scholarly journals Applications of Flow Cytometry to Clinical Microbiology

2000 ◽  
Vol 13 (2) ◽  
pp. 167-195 ◽  
Author(s):  
Alberto Álvarez-Barrientos ◽  
Javier Arroyo ◽  
Rafael Cantón ◽  
César Nombela ◽  
Miguel Sánchez-Pérez
Keyword(s):  
Flow Cytometry ◽  
Clinical Microbiology ◽  
Ex Vivo ◽  
Analytical Techniques ◽  
Clinical Samples ◽  
Clinical Microbiology Laboratory ◽  
Commercial Kits ◽  
Antimicrobial Treatments ◽  
Near Future

SUMMARY Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory.

Synthesis of a Novel Curcumin Derivative as a Potential Imaging Probe in Alzheimer’s Disease Imaging

2019 ◽  
Vol 16 (8) ◽  
pp. 723-731 ◽  
Author(s):  
Alexander Sturzu ◽  
Sumbla Sheikh ◽  
Hubert Kalbacher ◽  
Thomas Nägele ◽  
Christopher Weidenmaier ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Flow Cytometry ◽  
Transgenic Mice ◽  
Ex Vivo ◽  
Amyloid Plaques ◽  
Laser Scanning Microscopy ◽  
Β Amyloid ◽  
Curcumin Derivative

Background: Curcumin has been of interest in the field of Alzheimer’s disease. Early studies on transgenic mice showed promising results in the reduction of amyloid plaques.However, curcumin is very poorly soluble in aqueous solutions and not easily accessible to coupling as it contains only phenolic groups as potential coupling sites. For these reasons only few imaging studies using curcumin bound as an ester were performed and curcumin is mainly used as nutritional supplement. Methods: In the present study we produced an aminoethyl ether derivative of curcumin using a nucleophilic substitution reaction. This is a small modification and should not impact the properties of curcumin while introducing an easily accessible reactive amino group. This novel compound could be used to couple curcumin to other molecules using the standard methods of peptide synthesis. We studied the aminoethyl-curcumin compound and a tripeptide carrying this aminoethyl-curcumin and the fluorescent dye fluorescein (FITC-curcumin) in vitro on cell culture using confocal laser scanning microscopy and flow cytometry. Then these two substances were tested ex vivo on brain sections prepared from transgenic mice depicting Alzheimer-like β-amyloid plaques. Results: In the in vitro CLSM microscopy and flow cytometry experiments we found dot-like unspecific uptake and only slight cytotoxicity correlating with this uptake. As these measurements were optimized for the use of fluorescein as dye we found that the curcumin at 488nm fluorescence excitation was not strong enough to use it as a fluorescence marker in these applications. In the ex vivo sections CLSM experiments both the aminoethyl-curcumin and the FITC-curcumin peptide bound specifically to β- amyloid plaques. Conclusion: In conclusion we successfully produced a novel curcumin derivative which could easily be coupled to other imaging or therapeutic molecules as a sensor for amyloid plaques.

scholarly journals Using ex vivo culture to assess dynamic phenotype changes in human prostate macrophages following exposure to therapeutic drugs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Clovis Boibessot ◽  
France-Hélène Joncas ◽  
Aerin Park ◽  
Zohra Berrehail ◽  
Jean-François Pelletier ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Cell ◽  
Ex Vivo ◽  
Cell Phenotype ◽  
Gleason Grade ◽  
Tumor Resistance ◽  
Grade Group ◽  
In Vivo Models ◽  
Novel Associations

AbstractWithin the prostate tumor microenvironment (TME) there are complex multi-faceted and dynamic communication occurring between cancer cells and immune cells. Macrophages are key cells which infiltrate and surround tumor cells and are recognized to significantly contribute to tumor resistance and metastases. Our understanding of their function in the TME is commonly based on in vitro and in vivo models, with limited research to confirm these model observations in human prostates. Macrophage infiltration was evaluated within the TME of human prostates after 72 h culture of fresh biopsies samples in the presence of control or enzalutamide. In addition to immunohistochemistry, an optimized protocol for multi-parametric evaluation of cellular surface markers was developed using flow cytometry. Flow cytometry parameters were compared to clinicopathological features. Immunohistochemistry staining for 19 patients with paired samples suggested enzalutamide increased the expression of CD163 relative to CD68 staining. Techniques to validate these results using flow cytometry of dissociated biopsies after 72 h of culture are described. In a second cohort of patients with Gleason grade group ≥ 3 prostate cancer, global macrophage expression of CD163 was unchanged with enzalutamide treatment. However, exploratory analyses of our results using multi-parametric flow cytometry for multiple immunosuppressive macrophage markers suggest subgroup changes as well as novel associations between circulating biomarkers like the neutrophil to lymphocyte ratio (NLR) and immune cell phenotype composition in the prostate TME. Further, we observed an association between B7–H3 expressing tumor-associated macrophages and the presence of intraductal carcinoma. The use of flow cytometry to evaluate ex vivo cultured prostate biopsies fills an important gap in our ability to understand the immune cell composition of the prostate TME. Our results highlight novel associations for further investigation.

scholarly journals Characterisation of a New Human Alveolar Macrophage-Like Cell Line (Daisy)

Lung ◽  
2019 ◽  
Vol 197 (6) ◽  
pp. 687-698
Author(s):  
Laura R. Sadofsky ◽  
Yvette A. Hayman ◽  
Jesse Vance ◽  
Jorge L. Cervantes ◽  
Simon D. Fraser ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Ex Vivo ◽  
Human Macrophage ◽  
Macrophage Cell Line ◽  
Binding Assays ◽  
Macrophage Cell ◽  
Mitogenic Stimulation ◽  
Human Alveolar Macrophage

Abstract Purpose There is currently no true macrophage cell line and in vitro experiments requiring these cells currently require mitogenic stimulation of a macrophage precursor cell line (THP-1) or ex vivo maturation of circulating primary monocytes. In this study, we characterise a human macrophage cell line, derived from THP-1 cells, and compare its phenotype to the THP-1 cells. Methods THP-1 cells with and without mitogenic stimulation were compared to the newly derived macrophage-like cell line (Daisy) using microscopy, flow cytometry, phagocytosis assays, antigen binding assays and gene microarrays. Results We show that the cell line grows predominantly in an adherent monolayer. A panel of antibodies were chosen to investigate the cell surface phenotype of these cells using flow cytometry. Daisy cells expressed more CD11c, CD80, CD163, CD169 and CD206, but less CD14 and CD11b compared with mitogen-stimulated THP-1 cells. Unlike stimulated THP-1 cells which were barely able to bind immune complexes, Daisy cells showed large amounts of immune complex binding. Finally, although not statistically significant, the phagocytic ability of Daisy cells was greater than mitogen-stimulated THP-1 cells, suggesting that the cell line is more similar to mature macrophages. Conclusions The observed phenotype suggests that Daisy cells are a good model of human macrophages with a phenotype similar to human alveolar macrophages.

The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3090-3090 ◽  
Author(s):  
Folashade Otegbeye ◽  
Nathan Mackowski ◽  
Evelyn Ojo ◽  
Marcos De Lima ◽  
David N. Wald
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Ex Vivo ◽  
Leukemia Cells ◽  
Activating Receptor

Abstract Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2). Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

scholarly journals In Vitro and Ex Vivo Evaluation of Smart Infra-Red Fluorescent Caspase-3 Probes for Molecular Imaging of Cardiovascular Apoptosis

2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Manuelle Debunne ◽  
Christophe Portal ◽  
Bruno Delest ◽  
Ebba Brakenhielm ◽  
Françoise Lallemand ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Caspase 3 ◽  
Near Infrared ◽  
Ex Vivo ◽  
Annexin V ◽  
Fluorescent Signal ◽  
Infra Red ◽  
Infrared Probes

Purpose. The aim of this paper is to develop new optical bioprobes for the imaging of apoptosis. Procedure. We developed quenched near-infrared probes which become fluorescent upon cleavage by caspase-3, the key regulatory enzyme of apoptosis. Results. Probes were shown to be selectively cleaved by recombinant caspase-3. Apoptosis of cultured endothelial cells was associated with an increased fluorescent signal for the cleaved probes, which colocalized with caspase-3 and was reduced by the addition of a caspase-3 inhibitor. Flow cytometry demonstrated a similar profile between the cleaved probes and annexin V. Ex vivo experiments showed that sections of hearts obtained from mice treated with the proapoptotic drug doxorubicin displayed an increase in the fluorescent signal for the cleaved probes, which was reduced by a caspase-3 inhibitor. Conclusion. We demonstrated the capacity of these novel probes to detect apoptosis by optical imaging in vitro and ex vivo.

scholarly journals An alternative for urine cultures: Direct identification of uropathogens from urine by MALDI-TOF MS

2020 ◽  
Author(s):  
Arzu Akşit İlki ◽  
Sevim Özsoy ◽  
Gulşen Gelmez ◽  
Burak Aksu ◽  
Güner Söyletir
Keyword(s):  
Flow Cytometry ◽  
Antibiotic Susceptibility ◽  
Clinical Microbiology ◽  
Urine Samples ◽  
Clinical Microbiology Laboratory ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Susceptibility Tests ◽  
Tract Infections ◽  
Tof Ms

AbstractUrinary tract infections are one of the most common bacterial infections and rapid diagnosis of the infection is essential for appropriate antibiotic therapy. The goal of our study was to identify urinary pathogens directly by MALDI-TOF MS and to perform antibiotic susceptibility tests in order to shorten the period spent for culturing.Urine samples submitted for culture to the Clinical Microbiology Laboratory were enrolled in this study. Urine samples were screened for leukocyte and bacteria amount by flow cytometry. Samples with bacterial load of 106–107/mL were tested directly by MALDI-TOF MS and antibiotic susceptibility tests (AST) were performed.In total, 538 positive urine samples were evaluated in our study. MALDI-TOF MS identified the microorganism directly from the urine sample in 91.8% of these samples and the concordance rate of conventional identification and direct detection was 95.8% for Gram-negatives at the genus and species level. Escherichia coli (n:401) was the most frequently isolated microorganism, followed by Klebsiella pneumoniae (n:57). AST results were generated for 111 of these urine samples and the concordance was 90% and 87% for E. coli and K. pneumoniae, respectively.Our results showed that screening of urine samples with flow cytometry to detect positive samples and identification of uropathogens directly by MALDI-TOF MS with an accuracy of over 90% can be a suitable method particularly for Gram-negative bacteria in clinical microbiology laboratories.

scholarly journals Latest Trends in Electrochemical Sensors for Neurotransmitters: A Review

Sensors ◽  
2019 ◽  
Vol 19 (9) ◽  
pp. 2037 ◽  
Author(s):  
Tavakolian-Ardakani ◽  
Hosu ◽  
Cristea ◽  
Mazloum-Ardakani ◽  
Marrazza
Keyword(s):  
Electrochemical Sensors ◽  
Ex Vivo ◽  
Analytical Techniques ◽  
Substantial Improvement ◽  
Robust Detection ◽  
Brain Functions ◽  
The Brain ◽  
Made In

Neurotransmitters are endogenous chemical messengers which play an important role in many of the brain functions, abnormal levels being correlated with physical, psychotic and neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's disease. Therefore, their sensitive and robust detection is of great clinical significance. Electrochemical methods have been intensively used in the last decades for neurotransmitter detection, outclassing more complicated analytical techniques such as conventional spectrophotometry, chromatography, fluorescence, flow injection, and capillary electrophoresis. In this manuscript, the most successful and promising electrochemical enzyme-free and enzymatic sensors for neurotransmitter detection are reviewed. Focusing on the activity of worldwide researchers mainly during the last ten years (2010–2019), without pretending to be exhaustive, we present an overview of the progress made in sensing strategies during this time. Particular emphasis is placed on nanostructured-based sensors, which show a substantial improvement of the analytical performances. This review also examines the progress made in biosensors for neurotransmitter measurements in vitro, in vivo and ex vivo.

FRI0389 TOWARDS ONE-STAGE ARTICULAR CARTILAGE REPAIR USING DEVICE BASED ARTHROSCOPIC MECHANICAL RELEASE OF SYNOVIAL MESENCHYMAL STEM CELLS WITH CHONDROGENESIS AUGMENTATION WITH AUTOLOGOUS PLATELET CONCENTRATE WITHOUT CELL CULTURE EXPANSION

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 792-793
Author(s):  
A. Altaie ◽  
E. Jones ◽  
O. Wall ◽  
D. Mcgonagle
Keyword(s):  
Flow Cytometry ◽  
Synovial Fluid ◽  
Cartilage Repair ◽  
Ex Vivo ◽  
Synovial Cells ◽  
The Novel ◽  
Novel Device ◽  
Chondrogenic Potential ◽  
Before And After

Background:Synovial fluid contains resident mesenchymal stem cells (SF-MSCs) that are derived from the synovial membrane and may interact with superficial cartilage injury sites. We previously reported on a novel methodology for increasing the number of MSCs in the knee joints using synovial brushing combined with platelet lysate (PL) as a chondrogenic inducer [1, 2].Objectives:The purpose of this study was to evaluate autologous and allogenic PL as a chondrogenic inducer and the chondrogenic potential of the mobilised MSCs without further ex vivo expansion. The desired goal of the study was to provide in vitro proof of concept of direct chondrogensis without resort to MSC expansion protocols, since adequate MSCs towards repair could be mechanically procured in a minimally invasive fashion.Methods:SF-MSCs were derived from the joint cavity of patients undergoing arthroscopy procedures. For the mechanical release of MSCs ‘before’ and ‘after’ brushing the synovium with the novel device (Figure 1A), samples of irrigation fluid were collected and MSC numbers were evaluated by CFU-F assay and flow cytometry for stromal and immune populations. Standard chondrogenic assay was performed on uncultured and cultured expanded synovial MSCs. Pellet cultures were maintained in complete chondrogenic media (CCM), DMEM+50% autologous filtared platelet concentrate (fPC), 50% Stemulate (allogeneic human PL; Cook Regentec, Indianapolis, IN), or expansion media (control). Chondrogenesis was assessed by Glycosaminoglycan (GAG) andToluidine bluestaining. Autologous blood was processed through a gravity-based filtration system, HemaTrate®(HT; Cook Regentec, Indianapolis, IN), to produce a PC.Figure 1.Flow cytometry analysis of stromal and immune populations before’ and ‘after’ mechanically release of synovial with the novel device (CD90highCD45Lowin red circle) (A). Uncultured synovial cells after 21 days exposure to complete chondrogenic media Toulid Blue staining (B) Gags level (C) n=3.Results:Mechanically mobilized SF-MSC numbers increased as measured by CFU-F assay and flow cytometry for CD90HighCD45Lowcells (p<0.001), and CD14+HLA-DR+CD206+CD86+M2 macrophages also increased (p<0.05). The HT system significantly concentrated platelets and WBCs by 6- (p<0.0001) and 1.8-folds (p<0.001), respectively. Device-mobilized SF-MSC proliferation significantly increased after 6 days in DMEM + 10% PC (p<0.001) and correlated with PC platelet number (p<0.005). Autologous PC increased GAG levels compared to control (p<0.0001), and there was no significant difference compared to allogenic PL (p>0.5). Uncultured synovial cells produced significantly more GAG when cultured in CCM or DMEM + 50% autologous PC compared to control (p<0.0001). The GAG levels of uncultured synovial cells positively correlated with CFU-F (p<0.005). Chondrogenic potential of uncultured synovial cells that were mechanically mobilized with initial irrigation exhibited an increase (1.5-fold) in GAG levels (p<0.001) figure 1-B and also positively correlated with CFU-F (p<0.005).Conclusion:Synovial MSCs can be mechanically released in sufficient number to undergo in vitro chondrogenic induction with significant chondrogenic activity without the need for ex vivo culture expansion. In vitro, autologous PC can be used as chondrogenic inducer for uncultured SF-MSCs. The data presented here supports one stage arthroscopy procedures for cartilage repairReferences:[1]T.G. Baboolal, S.C. Mastbergen, E. Jones, S.J. Calder, F.P. Lafeber, D. McGonagle, Synovial fluid hyaluronan mediates MSC attachment to cartilage, a potential novel mechanism contributing to cartilage repair in osteoarthritis using knee joint distraction, Annals of the rheumatic diseases 75(5) (2016) 908-15.[2]A. Altaie, T.G. Baboolal, O. Wall, E. Jones, D. McGonagle, Platelet lysate enhances synovial fluid multipotential stromal cells functions: Implications for therapeutic use, Cytotherapy 20(3) (2018) 375-384.Disclosure of Interests:Ala Altaie: None declared, Elena Jones: None declared, Owen Wall: None declared, Dennis McGonagle Grant/research support from: Janssen Research & Development, LLC

scholarly journals Serum bactericidal test.

1988 ◽  
Vol 1 (1) ◽  
pp. 19-26 ◽  
Author(s):  
C W Stratton
Keyword(s):  
Clinical Trials ◽  
Clinical Utility ◽  
Clinical Microbiology ◽  
Additional Data ◽  
In Vitro Tests ◽  
Clinical Microbiology Laboratory ◽  
Microbiology Laboratory ◽  
New Information ◽  
Performance Results

The serum bactericidal test represents one of the few in vitro tests performed in the clinical microbiology laboratory that combines the interaction of the pathogen, the antimicrobial agent, and the patient. Although the use of such a test antedates the antimicrobial era, its performance, results, and interpretation have been subject to question and controversy. Much of the confusion concerning the serum bactericidal test can be avoided by an understanding of the various factors which influence bactericidal testing. In addition, the methodologic aspects of the serum bactericidal test have recently been addressed and should place this test on firmer ground. New information on the clinical utility of this test is becoming available; additional data are needed to establish more clearly the usefulness of the serum bactericidal test in specific infections. Such clinical trials from multiple centers will enable firmer recommendations for the future use of the serum bactericidal test.

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