A new alternative in vitro method for quantification of Toxoplasma gondii infectivity

An in vitro method to determine the infectious potency of an unknown suspension of the protozoan parasite Toxoplasma gondii based on kinetics of host cells lysis was developed. Mic1-3KO a mutant strain of T. gondii RH tachyzoites was inoculated in 25-cm2 flasks containing a 90% confluent monolayer of human foreskin fibroblasts. Lysis kinetics was monitored for infection ratios ranging from 1∶10? to 1∶10; we defined 10? tachyzoites/ml?1 as the threshold value for parasite egress. Results allowed us to build a calibration curve relating the initial infection ratios to the time needed to reach 10? tachyzoites/ml?1. Finally, we validated the method using a known mixture of dead and live parasites. This method was found to estimate with accuracy the initial ratio of infection of the unknown parasite suspension. This easy-to-use method is reproducible and can be applied to any T. gondii tachyzoite RH strain, genetically modified or not. This method is also suitable for testing promising candidates for an effective live vaccine.     

A simplified in vitro model of oxidant injury using vascular endothelial cells

Summary Oxidant injury of the vascular endothelium is considered an early event in the pathogenesis of atherosclerosis. The model of oxidant injury is crucial to the investigation of antioxidants. In the present study, a convenient in vitro model of oxidant injury induced by hydrogen peroxide (H₂O₂) was developed using bovine pulmonary artery endothelial cells (PAEC). Viability of PAEC grown in 96-well culture plates was determined with methylthiazol tetrazolium (MTT) colorimetric assay. Cell membrane integrity was measured by lactate dehydrogenase (LDH) release from PAEC grown in 24-well plates. Malondialdehyde (MDA, a product of lipid peroxidation) in PAEC grown in 6-well plates was detected by a thiobarbituric acid fluorometric assay. Incubation of H₂O₂ with PAEC caused a dose-dependent decrease of cell viability, an increase of LDH release, and an elevation of MDA production. MTT assay was convenient, quantitative, non-radioactive, and suitable for testing a large number of samples. The fluorometric assay for measuring MDA production in endothelial cells used 6-well plates instead of 80-cm² flasks employed by previous investigators. The use of multiwell culture plates in these assays made it possible for more samples to be tested in any single experiment. The three assays are reproducible with low intraplate and interplate coefficients of variation. This in vitro model is suitable for screening antioxidants and for studying pharmacodynamics at the cellular level.     

A new method for the quantification of chitin and chitosan in edible mushrooms

Along with β-glucans, chitin is the dominant component of the fungal cell wall. Chitosan, the deacetylated form of chitin, has found quite a number of biomedical and biotechnological applications recently. Mushroom chitin could be an important source for chitosan production. A direct determination of chitin and chitosan in mushrooms is of expedient interest. In this paper, a new method for the quantification of chitin and chitosan is described. This method is based on the specific reaction between polyiodide anions and chitosan and on measuring the optical density of the insoluble polyiodide–chitosan complex. After deacetylation, chitin can also be quantified. The specificity of the reaction is used to quantify the polymers in the presence of complex matrices. With this new spot assay, the chitin content of mycelia and fruiting bodies from several basidiomycetes and an ascomycete were analysed. The presented method could also be used for the determination in other samples as well. The chitin content of the analysed species varies between 0.4 and 9.8 g chitin per 100 g of dry mass. Chitosan could not be detected in our mushroom samples, indicating that the glucosamine units are mostly acetylated.     

A fluorometric assay for the measurement of endothelial cell density in vitro

Summary A fluorometric assay for determining endothelial cell numbers based on the endogenous enzyme acid phosphatase is described. In preliminary studies, three substrates—p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 2′-[2-benzthiazoyl]-6′-hydroxy-benthiazole phosphate (AttoPhos™)—were compared with respect to their kinetic, optimum assay conditions, sensitivity, and detection limits. Only AttoPhos™ was found to have a high degree of sensitivity, reliability, and reproducibility for measuring both high and low cell numbers in the same plate. In subsequent experiments, assay conditions were validated for measuring endothelial cell density in response to basic fibroblast growth factor and fumagillin. Furthermore, the AttoPhos™ assay revealed a linear correlation between acid phosphatase activity and cell number in many cell types, including BALB/3T3, CHO-K1, A431, MCF7, 2008, SK-OV-3, T47-D, and OVCAR-3. This assay is potentially valuable for use in many in vitro systems in which the quantitation of cell density and proliferation is necessary. The practical advantages of AttoPhos™ assay for measuring endothelial cell numbers include (1) nonradioactivity, (2) simplicity, (3) economy, (4) speed of assessment of proliferation of large number of samples, and (5) amenability to high-throughput drug screening.     

A new method for the synthesis of 2'-O-benzyladenosine using Mitsunobu reaction

A new method to introduce a benzyl group onto the 2'-OH of purine ribonucleoside is described. Thus, 6-chloropurine 3'-O-benzoylriboside and its 5'-O-trityl congener were condensed with benzyl alcohol using the Mitsunobu reaction to give the 2'-O-benzyl derivative. The yields were varied from 4.6 to 62.9% depending on the solvent used. The product was converted to adenosine, indicating that the stereochemistry at C-2' is retained.     

A spectrophotometric method for the quantification of 2-phenylethylamine in biological specimens

An efficient and specific extraction procedure is described for the isolation of 2-phenylethylamine (PEA) from biological material. The method employed, which involves n-hexane extraction from highly alkalinized samples, substantially eliminates most of catecholamines, indoleamines, their presumed metabolites and amino acid precursors as well as those of PEA itself.Characteristics of a chemical reaction used for the quantitation of this amine, which involves a pH and chloride ion dependent oxidation of this compound by Ce(SO₄)₂, are also described. This reaction could also be used for the quantitation of 2-hydroxy-2-phenylethylamine, phenylacetic acid, phenylacetaldehyde and phenylethanol. Using the described procedure, PEA levels were determined in different human, cat and rabbit organs, including brain, of nonpretreated animals as well as in human urine.     

A method for examining the endothelial cytoskeleton in situ using immunofluorescence

A simple technique is described for producing en face preparations of endothelial cells (EC) from large blood vessels fixed in situ that are suitable for studying the distribution of specific antigens in EC by immunofluorescence. This method has permitted us to examine the distribution of various components of the cytoskeleton, including microtubules (MT), centrioles, and microfilaments (MF) in EC in vivo.     

IL-2 rapidly induces natural killer cell adhesion to human endothelial cells. A potential mechanism for endothelial injury

NK cells promptly disappear from the circulation of patients treated with high dose i.v. rIL-2. To further study this process, we evaluated the effects of IL-2 (1000 U/ml) on normal donor PBMC incubated for 1 h on cultured human saphenous vein endothelial cells (EC). Although the NK activity of non-adherent PBMC recovered from flasks coated only with fibronectin increased in the presence of supplemental IL-2, the activity of cells recovered from flasks coated with EC decreased when IL-2 was added to the medium. The percentage of NK (CD16+) cells among the EC-non-adherent PBMC was reduced relative to that of the input cells when IL-2 was added. The percentage of CD16+ cells in the EC-adherent PBMC, as well as their NK activity, increased in the presence of added IL-2. Although EC had no effect on the lysis of labeled K-562 cells by unstimulated PBMC in cold target competition experiments, they were able to compete in cytolytic assays using PBMC previously activated by exposure to IL-2 for 1 h. EC were not lysed by these briefly activated PBMC in 3-h cytotoxicity assays but were lysed by these effectors in 18-h assays and in 3-h assays using PBMC pre-activated by more prolonged culture with IL-2. The ability of IL-2 to induce NK cell adhesion to EC was not blocked by a mixture of neutralizing antisera raised against rTNF-alpha, rIL-1 alpha, and rIL-1 beta, factors known to promote leukocyte adhesion to EC. We conclude that IL-2 rapidly induces NK cell adhesion to EC and propose that this effect accounts for the disappearance of circulating NK cells after the infusion of high doses of IL-2. In addition, these results suggest that NK cells activated by IL-2 in vivo may injure the endothelium and contribute to the extravasation of plasma and the retention of fluid characteristic of IL-2 treatment.     

Novel kinetics of single cell Ca2+ transients in stimulated hepatocytes and A10 cells measured using fura-2 and fluorescent videomicroscopy

The kinetics of agonist-induced increases in cytosolic free Ca2+ have been measured in single A10 vascular smooth muscle cells and rat hepatocytes using fluorescent videomicroscopy with fura-2 as a Ca2+ indicator. At high agonist concentrations there was no difference in the kinetics of the Ca2+ transient measured in vasopressin-stimulated single A10 cells or in cell populations. However, stimulation of single A10 cells with concentrations of vasopressin below 0.5 nM produced characteristic Ca2+ transients composed of two distinct peaks. The two peaks appeared to represent a temporal separation between release of intracellular Ca2+ and influx of extracellular Ca2+. The double transient was not observed in single rat hepatocytes stimulated with low concentrations of vasopressin or phenylephrine. In both A10 cells and hepatocytes, the initial rate of increase in Ca2+ concentrations in response to submaximal agonist concentrations was faster in single cells than in cell populations. This difference was due to asynchrony of the cellular response, where there was a latent period of variable length before onset of a rapid increase in Ca2+ concentration. The duration of the latent period was dependent on the agonist concentration, higher concentrations of agonist giving a reduced latent period. The hormone-stimulated Ca2+ transient measured in single hepatocytes with fura-2 was different from the series of transient spikes as previously reported using aequorin as the Ca2+ indicator, suggesting that fura-2 and aequorin may report different aspects of the Ca2+ response in stimulated cells. Collectively, these results demonstrate that measurement of Ca2+ transients in single cells provide novel information concerning the nature of the Ca2+ transient that is not apparent from studies with cell populations.     

A method for bacteriocin quantification

Different aspects of the most commonly used assay methods in the study of bacteriocins were examined. The conditions under which extraction and incubation (including exposure time) take place were analysed, and several different formal models that are usually employed to calculate ID50 were compared. As an alternative designed to overcome the problems which characterize the response of micro-organisms that are sensitive to bacteriocins, an operative procedure in a liquid medium and a modified re-parameterized logistic equation is proposed. When applied to the inhibition of Leuconostoc mesenteroides by nisin, the model allows an optimal experimental procedure to be defined.     

A new model of human aortic endothelial cells in vitro

Vascular endothelial cells play an important role in coagulation regulation of vascular tone and in a variety of synthetic and metabolic functions. Endothelial cells also have a pivotal role in immunological diseases atherogenesis and tumor angiogenesis. Endothelial cells are often used as system to study the pathophysiology of late complications in diabetes mellitus atherosclerotic damages and leukocyte adhesion in inflammatory diseases. Most of the studies have been performed on primary arterial and venous endothelial cell cultures with problems such as availability of autoptic material and reproducibility of cell cultures. We have isolated and characterized a novel system of proliferating long-term cultures of human aortic endothelial cells that maintain their differentiated characteristics for many generations in vitro. They produce antithrombotic and thrombotic factors such as t-PA and PAI-1 and respond to TNFalpha, an important factor correlated with the inflammatory process by modifying growth characteristics by producing cytokines such as GM-CSF by expressing ICAM-1 on the surface and by producing large amounts of nitric oxide and endothelin. This new system may be very useful to understand and study the molecular mechanisms involved in many vascular alteration pathologies and in the aging process.     

A new method for cell immobilization.

A new method for producing particles and membranes containing immobilized bacteria is presented. These immobilized bacteria display good stability over time making them well suited for use in a packed-bed reactor. Such a reactor is tested as a function of the different parameters of the system. The results are qualitatively similar to those obtained with purified enzyme reactors, but some discrepancies with the plug-flow model are noted. It is necessary to use a more sophisticated model in order to fit the experimental data.     

Lipid quantification method using FTIR spectroscopy applied on cancer cell extracts

Reprogramming energy metabolism constitutes one of the hallmarks of cancer. Changes in lipid composition of cell membranes also appear early in carcinogenesis. Quantification of various molecules such as lipids evidences the modifications in the metabolism of tumour cells and can serve as potential markers for cancer diagnosis and treatment. Fourier Transform Infrared (FTIR) spectroscopy is a powerful tool used for the detection and characterization of various types of molecules. This technique remains an attractive approach as it is cheap (equipment and reagents), does not require high grade solvents or expensive internal standards, equipment is widely available in standard laboratories and the method is robust and suitable for routine analyses. In this work we established partial least square (PLS) models based on FTIR spectra able to quantify lipids in complex mixtures such as cell extracts. In the first part, we attempted to build PLS models with FTIR spectra of 53 mixtures of 8 well-characterized pure lipids. Second, the PLS models were verified using FTIR spectra of mixtures that did not contribute to the calibration. The third step was the validation of the models on lipid cell extracts. In order to obtain reference values for cell extracts, high performance liquid chromatography was carried out by AVANTI. The lipid distribution were globally similar with both techniques, PLS models and chromatography. Finally, the models were applied to determine the lipid composition of cells exposed to four treatments. We could not evidence significant changes in the lipid composition of cell extracts after treatment, in terms of polar head groups. However, the models established in this study appear reliable and could be applied for high throughput measurements. This article is part of a Special Issue entitled Tools to study lipid functions.