scholarly journals SARS-CoV-2 Initiates Programmed Cell Death in Platelets

2021 ◽  
Author(s):  
Milka Koupenova ◽  
Heather A Corkrey ◽  
Olga Vitseva ◽  
Kahraman Tanriverdi ◽  
Mohan Somasundaran ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Caspase 3 ◽  
Infectious Virus ◽  
Morphological Changes ◽  
Enrichment Analysis ◽  
Extracellular Vesicle ◽  
Viral Rna ◽  
Transmission Electron

Rationale: COVID-19 is characterized by increased incidence of microthrombosis with hyperactive platelets sporadically containing viral RNA. It is unclear if SARS-CoV-2 directly alters platelet activation or if these changes are a reaction to infection-mediated global inflammatory alterations. Importantly, the direct effect of SARS-CoV-2 on platelets has yet to be studied. Objective: To characterize the direct SARS-CoV-2-platelet interactions using in vitro studies with purified infectious virions and samples from infected patients. Methods and Results: Platelet RNA analyzed by ARTIC v3 sequencing for SARS-CoV-2 showed presence of fragmented viral genome in all COVID-19 patients. Immunofluorescent imaging of platelets from COVID-19 patients confirmed presence of SARS-CoV-2 proteins, while there was no detection of viral RNA by RT-qPCR. Transmission electron microscopy (TEM) of platelets incubated with purified SARS-CoV-2 virions demonstrated rapid internalization and digestion leading to distinct morphological changes, and resulted in a release of extracellular vesicles. Interactions between SARS-CoV-2 and platelets occurred with or without ACE2 presence as measured by immunofluorescence. TEM showed that SARS-CoV-2 virions became internalized when they were attached to microparticles, bypassing the need for ACE2. Enrichment analysis of platelet-transcriptome from patients with acute COVID-19, compared to those with clinical thrombosis, suggested upregulation of pathways related to virally mediated cell death, specifically necroptosis and apoptosis. Platelets incubated with infectious virus appeared to undergo cell death in 30 min post-incubation as assessed by TEM and platelets from COVID-19 patients showed evidence of increased markers of apoptosis and necroptosis by WB. Immunofluorescence confirmed colocalization of SARS-CoV-2 with phospho-MLKL and Caspase-3 on non-permeabilized platelets in vitro and in COVID-19 platelets. Conclusions: Platelets internalize SARS-CoV-2 virions, directly or attached to microparticles, and viral internalization leads to rapid digestion, programmed cell death and extracellular vesicle release. During COVID-19, platelets mediate a rapid response to SARS-CoV-2 and this response can contribute to dysregulated immunity and thrombosis.

Dihydroartemisinin induces ER stress-dependent apoptosis of Echinococcus protoscoleces in vitro

2020 ◽  
Vol 52 (10) ◽  
pp. 1140-1147
Author(s):  
Rongji Ma ◽  
Wenjuan Qin ◽  
Yuanmao Xie ◽  
Ziwei Han ◽  
Shuojie Li ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Er Stress ◽  
Caspase 3 ◽  
Morphological Changes ◽  
Apoptotic Pathway ◽  
Homologous Protein ◽  
Caspase 12 ◽  
Transmission Electron ◽  
Stress Dependent

Abstract In this study, we investigated the effect of dihydroartemisinin on Echinococcus protoscoleces and explored the role of endoplasmic reticulum stress in this process. Echinococcus protoscoleces were collected and cultured in RPMI 1640 medium. Changes in the expressions of glucose-regulated protein 78 (GRP-78), caspase-12, and C/EBP homologous protein (CHOP) were assessed through confocal immunofluorescence and western blot analysis. Cell viability and morphological changes were observed under a light microscope. The ultrastructure of protoscoleces was observed by scanning electron microscopy and transmission electron microscopy. Caspase-3 activity was detected using an enzyme assay kit. After dihydroartemisinin treatment, the protoscoleces showed loss of viability, and morphological changes including soma contraction, blebs formation, hooks loss, microtrichia destruction, and development of lipid droplets was observed. The levels of caspase-12 and CHOP were increased within 2 days of dihydroartemisinin treatment. However, the levels of GRP-78, caspase-12, and CHOP were decreased in 4 days. Furthermore, caspase-3 activity was increased after treatment with different concentrations of dihydroartemisinin. Dihydroartemisinin can induce apoptosis in protoscoleces via the ER stress-caspase-3 apoptotic pathway in vitro. These results indicate that dihydroartemisinin is a potentially valuable therapeutic agent against echinococcosis.

scholarly journals The Sensitizer 2,4-Dinitrofluorobenzene Activates Caspase-3 and Induces Cell Death in a Skin Dendritic Cell Line

2003 ◽  
Vol 22 (1) ◽  
pp. 43-48 ◽  
Author(s):  
Maria Teresa Cruz ◽  
Carlos B. Duarte ◽  
Margarida Gonçalo ◽  
Américo Figueiredo ◽  
Arsélio P. Carvalho ◽  
...  
Keyword(s):  
Cell Death ◽  
Dendritic Cell ◽  
Cell Line ◽  
Caspase 3 ◽  
Morphological Changes ◽  
Mouse Skin ◽  
Annexin V ◽  
Tetrazolium Salt ◽  
Mtt Reduction

In this work, a dendritic cell line derived from mouse skin (FSDC) was used, as an in vitro experimental model, to evaluate the cytotoxic effect of two chemical sensitizers, a strong sensitizer (2,4-dinitrofluorobenzene, DNFB) and a weak sensitizer (2,4-dichloronitrobenzene, DCNB). The results indicated that DNFB reduces the cellular metabolism of FSDC, as evaluated by the reduction of the tetrazolium salt, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). All the DNFB concentrations tested, ranging from 5.2 μ M to 26 μM, significantly inhibited the MTT reduction after 1 hour of cell exposure to the sensitizer. In contrast, incubation of FSDC with the weak sensitizer DCNB had no significant effect on the MTT reduction assay. When the cells were incubated with DNFB (13 μ M), for 3 and 6 hours, morphological changes characteristics of cell death by apoptosis were observed, as assessed by propidium iodide (PI) DNA staining and annexin-V externalization analysis. These results correlate well with an increase of caspase-3-like activity after FSDC exposure to DNFB (13 μM) for 6 hours. Together, these results indicate that apoptotic death of skin dendritic cells occurs after exposure to the sensitizer DNFB, although necrotic cell death was also observed when the cells were incubated with high concentrations of DNFB (26 μM), or after long periods of cell exposure to the chemical DNFB (13 μM, for 6 hours).

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.

The P-MAPA immunomodulator partially prevents apoptosis induced by Zika virus infection in THP-1 cells

2020 ◽  
Vol 21 ◽  
Author(s):  
Morganna C. Lima ◽  
Elisa A. N. Azevedo ◽  
Clarice N. L. de Morais ◽  
Larissa I. O. de Sousa ◽  
Bruno M. Carvalho ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Virus Infection ◽  
Virus Replication ◽  
Zika Virus ◽  
Mammalian Cells ◽  
Caspase 3 ◽  
Neurological Complications ◽  
Zika Virus Infection

Background: Zika virus is an emerging arbovirus of global importance. ZIKV infection is associated with a range of neurological complications such as the Congenital Zika Syndrome and Guillain Barré Syndrome. Despite the magnitude of recent outbreaks, there is no specific therapy to prevent or to alleviate disease pathology. Objective: To investigate the role of P-MAPA immunomodulator in Zika-infected THP-1 cells. Methods: THP-1 cells were subjected at Zika virus infection (Multiplicity of Infection = 0.5) followed by treatment with P-MAPA for until 96 hours post-infection. After that, the cell death was analyzed by annexin+/ PI+ and caspase 3/ 7+ staining by flow cytometry. In addition, the virus replication and cell proliferation were accessed by RT-qPCR and Ki67 staining, respectively. Results: We demonstrate that P-MAPA in vitro treatment significantly reduces Zika virus-induced cell death and caspase-3/7 activation on THP-1 infected cells, albeit it has no role in virus replication and cell proliferation. Conclusions: Our study reveals that P-MAPA seems to be a satisfactory alternative to inhibits the effects of Zika virus infection in mammalian cells.

scholarly journals Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

2021 ◽  
Vol 22 (15) ◽  
pp. 7906
Author(s):  
Alexey A. Komissarov ◽  
Maria A. Karaseva ◽  
Marina P. Roschina ◽  
Andrey V. Shubin ◽  
Nataliya A. Lunina ◽  
...  
Keyword(s):  
Cell Death ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Proteolytic Enzymes ◽  
Morphological Changes ◽  
Human Cells ◽  
Mitochondrial Potential ◽  
3C Protease ◽  
Wide Range

Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD, including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we expressed 3Cpro in HEK293, HeLa, and A549 human cell lines to characterize 3Cpro-induced cell death morphologically and biochemically using flow cytometry and fluorescence microscopy. We found that dead cells demonstrated necrosis-like morphological changes including permeabilization of the plasma membrane, loss of mitochondrial potential, as well as mitochondria and nuclei swelling. Additionally, we showed that 3Cpro-induced cell death was efficiently blocked by ferroptosis inhibitors and was accompanied by intense lipid peroxidation. Taken together, these results indicate that 3Cpro induces ferroptosis upon its individual expression in human cells. This is the first demonstration that a proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.

scholarly journals TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection

2021 ◽  
Author(s):  
Hongli Zhou ◽  
Minyu Zhou ◽  
Yue Hu ◽  
Yanin Limpanon ◽  
Yubin Ma ◽  
...  
Keyword(s):  
Cell Death ◽  
Enrichment Analysis ◽  
Angiostrongylus Cantonensis ◽  
Gene Set Enrichment Analysis ◽  
Caspase 8 ◽  
Cns Injury ◽  
Vitro Assay ◽  
Tnf Α

AbstractAngiostrongylus cantonensis (AC) can cause severe eosinophilic meningitis or encephalitis in non-permissive hosts accompanied by apoptosis and necroptosis of brain cells. However, the explicit underlying molecular basis of apoptosis and necroptosis upon AC infection has not yet been elucidated. To determine the specific pathways of apoptosis and necroptosis upon AC infection, gene set enrichment analysis (GSEA) and protein–protein interaction (PPI) analysis for gene expression microarray (accession number: GSE159486) of mouse brain infected by AC revealed that TNF-α likely played a central role in the apoptosis and necroptosis in the context of AC infection, which was further confirmed via an in vivo rescue assay after treating with TNF-α inhibitor. The signalling axes involved in apoptosis and necroptosis were investigated via immunoprecipitation and immunoblotting. Immunofluorescence was used to identify the specific cells that underwent apoptosis or necroptosis. The results showed that TNF-α induced apoptosis of astrocytes through the RIP1/FADD/Caspase-8 axis and induced necroptosis of neurons by the RIP3/MLKL signalling pathway. In addition, in vitro assay revealed that TNF-α secretion by microglia increased upon LSA stimulation and caused necroptosis of neurons. The present study provided the first evidence that TNF-α was secreted by microglia stimulated by AC infection, which caused cell death via parallel pathways of astrocyte apoptosis (mediated by the RIP1/FADD/caspase-8 axis) and neuron necroptosis (driven by the RIP3/MLKL complex). Our research comprehensively elucidated the mechanism of cell death after AC infection and provided new insight into targeting TNF-α signalling as a therapeutic strategy for CNS injury.

scholarly journals Cell Death and Metabolic Stress in Gymnodinium catenatum Induced by Allelopathy

Toxins ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 506
Author(s):  
Leyberth José Fernández-Herrera ◽  
Christine Johanna Band-Schmidt ◽  
Tania Zenteno-Savín ◽  
Ignacio Leyva-Valencia ◽  
Claudia Judith Hernández-Guerrero ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Protein Content ◽  
Caspase 3 ◽  
Cellular Stress ◽  
Allelopathic Effect ◽  
Sod Activity ◽  
Free Media ◽  
O2 Production ◽  
Gymnodinium Catenatum

Allelopathy between phytoplankton species can promote cellular stress and programmed cell death (PCD). The raphidophyte Chattonella marina var. marina, and the dinoflagellates Margalefidinium polykrikoides and Gymnodinium impudicum have allelopathic effects on Gymnodinium catenatum; however, the physiological mechanisms are unknown. We evaluated whether the allelopathic effect promotes cellular stress and activates PCD in G. catenatum. Cultures of G. catenatum were exposed to cell-free media of C. marina var. marina, M. polykrikoides and G. impudicum. The mortality, superoxide radical (O2●−) production, thiobarbituric acid reactive substances (TBARS) levels, superoxide dismutase (SOD) activity, protein content, and caspase-3 activity were quantified. Mortality (between 57 and 79%) was registered in G. catenatum after exposure to cell-free media of the three species. The maximal O2●− production occurred with C. marina var. marina cell-free media. The highest TBARS levels and SOD activity in G. catenatum were recorded with cell-free media from G. impudicum. The highest protein content was recorded with cell-free media from M. polykrikoides. All cell-free media caused an increase in the activity of caspase-3. These results indicate that the allelopathic effect in G. catenatum promotes cell stress and caspase-3 activation, as a signal for the induction of programmed cell death.

scholarly journals In vitro antioxidant treatment recovers proliferative responses of anergic CD4+ lymphocytes from human immunodeficiency virus-infected individuals

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4746-4753 ◽  
Author(s):  
A Cayota ◽  
F Vuillier ◽  
G Gonzalez ◽  
G Dighiero
Keyword(s):  
Human Immunodeficiency Virus ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Redox Status ◽  
Therapeutic Strategies ◽  
Antioxidant Treatment ◽  
Immunodeficiency Virus ◽  
Cd4 Lymphocytes ◽  
Cellular Thiol

Oxidative stress has been proposed to be involved in the immunologic defeat observed in effector calls of the immune system as well as in lymphocyte cell death and viral replication in human immunodeficiency virus (HIV)-infected patients. Because thiol-containing antioxidants such as N-acetyl-L-cysteine have been shown to have beneficial effects on CD4+ lymphocyte survival and to inhibit programmed cell death and HIV-1 replication, they may play a role in therapeutic strategies of this disease. In this work we have studied the cellular thiol levels and the affect of in vitro antioxidant treatment of purified CD4+ lymphocytes from HIV-infected patients, and correlated these parameters to proliferative responses and programmed cell death. We show that CD4+ lymphocytes from HIV-infected patients display impaired proliferative responses and a significant decrease in cellular thiol levels, indicating a disturbed redox status. Interestingly, antioxidant treatment succeeded to restore defective proliferative responses to CD3- mediated activation in 8 of 11 patients (high antioxidant responders). In contrast to high responders, patients failing to respond to antioxidant treatment (low antioxidant responders), were characterized by an abnormal ratio of apoptotic cells, which was not affected by N- acetyl-L-cysteine and/or 2-beta-mercaptoethanol preincubation. These results demonstrate for the first time that antioxidant treatment is able to revert the impaired proliferative activity of CD4 cells from HIV-infected patients and could help designing therapeutic strategies with antioxidant drugs. However, this action is not observed in cells undergoing programmed cell death.

Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase-3-independent pathway

2004 ◽  
Vol 287 (4) ◽  
pp. H1730-H1739 ◽  
Author(s):  
Ron Zohar ◽  
Baoqian Zhu ◽  
Peter Liu ◽  
Jaro Sodek ◽  
C. A. McCulloch
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Cardiac Fibroblasts ◽  
Chromatin Condensation ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tunel Staining ◽  
Wild Type ◽  
Nuclear Fragmentation ◽  
A Cell

Reperfusion-induced oxidative injury to the myocardium promotes activation and proliferation of cardiac fibroblasts and repair by scar formation. Osteopontin (OPN) is a proinflammatory cytokine that is upregulated after reperfusion. To determine whether OPN enhances fibroblast survival after exposure to oxidants, cardiac fibroblasts from wild-type (WT) or OPN-null (OPN−/−) mice were treated in vitro with H2O2to model reperfusion injury. Within 1 h, membrane permeability to propidium iodide (PI) was increased from 5 to 60% in OPN−/−cells but was increased to only 20% in WT cells. In contrast, after 1–8 h of treatment with H2O2, the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained cells was more than twofold higher in WT than OPN−/−cells. Electron microscopy of WT cells treated with H2O2showed chromatin condensation, nuclear fragmentation, and cytoplasmic and nuclear shrinkage, which are consistent with apoptosis. In contrast, H2O2-treated OPN−/−cardiac fibroblasts exhibited cell and nuclear swelling and membrane disruption that are indicative of cell necrosis. Treatment of OPN−/−and WT cells with a cell-permeable caspase-3 inhibitor reduced the percentage of TUNEL staining by more than fourfold in WT cells but decreased staining in OPN−/−cells by ∼30%. Although the percentage of PI-permeable WT cells was reduced threefold, the percent of PI-permeable OPN−/−cells was not altered. Restoration of OPN expression in OPN−/−fibroblasts reduced the percentage of PI-permeable cells but not TUNEL staining after H2O2treatment. Thus H2O2-induced cell death in OPN-deficient cardiac fibroblasts is mediated by a caspase-3-independent, necrotic pathway. We suggest that the increased expression of OPN in the myocardium after reperfusion may promote fibrosis by protecting cardiac fibroblasts from cell death.

scholarly journals Caspase-mediated Cleavage of p130cas in Etoposide-induced Apoptotic Rat-1 Cells

2000 ◽  
Vol 11 (3) ◽  
pp. 929-939 ◽  
Author(s):  
Seunghyi Kook ◽  
Sang Ryeol Shim ◽  
Soo Jeon Choi ◽  
Joohong Ahnn ◽  
Jae Il Kim ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Focal Adhesion ◽  
Caspase 3 ◽  
Morphological Changes ◽  
Point Mutations ◽  
Membrane Blebbing ◽  
Adhesion Sites ◽  
Focal Adhesion Proteins ◽  
Induced Apoptosis

Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell–cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD416G and DSPD748G) in vitro, and point mutations substituting the Asp of DVPD416G and DSPD748G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD416G generated a 74-kDa fragment, which was in turn cleaved at DSPD748G, yielding 47- and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas–paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix.

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