Biomedical subjects

X Ronot

Publications and source records attributed to X Ronot.

62 records · Page 4Linked to original sources

PubMedDate not supplied

Human sperm mitochondrial function related to motility: a flow and image cytometric assessment.

Current evaluation of male fertility, routinely estimated by sperm count, motility, and morphology, provides only crude information about the fertility state of individuals. Both flow and image cytometry were applied to mitochondrial activity and sperm motility respectively. Sperm samples from fertile donors were concomitantly measured for Rhodamine 123 (Rh123) uptake (an estimation of mitochondrial activity), percentage of dead cells, and motility characteristics, such as percentage of motility, curvilinear velocity, and amplitude of lateral head displacement. These measurements were done under experimental conditions known to modulate sperm motility (temperature and time course survival in a capacitating medium). Bimodal distributions were found for Rh123 uptake. Flow cytometry-derived parameters were essentially time-dependent whereas motility characteristics were primarily temperature-dependent. Correlations were found between various flow cytometry-derived parameters and motility characteristics. Most of the correlations were obtained after a 24 h incubation in a capacitating medium. The most significant correlation in every experimental condition concerned the percentage of motile spermatozoa and the Rh123 uptakes. The drop in motility observed after a 24 h incubation was paralleled by a markedly lower drop in mitochondrial activity. The data suggest that these two complementary techniques represent an improvement in basic and/or clinical assessment of the functional spermatozoa status.

Flow Cytometry↗

PubMedDate not supplied

In vitro angiogenesis is modulated by the mechanical properties of fibrin gels and is related to alpha(v)beta3 integrin localization.

This study deals with the role of the mechanical properties of matrices in in vitro angiogenesis. The ability of rigid fibrinogen matrices with fibrin gels to promote capillarylike structures was compared. The role of the mechanical properties of the fibrin gels was assessed by varying concentration of the fibrin gels. When the concentration of fibrin gels was decreased from 2 mg/ml to 0.5 mg/ml, the capillarylike network increased. On rigid fibrinogen matrices, capillarylike structures were not formed. The extent of the capillarylike network formed on fibrin gels having the lowest concentration depended on the number of cells seeded. The dynamic analysis of capillarylike network formation permitted a direct visualization of a progressive stretching of the 0.5 mg/ml fibrin gels. This stretching was not observed when fibrin concentration increases. This analysis shows that 10 h after seeding, a prearrangement of cells into ringlike structures was observed. These ringlike structures grew in size. Between 16 and 24 h after seeding, the capillarylike structures were formed at the junction of two ringlike structures. Analysis of the alpha(v)beta3 integrin localization demonstrates that cell adhesion to fibrinogen is mediated through the alpha(v)beta3 integrin localized into adhesion plaques. Conversely, cell adhesion to fibrin shows a diffuse and dot-contact distribution. We suggest that the balance of the stresses between the tractions exerted by the cells and the resistance of the fibrin gels triggers an angiogenic signal into the intracellular compartment. This signal could be associated with modification in the alpha(v)beta3 integrin distribution.

Biomechanical Phenomena↗

PubMedDate not supplied

Age-related changes in rabbit articular chondrocytes.

Cell culture techniques have been used extensively in the study of the aging process at the cellular level. The "senescent" articular chondrocyte seems to be a good model to examine the responses to aging in osteoarthritis, one of the most frequent diseases of old age. Thus in vitro chondrocyte "senescence", established by weekly subculture was characterized by a declining proliferation rate during late passages, from a rapid growth rate in early subculture to a complete loss of the proliferation capacity after 8 +/- 1 passages. Flow cytometric analysis show a time course decrease in the fraction S and G2 + M during the subculture, and a concomitant enhancement in protein content related to the increase of cell size. The immunocytochemistry assays revealed an appearance of a rigid cytoarchitecture with an increase in the number, and organization, of three cytoskeletal components: actin, tubulin and vimentin. The cultured chondrocytes therefore undergo in vitro aging analogous to that described for diploid fibroblasts, and could constitute a cellular model for pharmacological and toxicological assays.

Aging↗

PubMedDate not supplied

A society of goal-oriented agents for the analysis of living cells.

This paper presents a new model for the segmentation and analysis of living cells. A multi-agent model has been developed for this application. It is based on a generic agent model, which is composed of different behaviors: perception, interaction and reproduction. The agent is further specialized to accomplish a specific goal. Different goals are defined from the different components of the cell images. The specialization specifies the parameters of the behaviors for the achievement of the agent's goal. From these goal-oriented agents, a society is defined, and it evolves dynamically as the agents are created and deleted. An internal manager is integrated in the agent to control the behavior's execution. It makes use of an event-driven scheme to manage the behavior priorities. The present design is mainly oriented toward image segmentation, however, it includes some features on tracking and motion analysis.

Animals↗

PubMedDate not supplied

Innocuousness and intracellular distribution of PKH67: a fluorescent probe for cell proliferation assessment.

PKH dyes were initially developed by Horan et al. to provide appropriate probes for in vitro and in vivo cell tracking. It has been reported for many cell types that PKH bind irreversibly to the cell membrane without significantly affecting cell growth. Thus, these probes provide an opportunity for long-term cell monitoring and the identification of cells of interest among a heterogeneous cell population. An important feature is that upon cell division, the probe is partitioned equally between each daughter cell, making it possible to quantify tell fluorescence by flow cytometry. In this situation. the flow cytometric study of PKH67 characteristics shows that this probe does not affect the main cell-functions such as viability or proliferation. Moreover, the intracellular distribution of PKH67 is demonstrated by following its kinetics of internalization by confocal microscopy. These results present PKH67 as a probe suitable for dynamic analysis of cell proliferation as well as the study of intracellular localization and membrane recycling mechanisms.

Animals↗

PubMedDate not supplied

Fluorescence-based assessment of LRP activity: a comparative study.

BACKGROUND: Broad resistance to anticancer drugs is a major cause of failure in cancer treatment. The Lung Resistance-related Protein (LRP) is a protein associated with drug resistance, which is involved in nucleo-cytoplasmic transport and is known to predict a poor response to chemotherapy in acute myeloid leukaemia. The only method allowing the detection of LRP activity is based on radio-labelled daunorubicin incorporation. Our goal was to develop a fluorescence-based assay to analyse LRP function. MATERIALS AND METHODS: We used human colon carcinoma cell lines treated with sodium butyrate (NaB) in order to induce LRP expression. Daunorubicin efflux in isolated nuclei was measured by flow cytometry, the localization and quantification of Daunorubicin analysed by confocal laser scanning microscopy (CLSM) and the diffusion coefficient of this drug estimated by Fluorescence Correlation Spectrometry (FCS). RESULTS: According to the method using [14C] Doxorubicin cells incubated with NaB displayed an efflux of Daunorubicin out of isolated nuclei demonstrated by flow cytometry or CLSM. The FCS method was able to evaluate kinetics of Daunorubicin molecules in nucleus and cytoplasm and showed a higher dispersion of Daunorubicin kinetics with cells previously NaB-treated. This argument is in favour of an increase of nucleo-cytoplasmic exchange. CONCLUSION: Using CLSM we showed that LRP was able to modify anticancer drug repartition in the cells. LRP activity assessment needs either isolated nuclei if flow cytometry is employed, or FCS, and only a few cells may be analysed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMedDate not supplied

Coexpression of multidrug resistance involve proteins: a flow cytometric analysis.

Cross resistance to multiple natural cytotoxic products represents a major obstacle in myeloblastic acute leukaemia (AML). Multidrug resistance (MDR) often involves overexpression of plasma membrane drug transporter P-glycoprotein (PGP) or the resistance associated protein (MRP). Recently, a protein overexpressed in a non-PGP MDR lung cancer cell line and termed lung resistance related protein (LRP) was identified. These proteins are known to be associated with a bad prognosis in AML. We have developed a triple indirect labelling analysed by flow cytometry to detect the coexpression of these proteins. Since no cell line expressing all three antigens is known, we mixed K562 cells (resistant to Adriblastine, PGP+, MRP-, LRP-) with GLC4 cells (resistant to Adriblastine, PGP-, MRP+, LRP+) to create a model system to test the method. The antibodies used were UIC2 for PGP, MRPm6 for MRP and LRP56 for LRP. They were revealed by Fab'2 coupled with Fluoresceine-isothiocyanate, Phycoerythrin or Tricolor with isotype specificity. Cells were fixed and permeabilized after PGP labelling because MRPm6 and LRP56 recognize intracellular epitopes. PGP and LRP were easily detected. MRP is expressed at relatively low levels and was more difficult to detect because in the triple labelling the non specific staining was higher than in a single labelling. Despite the increased background in the triple labelling we were able to detect coexpression of PGP, MRP, LRP by flow cytometry. This method appears to be very useful to detect coexpression of markers in AML. Such coexpression could modify the therapeutic approach with revertants.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMedDate not supplied

PKH26 probe in the study of the proliferation of chemoresistant leukemic sublines.

Proliferative status and multidrug resistance status are key predictors of therapeutic outcome in acute myeloid leukemia. Although classical methods for proliferative assessment such as tritiated thymidine or BrdUrd incorporation, are correlated with treatment outcome, they are time consuming and difficult to standardize. As an alternative, we have evaluated the use of a dye dilution method using PKH26 to determine rate and extent proliferation in drug sensitive and resistant cell lines. When cells labelled with this fluorescent membrane intercalating dye divide, each resulting daughter cell receives half of the dye. Using flow cytometric analysis, it is possible to estimate the number of cells having undergone different numbers of cell divisions. Four different questions were addressed in these studies: a) does PKH26 give stable and reproducible labelling? b) does labelling with PKH26 alter cellular proliferation characteristics? c) is PKH26 a substrate for PGP and MRP? d) does PKH26 labelling alter PGP expression and/or PGP activity? We found that PKH26 labelling is stable, reproducible and has no effect on cell proliferation. It does not modify PGP activity or expression, nor does it appear to be a substrate for PGP or MRP, since the rate of decrease in fluorescence intensity is similar for sensitive and resistant cells which are proliferating at the same rate. Using the dye dilution method, it is possible to simultaneously assess PGP, proliferative status, and level of PGP expression. We conclude that the methods developed here provide a simpler, more complete means for assessment of the effects of the drug therapy on sensitive and resistant cell populations in patients with hematologic malignancies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗