scholarly journals Investigating the Suitability of High Content Image Analysis as a Tool to Assess the Reversibility of Foamy Alveolar Macrophage Phenotypes In Vitro

Pharmaceutics ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 262 ◽  
Author(s):  
Ewelina Hoffman ◽  
Darragh Murnane ◽  
Victoria Hutter
Keyword(s):  
Image Analysis ◽  
Alveolar Macrophage ◽  
Morphological Changes ◽  
Phospholipid Content ◽  
Macrophage Phenotype ◽  
Drug Induced ◽  
Apoptosis Pathway ◽  
Foamy Macrophage ◽  
Macrophage Phenotypes

Many potential inhaled medicines fail during development due to the induction of a highly vacuolated or “foamy” alveolar macrophage phenotype response in pre-clinical studies. There is limited understanding if this response to an inhaled stimulus is adverse or adaptive, and additionally if it is a transient or irreversible process. The aim of this study was to evaluate whether high content image analysis could distinguish between different drug-induced foamy macrophage phenotypes and to determine the extent of the reversibility of the foamy phenotypes by assessing morphological changes over time. Alveolar-like macrophages derived from the human monocyte cell line U937 were exposed for 24 h to compounds known to induce a foamy macrophage phenotype (amiodarone, staurosporine) and control compounds that are not known to cause a foamy macrophage phenotype in vitro (fluticasone and salbutamol). Following drug stimulation, the cells were rested in drug-free media for the subsequent 24 or 48 h. Cell morphometric parameters (cellular and nuclear area, vacuoles numbers and size) and phospholipid content were determined using high content image analysis. The foamy macrophage recovery was dependent on the mechanism of action of the inducer compound. Amiodarone toxicity was associated with phospholipid accumulation and morphometric changes were reversed when the stimulus was removed from culture environment. Conversely cells were unable to recover from exposure to staurosporine which initiates the apoptosis pathway. This study shows that high content analysis can discriminate between different phenotypes of foamy macrophages and may contribute to better decision making in the process of new drug development.

scholarly journals Electrochemotherapy with Calcium Chloride and 17β-Estradiol Modulated Viability and Apoptosis Pathway in Human Ovarian Cancer

Pharmaceutics ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 19
Author(s):  
Zofia Łapińska ◽  
Michał Dębiński ◽  
Anna Szewczyk ◽  
Anna Choromańska ◽  
Julita Kulbacka ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Calcium Chloride ◽  
Morphological Changes ◽  
Cell Membrane Permeability ◽  
Immunocytochemical Staining ◽  
Ovarian Cancer Cells ◽  
Apoptosis Pathway ◽  
17Β Estradiol

Estrogens (Es) play a significant role in the carcinogenesis and progression of ovarian malignancies. Depending on the concentration, Es may have a protective or toxic effect on cells. Moreover, they can directly or indirectly affect the activity of membrane ion channels. In the presented study, we investigated in vitro the effectiveness of the ovarian cancer cells (MDAH-2774) pre-incubation with 17β-estradiol (E2; 10 µM) in the conventional chemotherapy (CT) and electrochemotherapy (ECT) with cisplatin or calcium chloride. We used three different protocols of electroporation including microseconds (µsEP) and nanoseconds (nsEP) range. The cytotoxic effect of the applied treatment was examined by the MTT assay. We used fluorescent staining and holotomographic imaging to observe morphological changes. The immunocytochemical staining evaluated the expression of the caspase-12. The electroporation process’s effectiveness was analyzed by a flow cytometer using the Yo-Pro™-1 dye absorption assay. We found that pre-incubation of ovarian cancer cells with 17β-estradiol may effectively enhance the chemo- and electrochemotherapy with cisplatin and calcium chloride. At the same time, estradiol reduced the effectiveness of electroporation, which may indicate that the mechanism of increasing the effectiveness of ECT by E2 is not related to the change of cell membrane permeability.

scholarly journals The anti-ovarian carcinoma activity of L-amino Acid Oxidase from Crotalus adamanteus venom in vivo and in vitro

2021 ◽  
Author(s):  
Li Bo ◽  
Yan Xiong ◽  
Qiyi He ◽  
Xiaodong Yu ◽  
Bo Li ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Amino Acid ◽  
Cancer Cells ◽  
Morphological Changes ◽  
Complex Mixture ◽  
Ovarian Cancer Cells ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Apoptosis Pathway

Abstract The anti-tumor potential of animal toxins has fully attracted the attention of researchers. Snake venoms is a complex mixture of different components and has revealed high toxicity on normal and tumoral tissues or cells. The snake venom L-Amino-acid oxidase (svLAAO) has grown up to be a critical research target in molecular biology sciences and medicine sciences since widespread presence and various biological roles, including antitumor application. We found that Crotalus adamanteus (C. adamanteus) venom LAAO significantly decreased the viability of ovarian cancer cells and caused morphological changes preceded cell death. Cell experiments confirmed that C. adamanteus venom LAAO caused alterations of intrinsic or extrinsic apoptosis pathway-related genes in ovarian cancer cells. Animal experiments and histological analysis also proved that C. adamanteus venom LAAO could effectively inhibit the damage of ovarian cancer to tissues. The major apoptosis induction of C. adamanteus venom LAAO on ovarian cancer cells can be blocked by catalase, suggesting that the cytotoxicity of C. adamanteus venom LAAO on ovarian cancer cells was mainly mediated by H2O2. Our preliminary results revealed that C. adamanteus venom LAAO may induce apoptosis of ovarian cancer cells through the death receptor pathway and mitochondrial pathway. It is inferred that C. adamanteus venom LAAO will be some advantages in New Drug Research and Development of antitumor drugs in the future. Nevertheless, extra studies on the pharmacological actions and molecular mechanism of svLAAO in anti-cancer are necessary in order to better promote its application.

Characterization and quantitation of morphological changes induced in bovine endothelial cells by interleukin-2 (IL-2)

1986 ◽  
Vol 44 ◽  
pp. 322-323
Author(s):  
C. D. Bucana ◽  
J. Trial ◽  
A. C. Papp ◽  
K. K. Wu
Keyword(s):  
Endothelial Cells ◽  
Image Analysis ◽  
Arachidonic Acid ◽  
Ex Vivo ◽  
Morphological Changes ◽  
Interleukin 2 ◽  
Bovine Endothelial Cells ◽  
Cell Changes ◽  
Dose Dependent

Endothelial cells respond to endotoxins, arachidonic acid, and interferon-alpha by exhibiting a time- and dose-dependent production of prostacyclin which is accompanied by morphological changes in the endothelial cells. Recent studies showed that IL-2 also stimulates production of prostacyclin by endothelial cells. Therefore, the effect of interleukin-2 on the morphology of endothelial cells in vitro and ex vivo was investigated by LM, SEM and TEM. Quantitation of the cell changes in vitro was performed by image analysis and was correlated with time- and dosedependent production of prostacyclin.

scholarly journals High Content Image Analysis as a Tool to Morphologically Distinguish Macrophage Activation and Determine Its Importance for Foamy Alveolar Macrophage Responses

2021 ◽  
Vol 12 ◽  
Author(s):  
Ewelina Hoffman ◽  
Paulina Napieralska ◽  
Rhamiya Mahendran ◽  
Darragh Murnane ◽  
Victoria Hutter
Keyword(s):  
Image Analysis ◽  
Alveolar Macrophage ◽  
Safety Assessment ◽  
Macrophage Activation ◽  
Activated Macrophages ◽  
Analysis Techniques ◽  
Arginase 1 ◽  
Ifn Γ ◽  
Macrophage Phenotypes ◽  
Image Analysis Techniques

IntroductionLung diseases are an increasing global health burden affecting millions of people worldwide. Only a few new inhaled medicines have reached the market in the last 30 years, in part due to foamy alveolar macrophage (FAM) responses observed in pre-clinical rat studies. The induction mechanism and signaling pathways involved in the development of highly vacuolated ‘foamy’ phenotype is not known. Furthermore, it has not been determined if these observations are adaptive or adverse responses.AimTo determine if high content image analysis techniques can distinguish between alveolar macrophage activation (LPS/IFN-γ activated and IL-4 activated macrophages) and if this could be applied to understanding the generation of ‘foamy’ macrophage phenotypes.MethodsNR8383 rat alveolar macrophages were stimulated with a mix of cytokines (LPS/IFN-γ or IL-4) for 24 h. The cells were further exposed to FAM inducing-compounds amiodarone and staurosporine. Following 24 h incubation, phagocytosis and lipid accumulation were measured using flow cytometry and high content image analysis techniques. The alveolar macrophages responses after exposure to cytokines were assessed by evaluation: (i) cell surface and biochemical markers such as: nitric oxide production, arginase-1 activity and MRC-1 receptor expression (ii) cellular morphology (iii) cellular functionality (phagocytic activity and lipids accumulation).ResultsMacrophages activated with LPS/IFN-γ showed distinct morphological (increased vacuolation) features and functionality (increased lipidosis, decreased phagocytic activity). Foamy macrophage phenotypes induced by amiodarone also displayed characteristics of proinflammatory macrophages (significantly increased nitric oxide production, increased vacuolation and lipidosis and decreased phagocytosis). In contrast, staurosporine treatment resulted in increased NO production, as well as arginase-1 activity.ConclusionHigh content image analysis was able to determine distinct differences in morphology between non-activated and LPS/IFN-γ activated macrophages, characterized by increased vacuolation and lipidosis. When exposed to compounds that induce a FAM phenotype, healthy non-activated macrophages displayed proinflammatory (amiodarone) or pro-apoptotic (staurosporine) characteristics but these responses were independent of a change in activation status. This technique could be applied in early drug discovery safety assessment to identify immune responses earlier and increase the understanding of alveolar macrophage responses to new molecules challenge in development of new inhalation therapies, which in turn will enhance decision-making in an early safety assessment of novel drug candidates.

Correlation Between Cellular Functions and Morphological Changes of Human Trophoblast in Vitro

1975 ◽  
Vol 33 ◽  
pp. 600-601
Author(s):  
John C. Garancis ◽  
Robert O. Hussa ◽  
Michael T. Story ◽  
Donald Yorde ◽  
Roland A. Pattillo
Keyword(s):  
Electron Microscopy ◽  
Cellular Proliferation ◽  
Morphological Changes ◽  
Culture Fluid ◽  
Cellular Functions ◽  
Human Trophoblast ◽  
Transmission Electron ◽  
Marked Activity ◽  
Malignant Trophoblast

Human malignant trophoblast cells in continuous culture were incubated for 3 days in medium containing 1 mM N6-O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) and 1 mM theophylline. The culture fluid was replenished daily. Stimulated cultures secreted many times more chorionic gonadotropin and estrogens than did control cultures in the absence of increased cellular proliferation. Scanning electron microscopy revealed remarkable surface changes of stimulated cells. Control cells (not stimulated) were smooth or provided with varying numbers of microvilli (Fig. 1). The latter, usually, were short and thin. The surface features of stimulated cells were considerably different. There was marked increase of microvilli which appeared elongated and thick. Many cells were covered with confluent polypoid projections (Fig. 2). Transmission electron microscopy demonstrated marked activity of cytoplasmic organelles. Mitochondria were increased in number and size; some giant forms with numerous cristae were observed.

A Hexagonal (II) Phase Phospholipid Neutralization Assay for Lupus Anticoagulant Identification

1993 ◽  
Vol 70 (05) ◽  
pp. 787-793 ◽  
Author(s):  
Douglas A Triplett ◽  
Linda K Barna ◽  
Gail A Unger
Keyword(s):  
Hemophilia A ◽  
Clinical Laboratory ◽  
Neutralization Assay ◽  
Confirmatory Test ◽  
Specific Factor ◽  
Clinical Settings ◽  
Drug Induced ◽  
Variable Screening ◽  
Routine Clinical Laboratory

SummaryLupus anticoagulants (LAs) are immunoglobulins (IgG, IgM, or both) which interfere with in vitro phospholipid (PL) dependent tests of coagulation (e.g. APTT, dilute PT, dilute Russell Viper Venom Time). These antibodies may be identified in a wide variety of clinical settings. With the exception of heparinized patient samples, the presence of LAs is often the most common cause of an unexplained APTT in a routine clinical laboratory. The diagnosis of LAs is difficult due to variable screening reagent sensitivity and intrinsic heterogeneity of LAs. Recently, Rauch and colleagues have shown human monoclonal hybridoma LAs were inhibited by hexagonal (II) phase PLs. In contrast, lamellar phase PLs had no effect. We have evaluated a new assay system, Staclot LA®, which utilizes a hexagonal (II) phase PL (egg phosphatidylethanolamine [EPE]) as a confirmatory test for LAs. Plasma samples from the following patient populations were studied: LA positive, heparinized, oral anticoagulated, hemophilia A and B, and specific factor inhibitors (factors V, VIII, IX). Unlike previous studies, the LA positive patients were a mixed population including: autoimmune diseases, drug-induced, and post-infection. Our findings confirm the specificity of hexagonal (II) phase PL neutralization of LAs.

Effect of Temperature, Velocity Gradient and I.V. Heparin on in Vitro Blood Coagulation and Platelet Aggregation

1967 ◽  
Vol 17 (01/02) ◽  
pp. 112-119 ◽  
Author(s):  
L Dintenfass ◽  
M. C Rozenberg
Keyword(s):  
Blood Coagulation ◽  
Velocity Gradient ◽  
Elevated Temperatures ◽  
Morphological Changes ◽  
Effect Of Temperature ◽  
Clotting Time ◽  
Progressive Change ◽  
Aggregation Of Platelets ◽  
Microscopic Techniques

SummaryA study of blood coagulation was carried out by observing changes in the blood viscosity of blood coagulating in the cone-in-cone viscometer. The clots were investigated by microscopic techniques.Immediately after blood is obtained by venepuncture, viscosity of blood remains constant for a certain “latent” period. The duration of this period depends not only on the intrinsic properties of the blood sample, but also on temperature and rate of shear used during blood storage. An increase of temperature decreases the clotting time ; also, an increase in the rate of shear decreases the clotting time.It is confirmed that morphological changes take place in blood coagula as a function of the velocity gradient at which such coagulation takes place. There is a progressive change from the red clot to white thrombus as the rates of shear increase. Aggregation of platelets increases as the rate of shear increases.This pattern is maintained with changes of temperature, although aggregation of platelets appears to be increased at elevated temperatures.Intravenously added heparin affects the clotting time and the aggregation of platelets in in vitro coagulation.

Cytotoxic Effect of the Combination of Gemcitabine and Atorvastatin Loaded in Microemulsion on the HCT116 Colon Cancer Cells

2017 ◽  
Vol 9 (2) ◽  
Author(s):  
Mayson H. Alkhatib ◽  
Dalal Al-Saedi ◽  
Wadiah S. Backer
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Anticancer Drugs ◽  
Therapeutic Potential ◽  
Morphological Changes ◽  
In Vitro Study ◽  
Colon Cancer Cells ◽  
Apoptosis Detection ◽  
Hct116 Cells

The combination of anticancer drugs in nanoparticles has great potential as a promising strategy to maximize efficacies by eradicating resistant, reduce the dosage of the drug and minimize toxicities on the normal cells. Gemcitabine (GEM), a nucleoside analogue, and atorvastatin (ATV), a cholesterol lowering agent, have shown anticancer effect with some limitations. The objective of this in vitro study was to evaluate the antitumor activity of the combination therapy of GEM and ATVencapsulated in a microemulsion (ME) formulation in the HCT116 colon cancer cells. The cytotoxicity and efficacy of the formulation were assessed by the 3- (4,5dimethylthiazole-2-yl)-2,5-diphyneltetrazolium bromide (MTT) assay. The mechanism of cell death was examined by observing the morphological changes of treated cells under light microscope, identifying apoptosis by using the ApopNexin apoptosis detection kit, and viewing the morphological changes in the chromatin structure stained with 4′,6-diamidino-2-phenylindole (DAPI) under the inverted fluorescence microscope. It has been found that reducing the concentration of GEM loaded on ME (GEM-ME) from 5μM to 1.67μM by combining it with 3.33μM of ATV in a ME formulation (GEM/2ATV-ME) has preserved the strong cytotoxicity of GEM-ME against HCT116 cells. The current study proved that formulating GEM with ATV in ME has improved the therapeutic potential of GEM and ATV as anticancer drugs.

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