Cytometric approach for a rapid evaluation of susceptibility of Candida strains to antifungals.

OBJECTIVE: To achieve a fast and reliable determination of the susceptibility of Candida strains to amphotericin B (Am B), fluconazole (Flu) and 5-fluorocytosine (5-FC), using cytometric methods as an alternative to the classical dilution method. METHODS: Twenty-three clinical isolates of Candida with different susceptibility patterns were treated for 1 h with two concentrations each of Am B (2 and 8 mg/L), Flu (8 and 64 mg/L) and 5-FC (4 and 32 mg/L), followed by staining with three different fluorochromes, under conditions previously defined through an optimisation study. These were 1 mg/L propidium iodide (PI)/10(6) cells for 30 min at 30 degrees C (a marker that only penetrates cells with severe lesions of the membrane); 0.5 microM FUN-1/10(6) cells for 30 min at 30 degrees C (a fluorescent probe which after entering the yeast cell is converted, by metabolically active yeasts, from a diffuse cytosolic pool with a yellow-green fluorescence into red cylindrical intravacuolar structures) and 0.25 microM of JC-1/10(6) cells for 15 min at 37 degrees C (a monomer that changes reversibly from green to red the J-aggregates, with the increased membrane potential). About 50 000 yeast cells were analysed by flow cytometry (FCM), at FL3 (red, 620 nm) for PI and FL2 (yellow-green, 575 nm) for FUN-1 and the ratio of FL3 to FL1 was determined (red, 620 nm/green, 525 nm) for JC-1; 200 cells of each suspension were also analysed by epifluorescence microscopy (EPM). Viability studies were performed in parallel to count the number of colony forming units. RESULTS: Susceptible (S) strains exposed to Am B and stained with JC-1 showed a dose-dependent decrease in the mitochondrial potential, i.e. a decreased ratio between red/green fluorescence by FCM and a decrease in J-aggregates by EPM. Neither FUN-1 nor PI was useful in the study of Am B activity. Susceptibility to Flu and 5-FC could be detected with FUN-1 staining: metabolic changes were detected by an increase in yellow-green intensity of fluorescence by FCM or a decrease of cylindrical intravacuolar structure formation by EPM, although no decrease in total viability was registered. Staining with JC-1 could predict resistance to both drugs, but did not allow distinction between sensitive dose-dependent strains (S-DD) or intermediate (I) resistance to Flu or 5-FC, respectively, from S strains. PI did not stain Candida cells treated with Flu or 5-FC under our experimental conditions. CONCLUSION: Susceptibility patterns of Candida strains to Am B can be determined by using JC-1, and to Flu and 5-FC by using FUN-1. PI was not a useful probe with which to study the effect of such antifungals under the conditions described here.

Assessment of mitochondrial membrane potential in yeast cell populations by flow cytometry.

In yeast the use of rhodamine 123 (Rh123) has been restricted to the evaluation of mitochondrial respiratory function including the discrimination between respiratory-competent and -deficient cells. This study describes the optimization and validation of a low-concentration Rh123 staining protocol for the flow-cytometric assessment of mitochondrial membrane potential (Delta Psi m) changes in whole yeast cells. The optimized protocol was validated by the use of compounds that specifically affect mitochondrial energetics. Epifluorescence microscopy was used to monitor Rh123 distribution within the cell. Incubation of yeast cell suspensions with Rh123 (50 nM, 10 min) gave minimal non-specific binding and cytotoxicity of the dye. The ratio (R) between the green fluorescence and forward scatter (both measured as log values) was used to measure Delta Psi m with only little dependence on cell 'volume' and mitochondrial concentration. Cells treated with mitochondrial membrane de- or hyper-polarizing agents displayed a decrease and an increase of R values respectively, indicating that changes of the Rh123 distribution in cells indicate variations in the Delta Psi m. Live and dead cells also displayed significantly different R values. The method described here allows assessment of Delta Psi m changes in whole yeast cells in response to a given drug. Moreover, the relationship between drug effects and disorders of mitochondrial energetics might be addressed.

Flow cytometric analysis of chronic and acute toxicity of copper(II) on the marine dinoflagellate Amphidinium carterae.

BACKGROUND: Copper(II) is a heavy metal whose levels have increased in some marine ecosystems to polluting levels. Dinoflagellates, an important phytoplankton group, are at the base of aquatic food chains and bioaccumulation of copper by these microorganisms can result in complex ecosystem alterations, so we investigated how copper disturbs those cells. METHODS: Cytotoxic effects of sublethal and lethal copper concentrations ranging from 4.2 nM (control condition) to 3.13 microM estimated labile copper were studied in batch cultures of Amphidinium carterae. Cell morphology, motility, autofluorescence, and fluorescein diacetate (FDA)-dependent fluorescence generation were evaluated by flow cytometry (FCM) and microscopy. RESULTS: Exposure of A. carterae to toxic levels of copper impaired cell mobility, delayed cell proliferation, led to increased green autofluorescence, and at 3.13 microM labile copper also induced encystment and death. Chlorophyll fluorescence, however, was not affected. Kinetic FCM assay of FDA-dependent fluorescence generation showed a dose-dependent enhancement of fluorescein fluorescence immediately after copper addition and in cultures with sustained exposure to this toxicant. CONCLUSIONS: Our data suggest that copper toxicity occurs quickly at the membrane level in relation to oxidative stress generation. Based on fluorescence kinetic studies, the Na(+)/H(+) antiporter seemed to be affected by copper, thereby affecting intracellular pH.

Fetal adenomas and minimally invasive follicular carcinomas of the thyroid frequently display a triploid or near triploid DNA pattern.

The ploidy pattern and the percentage of S-phase cells were investigated by means of flow cytometry using fresh or frozen samples in a series of 143 tumors and tumor-like lesions of the thyroid in an attempt to find whether there is any relationship between the histological characteristics of the lesions and their DNA content. The percentages of aneuploidy cases per category were: nodular goiter, 18.5% (15/81); fetal adenoma (including cases with trabecular/solid growth pattern), 58.3% (14/24); follicular adenoma other than fetal adenoma, 0% (0/18); papillary carcinoma, 11.1% (1/9); and minimally invasive follicular carcinoma, 57.1% (4/7). Regardless of the histological category, aneuploid lesions had a significantly higher (P < 0.001) percentage of S-phase cells (7.3%) than diploid lesions (4.1%). All the six cases with a DNA content within the triploid range were fetal adenomas, but one was a follicular carcinoma displaying a fetal adenoma-like growth pattern. The other three follicular carcinomas with an aneuploid DNA pattern also displayed foci of fetal adenoma-like growth pattern. Image cytometry of the four aneuploid follicular carcinomas showed similar DNA indexes in the peripheral, invasive foci of the lesions and in the central fetal adenoma-like areas. These results demonstrate that aneuploidy in benign tumors is restricted to adenomas displaying a fetal or fetal/embryonal growth pattern and support the concept that chromosome instability is a major pathway of tumorigenesis in thyroid follicular neoplasms.

Cell cycle analysis of yeasts.

Staining protocols generally designed for the flow cytometric analysis of the cell cycle in mammalian cells are frequently not satisfactory for quantification of the various cell-cycle phases in yeasts. High CVs limit the accuracy of DNA content measurement and estimates of populations in cell-cycle compartments. This unit describes a staining procedure for yeasts using the sensitive nucleic acid stain SYBR Green I, which binds to double-stranded DNA with high selectivity and which has a much higher fluorescence quantum yield upon binding than most other commonly used fluorophores. The properties of this dye combined with optimized sample processing provide high-resolution DNA analysis, with half-peak CVs around 3 to 4% and clear-cut identification of the S phase.

Antifungal activity of local anesthetics against Candida species.

OBJECTIVE: To evaluate the activity of benzydamine, lidocaine, and bupivacaine, three drugs with local anesthetic activity, against Candida albicans and non-albicans strains and to clarify their mechanism of activity. METHODS: The minimal inhibitory concentration (MIC) was determined for 20 Candida strains (18 clinical isolates and two American Type Culture Collection strains). The fungistatic activity was studied with the fluorescent probe FUN-1 and observation under epifluorescence microscopy and flow cytometry. The fungicidal activity of the three drugs was assayed by viability counts. Membrane alterations induced in the yeast cells were evaluated by staining with propidium iodide, by quantitation of intracellular K+ leakage and by transmission electron microscopy of intact yeast cells and prepared spheroplasts. RESULTS: The MIC ranged from 12.5-50.0 microg/mL, 5.0-40.0 mg/mL, and 2.5-10.0 mg/mL for benzydamine, lidocaine, and bupivacaine, respectively. The inhibitory activity of these concentrations could be detected with the fluorescent probe FUN-1 after incubation for 60 minutes. A very fast fungicidal activity was shown by 0.2, 50, and 30 mg/mL of benzydamine, lidocaine, and bupivacaine, respectively. CONCLUSIONS: At lower concentrations, the tested drugs have a fungistatic activity, due to yeast metabolic impairment, while at higher concentrations they are fungicidal, due to direct damage to the cytoplasmic membrane.

Rapid detection of efflux pumps and their relation with drug resistance in yeast cells.

BACKGROUND: Cell drug resistance can be due to the presence of active efflux pumps (AEP). Identification of yeast cells with a resistance phenotype is important either from a clinical, agricultural or biotechnological point of view. Rapid and reliable methods to detect AEP can be therefore very useful. METHODS: Some yeast cells change their staining by calcein-AM, BCECF-AM, rhodamine 123 and DiOC(5), when pretreated with verapamil, CCCP or ATP depletion, or when pretreated with specific antimicrobial agents. This fact may be interpreted as an indication of the presence/absence of AEP. Six yeast species were tested with a flow cytometric method (FCM) and an epifluorescence microscopic method (EFM), and ten other species were evaluated only by EFM. The minimum inhibitory concentration (MIC) of penconazol, benomyl and cycloheximide for Saccharomyces cerevisiae and Kluyveromyces marxianus, were determined by growth inhibition on solid medium and were compared to the staining changes detected by FCM. RESULTS: The FCM and the EFM allowed the detection of AEP in all the yeast species tested. High MIC values for a drug were related with the presence of at least one AEP indicated by the cytometric data. CONCLUSIONS: The FCM revealed to be a robust assay whereas the EFM can be used as a preliminary test. It is possible to identify resistance/sensitivity patterns in yeast cells through cytometric detection methods of different efflux pumping systems.

Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo-3 and CD41.

BACKGROUND: Platelet activation plays a major role in the physiology and pathology of hemostasis. Flow cytometry is a promising approach for the structural and functional analysis of platelets. However, the choice of adequate biological parameters and most technical issues are still under discussion. A rise in cytosolic free Ca2+ is a key early event that follows platelet stimulation and precedes several activation responses, including shape change, aggregation, secretion, and expression of procoagulant activity. Our objective was to set up a fast and sensitive flow cytometric method to determine the kinetics of intracellular Ca2+ mobilization in platelets, which could be performed with the least artifactual perturbation of platelet function. METHODS: Anticoagulated blood was diluted in Tyrode's buffer and incubated with Fluo-3-acetoxymethyl ester prior to staining with phycoerytrin-conjugated antiplatelet GPIIb/IIIa complex monoclonal antibody. Platelets were identified by a gate including only CD41+ events. After the determination of baseline Fluo-3 green fluorescence on a flow cytometer (EPICS XL-MCL, Coulter Electronics, Hialeah, FL), adequate agonists were added and time-dependent changes in Fluo-3 fluorescence were recorded on-line for up to 3 min. RESULTS: In these conditions, a very fast and transient increase of cytosolic-free Ca2+ was observed following the addition of thrombin, a strong platelet agonist. Stimulation with adenosine diphosphate (ADP), a weak agonist, also resulted in evident increase of Ca2+ levels. CONCLUSIONS: Our results show that this flow cytometric kinetic method provides a simple and sensitive tool to assess in vitro the time course and intensity of signal transduction responses to different platelet agonists under near physiological conditions. In this way, it may be useful to evaluate the degree of platelet reactivity and thus to monitor antiplatelet therapy.

Flow cytometric assessment of cell structural and functional changes induced by acetic acid in the yeasts Zygosaccharomyces bailii and Saccharomyces cerevisiae.

Flow cytometry (FCM) was used with different viability dyes to assess changes in cell structure and function induced by acetic acid (AA) in populations of Zygosaccharomyces bailii (AA resistant) and Saccharomyces cerevisiae (AA sensitive). Kinetic changes in esterase activity, intracellular dye processing, and membrane integrity were monitored, and to detect those changes we used three assays involving fluorescein diacetate hydrolysis, FUN-1 processing, and propidium iodide exclusion, respectively. In S. cerevisiae, the decrease in the ability to process FUN-1 preceded the decrease in esterase activity, and there was loss of cell membrane integrity after incubation with AA. In Z. bailii, with higher AA concentrations, there was a similar decrease in the ability to process FUN-1, which also preceded the loss of cell membrane integrity. Changes in esterase activity in this yeast induced by AA treatment could not be monitored because the changes occurred independently of the presence of the acid. For control samples (untreated cells killed with 10% v/v of AA), the percentages of nonaltered cells as estimated by FCM and percentages of viable cells as estimated by colony forming unit (CFU) counts were identical. However, for cell samples treated for short periods with 3% (v/v) or less of AA, none of the dyes produced FCM results comparable to those produced by CFU counts.

Ki67 labelling index in gastric carcinomas. An immunohistochemical study using double staining for the evaluation of the proliferative activity of diffuse-type carcinomas.

The aim of the present study was to clarify the conflicting recorded data on the proliferative features of gastric carcinoma. The Ki67 labelling index (Ki67 LI) was evaluated using MIB-1 in 43 carcinomas (24 diffuse and 19 intestinal). In 18 cases, differential counts were performed in superficial and deep layers. In ten diffuse carcinomas with a prominent desmoplastic response, Ki67 LI was evaluated in sections double-stained with MIB-1 and CAM5.2. Flow cytometry was performed in 26 cases. Ki67 LI of diffuse carcinomas (36.3 +/- 19.0) was not significantly different from that of intestinal carcinomas (28.2 +/- 18.5). Ki67 LI was significantly higher (P = 0.006) in superficial than in deep areas (41.9 +/- 22.7 and 29.7 +/- 19.7, respectively) regardless of histological tumour type. No significant relationship was observed between Ki67 LI and wall invasion, lymph node metastasis, vascular invasion or ploidy. The following conclusions were drawn: double immunostaining techniques are apparently the best way to overcome the underestimation of cell proliferation in diffuse gastric carcinomas with a prominent desmoplastic response; the diffuse and intestinal types of gastric carcinoma have proliferation rates within the same range, even when the comparison is restricted to diploid tumours; and, finally, the major pool of proliferating cells resides in the superficial areas of gastric carcinomas, regardless of the histotype, which should be taken into consideration when overall counts are performed, using either immunohistochemical markers in tissue sections or suspensions of nuclei in flow cytometry.

Signet ring cell carcinoma of the stomach: a morphometric, ultrastructural, and DNA cytometric study.

The present study was undertaken to determine whether or not signet ring cell (diffuse, isolated cell) gastric carcinomas display a specific profile at the ultrastructural, morphometric, and DNA cytometric levels. Thirty-two cases of gastric carcinoma and 8 cases of peptic ulcer (control group) were studied with electron microscopy, morphometry, flow cytometry, and image cytometry. Despite the ultrastructural cellular heterogeneity of signet ring cell carcinomas, the neoplastic cells display fairly constant morphometric features: The cellular and nuclear volumes are significantly smaller than those of the other types of gastric carcinomas and closely resemble those of normal foveolar cells. The relatively small size of signet ring cell carcinoma nuclei fits with the high percentage of the cases of this type of gastric carcinoma that are either diploid or nearly diploid. There is a relationship between the infiltrative pattern of growth of gastric carcinoma (regardless of histologic subtype and ultrastructural cell differentiation) and the small size of neoplastic cells and their nuclei.

c-erbB-2 expression in primary gastric carcinomas and their metastases.

In an attempt to evaluate the relationship between c-erbB-2 expression and/or gene amplification, DNA ploidy and morphology, wall penetration, lymphatic permeation, and vascular invasion, we studied a series of 87 primary gastric carcinomas and their respective metastases (n = 335) using immunohistochemistry and performed DNA analysis of 30 primary tumors and 10 metastases from eight cases. Flow cytometry of fresh or frozen material was performed in 79 primary tumors. Five out of 87 primary tumors (5.7%) and 17 out of 335 lymph node metastases (5.1%) showed unequivocal membrane immunostaining for c-erbB-2. Seven out of 30 primary tumors (23.3%) showed gene amplification while amplification was identified in four out of 10 metastases (40.0%) from three patients. Eight tumors (9.2%) showed c-erbB-2 protein immunoreactivity, gene amplification, or both. One of these cases showed c-erbB-2 protein immunoreactivity only in the metastatic deposits, while gene amplification could be identified in the primary tumor. Three primary tumors showed gene amplification, but immunoreactive cells could not be identified. In no case was protein overexpression identified in the absence of gene amplification. Five cases with c-erbB-2 expression/amplification were well/moderately differentiated, and all the eight cases with c-erbB-2 expression/amplification disclosed aggressive features. Lymphatic permeation/lymph node metastases were found in all the cases and seven cases showed vascular invasion as well. In one case, there was also a liver metastasis. Two cases were early gastric carcinomas (T1sm) showing lymphatic permeation/nodal metastases and venous invasion. Six cases were aneuploid.(ABSTRACT TRUNCATED AT 250 WORDS)