Ethinylestradiol and estradiol have different effects on oxidative stress and nitric oxide synthesis in human endothelial cell cultures

Study objective: To compare the effects of ethinylestradiol (EE) and 17 beta-estradiol (E(2)) on nitric oxide (NO) production and protection against oxidative stress in human endothelial cell cultures. Design: Experimental study. Settings: Research laboratory. Material: Human ECV304 endothelial cell cultures. Intervention(s): The NO synthesis was determined by flow cytometry, and oxidative stress was determined by a cell viability assay, after exposure to hydrogen peroxide (H(2)O(2)) and stimulation of endothelial cells with EE at concentrations similar to those of a contraceptive containing 30 mu g EE. Main Outcome Measure(s): The effects of EE were compared with those of E(2) at concentrations similar to those occurring during the follicular phase. Result(s): Ethinylestradiol did not increase NO synthesis and did not protect cells against oxidative stress. The viability of the cells incubated with E(2) in combination with H(2)O(2) was greater than the viability obtained with H(2)O(2) only or with H(2)O(2) in combination with EE. The cells stimulated with E(2) presented a significant increase in NO production compared with control. Conclusion(s): In contrast to the effects of E(2), EE did not protect human ECV304 endothelial cells against oxidative stress and did not increase their production of NO. (Fertil Steril (R) 2010; 94: 1578-82. (C) 2010 by American Society for Reproductive Medicine.)

Effect of BCG stimulus on proinflammatory cytokine production in laryngeal cancer

Background Evaluate the production of TNF and IL-6 in the supernatant of peripheral blood mononuclear cell (PBMC) cultures of patients with supraglottic laryngeal cancer before and after surgical treatment. Materials and methods Adherent cell cultures were stimulated with LPS and BCG. Fourteen patients with advanced supraglottic laryngeal cancer were studied. Cytokine concentration was determined by ELISA in supernatants of mononuclear cell cultures. Results In non-stimulated cultures, lower TNF cytokine levels were detected during the late postoperative (LP) period compared to control (P = 0.02). LP TNF and IL-6 levels were high in cultures stimulated with LPS compared with the preoperative period (PREOP) (P = 0.007; P = 0.008, respectively). Stimulation with BCG led to increased levels of TNF and IL-6 during the LP period compared to control (P = 0.001; P = 0.04, respectively). Conclusions BCG is able to modulate the immune response of patients with advanced supraglottic laryngeal cancer in vitro, increasing the secretion of TNF and IL-6 by macrophages during the postoperative period.

Heat-killed Enterococcus faecalis Alters Nitric Oxide and CXCL12 Production but not CXCL8 and CCL3 Production by Cultured Human Dental Pulp Fibroblasts

Introduction: Fibroblasts are the most abundant cells in dental pulp. To investigate their capacity to produce the chemokines CCL3, CXCL8, and CXCL12 as well as nitric oxide (NO), we evaluated the production of these mediators in supernatants of cultured human dental pulp fibroblasts (HDPF) stimulated by heat-killed Enterococcus faecalis (HKEF). Methods: Primary cultures of HDPF were stimulated with medium alone or HKEF (1:1, 10:1, or 100:1 bacteria:fibroblast) for 1, 6, and 24 hours. Chemokines and NO were assessed through enzyme-linked immunosorbent assay and Griess reaction, respectively. Statistical analysis was performed by using analysis of variance and Tukey post test. Results: CCL3 was not detected, whereas constitutive CXCL8 was not affected. Production of CXCL12 was increased at 1 and 6 hours, and NO was increased at the concentration of 1:1 bacteria:fibroblast at 24 hours. Viability and proliferation assays did not reveal cell number differences. Conclusions: These findings demonstrate that heat-killed E. faecalis is able to increase production of CXCL12 and NO by HDPF. (J Endod 2010;36:91-94)

An easy and reliable method for establishment and maintenance of leaf and root cell cultures of Arabidopsis thaliana

Cell suspension cultures are useful for a wide range of biochemical and physiological studies, yet their production can be technically demanding and often unreliable. Here we describe a protocol for producing Arabidopsis cell suspension cultures that is reliable and easy to use.

Growth of papaya nodal and rooted shoot cultures as affected by ACC, STS, culture vessel size and incubation conditions

Nodal shoot cultures of 'Clone 003', a selected Australian papaya cultivar, were cultured on modified De Fossard medium supplemented with chemicals that either promote ethylene evolution or inhibit action while in culture. Nodal shoot cultures grown in the presence of 1-aminocyclopropane carboxylic acid (ACC, 1.0 mM) resulted in a significant reduction in percent fresh and dry weights, shoot length, leaf area, petiole length and chlorophyll content, but leaf development was significantly increased. In contrast, nodal cultures grown in the presence of silver thiosulphate (STS, 0.5 mM) significantly produced the highest percentage of fresh and dry weights, shoot length, leaf production, leaf area expansion, petiole length and leaf chlorophyll content. Nodal cultures and rooted whole plantlets placed in medium-sized (125 mL) culture vessels had significantly better growth than those cultures placed in small (70 mL) or in large (250 mL) vessels. Cultures grown in medium-sized vessels had higher fresh and dry weights, longer shoots, more leaves and larger leaf area than those cultures placed in smaller or larger vessels. Similarly, values for said growth parameters and for chlorophyll content of the nodal and rooted whole plantlets were higher when they were incubated under high light intensity of 120 mumol m(-2)s(-1) at a prevailing temperature of either 20+/-1 C or 25+/-1 C.

Biotoxicity assays of quantum dots in in vitro cultures of Medicago sativa and Medicago truncatula

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia

Protein-Polysaccharides of trametes versocolor: production and biological activities

Extracellular-(E-PPS) and intracellular-protein-polysaccharides (I-PPS) complexes were produced by Trametes versicolor in submerged cultures with different carbon sources. The highest extracellular-(EPS) and intracellular-polysaccharide (IPS) concentration in the complexes was obtained with tomato pomace culture. DPPH radical scavenging for E-PPS and I-PPS produced by liter of culture was equivallent to 2.115 +/- A 0.227 and 1.374 +/- A 0.364 g of ascorbic acid, respectively. These complexes showed a protector effect in the oxidation of erythrocyte membranes and had ability to inhibit the hemolysis and methemoglobin synthesis in stressed erythrocytes. These results suggest that extracellular- and intracellular- polysaccharides produced are important bioactive compounds with medicinal potential.

In Situ Near-Infrared (NIR) Versus High-Throughput Mid- Infrared (MIR) Spectroscopy to Monitor Biopharmaceutical Production

The development of biopharmaceutical manufacturing processes presents critical constraints, with the major constraint being that living cells synthesize these molecules, presenting inherent behavior variability due to their high sensitivity to small fluctuations in the cultivation environment. To speed up the development process and to control this critical manufacturing step, it is relevant to develop high-throughput and in situ monitoring techniques, respectively. Here, high-throughput mid-infrared (MIR) spectral analysis of dehydrated cell pellets and in situ near-infrared (NIR) spectral analysis of the whole culture broth were compared to monitor plasmid production in recombinant Escherichia coil cultures. Good partial least squares (PLS) regression models were built, either based on MIR or NIR spectral data, yielding high coefficients of determination (R-2) and low predictive errors (root mean square error, or RMSE) to estimate host cell growth, plasmid production, carbon source consumption (glucose and glycerol), and by-product acetate production and consumption. The predictive errors for biomass, plasmid, glucose, glycerol, and acetate based on MIR data were 0.7 g/L, 9 mg/L, 0.3 g/L, 0.4 g/L, and 0.4 g/L, respectively, whereas for NIR data the predictive errors obtained were 0.4 g/L, 8 mg/L, 0.3 g/L, 0.2 g/L, and 0.4 g/L, respectively. The models obtained are robust as they are valid for cultivations conducted with different media compositions and with different cultivation strategies (batch and fed-batch). Besides being conducted in situ with a sterilized fiber optic probe, NIR spectroscopy allows building PLS models for estimating plasmid, glucose, and acetate that are as accurate as those obtained from the high-throughput MIR setup, and better models for estimating biomass and glycerol, yielding a decrease in 57 and 50% of the RMSE, respectively, compared to the MIR setup. However, MIR spectroscopy could be a valid alternative in the case of optimization protocols, due to possible space constraints or high costs associated with the use of multi-fiber optic probes for multi-bioreactors. In this case, MIR could be conducted in a high-throughput manner, analyzing hundreds of culture samples in a rapid and automatic mode.

In situ NIR spectroscopy monitoring of plasmid production processes effect of producing strain, medium composition and the cultivation strategy

BACKGROUNDWhile the pharmaceutical industry keeps an eye on plasmid DNA production for new generation gene therapies, real-time monitoring techniques for plasmid bioproduction are as yet unavailable. This work shows the possibility of in situ monitoring of plasmid production in Escherichia coli cultures using a near infrared (NIR) fiber optic probe. RESULTSPartial least squares (PLS) regression models based on the NIR spectra were developed for predicting bioprocess critical variables such as the concentrations of biomass, plasmid, carbon sources (glucose and glycerol) and acetate. In order to achieve robust models able to predict the performance of plasmid production processes, independently of the composition of the cultivation medium, cultivation strategy (batch versus fed-batch) and E. coli strain used, three strategies were adopted, using: (i) E. coliDH5 cultures conducted under different media compositions and culture strategies (batch and fed-batch); (ii) engineered E. coli strains, MG1655endArecApgi and MG1655endArecA, grown on the same medium and culture strategy; (iii) diverse E. coli strains, over batch and fed-batch cultivations and using different media compositions. PLS models showed high accuracy for predicting all variables in the three groups of cultures. CONCLUSIONNIR spectroscopy combined with PLS modeling provides a fast, inexpensive and contamination-free technique to accurately monitoring plasmid bioprocesses in real time, independently of the medium composition, cultivation strategy and the E. coli strain used.

A systems biology framework for pathway level culture media engineering: pplication to Pichia pastoris cultures

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica

Investigation of the regulation mechanisms for bioplastics production from industrial residues

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Constraint-based modelling of mixed microbial populations: Application to polyhydroxyalkanoates production

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica

Microbial contribution to biofuels production

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica

Production of cytokine and chemokines by human mononuclear cells and whole blood cells after infection with Trypanosoma cruzi

INTRODUCTION: The innate immune response is the first mechanism of protection against Trypanosoma cruzi, and the interaction of inflammatory cells with parasite molecules may activate this response and modulate the adaptive immune system. This study aimed to analyze the levels of cytokines and chemokines synthesized by the whole blood cells (WBC) and peripheral blood mononuclear cells (PBMC) of individuals seronegative for Chagas disease after interaction with live T. cruzi trypomastigotes. METHODS: IL-12, IL-10, TNF-α, TGF-β, CCL-5, CCL-2, CCL-3, and CXCL-9 were measured by ELISA. Nitrite was determined by the Griess method. RESULTS: IL-10 was produced at high levels by WBC compared with PBMC, even after incubation with live trypomastigotes. Production of TNF-α by both PBMC and WBC was significantly higher after stimulation with trypomastigotes. Only PBMC produced significantly higher levels of IL-12 after parasite stimulation. Stimulation of cultures with trypomastigotes induced an increase of CXCL-9 levels produced by WBC. Nitrite levels produced by PBMC increased after the addition of parasites to the culture. CONCLUSIONS: Surface molecules of T. cruzi may induce the production of cytokines and chemokines by cells of the innate immune system through the activation of specific receptors not evaluated in this experiment. The ability to induce IL-12 and TNF-α contributes to shift the adaptive response towards a Th1 profile.

Effect of fetal bovine serum on Candida glabrata cultures

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)