scholarly journals Comparison of Apoptosis Detection Markers Combined with Macrophage Immunostaining to Study Phagocytosis of Apoptotic Cells in Situ

2006 ◽  
Vol 1 ◽  
pp. 117727190600100 ◽  
Author(s):  
Dorien M. Schrijvers ◽  
Guido R.Y. De Meyer ◽  
Mark M. Kockx ◽  
Arnold G. Herman ◽  
Wim Martinet
Keyword(s):  
Lupus Erythematosus ◽  
Caspase 3 ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Apoptotic Cells ◽  
Immunological Responses ◽  
Apoptosis Detection ◽  
Apoptosis Markers ◽  
Suitable Marker ◽  
Parp 1

Efficient phagocytosis of cells undergoing apoptosis by macrophages is important to prevent immunological responses and development of chronic inflammatory disorders such as systemic lupus erythematosus, cystic fibrosis and atherosclerosis. To study phagocytosis of apoptotic cells (AC) by macrophages in tissue, we validated different apoptosis markers (DNA fragmentation, caspase-3 activation and cleavage of its substrate poly(ADP-ribose)polymerase-1) in combination with macrophage immunostaining. Human tonsils were used as a model because they show a high apoptosis frequency under physiological conditions as well as efficient phagocytosis of AC by macrophages. On the other hand, advanced human atherosclerotic plaques were examined since plaques show severely impaired phagocytosis of AC. Our results demonstrate that the presence of non-phagocytized terminal deoxynucleotidyl transferase end labelling (TUNEL)-positive AC represents a suitable marker of poor phagocytosis by macrophages in situ. Other markers for apoptosis, such as cleavage of caspase-3 or PARP-1, should not be used to assess phagocytosis efficiency, because activation of the caspase cascade and cleavage of their substrates can occur in AC when they have not yet been phagocytized by macrophages.

scholarly journals Presence of double-strand breaks with single-base 3' overhangs in cells undergoing apoptosis but not necrosis.

1996 ◽  
Vol 135 (5) ◽  
pp. 1369-1376 ◽  
Author(s):  
V V Didenko ◽  
P J Hornsby
Keyword(s):  
Dna Damage ◽  
Double Strand Breaks ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Apoptotic Cells ◽  
Dna Fragments ◽  
Strand Breaks ◽  
Single Base ◽  
Double Stranded Dna

Apoptotic cells in rat thymus were labeled in situ in paraffin-embedded and frozen tissue sections by ligation of double-stranded DNA fragments containing digoxigenin or Texas red. Two forms of double-stranded DNA fragments were prepared using the polymerase chain reaction: one was synthesized using Taq polymerase, which yields products with single-base 3' overhangs, and one using Pfu polymerase, which produces blunt-ended products. Both types of fragment could be ligated to apoptotic nuclei in thymus, indicating the presence in such nuclei of DNA double-strand breaks with single-base 3' overhangs as well as blunt ends. However, in nuclei with DNA damage resulting from a variety of nonapoptotic processes (necrosis, in vitro autolysis, peroxide damage, and heating) single-base 3' overhangs were either nondetectable or present at much lower concentrations than in apoptotic cells. Blunt DNA ends were present in such tissues, but at lower concentrations than in apoptotic cells. In contrast, in all of these forms of DNA damage, nuclei contained abundant 3'-hydroxyls accessible to labeling with terminal deoxynucleotidyl transferase. Thus, although single-base 3' overhangs and blunt ends are present in apoptotic nuclei, the specificity of the in situ ligation of 3'-overhang fragments to apoptotic nuclei indicates that apoptotic cells labeled in this way can readily be distinguished from cells with nonapoptotic DNA damage. These data are consistent with the involvement of an endonuclease similar to DNase I in apoptosis, which is predicted to leave short 3' overhangs as well as blunt ends in digestion of chromatin.

scholarly journals Hepatocarcinogenesis Prevention by Pirfenidone Is PPARγ Mediated and Involves Modification of Nuclear NF-kB p65/p50 Ratio

2021 ◽  
Vol 22 (21) ◽  
pp. 11360
Author(s):  
Jorge Antonio Silva-Gomez ◽  
Marina Galicia-Moreno ◽  
Ana Sandoval-Rodriguez ◽  
Hipolito Otoniel Miranda-Roblero ◽  
Silvia Lucano-Landeros ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Experimental Studies ◽  
Tissue Samples ◽  
P53 Expression ◽  
Fischer 344 ◽  
Apoptosis Markers ◽  
Anti Inflammatory ◽  
Pparγ Expression ◽  
Parp 1 ◽  
Tgf Β1

Targeted therapies for regulating processes such as inflammation, apoptosis, and fibrogenesis might modulate human HCC development. Pirfenidone (PFD) has shown anti-fibrotic and anti-inflammatory functions in both clinical and experimental studies. The aim of this study was to evaluate PPARγ expression and localization in samples of primary human tumors and assess PFD-effect in early phases of hepatocarcinogenic process. Human HCC tissue samples were obtained by surgical resection. Experimental hepatocarcinogenesis was induced in male Fischer-344 rats. TGF-β1 and α-SMA expression was evaluated as fibrosis markers. NF-kB cascade, TNFα, IL-6, and COX-2 expression and localization were evaluated as inflammation indicators. Caspase-3, p53, and PARP-1 were used as apoptosis markers, PCNA for proliferation. Finally, PPARα and PPARγ expression were evaluated to understand the effect of PFD on the activation of such pathways. PPARγ expression was predominantly localized in cytoplasm in human HCC tissue. PFD was effective to prevent histopathological damage and TGF-β1 and α-SMA overexpression in the experimental model. Anti-inflammatory effects of PFD correlate with diminished IKK and decrease in both IkB-phosphorylation/NF-kB p65 expression and p65-translocation into the nucleus. Pro-apoptotic PFD-induced effects are related with p53 expression, Caspase-3 p17 activation, and PARP-1-cleavage. In conclusion, PFD acts as a tumor suppressor by preventing fibrosis, reducing inflammation, and promoting apoptosis in MRHM.

scholarly journals Host-cell apoptosis inTaenia solium-induced brain granulomas in naturally infected pigs

Parasitology ◽  
2008 ◽  
Vol 135 (10) ◽  
pp. 1237-1242 ◽  
Author(s):  
C. S. SIKASUNGE ◽  
I. K. PHIRI ◽  
M. V. JOHANSEN ◽  
A. L. WILLINGHAM ◽  
P. S. LEIFSSON
Keyword(s):  
T Lymphocytes ◽  
Normal Cell ◽  
Taenia Solium ◽  
Cell Types ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Tunel Staining ◽  
Apoptotic Cells ◽  
Host Cell Apoptosis ◽  
Cellular Immune

SUMMARYTo assess whether apoptosis occurs in pig brain granulomas due toTaenia soliumcysticerci, brain tissues from 30 pigs naturally infected withT. soliumcysticercosis were evaluated by terminal deoxynucleotidyl transferase-end labelling (TUNEL) staining. In addition, tissues were stained with CD3 marker to identify T lymphocytes. Examination of TUNEL-stained tissues showed apoptotic cells in early lesions that contained viable cysticerci. Apoptotic cells were primarily found interspersed with normal cell types, and were mostly located in the inflammatory infiltrate. Late or advanced granulomas with disintegrated scolices did not show TUNEL-positive cells. CD3+ cells were found in both early and advanced lesions and apoptosis mainly co-localized with CD3+ T lymphocytes. This suggests that these cells are constantly undergoing apoptosis and thus die as soon as they arrive at the site of infection. Apoptosis indeed may be one way by whichT. soliumcysticerci down-regulate the host's cellular immune response in early cysticercosis. Therefore, further research is needed to establish if other cells besides T-lymphocytes are also a target for destruction by cysticerci in early cysticercosis as well as studies to assess if cysteine protease is expressed by viable cysticerciin situ.

270. TGF-β and ovarian follicle development

2008 ◽  
Vol 20 (9) ◽  
pp. 70
Author(s):  
D. A. Rosairo ◽  
J. K. Findlay ◽  
A. E. Drummond
Keyword(s):  
Ovarian Follicle ◽  
Combined Treatment ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Preantral Follicles ◽  
Ovarian Follicle Development ◽  
Apoptosis Detection ◽  
Rat Ovary ◽  
Tgf Β1

The role TGF-β plays in ovarian follicular growth and differentiation was investigated using a ‘physiological' culture system. TGF- β ligand and receptors are present in the rat ovary from 4 days after birth. Therefore we established organ cultures with these ovaries in order to assess the potential impact of TGF- β1 on follicle growth and transition from the primordial through to the primary and preantral stages of development. Whole ovaries were isolated and cultured for 10 days on floating filters with the addition of supplemented DMEM/Hams F-12 media and either FSH (100ng/mL), TGF- β1 (10ng/mL), or a combination of the two. Media as well as treatments were refreshed every second day. At the end of the culture period, ovaries were fixed in 10% formalin, embedded in paraffin and sectioned at 5µm. Sections were used for morphological assessment and ovarian follicle counting with three serial sections mounted/slide and every alternate slide used for counting of primordial, primary and preantral follicles. An evaluation of atresia by the detection of apoptotic cells was undertaken using terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick-end labelling (TUNEL) via the ApopTag® Peroxidase in situ apoptosis detection kit. Results gathered from this study show preantral follicle numbers declined significantly when treated with the combination of FSH and TGF- β1, consistent with our morphological appraisal of atresia where the combined treatment appeared to produce more apoptotic follicles than healthy follicles, suggesting an increase in atretic primary and preantral follicles. These preliminary findings suggest an inhibitory role for TGF- β1 in the presence of FSH, resulting in fewer follicles making the transition from the primary to the preantral stage. Further studies are required to test the effects of other TGF-β superfamily members on follicle transition in vitro. Supported by the NHMRC of Australia (Regkeys 241000, 441101, 465415, 198705)

Inhibition of Histone Deacetylase 6 Protects Hippocampal Cells Against Mitochondria-mediated Apoptosis in a Model of Severe Oxygen-glucose Deprivation

2019 ◽  
Vol 19 (9) ◽  
pp. 673-682 ◽  
Author(s):  
Panpan Chang ◽  
Yuzi Tian ◽  
Aaron M. Williams ◽  
Umar F. Bhatti ◽  
Baoling Liu ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Cytochrome C ◽  
Cell Viability ◽  
Histone Deacetylase ◽  
Caspase 3 ◽  
Cell Counting ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Histone Deacetylase 6 ◽  
Phosphorylated Akt ◽  
Ldh Release

Background: Histone deacetylase (HDAC) 6 inhibitors have demonstrated significant protective effects in traumatic injuries. However, their roles in neuroprotection and underlying mechanisms are poorly understood. This study sought to investigate the neuroprotective effects of Tubastatin A (Tub-A), an HDAC6 inhibitor, during oxygenglucose deprivation (OGD) in HT22 hippocampal cells. Methods: HT22 hippocampal cells were exposed to OGD. Cell viability and cytotoxicity were assessed by cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) release assay. Cellular apoptosis was assessed by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Mitochondria membrane potential was detected using JC-1 dye. Expressions of acetylated α-tubulin, α-tubulin, cytochrome c, VDAC, Bax, Bcl- 2, cleaved caspase 3, phosphorylated Akt, Akt, phosphorylated GSK3β and GSK3β were analyzed by Western blot analysis. Results: Tub-A induced acetylation of α-tubulin, demonstrating appropriate efficacy. Tub-A significantly increased cell viability and attenuated LDH release after exposure to OGD. Furthermore, Tub-A treatment blunted the increase in TUNEL-positive cells following OGD and preserved the mitochondrial membrane potential. Tub-A also attenuated the release of cytochrome c from the mitochondria into the cytoplasm and suppressed the ratio of Bax/Bcl-2 and cleaved caspase 3. This was mediated, in part, by the increased phosphorylation of Akt and GSK3β signaling pathways. Conclusion: HDAC 6 inhibition, using Tub-A, protects against OGD-induced injury in HT22 cells by modulating Akt/GSK3β signaling and inhibiting mitochondria-mediated apoptosis.

scholarly journals Apoptotic Effects of Xanthium strumarium via PI3K/AKT/mTOR Pathway in Hepatocellular Carcinoma

2019 ◽  
Vol 2019 ◽  
pp. 1-13
Author(s):  
Juyoung Kim ◽  
Kyung Hee Jung ◽  
Hyung Won Ryu ◽  
Doo-Young Kim ◽  
Sei-Ryang Oh ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Growth ◽  
Caspase 3 ◽  
Mtor Pathway ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Mechanistic Study ◽  
Migration And Invasion ◽  
Xanthium Strumarium ◽  
Apoptotic Effects ◽  
Hcc Cells

Xanthium strumarium (XS) has been traditionally used as a medicinal herb for treating inflammatory diseases, such as appendicitis, chronic bronchitis, rheumatism, and rhinitis. In this study, we yielded ethanol extracts from XS and investigated whether they could inhibit the progression of hepatocellular carcinoma (HCC) and its underlying mechanism. The XS-5 and XS-6 extracts dose-dependently inhibited the growth and proliferation in HCC cell lines. The apoptotic effects of them were observed via increased levels of cleaved caspase-3 and cleaved PARP, as well as elevated numbers of terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling- (TUNEL-) positive apoptotic cells. They also decreased XIAP and Mcl-1 expression via loss of mitochondrial membrane potential. Additionally, they inhibited the invasion and migration of HCC cells. In an ex vivo model, the extracts significantly inhibited tumor cell growth and induced apoptosis by increasing the expression of the cleaved caspase-3. A mechanistic study revealed that they effectively suppressed PI3K/AKT/mTOR signaling pathways in HCC cells. Taken together, our findings demonstrate that they could efficiently not only induce apoptosis but also inhibit cell growth, migration, and invasion of human HCC cells by blocking the PI3K/AKT/mTOR pathway. We suggest XS-5 and XS-6 as novel natural anti-HCC agents.

scholarly journals Pyrido[2′,1′:2,3]imidazo[4,5-c]isoquinolin-5-amines as Potential Cytotoxic Agents against Human Neuroblastoma

2021 ◽  
Vol 14 (8) ◽  
pp. 750
Author(s):  
Zahira Tber ◽  
Mohammed Loubidi ◽  
Jabrane Jouha ◽  
Ismail Hdoufane ◽  
Mümin Alper Erdogan ◽  
...  
Keyword(s):  
Cell Line ◽  
Drug Exposure ◽  
Caspase 3 ◽  
Biological Activities ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Cytotoxic Agents ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma ◽  
Parp 1

We report herein the evaluation of various pyrido[2′,1′:2,3]imidazo[4,5-c]isoquinolin-5-amines as potential cytotoxic agents. These molecules were obtained by developing the multicomponent Groebke–Blackburn–Bienaymé reaction to yield various pyrido[2′,1′:2,3]imidazo[4,5-c]quinolines which are isosteres of ellipticine whose biological activities are well established. To evaluate the anticancer potential of these pyrido[2′,1′:2,3]imidazo[4,5-c]isoquinolin-5-amine derivatives in the human neuroblastoma cell line, the cytotoxicity was examined using the WST-1 assay after 72 h drug exposure. A clonogenic assay was used to assess the ability of treated cells to proliferate and form colonies. Protein expressions (Bax, bcl-2, cleaved caspase-3, cleaved PARP-1) were analyzed using Western blotting. The colony number decrease in cells was 50.54%, 37.88% and 27.12% following exposure to compounds 2d, 2g and 4b respectively at 10 μM. We also show that treating the neuroblastoma cell line with these compounds resulted in a significant alteration in caspase-3 and PARP-1 cleavage.

scholarly journals Phloretin Ameliorates Testosterone-Induced Benign Prostatic Hyperplasia in Rats by Regulating the Inflammatory Response, Oxidative Stress and Apoptosis

Life ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 743
Author(s):  
Chao Yu Hsu ◽  
Yi Sheng Lin ◽  
Wei Chun Weng ◽  
Lauren Panny ◽  
Hsiang Lai Chen ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Weight Ratio ◽  
Nuclear Antigen ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Body Weight Ratio ◽  
Proliferative Cell Nuclear Antigen ◽  
Prostate Weight ◽  
Prostatic Enlargement ◽  
Inflammatory Proteins ◽  
Proliferative Cell

The inflammatory process is proposed to be one of the factors to benign prostatic enlargement (BPH), and this is the first study examining the anti-inflammatory ability of phloretin in treating rats with testosterone-induced BPH. BPH would be induced by testosterone (10 mg/kg/day testosterone subcutaneously for 28 days), and the other groups of rats were treated with phloretin 50 mg/kg/day or 100 mg/kg/day orally (phr50 or phr100 group) after induction. Prostate weight and prostate weight to body weight ratio were significantly reduced in the Phr100 group. Reduced dihydrotestosterone without interfering with 5α-reductase was observed in the phr100 group. In inflammatory proteins, reduced IL-6, IL-8, IL-17, NF-κB, and COX-2 were seen in the phr100 group. In reactive oxygen species, malondialdehyde was reduced, and superoxide dismutase and glutathione peroxidase were elevated in the phr100 group. In apoptotic assessment, elevated cleaved caspase-3 was observed in rats of the phr100 group. Enhanced pro-apoptotic Bax and reduced anti-apoptotic Bc1-2 could be seen in the phr100 group. In histological stains, markedly decreased glandular hyperplasia and proliferative cell nuclear antigen were observed with reduced expression in the phr100 group. Meanwhile, positive cells of terminal deoxynucleotidyl transferase dUTP nick end labeling were increased in the phr100 group. In conclusion, the treatment of phloretin 100 mg/kg/day could ameliorate testosterone-induced BPH.

scholarly journals Apoptotic Cells, Including Macrophages, Are Prominent in Theiler's Virus-Induced Inflammatory, Demyelinating Lesions

2003 ◽  
Vol 77 (7) ◽  
pp. 4383-4388 ◽  
Author(s):  
Brian P. Schlitt ◽  
Matthew Felrice ◽  
Mary Lou Jelachich ◽  
Howard L. Lipton
Keyword(s):  
Central Nervous System ◽  
T Cells ◽  
Nervous System ◽  
In Situ Hybridization ◽  
Apoptotic Cells ◽  
Theiler's Virus ◽  
Demyelinating Lesions ◽  
Encephalomyelitis Virus ◽  
Mouse Central Nervous System

ABSTRACT Theiler's murine encephalomyelitis virus (TMEV) persists in the mouse central nervous system principally in macrophages, and infected macrophages in culture undergo apoptosis. We have detected abundant apoptotic cells in perivascular cuffs and inflammatory, demyelinating lesions of SJL mice chronically infected with TMEV. T cells comprised 74% of apoptotic cells, while 8% were macrophages, 0.6% were astrocytes, and ∼17% remained unidentified. In situ hybridization revealed viral RNA in ∼1% of apoptotic cells.

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