scholarly journals Dose Dependent Antimicrobial Cellular Cytotoxicity—Implications for ex vivo Diagnostics

2021 ◽  
Vol 12 ◽  
Author(s):  
Ana Copaescu ◽  
Phuti Choshi ◽  
Sarah Pedretti ◽  
Effie Mouhtouris ◽  
Jonathan Peter ◽  
...  
Keyword(s):  
Cell Viability ◽  
Elispot Assay ◽  
Ex Vivo ◽  
Cellular Cytotoxicity ◽  
Cell Cytotoxicity ◽  
Viability Staining ◽  
Drug Concentrations ◽  
Ldh Assay ◽  
Ifn Γ ◽  
Dose Dependent

Introduction:Ex vivo and in vitro diagnostics, such as interferon-γ (IFN-γ) release enzyme linked ImmunoSpot (ELISpot) and flow cytometry, are increasingly employed in the research and diagnostic setting for severe T-cell mediated hypersensitivity. Despite an increasing use of IFN-γ release ELISpot for drug causality assessment and utilization of a range of antimicrobial concentrations ex vivo, data regarding antimicrobial-associated cellular cytotoxicity and implications for assay performance remain scarcely described in the literature. Using the measurement of lactate dehydrogenase (LDH) and the 7-AAD cell viability staining, we aimed via an exploratory study, to determine the maximal antimicrobial concentrations required to preserve cell viability for commonly implicated antimicrobials in severe T-cell mediated hypersensitivity.Method: After an 18-h incubation of patient peripheral blood monocytes (PBMCs) and antimicrobials at varying drug concentrations, the cell cytotoxicity was measured in two ways. A colorimetric based assay that detects LDH activity and by flow cytometry using the 7-AAD cell viability staining. We used the PBMCs collected from three healthy control participants with no known history of adverse drug reaction and two patients with a rifampicin-associated drug reaction with eosinophilia and systemic symptoms (DRESS), confirmed on IFN-γ ELISpot assay. The PBMCs were stimulated for the investigated drugs at the previously published drug maximum concentration (Cmax), and concentrations 10- and 100-fold above.Results: In a human immunodeficiency virus (HIV) negative and a positive rifampicin-associated DRESS with positive ex vivo IFN-γ ELISpot assay, use of 10- and 100-fold Cmax drug concentrations decreased spot forming units/million cells by 32–100%, and this corresponded to cell cytotoxicity of more than 40 and 20% using an LDH assay and 7-AAD cell viability staining, respectively. The other antimicrobials (ceftriaxone, flucloxacillin, piperacillin/tazobactam, and isoniazid) tested in healthy controls showed similar dose-dependent increased cytotoxicity using the LDH assay, but cytotoxicity remained lower than 40% for all Cmax and 10-fold Cmax drug concentrations except flucloxacillin. All 100-fold Cmax concentrations resulted in cell death >40% (median 57%), except for isoniazid. 7-AAD cell viability staining also confirmed an increase in lymphocyte death in PBMCs incubated with 10-fold and 100-fold above Cmax for ceftriaxone, and flucloxacillin; however, piperacillin/tazobactam and isoniazid indicated no differences in percentages of viable lymphocytes across concentrations tested.Conclusion: The LDH cytotoxicity and 7-AAD cell viability staining techniques both demonstrate increased cell death corresponding to a loss in ELISpot sensitivity, with use of higher antimicrobial drug concentrations for ex vivo diagnostic IFN-γ ELISpot assays. For all the antimicrobials evaluated, the use of Cmax and 10-fold Cmax concentrations impacts cell viability and potentially affects ELISpot performance. These findings inform future approaches for ex vivo diagnostics such as IFN-γ release ELISpot.

scholarly journals Dose Dependent Antimicrobial Cellular Cytotoxicity – Implications for ex vivo diagnostics

2021 ◽  
Vol 147 (2) ◽  
pp. AB246
Author(s):  
Ana Copaescu ◽  
Phuti Choshi ◽  
Sarah Pedretti ◽  
Effie Mouhtouris ◽  
Jonny Peter ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Cellular Cytotoxicity ◽  
Dose Dependent

scholarly journals Evaluation of the Modified ELISPOT Assay for Gamma Interferon Production in Cancer Patients Receiving Antitumor Vaccines

2000 ◽  
Vol 7 (2) ◽  
pp. 145-154 ◽  
Author(s):  
Tadao Asai ◽  
Walter J. Storkus ◽  
Theresa L. Whiteside
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocyte ◽  
Elispot Assay ◽  
Ex Vivo ◽  
Peptide Vaccine ◽  
Precursor Cells ◽  
Gamma Interferon ◽  
Patients With Cancer ◽  
Ifn Γ

ABSTRACT Frequencies of vaccine-responsive T-lymphocyte precursors in peripheral blood mononuclear cells (PBMC) prior to and after administration of peptide-based vaccines in patients with cancer can be measured by limiting-dilution assays (LDA) or by ELISPOT assays. We have used a modified version of the ELISPOT assay to monitor changes in the frequency of gamma interferon (IFN-γ)-producing T cells in a population of lymphocytes responding to a relevant peptide or a nonspecific stimulator, such as phorbol myristate acetate-ionomycin. Prior to its use for monitoring of patient samples, the assay was validated and found to be comparable to the LDA performed in parallel, using tumor-reactive cytolytic T-lymphocyte (CTL) lines. The sensitivity of the ELISPOT assay was found to be 1/100,000 cells, with an interassay coefficient of variation of 15%, indicating that it could be reliably used for monitoring of changes in the frequency of IFN-γ-secreting responder cells in noncultured or cultured lymphocyte populations. To establish that the assay is able to detect the T-cell precursor cells responsive to the vaccine, we used CD8+T-cell populations positively selected from PBMC of HLA-A2+patients with metastatic melanoma, who were treated with dendritic cell-based vaccines containing gp100, MELAN-A/MART-1, tyrosinase, and influenza virus matrix peptides. The frequency of peptide-specific responder T cells ranged from 0 to 1/2,600 before vaccination and increased by at least 1 log unit after vaccination in two patients, one of whom had a clinical response to the vaccine. However, no increases in the frequency of peptide-responsive T cells were observed in noncultured PBMC or PBMC cultured in the presence of the relevant peptides after the melanoma patients enrolled in another trial were treated with the intramuscular peptide vaccine plus MF59 adjuvant. Thus, while the ELISPOT assay was found to be readily applicable to assessments of frequencies of CTL precursors of established CTL lines and ex vivo-amplified PBMC, its usefulness for monitoring of fresh PBMC in patients with cancer was limited. In many of these patients antitumor effector T cells are present at frequencies of lower than 1/100,000 in the peripheral circulation. Serial monitoring of such patients may require prior ex vivo amplification of specific precursor cells.

scholarly journals Early Gamma Interferon and Interleukin-2 Responses to Vaccination Predict the Late Resting Memory in Malaria-Naïve and Malaria-Exposed Individuals

2006 ◽  
Vol 74 (11) ◽  
pp. 6331-6338 ◽  
Author(s):  
Philip Bejon ◽  
Sheila Keating ◽  
Jedidah Mwacharo ◽  
Oscar K. Kai ◽  
Susanna Dunachie ◽  
...  
Keyword(s):  
Elispot Assay ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Gamma Interferon ◽  
Long Term Memory ◽  
Effector Cells ◽  
Ifn Γ ◽  
Relationship Of ◽  
The Relationship

ABSTRACT Two different cell populations respond to potent T-cell-inducing vaccinations. The induction and loss of effector cells can be seen using an ex vivo enzyme-linked immunospot (ELISPOT) assay, but the more durable resting memory response is demonstrable by a cultured ELISPOT assay. The relationship of the early effector response to durable resting memory is incompletely understood. Effector phenotype is usually identified by gamma interferon (IFN-γ) production, but interleukin-2 (IL-2) has been specifically linked to the differentiation of memory cells. Here, IFN-γ- and IL-2-secreting effector cells were identified by an ex vivo ELISPOT assay 1 week after vaccination and compared with the resting memory responses detected by a cultured ELISPOT assay 3 months later. The different kinetics and induction of IL-2 by different vaccines and natural exposure are described. Furthermore, both early IFN-γ and IL-2 production independently predicted subsequent memory responses at 3 months in malaria-naïve volunteers, but only IFN-γ predicted memory in malaria-exposed volunteers. However, dual ELISPOT assays were also performed on malaria-exposed volunteers to identify cells producing both cytokines simultaneously. This demonstrated that double-cytokine-producing cells were highly predictive of memory. This assay may be useful in predicting vaccinations most likely to generate stable, long-term memory responses.

scholarly journals Frequencies of Ex Vivo-Activated Human Immunodeficiency Virus Type 1-Specific Gamma-Interferon-Producing CD8+ T Cells in Infected Children Correlate Positively with Plasma Viral Load

2002 ◽  
Vol 76 (24) ◽  
pp. 12414-12422 ◽  
Author(s):  
Florence Buseyne ◽  
Daniel Scott-Algara ◽  
Françoise Porrot ◽  
Béatrice Corre ◽  
Nassima Bellal ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Viral Load ◽  
Elispot Assay ◽  
Ex Vivo ◽  
Cell Subset ◽  
Gamma Interferon ◽  
Functional Assay ◽  
Immunodeficiency Virus ◽  
Ifn Γ

ABSTRACT HIV-specific CD8+ T cells are critical in controlling human immunodeficiency virus (HIV) replication. We present the evaluation of a gamma-interferon (IFN-γ)-based enzyme linked immunospot (ELISPOT) assay for the quantification of HIV-specific CD8+ T cells from HIV-infected children. We studied 20 HLA-A∗0201-positive HIV-infected children. The IFN-γ production in response to stimulation with two HLA-A∗0201-restricted immunodominant CD8 epitopes (SLYNTVATL [SL9] in Gag and ILKEPVHGV [IV9] in Pol) was tested using the ELISPOT assay. The results were compared to labeling with the corresponding tetramers. Among the 20 children, 18 had detectable responses against the SL9 and/or the IV9 epitope using the ELISPOT assay (medians, 351 and 134 spot-forming cells/106 peripheral blood mononuclear cells, respectively). Comparison of results from the tetramer and ELISPOT assays suggests that only a fraction of HIV-specific CD8+ T cells were able to produce IFN-γ. Most importantly, we found that the frequencies of IFN-γ-producing CD8+ T cells were positively correlated with the viral load whereas the frequencies of tetramer-binding CD8+ T cells were not. The high sensitivity of the ELISPOT assay and the fact that this functional assay provided information different from that of tetramer labeling support its use for measurement of HIV-specific CD8+ T cells. In conclusion, our results show that the ex vivo-activated IFN-γ-producing HIV-specific CD8+-T-cell subset is dependent upon continuous antigenic stimulation.

The effect of tobacco smoke exposure on the generation of reactive oxygen species and cellular membrane damage using co-culture model of blood brain barrier with astrocytes

2017 ◽  
Vol 33 (6) ◽  
pp. 530-536 ◽  
Author(s):  
Seung-Beom Seo ◽  
Eun Sang Choe ◽  
Kwang-Sik Kim ◽  
Soon-Mi Shim
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Viability ◽  
Tobacco Smoke ◽  
Membrane Damage ◽  
Cellular Membrane ◽  
Dependent Manner ◽  
Oxygen Species ◽  
Cell Cytotoxicity ◽  
Culture Model ◽  
Dose Dependent

Brain tissue is known to be vulnerable to the exposure by tobacco smoke. Tobacco smoke can induce generation of reactive oxygen species (ROS), causing inflammatory activity and blood–brain barrier (BBB) impairment. The aim of the present study was to investigate the effect of tobacco smoke on cell cytotoxicity, generation of ROS, and cellular membrane damage in astrocytes and BBB using a co-culture system. Cell viability of U373MG cells was reduced in a dose-dependent manner, ranging from 96.7% to 40.3% by tobacco smoke condensate (TSC). Cell viability of U373MG co-cultured with human brain microvascular endothelial cells (HBMECs) was 104.9% at the IC50 value of TSC. Trans-epithelial electric resistance values drastically decreased 80% following 12-h incubation. The value was maintained until 48 h and then increased at 72-h incubation (85%). It then decreased to 75% at 120 h. Generation of ROS increased in a dose-dependent manner, ranging from 102.7% to 107.9%, when various concentrations of TSC (4–16 mg/mL) were administered to the U373MG monoculture. When TSC was added into U373MG co-cultured with HBMECs, production of ROS ranged from 101.7% to 102.6%, slightly increasing over 12 h. Maximum exposure-generated ROS of 104.8% was reached at 24 h. Cell cytotoxicity and oxidative stress levels in the U373MG co-culture model system with HBMECs were lower than U373MG monoculture. HBMECs effectively acted as a barrier to protect the astrocytes (U373MG) from toxicity of TSC.

scholarly journals Effect of Local Anesthetics on Human Mesenchymal Stromal Cell Secretion

Nano LIFE ◽  
2015 ◽  
Vol 05 (02) ◽  
pp. 1550001 ◽  
Author(s):  
Andrea Gray ◽  
Ileana Marrero-Berrios ◽  
Mehdi Ghodbane ◽  
Timothy Maguire ◽  
Jonathan Weinberg ◽  
...  
Keyword(s):  
Cell Viability ◽  
Pathway Analysis ◽  
Local Anesthetics ◽  
Mesenchymal Stromal Cell ◽  
Stromal Cell ◽  
Secretory Function ◽  
Variable Effect ◽  
Tnf Α ◽  
Ifn Γ ◽  
Dose Dependent

Anti-fibrotic and tissue regenerative mesenchymal stromal cell (MSC) properties are largely mediated by secreted cytokines and growth factors. MSCs are implanted to augment joint cartilage replacement and to treat diabetic ulcers and burn injuries simultaneously with local anesthetics, which reduce pain. However, the effect of anesthetics on therapeutic human MSC secretory function has not been evaluated. In order to assess the effect of local anesthetics on the MSC secretome, a panel of four anesthetics with different potencies — lidocaine, procaine, ropivacaine and bupivacaine — was evaluated. Since injured tissues secrete inflammatory cytokines, the effects of anesthetics on MSCs stimulated with tumor necrosis factor (TNF)-α and interferon (IFN)-γ were also measured. Dose dependent and anesthesia specific effects on cell viability, post exposure proliferation and secretory function were quantified using alamar blue reduction and immunoassays, respectively. Computational pathway analysis was performed to identify upstream regulators and molecular pathways likely associated with the effects of these chemicals on the MSC secretome. Our results indicated while neither lidocaine nor procaine greatly reduced unstimulated cell viability, ropivacaine and bupivacaine induced dose dependent viability decreases. This pattern was exaggerated in the simulated inflammatory environment. The reversibility of these effects after withdrawal of the anesthetics was attenuated for TNF-α/IFN-γ-stimulated MSCs exposed to ropivacaine and bupivacaine. In addition, secretome analysis indicated that constitutive secretion changes were clearly affected by both anesthetic alone and anesthetic plus TNFα/IFNγ cell stimulation, but the secretory pattern was drug specific and did not necessarily coincide with viability changes. Pathway analysis identified different intracellular regulators for stimulated and unstimulated MSCs. Within these groups, ropivacaine and bupivacaine appeared to act on MSCs similarly via the same regulatory mechanisms. Given the variable effect of local anesthetics on MSC viability and function, these studies underscore the need to evaluate MSC in the presence of medications, such as anesthetics, that are likely to accompany cell implantation.

scholarly journals Corrigendum: Dose Dependent Antimicrobial Cellular Cytotoxicity—Implications for ex vivo Diagnostics

2021 ◽  
Vol 12 ◽  
Author(s):  
Ana Copaescu ◽  
Phuti Choshi ◽  
Sarah Pedretti ◽  
Effie Mouhtouris ◽  
Jonathan Peter ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Cellular Cytotoxicity ◽  
Dose Dependent

Adenosine Diphosphate (ADP)-Induced ⍺-Granules Release from Platelets of Native Whole Blood Is Reduced by Ticlopidine but Not by Aspirin or Dipyridamole

1986 ◽  
Vol 56 (02) ◽  
pp. 147-150 ◽  
Author(s):  
V Pengo ◽  
M Boschello ◽  
A Marzari ◽  
M Baca ◽  
L Schivazappa ◽  
...  
Keyword(s):  
Whole Blood ◽  
Light Transmission ◽  
Ex Vivo ◽  
Early Stage ◽  
Shape Change ◽  
Adenosine Diphosphate ◽  
Dose Dependent ◽  
Plateletderived Growth Factor ◽  
Platelet Shape

SummaryA brief contact between native whole blood and ADP promotes a dose-dependent release of platelet a-granules without a fall in the platelet number. We assessed the “ex vivo” effect of three widely used antiplatelet drugs, aspirin dipyridamole and ticlopidine, on this system. Aspirin (a single 800 mg dose) and dipyridamole (300 mg/die for four days) had no effect, while ticlopidine (500 mg/die for four days) significantly reduced the a-granules release for an ADP stimulation of 0.4 (p <0.02), 1.2 (p <0.01) and 2 pM (p <0.01). No drug, however, completeley inhibits this early stage of platelet activation. The platelet release of α-granules may be related to platelet shape change of the light transmission aggregometer and may be important “in vivo” by enhancing platelet adhesiveness and by liberating the plateletderived growth factor.

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