scholarly journals Flow Cytometric Assessment of Susceptibilities of Streptococcus pyogenes to Erythromycin and Rokitamycin

2003 ◽  
Vol 47 (1) ◽  
pp. 408-412 ◽  
Author(s):  
Pier Carlo Braga ◽  
Cinzia Bovio ◽  
Maria Culici ◽  
Monica Dal Sasso
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Microscopy ◽  
Streptococcus Pyogenes ◽  
Single Cells ◽  
Membered Ring ◽  
Flow Cytometric ◽  
Live Bacteria ◽  
Erythromycin A ◽  
Set Up ◽  
Syto 9

ABSTRACT The effects of erythromycin (a 14-membered ring macrolide) and rokitamycin (a 16-membered ring macrolide) on the viability of the Streptococcus pyogenes M phenotype were studied by means of flow cytometry and fluorescence microscopy by using a combination of two fluorochromes (syto 9 and propidium iodide) that stains live bacteria green and dead bacteria red. In order to apply the flow cytometry, a bacterial sonication procedure was expressly set up to separate single cells from the long, intralaced S. pyogenes chains of up to 30 to 40 cells that have previously prevented the application of flow cytometry to this type of bacteria. The association of flow cytometry using an appropriate sonication procedure, together with a combination of fluorescent probes, offered the possibility of very quickly investigating the different microbiological effects of rokitamycin at 2 μg/ml, which was active on the S. pyogenes M phenotype, and of erythromycin at doses of up to 32 μg/ml, which was not.

Antiphospholipid Antibodies Impair the Clearance of Apoptotic Platelets.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3948-3948
Author(s):  
Silvia S. Pierangeli ◽  
Mariano E. Vega-Ostertag
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Microscopy ◽  
Immune Responses ◽  
Antiphospholipid Antibodies ◽  
Human Platelet ◽  
Human Platelets ◽  
Platelet Destruction ◽  
Outer Leaflet ◽  
Platelet Antibodies ◽  
Flow Cytometric

Abstract Background: Thrombocytopenia is frequent in patients with the Antiphospholipid Syndrome (APS). The mechanism(s) that lead to abnormal platelet destruction is not understood. Phosphatidylserine (PS) that is exposed in the outer leaflet of the membrane of aged human platelets (AHP) and β2glycoprotein I (β2GPI) mediate their phagocytosis by macrophages without inducing inflammatory or immune responses. We hypothesized that antiphospholipid antibodies (aPL) affect the clearance of AHP and induce immunogenicity of AHP leading to the production of anti-platelet antibodies. Methods: To examine that question, we studied phagocytosis of AHP by macrophages in the presence of β2GPI, IgG aPL or control IgG (IgG-NHS). AHP labeled with CM-Orange, were incubated with β2GPI and aPL IgG or with IgG-NHS, and added to a monolayer of cultured phagocytes. The fluorescent-positive phagocytes (FPPC) were then tested by fluorescence microscopy. Then the cells were trypsinized and analyzed by flow cytometry. We also examined the immunogenicity (production of anti-platelet antibodies) of AHP treated with IgG aPL and with IgG-NHS by immunizing Balb/c mice with AHP and IgG-aPL or IgG-NHS. The patterns of reactivity of the sera of the immunized mice was examined by immunoblot of human platelet lysates. Results: aPL IgG produced a significantly lower % FPPC compared to the IgG-NHS-treated cells (15.33 ± 5.31 vs 89.0 ± 7.94, respectively). This was confirmed in the flow cytometric studies: IgG aPL produced significantly lower % of FPPC when compared to IgG-NHS-treated platelets (42.49 ±11.77 vs 78.11± 5.43, respectively).. Mice immunized with AHP and aPL IgG produced significantly higher titers of anti-platelet antibodies (as detected by ELISA) when compared to mice immunized with IgG-NHS (p=0.0033). Furthermore, there was a different pattern of reactivity of the sera with respect to recognition of platelet antigens, when sera of immunized mice were analyzed by immunoblot. Conclusions: The data indicate that aPL impair the clearance of apoptotic platelets and affect their immunogenicity. This may lead to the production of anti-platelet antibodies and thrombocytopenia in APS.

scholarly journals In Vitro Activity of the New Ketolide Telithromycin Compared with Those of Macrolides against Streptococcus pyogenes: Influences of Resistance Mechanisms and Methodological Factors

2000 ◽  
Vol 44 (11) ◽  
pp. 2999-3002 ◽  
Author(s):  
Pascale Bemer-Melchior ◽  
Marie-Emmanuelle Juvin ◽  
Sandrine Tassin ◽  
Andre Bryskier ◽  
Gian Carlo Schito ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Ambient Air ◽  
Resistance Mechanisms ◽  
Test Methods ◽  
Membered Ring ◽  
Broth Microdilution ◽  
Broth Microdilution Method ◽  
Resistant Strains ◽  
Erythromycin A

ABSTRACT One hundred and seven clinical isolates of Streptococcus pyogenes, 80 susceptible to macrolides and 27 resistant to erythromycin A (MIC >0.5 μg/ml), were examined. The erythromycin A-lincomycin double-disk test assigned 7 resistant strains to the M-phenotype, 8 to the inducible macrolide, lincosamide, and streptogramin B resistance (iMLSB) phenotype, and 12 to the constitutive MLSB resistance (cMLSB) phenotype. MICs of erythromycin A, clarithromycin, azithromycin, roxithromycin, and clindamycin were determined by a broth microdilution method. MICs of telithromycin were determined by three different methods (broth microdilution, agar dilution, and E-test methods) in an ambient air atmosphere and in a 5 to 6% CO2 atmosphere. Erythromycin A resistance genes were investigated by PCR in the 27 erythromycin A-resistant isolates. MICs of erythromycin A and clindamycin showed six groups of resistant strains, groups A to F. iMLSB strains (A, B, and D groups) are characterized by two distinct patterns of resistance correlated with genotypic results. A- and B-group strains were moderately resistant to 14- and 15-membered ring macrolides and highly susceptible to telithromycin. All A- and B-group isolates harbored erm TR gene, D-group strains, highly resistant to macrolides and intermediately resistant to telithromycin (MICs, 1 to 16 μg/ml), were all characterized by having the ermB gene. All M-phenotype isolates (C group), resistant to 14- and 15-membered ring macrolides and susceptible to clindamycin and telithromycin, harbored the mefA gene. All cMLSB strains (E and F groups) with high level of resistance to macrolides, lincosamide, and telithromycin had the ermB gene. The effect of 5 to 6% CO2 was remarkable on resistant strains, by increasing MICs of telithromycin from 1 to 6 twofold dilutions against D-E- and F-group isolates.

The Shaving Reaction Is Facilitated through Trogocytosis: Concerted Transfer of Both Rituximab (RTX) and the Membrane Dye PKH26 from B Lymphocytes to Macrophages Is Demonstrable by Flow Cytometry, Fluorescence Microscopy, and High Resolution Digital Imaging in a Flow Cytometric Environment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2096-2096 ◽  
Author(s):  
David A. Mack ◽  
Paul V. Beum ◽  
Margaret A. Lindorfer ◽  
Andrew W. Pawluczkowycz ◽  
Ronald P. Taylor
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
High Resolution ◽  
Fluorescence Microscopy ◽  
B Cell ◽  
B Lymphocytes ◽  
Digital Imaging ◽  
Antigenic Modulation ◽  
Raji Cells ◽  
Flow Cytometric

Abstract Opsonization of CD20+ B lymphocytes with the anti-CD20 mAb RTX can promote substantial loss of B cell-bound RTX and CD20 in the presence of acceptor monocytes (Beum et al., J Immunol, 2006). This process of antigenic modulation, or shaving, occurs when patients with chronic lymphocytic leukemia are treated with the standard RTX dose of 375 mg/m2, and represents an escape mechanism for mAb-targeted malignant cells. Shaving in vitro, and likely in vivo, is mediated by mononuclear phagocytic cells: Fc gamma receptors on these cells bind and take up CD20-RTX immune complexes. This process may occur by trogocytosis, in which donor and acceptor cells form immunologic synapses, allowing membrane fragments and cell-surface proteins to be removed from donor cells and transferred to and internalized by acceptor cells. To investigate this hypothesis, Raji cells were stained with the lipophilic fluorescent reporter membrane dye PKH26, and opsonized with RTX or Alexa (Al) 488-conjugated RTX. The cells were incubated with retinoic acid-treated THP-1 cells, a human monocyte/macrophage cell line. THP-1 cells in the mixtures were then stained for CD11b and CD14 for identification. Quantitative analysis of cells by flow cytometry and high resolution digital imaging in a flow cytometric environment (ImageStream, AMNIS Corp.) demonstrated time-dependent transfer of PKH26 from RTX-opsonized Raji cells to THP-1 cells, as illustrated in a representative kinetics experiment in the table. Fluorescence intensity values are given for THP-1 cells after incubation with naïve (control), or RTX-opsonized Raji cells, both of which were PKH26 labeled. Molecules of equivalent soluble fluorochrome (MESF) units or geometric mean fluorescence (GMF) units are used, based on flow cytometry or ImageStream analysis, respectively. PKH26 Fluorescence Intensities Acquired on THP-1 Cells after Incubation with Control or RTX-Opsonized PKH26-Labeled Raji Cells Time of Incubation of Raji and THP-1 Cells, min <1 5 15 45 Control (no RTX on Raji cells) MESF 170 240 260 630 GMF 900 900 1300 3300 Experimental (RTX-opsonized Raji Cells) MESF 700 1200 1700 4300 GMF 3500 6100 8300 19000 Based on the PKH26 signal, a small but readily demonstrable portion of Raji cell membrane is transferred from RTX-opsonized cells to THP-1 cells. Transfer corresponds to only ~5–10% of total PKH26 on Raji cells, which is reasonable as B cells are left intact after shaving. However, we observed >65% transfer of Al488 RTX from B cells to THP-1 cells. Results were confirmed by fluorescence microscopy and by inspection of ImageStream digital images of THP-1 and Raji cells. We believe that transfer of RTX and CD20 from RTX-opsonized B cells to acceptor monocytes/macrophages proceeds via trogocytosis, and plays an important role in the resistance of some B-cell malignancies to RTX therapy. Based on reports of antigenic modulation of targets for other immunotherapeutic mAbs used in cancer treatment, the trogocytic mechanism we document for RTX is likely to underlie antigenic modulation promoted by these mAbs as well.

scholarly journals Dynamic assay of enzyme activities in single cells by flow cytometry.

1979 ◽  
Vol 27 (12) ◽  
pp. 1644-1646 ◽  
Author(s):  
F Dolbeare
Keyword(s):  
Flow Cytometry ◽  
Enzyme Activities ◽  
Single Cells ◽  
Lactic Dehydrogenase ◽  
Sodium Lactate ◽  
Specific Enzyme ◽  
Fluorogenic Substrates ◽  
Fluorescence Measurements ◽  
Flow Cytometric ◽  
Dynamic Assay

Three enzymes in single cells were assayed dynamically by flow cytometry using four fluorogenic substrates. Acid phosphatase was determined with 7-bromo-3-hydroxy-2-naphtho-o-anisidine (naphthol AS-BI) phosphate and 4-methylumbelliferone (MU) phosphate, neutral esterase with fluorescein diacetate, and lactic dehydrogenase with NAD-sodium lactate. Fluorescence measurements obtained with the flow cytometer were converted into relative specific enzyme activities for single cells with molar fluorescence coefficients determined with a spectrofluorometer. Specific activities obtained from spectrofluorometric data were compared with activities calculated from flow cytometeric data. Flow cytometric assays gave lower specific single cell activities for 4-methylumbelliferone phosphate hydrolysis and for lactic dehydrogenase than did similar assays by standard spectrofluorometry. Product diffusion may be the greatest cause for this discrepancy.

scholarly journals Antibacterial activity of RU 64004 (HMR 3004), a novel ketolide derivative active against respiratory pathogens.

1997 ◽  
Vol 41 (10) ◽  
pp. 2149-2158 ◽  
Author(s):  
C Agouridas ◽  
A Bonnefoy ◽  
J F Chantot
Keyword(s):  
Antibacterial Activity ◽  
Streptococcus Pyogenes ◽  
Respiratory Infections ◽  
Membered Ring ◽  
Penicillin G ◽  
Respiratory Pathogens ◽  
Bacterial Strains ◽  
Good Antibacterial Activity ◽  
Resistant Strains ◽  
Erythromycin A

The antibacterial activity of RU 64004, a new ketolide, was evaluated against more than 600 bacterial strains and was compared with those of various macrolides and pristinamycin. RU 64004 had good activity against multiresistant pneumococci, whether they were erythromycin A resistant or not, including penicillin-resistant strains. RU 64004 inhibited 90% of pneumococci resistant to erythromycin A and penicillin G at 0.6 and 0.15 microg/ml, respectively. Unlike macrolides, RU 64004 did not induce the phenotype of resistance to macrolides-lincosamides-streptogramin B. Its good antibacterial activity against multiresistant pneumococci ran in parallel with its well-balanced activity against all bacteria involved in respiratory infections (e.g., Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pyogenes). In contrast to all comparators (14- and 16-membered-ring macrolides and pristinamycin), RU 64004 displayed high therapeutic activity in animals infected with all major strains, irrespective of the phenotypes of the strains. The results suggest that RU 64004 has potential for use in the treatment of infections caused by respiratory pathogens including multiresistant pneumococci.

scholarly journals O-GlcNAcylation in early stages of chronic lymphocytic leukemia; protocol development for flow cytometry

2021 ◽  
pp. 1-10
Author(s):  
Viktória Temesfői ◽  
Kinga Molnár ◽  
Péter Kaltenecker ◽  
Barbara Réger ◽  
Árpád Szomor ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Lymphocyte Count ◽  
Blood Parameters ◽  
Cell Number ◽  
Metabolic Changes ◽  
Protein Glycosylation ◽  
Lymphocytic Leukemia ◽  
Tween 20 ◽  
Protocol Development ◽  
Flow Cytometric

BACKGROUND: Recent studies proved that metabolic changes in malignant disorders have an impact on protein glycosylation, however, only a few attempts have been made so far to use O-GlcNAc analysis as a prognostic tool. Glucose metabolism is reported to be altered in hematological malignancies thus, we hypothesized that monitoring intracellular O-GlcNAc levels in Rai stage 0-I (Binet A) CLL patients could give deeper insights regarding subtle metabolic changes of progression which are not completely detected by the routine follow-up procedures. OBJECTIVE: In this proof of concept study we established a flow cytometric detection method for the assessment of O-GlcNAcylation as a possible prognostic marker in CLL malignancy which was supported by fluorescence microscopy. METHODS: Healthy volunteers and CLL patients were recruited for this study. Lymphocytes were isolated, fixed and permeabilised by various methods to find the optimal experimental condition for O-GlcNAc detection by flow cytometry. O-GlcNAc levels were measured and compared to lymphocyte count and various blood parameters including plasma glucose level. RESULTS: The protocol we developed includes red blood cell lysis, formalin fixation, 0.1% Tween 20 permeabilisation and employs standardized cell number per sample and unstained controls. We have found significant correlation between O-GlcNAc levels and WBC (R2= 0.8535, p< 0.0029) and lymphocyte count (R2= 0.9225, p< 0.0006) in CLL patients. Interestingly, there was no such correlation in healthy individuals (R2= 0.05664 for O-GlcNAc vs WBC and R2= 0.04379 for O-GlcNAc vs lymphocytes). CONCLUSION: Analyzing O-GlcNAc changes in malignant disorders, specifically in malignant hematologic diseases such as CLL, could be a useful tool to monitor the progression of the disease.

scholarly journals Cord Blood Extracellular Vesicles Analyzed by Flow Cytometry with Thresholding Using 405 nm or 488 nm Laser Leads to Concurrent Results

Diagnostics ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1320
Author(s):  
Kristýna Pekárková ◽  
Jakub Soukup ◽  
Marie Kostelanská ◽  
Jan Širc ◽  
Zbyněk Straňák ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cord Blood ◽  
Extracellular Vesicles ◽  
Correlation Coefficients ◽  
Flow Cytometer ◽  
Liquid Biopsies ◽  
Physiological Conditions ◽  
Flow Cytometric ◽  
Term Births ◽  
And Control

Extracellular vesicles (EVs) from liquid biopsies are extensively analyzed by flow cytometry, a technology that is continuously evolving. Thresholding utilizing a violet 405 nm laser side scatter (VSSC) has recently been implemented. Here, we collected set of large EV (lEV) samples from cord blood, which we analyzed using a standard flow cytometer improved via a 405 nm laser side scatter. Samples were analyzed using two distinct thresholding methods—one based on VSSC, and one based on VSSC combined with fluorescence thresholding on stained phosphatidylserine. Through these thresholding methods, we compared lEVs from pre-term births and control cord blood. Double-labeled lEVs with platelet CD36+/CD41+, activated platelet CD41+/CD62P+ and endothelial CD31+/CD105+ antibodies were used. Apart from comparing the two groups together, we also correlated measured lEVs with the thresholding methods. We also correlated the results of this study with data analyzed in our previous study in which we used a conventional 488 nm laser SSC. We did not find any difference between the two cord blood groups. However, we found highly concurrent data via our correlation of the thresholding methods, with correlation coefficients ranging from 0.80 to 0.96 even though the numbers of detected lEVs differed between thresholding methods. In conclusion, our approaches to thresholding provided concurrent data and it seems that improving the cytometer with the use of a VSSC increases its sensitivity, despite not being particularly critical to the validity of flow cytometric studies that compare pathological and physiological conditions in liquid biopsies.

scholarly journals Flow cytometric measurements as a proxy for sporulation intensity in the cultured macroalga Ulva (Chlorophyta)

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Omri Nahor ◽  
Cristina F. Morales-Reyes ◽  
Gianmaria Califano ◽  
Thomas Wichard ◽  
Alexander Golberg ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Life Cycle ◽  
Abiotic Factors ◽  
Physiological Status ◽  
Seaweed Cultivation ◽  
Reproductive Growth ◽  
Biotic And Abiotic Factors ◽  
Flow Cytometric ◽  
Abiotic Stimuli ◽  
Fundamental Shift

Abstract Controlling the life cycle of the green macroalga Ulva (Chlorophyta) is essential to maintain its efficient aquaculture. A fundamental shift in cultivation occurs by transforming the thallus cells into gametangia and sporangia (sporulation), with the subsequent release of gametes and zoids. Sporulation occurrence depends on algal age and abiotic stimuli and is controlled by sporulation inhibitors. Thus, quantification of sporulation intensity is critical for identifying the biotic and abiotic factors that influence the transition to reproductive growth. Here, we propose to determine the sporulation index by measuring the number of released gametes using flow cytometry, in proportion to the total number of thallus cells present before the occurrence of the sporulation event. The flow cytometric measurements were validated by manually counting the number of released gametes. We observed a variation in the autofluorescence levels of the gametes which were released from the gametangia. High autofluorescence level correlated to phototactically active behaviour of the gametes. As autofluorescence levels varied between different groups of gametes related to their mobility, flow cytometry can also determine the physiological status of the gametes used as feedstock in seaweed cultivation.

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