Screening and Detection of Apoptosis
ABSTRACT Since programmed cell death was first described by the electron microscopic cellular changes demonstrating an organized form of cell death over 30 years ago, it has undergone a great deal of scrutiny as a potential target for several diseases including cancer. The techniques for the study of apoptosis have evolved accordingly. Methodologies for the study of apoptosis were examined by a MEDLINE search of the English-language literature and are summarized in this review. This review discusses the various ways to study apoptosis with specific assays, reagents, and molecules. The particular advantages and disadvantages of each method are reviewed.
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- "Monoclonal antibodies are available against all the caspases for immunohistochemistry , ELISA, and even some for flow cytometry (i.e., active caspase 3) . Each method that is able to detect and verify apoptosis has advantages and disadvantages, and it is best to confirm apoptosis with multiple complementary techniques . Inhibition of apoptosis seems to improve hepatocyte survival and prevent reperfusion injury. "
ABSTRACT: Background: The aim of this experimental study was to investigate the role of apigenin in liver apoptosis, in an experimental model of hepatic ischemia-reperfusion in rats. Materials and methods: Forty-eight Wistar rats (apigenin and control groups), 14 to 16 weeks old and weighing 220 to 350 g, were used. They were all subjected to hepatic ischemia by occlusion of the hepatic artery and portal vein for 45 minutes and reperfusion was followed for 60, 120, and 240 minutes. Apigenin was administrated intraperitoneally. Liver tissues were used for the detection of apoptosis by TUNEL assay and caspase 3 antibodies. Expression analysis of Fas/FasL genes was evaluated by real time PCR. Results: The expression analysis of Fas and FasL genes was increasing during reperfusion (significantly in the group of 240 minutes of reperfusion). It was in the same group that apigenin decreased Fas receptor levels and inhibited apoptosis as confirmed by TUNEL assay and caspase 3 antibodies. Conclusions: The effects of apigenin in the Fas/FasL mediated pathway of apoptosis, in the hepatic ischemia-reperfusion, seem to have a protective result on the hepatic cell.BioMed Research International 07/2014; 2014:157216. DOI:10.1155/2014/157216 · 2.71 Impact Factor
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- "Apoptosis can be induced by two main pathways: the intrinsic (mitochondrial), in which Bax is one of the most important pro-apoptotic protein, and the extrinsic pathways.  In addition, there is a close relationship between these apoptosis-related pathways and inflammatory pathways. TNF-α, an important pro-inflammatory cytokine, is involved in the activation of apoptosis, while NF-κB has an anti-apoptotic function, activating the expression of other members of the Bcl-2 family, such as Bcl-2, which prevents cell death , . "
ABSTRACT: BackgroundCrohn’s disease (CD) is associated with complex pathogenic pathways involving defects in apoptosis mechanisms. Recently, mesenteric adipose tissue (MAT) has been associated with CD ethiopathology, since adipose thickening is detected close to the affected intestinal area. However, the potential role of altered apoptosis in MAT of CD has not been addressed.AimsTo evaluate apoptosis in the intestinal mucosa and MAT of patients with CD.MethodsSamples of intestinal mucosa and MAT from patients with ileocecal CD and from non-inflammatory bowel diseases patients (controls) were studied. Apoptosis was assessed by TUNEL assay and correlated with the adipocytes histological morphometric analysis. The transcriptional and protein analysis of selected genes and proteins related to apoptosis were determined.ResultsTUNEL assay showed fewer apoptotic cells in CD, when compared to the control groups, both in the intestinal mucosa and in MAT. In addition, the number of apoptotic cells (TUNEL) correlated significantly with the area and perimeter of the adipose cells in MAT. Transcriptomic and proteomic analysis reveal a significantly lower transcript and protein levels of Bax in the intestinal mucosa of CD, compared to the controls; low protein levels of Bax were found localized in the lamina propria and not in the epithelium of this tissue. Furthermore, higher level of Bcl-2 and low level of Caspase 3 were seen in the MAT of CD patients.ConclusionThe defective apoptosis in MAT may explain the singular morphological characteristics of this tissue in CD, which may be implicated in the pathophysiology of the disease.PLoS ONE 06/2014; 9(6):e98547. DOI:10.1371/journal.pone.0098547 · 3.23 Impact Factor
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- "Apoptosis plays an important role in the control and regulation of the immune response ,  and DCs have been shown to have a high turnover rate . Frh-imdDCs undergo apoptosis in a time-dependent manner in the absence of specific stimuli . "
ABSTRACT: Dendritic cells play a key role in the immune system, in the sensing of foreign antigens and triggering of an adaptive immune response. Cryopreservation of human monocytes was investigated to understand its effect on differentiation into immature monocyte-derived dendritic cells (imdDCs), the response to inflammatory stimuli and the ability to induce allogeneic lymphocyte proliferation. Cryopreserved (crp)-monocytes were able to differentiate into imdDCs, albeit to a lesser extent than freshly (frh)-obtained monocytes. Furthermore, crp-imdDCs had lower rates of maturation and cytokine/chemokine secretion in response to LPS than frh-imdDCs. Lower expression of Toll-like receptor 4 (at 24 and 48 h) and higher susceptibility to apoptosis in crp-imdDCs than in fresh cells would account for the impaired maturation and cytokine/chemokine secretion observed. A mixed leukocyte reaction showed that lymphocyte proliferation was lower with crp-imdDCs than with frh-imdDCs. These findings suggested that the source of monocytes used to generate human imdDCs could influence the accuracy of results observed in studies of the immune response to pathogens, lymphocyte activation, vaccination and antigen sensing. It is not always possible to work with freshly isolated monocytes but the possible effects of freezing/thawing on the biology and responsiveness of imdDCs should be taken into account.PLoS ONE 07/2013; 8(7):e71291. DOI:10.1371/journal.pone.0071291 · 3.23 Impact Factor