scholarly journals Flow Cytometric Determination of Panton-Valentine Leucocidin S Component Binding

2001 ◽  
Vol 69 (4) ◽  
pp. 2390-2395 ◽  
Author(s):  
Valérie Gauduchon ◽  
Sandra Werner ◽  
Gilles Prévost ◽  
Henri Monteil ◽  
Didier A. Colin
Keyword(s):  
Flow Cytometry ◽  
Polymorphonuclear Neutrophils ◽  
Mutant Form ◽  
Binding Studies ◽  
Flow Cytometric ◽  
Kinase C ◽  
Specific Affinity ◽  
Substitution Mutant ◽  
Protein Kinase C Activation

ABSTRACT The binding of the S component (LukS-PV) from the bicomponent staphylococcal Panton-Valentine leucocidin to human polymorphonuclear neutrophils (PMNs) and monocytes was determined using flow cytometry and a single-cysteine substitution mutant of LukS-PV. The mutant was engineered by replacing a glycine at position 10 with a cysteine and was labeled with a fluorescein moiety. The biological activity of the mutant was identical to that of the native protein. It has been shown that LukS-PV has a high affinity for PMNs (K d = 0.07 ± 0.02 nM, n = 5) and monocytes (Kd = 0.020 ± 0.003 nM,n = 3) with maximal binding capacities of 197,000 and 80,000 LukS-PV molecules per cell, respectively. The nonspecifically bound molecules of LukS-PV do not form pores in the presence of the F component (LukF-PV) of leucocidin. LukS-PV and HlgC share the same receptor on PMNs, but the S components of other staphylococcal leukotoxins, HlgA, LukE, and LukM, do not compete with LukS-PV for its receptor. Extracellular Ca2+ at physiological concentrations (1 to 2 nM) has only a slight influence on the LukS-PV binding, in contrast to its complete inhibition by Zn2+. The down-regulation by phorbol 12-myristate 13-acetate (PMA) of the binding of LukS-PV was blocked by staurosporine, suggesting that the regulatory effect of PMA depends on protein kinase C activation. The labeled mutant form of LukS-PV has proved very useful for detailed binding studies of circulating white cells by flow cytometry. LukS-PV possesses a high specific affinity for a unique receptor on PMNs and monocytes.

scholarly journals Flow cytometric analysis of herpes simplex virus type 1 susceptibility to acyclovir, ganciclovir, and foscarnet.

1997 ◽  
Vol 41 (12) ◽  
pp. 2686-2692 ◽  
Author(s):  
I Pavić ◽  
A Hartmann ◽  
A Zimmermann ◽  
D Michel ◽  
W Hampl ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cytopathic Effect ◽  
Flow Cytometric Analysis ◽  
Flow Cytometric ◽  
Resistant Strains ◽  
Simplex Virus

We established a quantitative flow cytometric method for determination of herpes simplex virus type 1 (HSV-1) susceptibility to acyclovir (ACV), ganciclovir, and foscarnet in vitro. Susceptibility was defined in terms of the drug concentration which reduced the number of cells expressing HSV-1 glycoprotein C (gpC) with a fluorescence intensity of > or =10(2) by 50% (IC50). Flow cytometry allowed us to use a high (1.0) as well as a low (0.005) multiplicity of infection, and determination of the IC50 was possible after one or more viral replicative cycles. IC50s were dependent on virus input and on time postinfection. In mixture experiments, 1 to 2% resistant viruses added to a sensitive strain could be detected. The results obtained by flow cytometry showed a good qualitative correlation with those achieved by cytopathic effect inhibitory assay. However, flow cytometry might detect more quantitative differences in drug susceptibility, especially among resistant strains, as confirmed also by determination of intracellular drug phosphorylation. The mean IC50s for ACV-sensitive strains were 0.45 to 1.47 microM, and those for ACV-resistant strains were between 140 and 3,134 microM. Flow cytometric analysis was fast and accurate, automatizable, and highly reproducible. Flow cytometry may be a more powerful tool than standard cytopathic effect-based assays and could have advantages for the detection of low levels of drug resistance or mixtures of sensitive and resistant virus strains.

scholarly journals Protein kinase C activation alters the sensitivity of agonist-stimulated endothelial-cell prostacyclin production to intracellular Ca2+

1989 ◽  
Vol 262 (2) ◽  
pp. 431-437 ◽  
Author(s):  
T D Carter ◽  
T J Hallam ◽  
J D Pearson
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Threshold Value ◽  
Kinase C ◽  
Lag Period ◽  
Chronic Activation ◽  
Transduction Signal ◽  
Prostacyclin Production ◽  
Protein Kinase C Activation

Agonist-stimulated release of prostacyclin (PGI2) from endothelial cells requires elevation of the concentration of intracellular ionized calcium ([Ca2+]i) above a threshold value, and raised [Ca2+]i provides a sufficient transduction signal to account for the extent of PGI2 production. However, chronic activation of protein kinase C has been reported separately to potentiate PGI2 release, but to depress agonist-induced elevations of [Ca2+]i. We show here that pretreatment with phorbol 12-myristate 13-acetate (PMA) dose-dependently induces PGI2 release over many minutes after a significant lag period without any change in [Ca2+]i. In addition, PMA potentiates the transient release of PGI2 in response to agonists in a complex manner depending on the time of pre-incubation and the concentrations of both PMA and agonist. Concomitant measurement of [Ca2+]i and PGI2 release demonstrates that PMA pretreatment dose-dependently inhibits both the peak [Ca2+]i transient and the subsequent steady-state elevation of [Ca2+]i in response to agonists. Determination of the quantitative [Ca2+]i/PGI2 dose/response relationship, when PGI2 release is driven purely by elevating [Ca2+]i with ionomycin, demonstrates that PMA also enhances the Ca2+-sensitivity of PGI2 release. The observed effects of PMA on PGI2 release can be explained quantitatively by its abilities to lower the threshold [Ca2+]i required for PGI2 synthesis and to depress the peak [Ca2+]i evoked by agonist. We propose that these effects are due respectively to actions of PMA on phospholipase A2 and on a G-protein (Gp) that couples activated receptors to phospholipase C.

scholarly journals Quantitative Flow Cytometric Evaluation of MaximalCryptosporidium parvum Oocyst Infectivity in a Neonate Mouse Model

2000 ◽  
Vol 66 (10) ◽  
pp. 4315-4317 ◽  
Author(s):  
Agnès Delaunay ◽  
Gilles Gargala ◽  
Xunde Li ◽  
Loic Favennec ◽  
Jean Jacques Ballet
Keyword(s):  
Flow Cytometry ◽  
Mouse Model ◽  
Risk Evaluation ◽  
Detection Sensitivity ◽  
Contaminated Water ◽  
Suckling Mouse ◽  
Flow Cytometric ◽  
Waterborne Transmission ◽  
Flow Cytometric Evaluation

ABSTRACT The importance of waterborne transmission of Cryptosporidium parvum to humans has been highlighted by recent outbreaks of cryptosporidiosis. The first step in a survey of contaminated water currently consists of counting C. parvum oocysts. Data suggest that an accurate risk evaluation should include a determination of viability and infectivity of counted oocysts in water. In this study, oocyst infectivity was addressed by using a suckling mouse model. Four-day-old NMRI (Naval Medical Research Institute) mice were inoculated per os with 1 to 1,000 oocysts in saline. Seven days later, the number of oocysts present in the entire small intestine was counted by flow cytometry using a fluorescent, oocyst-specific monoclonal antibody. The number of intestinal oocysts was directly related to the number of inoculated oocysts. For each dose group, infectivity of oocysts, expressed as the percentage of infected animals, was 100% for challenge doses between 25 and 1,000 oocysts and about 70% for doses ranging from 1 to 10 oocysts/animal. Immunofluorescent flow cytometry was useful in enhancing the detection sensitivity in the highly susceptible NMRI suckling mouse model and so was determined to be suitable for the evaluation of maximal infectivity risk.

Flow cytometric determination of DNA content in isolated nuclei of cereals

Genome ◽  
1988 ◽  
Vol 30 (4) ◽  
pp. 565-569 ◽  
Author(s):  
E. Hülgenhof ◽  
R. A. Weidhase ◽  
R. Schlegel ◽  
A. Tewes
Keyword(s):  
Flow Cytometry ◽  
Dna Content ◽  
Internal Standard ◽  
Aegilops Speltoides ◽  
Isolated Nuclei ◽  
Triticum Timopheevi ◽  
Flow Cytometric ◽  
Nuclear Isolation ◽  
Cereal Plants

Isolated nuclei from cereal plants were used for quantitative determination of DNA per nucleus by flow cytometry. The technique is based on enhanced fluorescence of ethidium bromide and olivomycin when they bind to DNA. Nuclei were isolated from protoplasts derived from leaves of seedlings. The diploid Hordeum vulgare cv. Trumpf was used as an internal standard. Analysis of nuclei from several cultivars of hexaploid wheat (Triticum aestivum), Triticum durum var. hordeiforme, T. durum var. alexandrinum, Triticum araraticum, Triticum timopheevi, Triticum monococcum, Aegilops speltoides, Secale cereale, and one hexaploid and one octoploid triticale revealed significant intra- and inter-specific differences in DNA values per 2C nucleus. The advantages of the procedure are discussed along with its utilization for quantitative DNA measurement in Gramineae.Key words: nuclear isolation, flow cytometry, DNA content, cereals.

scholarly journals Determination of the Viability of Aeromonas hydrophila in Different Types of Water by Flow Cytometry, and Comparison with Classical Methods

2005 ◽  
Vol 71 (12) ◽  
pp. 7948-7954 ◽  
Author(s):  
Anna Pianetti ◽  
Tania Falcioni ◽  
Francesca Bruscolini ◽  
Luigia Sabatini ◽  
Elivio Sisti ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Aeromonas Hydrophila ◽  
Flow Cytometric Analysis ◽  
Staining Technique ◽  
Plate Count ◽  
Physiological Status ◽  
Traditional Methods ◽  
Flow Cytometric ◽  
Bacterial Content

ABSTRACT The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.

scholarly journals Flow Cytometric Determination of the Effects of Antibacterial Agents on Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. capricolum, and Mycoplasma mycoides subsp. mycoides Large Colony Type

2006 ◽  
Vol 50 (8) ◽  
pp. 2845-2849 ◽  
Author(s):  
Patricia Assunção ◽  
Nuno T. Antunes ◽  
Ruben S. Rosales ◽  
Carlos Poveda ◽  
Jose B. Poveda ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Antibacterial Agents ◽  
Mycoplasma Species ◽  
Colony Type ◽  
Flow Cytometric ◽  
Mycoplasma Capricolum ◽  
Large Colony ◽  
Mycoplasma Mycoides

ABSTRACT Flow cytometry together with SYBR green I and propidium iodide was used to study the effects of enrofloxacin, ciprofloxacin, gentamicin, chloramphenicol, oxytetracycline, and tylosin on four mycoplasma species. Inhibition of mycoplasma growth could be detected by as early as 3 h after the start of treatment. The strongest effect was observed with enrofloxacin- and ciprofloxacin-treated cells.

scholarly journals Safe Determination of Susceptibility of Mycobacterium tuberculosis to Antimycobacterial Agents by Flow Cytometry

1999 ◽  
Vol 37 (3) ◽  
pp. 479-483 ◽  
Author(s):  
Andrea V. Moore ◽  
Scott M. Kirk ◽  
Steven M. Callister ◽  
Gerald H. Mazurek ◽  
Ronald F. Schell
Keyword(s):  
Flow Cytometry ◽  
Mycobacterium Tuberculosis ◽  
Flow Cytometric Analysis ◽  
Test Methods ◽  
Susceptibility Test ◽  
Flow Cytometric ◽  
Proportion Method ◽  
Bactec System ◽  
Drug Free

We showed previously that susceptibility testing forMycobacterium tuberculosis labeled with fluorescein diacetate could be accomplished rapidly by using flow cytometry. However, safety was a major concern because mycobacteria were not killed prior to flow cytometric analysis. In this study, we developed a biologically safe flow cytometric susceptibility test that depends on detection and enumeration of actively growing M. tuberculosis organisms in drug-free and antimycobacterial agent-containing medium. The susceptibilities of 17 clinical isolates of M. tuberculosis to ethambutol, isoniazid, and rifampin were tested by the agar proportion and flow cytometric methods. Subsequently, all flow cytometric susceptibility test samples were inactivated by exposure to paraformaldehyde before analysis with a flow cytometer. Agreement between the results from the two methods was 98%. In addition, the flow cytometric results were available 72 h after the initiation of testing. The flow cytometric susceptibility assay is safe, simple to perform, and more rapid than conventional test methods, such as the BACTEC system and the proportion method.

Development and Evaluation of a Novel Flow Cytometric Assay for Determination of Fcγ Receptor IIIa-158 V/F Dimorphism.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1366-1366
Author(s):  
Sebastian Boettcher ◽  
Matthias Ritgen ◽  
Christiane Pott ◽  
Andreas Humpe ◽  
Michael Kneba
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Single Agent ◽  
Amino Acid Position ◽  
Taqman Assay ◽  
Flow Cytometric Assay ◽  
Fcγ Receptor ◽  
Color Flow ◽  
Flow Cytometric

Abstract A recently described dimorphism at position 559 of FCGR3A gene results in two allotypes of Fcγ receptor IIIa (Fcγ RIIIa) with phenylalanine (F) or valine (V) at amino acid position 158. It has been shown that Fcγ RIIIa-VV homozygous patients with follicular lymphoma respond better to Rituximab as a single agent than F carriers. However, there is evidence that this disadvantage in F carriers can be overcome by higher Rituximab concentrations or by the use of defucosylated therapeutic antibodies. Therefore rapid, widely applicable, and cost-effective methods for the determination of this Fcγ RIIIa dimorphism are necessary. Currently available methods to determine this dimorphism are based on PCR techniques. To simplify the determination of Fcγ RIIIa-V/F dimorphism we developed a novel flow cytometric approach using known differences in epitope recognition of monoclonal antibodies (moabs) to Fcγ RIIIa. The moab MEM-154 recognizes Fcγ RIIIa-V only, whereas moab 3G8 recognizes Fcγ RIIIa irrespective of the dimorphism. We determined the expression level of epitopes recognized by 3G8 and MEM-154 in NK cells of 35 healthy donors (FF 14; V/F 17; VV 4) by three color flow cytometry and secondary immunofluorescence. Results were compared to genotypes determined by a TaqMan assay using allele specific fluorochrome labelled probes. Donors genotyped as Fcγ RIIIa FF, V/F, and VV demonstrated overlapping immunofluorescence levels detected by both 3G8 and MEM-154. However, the ratio of fluorescence measured using MEM-154 divided by the immunofluorescence measured using 3G8 was 100% accurate for predicting genotypes. Ratios below 0.05 were measured in Fcγ RIIIa FF individuals, ratios between 0.1 and 0.5 were measured in heterozygotes, whereas ratios higher than 0.6 were found in Fcγ RIIIa VV individuals only. Quantitative flow cytometry demonstrated a great variation in Fcγ RIIIa expression on NK cells between individuals with identical Fcγ R IIIa genotype explaining the failure to predict the genotype by a single Fcγ R IIIa moab only. This novel flow cytometric assay is cost-efficient, easy to implement, reliable and uses standard flow cytometric techniques. Compared to known methods to determine the dimorphism it is faster and applicable in laboratories without sophisticated PCR technology.

Detection of mastitis in the bovine mammary gland by flow cytometry at early stages

2008 ◽  
Vol 75 (2) ◽  
pp. 225-232 ◽  
Author(s):  
Cordula Koess ◽  
Joern Hamann
Keyword(s):  
Flow Cytometry ◽  
Cell Count ◽  
Early Stage ◽  
Cell Types ◽  
Subclinical Mastitis ◽  
Polymorphonuclear Neutrophils ◽  
Cross Sectional ◽  
Flow Cytometric Method ◽  
Flow Cytometric ◽  
Fast Flow

Subclinical mastitis is a costly disease and its diagnosis is difficult. Besides the somatic cell count (SCC) and bacteriology, the differential inflammatory cell count (DICC) is a meaningful tool for mastitis detection. As microscopy is very subjective because of the low number of events to be counted, flow cytometry has often been proposed for the differentiation of milk cells. The objective of this study was to determine whether it is possible to identify subclinical mastitis in cattle at an early stage by a simple and fast flow cytometric method. The aim was to identify the main leucocyte populations in flow cytometric dotplots (polymorphonuclear neutrophils (PMN), lymphocytes and macrophages) and, with these, to elaborate a method of mastitis prognostics. Milk from 15 German Holstein cows was sampled in cross-sectional studies and SCC determined. After preparation, the milk cells were incubated with different specific antibodies that bind to different cell types and also to propidium iodide (PI), which differs between viable and non-viable cells. This procedure made it possible to localize cell types in a flow cytometric dot plot and to differentiate between viable and non-viable PMN. Percentages of viable PMN can be determined by a procedure consisting of a simple centrifugation, incubation with PI, and flow cytometric measurement. So it is possible to quickly determine the stage of the inflammation even in quarters with a low SCC.

Quantitative determination of CD4/CD8 molecules by a cell marker ELISA

1994 ◽  
Vol 40 (1) ◽  
pp. 38-42 ◽  
Author(s):  
L Franke ◽  
E Nugel ◽  
W D Döcke ◽  
T Porstmann
Keyword(s):  
Flow Cytometry ◽  
T Lymphocytes ◽  
Flow Cytometric Analysis ◽  
Sample Volume ◽  
Small Sample ◽  
Cell Marker ◽  
Cell Counts ◽  
Flow Cytometric ◽  
A Cell

Abstract Determination of percentages of CD4+ and CD8+ T cells from patients with human immunodeficiency virus infection is usually done by flow cytometric analysis. We compared a cell marker ELISA with flow cytometry for quantitation of CD4 and CD8 molecules on T lymphocytes, and correlated the values both with the number of CD4+ and CD8+ T lymphocytes and with clinical data. Results by cell marker ELISA (y) correlated well with those by flow cytometric analysis (x); r = 0.69, P < 0.001 (y = 0.01x + 3.9) for CD4; r = 0.81, P < 0.001 (y = 0.03x + 5.4) for CD8; n = 343. The ELISA detected changes in numbers of CD8 molecules on the cells earlier than flow cytometry recognized changes in CD8+ T-cell counts. The advantages of the ELISA are the small sample volume required (0.5 mL of blood), its internal standardization by a CD4+/CD8+ cell line, and its simple and fast performance. The cell marker ELISA appears to be an efficient alternative to flow cytometry.

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