agarose medium, while for the dynamics assay both cells were maintained in suspension. NK activity from patients with vivax malaria presented a kinetics profile faster than those with falciparum malaria. NK cytotoxicity positively correlated with parasitemia in falciparum malaria. The dynamics of NK cytotoxicity of healthy individuals was elevated at the beginning of the process and then significantly decreased. In contrast, malaria patients presented successive peaks of NK activity. Our results confirmed the occurrence of alteration in NK cell function during malaria, and added new data about the NK cytotoxicity process.

Kinetics of IFN-gamma, TNF-alpha, IL-10 and IL-4 production by mononuclear cells stimulated with gp43 peptides, in patients cured of paracoccidioidomycosis

We analyzed the kinetics of cytokine production by mononuclear cells from 17 patients who had been treated for paracoccidioidomycosis, using the stimulus of gp43 peptide groups (43kDa glycoprotein of Paracoccidioides brasiliensis) at 0.1 and 1µM, gp43 (1µg/ml) and crude Paracoccidioides brasiliensis antigen (PbAg; 75µg/ml). IFN-gamma production was a maximum at 144 hours in relation to the G2 and G8 peptide groups at 1µM and was greatest at 144 hours when stimulated by gp43 and by PbAg. The maximum TNF-alpha production was at 144 hours for the G2 group (0.1µM) and for gp43. IL-10 production was highest after 48 and 72 hours for G7 and G6 at 1µM, respectively. We also suggest the best time for analysis of IL4 production. These results may contribute towards future studies with gp43 peptides and encourage further investigations with the aim of understanding the influence of these peptides on the production of inflammatory and regulatory cytokines.

Marine toxins in bivalves: accumulation, kinetics and subcellular responses

Sociedade Polis Litoral Ria Formosa,Projects Quasus and Project Toxigest financed by PROMAR (2007-2013)

Influence of the molecular weight in mechanical properties and degradation kinetics of chitosan inverted colloidal crystals

Tissue engineering arises from the need to regenerate organs and tissues, requiring the development of scaffolds, which can provide an optimum environment for tissue growth. In this work, chitosan with different molecular weights was used to develop biodegradable 3D inverted colloidal crystals (ICC) structures for bone regeneration, exhibiting uniform pore size and interconnected network. Moreover, in vitro tests were conducted by studying the influence of the molecular weight in the degradation kinetics and mechanical properties. The production of ICC included four major stages: fabrication of microspheres; assembly into a cohesive structure, polymeric solution infiltration and microsphere removal. Chitosan’s degree of deacetylation was determined by infrared spectroscopy and molecular weight was obtained via capillary viscometry. In order to understand the effect of the molecular weight in ICC structures, the mass loss and mechanical properties were analyzed after degradation with lysozyme. Structure morphology observation before and after degradation was performed by scanning electron microscopy. Cellular adhesion and proliferation tests were carried out to evaluate ICC in vitro response. Overall, medium molecular weight ICC revealed the best balance in terms of mechanical properties, degradation rate, morphology and biological behaviour.

Evaluation of CD4+CD25+ T lymphocyte response time kinetics in patients with chronic Chagas disease after in vitro stimulation with recombinant Trypanosoma cruzi antigens

Introduction CD4+CD25+ T lymphocytes have been implicated in the regulation of host inflammatory response against Trypanosoma cruzi, and may be involved in the clinical course of the disease. Methods Peripheral blood mononuclear cells from patients with chronic Chagas disease were cultured in the presence of T. cruzi recombinant antigens and assayed for lymphocytes at distinct time points. Results It was possible to differentiate clinical forms of chronic Chagas disease at days 3 and 5 according to presence of CD4+CD25+ T cells in cell cultures. Conclusions Longer periods of cell culture proved to be potentially valuable for prospective evaluations of CD4+CD25+ T lymphocytes in patients with chronic Chagas disease.

Induction and nosocomial dissemination of carbapenem and polymyxin-resistant Klebsiella pneumoniae

INTRODUCTION:Polymyxins are antimicrobial agents capable of controlling carbapenemase-producing Klebsiella pneumoniae infection.METHODS: We report a cluster of four patients colonized or infected by polymyxin-resistant and Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae.RESULTS: Three patients were hospitalized in adjacent wards, and two were admitted to the intensive care unit. The index case maintained prolonged intestinal colonization by KPC-producing K. pneumoniae. Three patients received polymyxin B before the isolation of polymyxin-resistant K. pneumoniae.CONCLUSIONS: Colonization by KPC-producing K. pneumoniae and previous use of polymyxin B may be causally related to the development of polymyxin-resistant microorganisms.

Vancomycin monitoring in term newborns: comparison of peak and trough serum concentrations determined by high performance liquid chromatography and fluorescence polarization immunoassay

INTRODUCTION: Peak and trough serum concentrations of vancomycin were determined in term newborn infants with confirmed or suspected Staphylococcus sp sepsis by high performance liquid chromatography and flourescence polarization immunoassay. OBJECTIVE: To statistically compare the results of the high performance liquid chromatography and flourescence polarization immunoassay techniques for measuring serum vancomycin concentrations. METHODS: Eighteen peak and 20 trough serum samples were assayed for vancomycin concentrations using high performance liquid chromatography and flourescence polarization immunoassay from October 1995 to October 1997. RESULTS: The linear correlation coefficients for high performance liquid chromatography and flourescence polarization immunoassay were 0.27 (peak, P = 0.110) and 0.26 (trough, P = 0.1045) respectively, which were not statistically significant. CONCLUSION: There was wide variation in serum vancomycin concentrations determined by high performance liquid chromatography as compared with those determined by flourescence polarization immunoassay. There was no recognizable pattern in the variability; in an apparently random fashion, the high performance liquid chromatography measurement was sometimes substantially higher than the flourescence polarization immunoassay measurement, and at other times it was substantially lower.

Induction of experimental mammary carcinogenesis in rats with 7,12-dimethylbenz(a)anthracene

PURPOSE: To test an experimental model of chemical mammary carcinogenesis induction in rats. METHODS: Twenty young virgin Sprague-Dawley female rats, aged 47 days, received 20 mg of 7,12-dimethylbenz(a)anthracene (DMBA) intragastrically by gavage. Afterwards, at 8 and 13 weeks, their mammary glands were examined. At the end of the experiment, the animals were sacrificed, and the mammary tumors were measured and weighed. Tumor fragments were analyzed using light microscopy. RESULTS: Eight weeks after DMBA injection, 16 rats presented at least 1 breast tumor (80%). After 13 weeks, all of them (100%) developed breast carcinomas that were confirmed by histopathological analysis. CONCLUSION: This experimental animal model of chemical mammary induced carcinogenesis is feasible and can be used in further experiments on the role of tumorigenic biomodulator substances.

Renal and urinary findings in 20 patients with Williams-Beuren syndrome diagnosed by fluorescence in situ hybridization (FISH)

PURPOSE: Williams-Beuren syndrome is a rare multiple anomalies/mental retardation syndrome caused by deletion of contiguous genes at chromosome region 7q11.23. The aim of this work was to determine the frequency and the types of renal and urinary tract anomalies in 20 patients with Williams-Beuren syndrome. METHODS: The fluorescence in situ hybridization test using a LSI Williams syndrome region DNA probe was performed for all 20 patients to confirm the diagnosis of Williams-Beuren syndrome. A prospective study was performed in order to investigate renal and urinary aspects using laboratory assays to check renal function, ultrasonography of the kidneys and urinary tract, voiding cystourethrogram and urodynamics. RESULTS: Deletion of the elastin gene (positive fluorescence in situ hybridization test) was found in 17 out of 20 patients. Renal alterations were diagnosed in 5 of 17 (29%) the patients with the deletion and in 1 of 3 patients without the deletion. Fourteen patients with the deletion presented dysfunctional voiding. Arterial hypertension was diagnosed in 3 patients with deletions and 1 of these presented bilateral stenosis of the renal arteries. CONCLUSIONS: Due to the high incidence of renal and urinary abnormalities in Williams-Beuren syndrome, performing a systematic laboratory and sonographic evaluation of the patients is recommended.

Effect of surface plasmon resonance in TiO2/Au thin films on the fluorescence of self-assembled CdTe QDs structure

The exceptional properties of localised surface plasmons (LSPs), such as local field enhancement and confinement effects, resonant behavior, make them ideal candidates to control the emission of luminescent nanoparticles. In the present work, we investigated the LSP effect on the steady-state and time-resolved emission properties of quantum dots (QDs) by organizing the dots into self-assembled dendrite structures deposited on plasmonic nanostructures. Self-assembled structures consisting of water-soluble CdTe mono-size QDs, were developed on the surface of co-sputtered TiO2 thin films doped with Au nanoparticles (NPs) annealed at different temperatures. Their steady-state fluorescence properties were probed by scanning the spatially resolved emission spectra and the energy transfer processes were investigated by the fluorescence lifetime imaging (FLIM) microscopy. Our results indicate that a resonant coupling between excitons confined in QDs and LSPs in Au NPs located beneath the self-assembled structure indeed takes place and results in (i) a shift of the ground state luminescence towards higher energies and onset of emission from excited states in QDs, and (ii) a decrease of the ground state exciton lifetime (fluorescence quenching).

Molecular regulation of flowering induction in Quercus suber

Dissertação de mestrado em Plant Molecular Biology, Biotechnology and Bioentrepeneurship

Fluorescence-based approaches towards the assessment of structural and functional alterations in lysosomes and vacuoles induced by lactoferrin and acetic acid

Dissertação de mestrado em Biofísica e Bionanossistemas

Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: the effect of pH, dextran sulfate and probe concentration

Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300 nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria.

FISHji: new ImageJ macros for the quantification of fluorescence in epifluorescence images

Fluorescence in situ hybridization (FISH) is based on the use of fluorescent staining dyes, however, the signal intensity of the images obtained by microscopy is seldom quantified with accuracy by the researcher. The development of innovative digital image processing programs and tools has been trying to overcome this problem, however, the determination of fluorescent intensity in microscopy images still has issues due to the lack of precision in the results and the complexity of existing software. This work presents FISHji, a set of new ImageJ methods for automated quantification of fluorescence in images obtained by epifluorescence microscopy. To validate the methods, results obtained by FISHji were compared with results obtained by flow cytometry. The mean correlation between FISHji and flow cytometry was high and significant, showing that the imaging methods are able to accurately assess the signal intensity of fluorescence images. FISHji are available for non-commercial use at http://paginas.fe.up.pt/nazevedo/.