Detection Of Apoptotic Cells Research Articles

Roseotoxin B alleviates cholestatic liver fibrosis through inhibiting PDGF-B/PDGFR-\u03b2 pathway in hepatic stellate cells

Identifying effective anti-fibrotic therapies is a major clinical need that remains unmet. In the present study, roseotoxin B was shown to possess an improving effect on cholestatic liver fibrosis in bile duct–ligated mice, as proved by histochemical and immunohistochemical staining, hepatic biochemical parameters, and TUNEL apoptotic cell detection in tissue sections. Using cellular thermal shift assay, computational molecular docking, microscale thermophoresis technology, and surface plasmon resonance biosensor, we confirmed that PDGFR-β was a direct target of roseotoxin B in fibrotic livers. Of note, human tissue microarrays detected pathologically high expression of p-PDGFR-β in liver samples of ~80% of patients with liver fibrosis and cirrhosis. PDGF-B/PDGFR-β pathway promotes transdifferentiation and excessive proliferation of hepatic stellate cells (HSCs), which is a very crucial driver for liver fibrosis. Meaningfully, roseotoxin B blocked the formation of PDGF-BB/PDGFR-ββ complex by targeting the D2 domain of PDGFR-β, thereby inhibiting the PDGF-B/PDGFR-β pathway in HSCs. In summary, our study provided roseotoxin B as a unique candidate agent for the treatment of liver fibrosis.

Recognition of Apoptotic Cells by Viruses and Cytolytic Lymphocytes: Target Selection in the Fog of War.

Viruses and cytolytic lymphocytes operate in an environment filled with dying and dead cells, and cell fragments. For viruses, irreversible fusion with doomed cells is suicide. For cytotoxic T lymphocyte and natural killer cells, time and limited lytic resources spent on apoptotic targets is wasteful and may result in death of the host. We make the case that the target membrane cytoskeleton is the best source of information regarding the suitability of potential targets for engagement for both viruses and lytic effector cells, and we present experimental evidence for detection of apoptotic cells by HIV, without loss of infectivity.

A novel mitochondria-targeting method using special staining for the detection of apoptotic hepatocytes

ABSTRACT Early detection of apoptotic cells on histological slides is of major importance for both diagnostic and research areas. In the current study, the aim was to propose a convenient method to stain the mitochondria and establish whether hepatocytes undergoing apoptosis can be identified in tissue sections using the proposed method. Liver tissue from five adult chinchillas was fixed with 10% neutral buffered formalin for Goldner’s trichrome (GT) and Groat’s iron hematoxylin and eosin (HE) stains and with Kolster’s fixative for the Heidenhain’s iron hematoxylin procedure. The HE and GT-stained sections showed the morphological features consistent with apoptosis i.e., homogenous intensely acidophilic cytoplasm, cell shrinkage with an irregular outline, nuclear shrinkage with cloudy karyoplasm, and karyopyknosis in the late stage. Sections stained with Heidenhain’s iron hematoxylin method was used to pinpoint mitochondria and revealed cells which were undergoing the first stages of the apoptosis process i.e., disappearance of mitochondria from the cell, chromatin condensation and margination, paracentral localization of nucleoli, and vacuolated nuclei. In more advanced stages of apoptosis, cells presented significant nuclear and cytoplasmic changes. It was concluded that this is the first report targeting the mitochondria, by performing inexpensive histological staining techniques, in order to assess dead cells in situ.

LncRNA RMST-mediated miR-107 transcription promotes OGD-induced neuronal apoptosis via interacting with hnRNPK

The long noncoding RNA (lncRNA) rhabdomyosarcoma 2-associated transcript (RMST) silencing has been demonstrated to protect against ischemic brain injury in vivo and neuron injury in vitro. However, its underlying mechanisms in the progression of ischemic stroke have not been well explored. The expression of RMST in oxygen-glucose deprivation (OGD)-treated HT-22 hippocampal neuron cell line was examined using quantitative Real-Time PCR (qRT-PCR). CCK-8 cell viability and apoptotic cell detection using Annexin V-FITC and PI staining coupled with flow cytometry were performed to determine the pro-apoptotic role of RMST in HT-22 hippocampal neuron cell line. Furthermore, RNA pull-down, RNA immunoprecipitation (RIP), coimmunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP) and dual-Luciferase reporter assays were performed to determine the mechanism of RMST in OGD-induced HT-22 cell apoptosis. In the results, RMST was highly expressed in OGD-treated HT-22 cells. Altered RMST expression led to marked changes in HT-22 cell proliferation and apoptosis. Mechanistically, RMST indirectly activated p53/miR-107 signaling pathway via interacting with heterogeneous nuclear ribonucleoprotein K (hnRNPK) and fulfilled its pro-apoptotic function in HT-22 cells. In conclusion, our data indicated that the RMST/hnRNPK/p53/miR-107/Bcl2l2 axis plays an important role in regulating neuronal apoptosis.

Bioluminescence Resonance Energy Transfer (BRET) Coupled Near-Infrared Imaging of Apoptotic Cells.

Detection of apoptotic cells is crucial for understanding the mechanism of diseases and for therapy development. So far, visible-emitting fluorescent probes such as FITC-labeled Annexin V has been widely used for the detection of apoptotic cells. However, such probes cannot be applied to noninvasive imaging in the near-infrared (NIR) region. Compared with visible light, NIR light is highly permeable in turbid biological samples and tissues. In addition, NIR optical imaging has several advantages such as lower autofluorescence and scattering from biological samples, leading to clearer images with high signal to background ratios. Here, we describe the synthesis and application of bioluminescence resonance energy transfer (BRET)-coupled quantum dots (QDs) for the NIR optical imaging of apoptotic cells.

Potential Impact of DARC Technology in Neuroprotective Therapies

The ability to use and control primary neuroprotective agents is a promising approach for future treatment designs in neurodegenerative diseases, particularly in glaucoma. Previous drug and surgical approaches aim to prevent secondary neuroprotection by reducing intraocular pressure. Imaging procedures play a fundamental role in diagnostics and therapy monitoring. The potential importance of DARC technology (= detection of apoptotic retinal cells: visualisation of retinal cells by fluorescence-labeled annexin V = ANX776 using the cSLO technology) in everyday clinical practice lies in its ease of implementation. The necessary HRT is available in many ophthalmology centres. The use of a fluorescent marker is similar to that of conventional fluorescein angiography. The most important limitation is the individually fluctuating physiological apoptosis of the human retinal ganglion cells, which has to be standardised before this method can be used in everyday clinical practice.

Propranolol inhibits catecholestrogen-induced human endometrial stromal cell survival mediated by p38 MAPK signaling: potential therapy for endometriosis

Catecholestrogens (CCEs), 2-Hydroxyestradiol (OHE2) and 4-OHE2, are biologically active metabolites of 17β-estradiol (E2). Studies indicate that local increases in E2 production as well as aberrant expression of E2 metabolizing enzymes enhance local generation of CCEs in women with endometriosis. CCEs have low binding affinity to estrogen receptors whereas CCEs display high binding affinity to β2-adrenergic receptor (AR), which induce uterine endothelial cell proliferation during gestation. Our recent data demonstrated β2-AR expression in eutopic and ectopic endometrial tissue. In addition, binding of CCEs to β-AR enhances human endometrial stromal cell (HESC) viability, suggesting contribution of CCEs to the pathogenesis of endometriosis. Thus, we tested the hypothesis that the mechanism of CCE-enhanced HESC viability involves alterations in either proliferation or apoptosis mediated by β-AR-induced common intracellular signaling pathways, i.e. AKT, MAPK and/or NFκB. BrdU, Apoptotic Cell Detection ELISA, q-PCR, Western blot and XTT analyses were performed on cultured HESCs derived from endometrial biopsies. Cultured HESCs treated with 10-8 M E2 or 2-OHE2 or 4-OHE2 were measured by BrdU for proliferation and ELISA for apoptosis (n=3 with quadruplicate). Total RNA from cultured HESCs was isolated and pro-apoptotic, anti-apoptotic, and proliferation markers were evaluated by q-PCR (n=5 with duplicate). Total and phosphorylated AKT, p38 and ERK1/2 MAPKs, and NFκB levels were detected in lysates of cultured HESCs (n=3 with triplicate) treated for 10 min with vehicle (control) or 10-8 M E2 or 2-OHE2 or 4-OHE2 ± 2x10-5 M non-specific β-antagonist (propranolol). Subsequently, XTT assays were conducted with p38 MAPK inhibitor to assess the role of p38 MAPK on CCE-induced HESC viability (n=4 with triplicate). Results were analyzed by One-way ANOVA and post hoc Tukey test. An increased HESC proliferation index by E2 and 4-OHE2 (P<0.05 and P<0.05, respectively) and decreased apoptosis were detected in HESCs treated with 2-OHE2 and 4-OHE2 vs. control (P<0.01 and P<0.01, respectively). Analysis of apoptotic markers by q-PCR revealed a significant decrease in Bax mRNA expression in response to 2-OHE2 treatment vs. control (P<0.01). Among the several intracellular signaling cascades analyzed, only phosphorylation levels of p38 MAPK were increased by either treatment with 2-OHE2 or 4-OHE2 (P<0.05 and P<0.05 vs. control, respectively), but not with E2. β-AR antagonism with propranolol mitigated this increased phosphorylation in p38 MAPK levels (P<0.05 and P<0.01, respectively) and inhibition of p38 MAPK by SB203580 blocked CCE-induced HESC survival (P<0.05 and P<0.001, respectively). These data indicate that induction of endometrial stromal cell viability by CCE-β-AR interactions results from an imbalance in both proliferation and anti-apoptotic mechanisms and is specifically mediated by the activation of p38 MAPK signaling. These data also suggest that inhibition of β2-ARs with propranolol may be a novel treatment option in endometriosis.

Ultrasensitive amperometric cytosensor for drug evaluation with monitoring early cell apoptosis based on Cu2O@PtPd nanocomposite as signal amplified label

It is significant to monitor the early stage of apoptosis and evaluate the therapeutic effect of anticancer drugs in the diagnostic of cancer. Here, an electrochemical cytosensor with signal amplified strategy was constructed to quantitatively detect the concentration of early apoptotic cells and respond to therapy of anticancer drugs. To fabricate the platform, cuprous oxide (Cu2O) cube was utilized as a large platform for loading platinum-palladium nanoparticles (PtPd NPs) to prepare the Cu2O@PtPd nanocomposites. The formed nanocomposite was not only acted as signal amplified molecules towards the reduction of H2O2 by synergistic effect but also possessed large specific area to conjugate concanavalin A (Con A), which specifically interacted with the cell-surface carbohydrate. What’s more, the porous graphene oxide-Au (PGO-Au) nanocomposites were modified onto working electrode to improve the efficiency of electron transfer. Meanwhile, the PGO-Au nanocomposite was served as effective matrix to couple annexin V through Au-N bond to capture apoptotic cells owing to the specific interaction between phosphatidylserine (PS) on the cell surface and annexin V. In this system, A549 cell was chosen as model cells and cisplatin acted as apoptotic inducer. The proposed amperometric cytosensor exhibited a wide range detection of early apoptotic cells from 50 to 5 × 107 cells mL−1. Meanwhile, the detection limit was down to 20 cells mL−1. It also showed high specificity, acceptable sensitivity and good reproducibility, which had promising potential for the early evaluation of therapeutic effect.

Abstract 2303: In vitro evaluation of TCR efficacy and toxicity using 3D spheroid models

Abstract Clinical studies using TCR-transgenic T cells for adoptive T cell therapy revealed the efficacy of this therapeutic approach but also uncovered possible toxic effects against healthy tissues, such as neuronal or cardiac cells, caused by on-target/off-tumor or off-target toxicity (Morgan et al., J Immunother. 2013; Linette et al., Blood 2013). Due to the lack of adequate in vivo models for prediction of TCR efficacy or potential TCR-mediated toxicity against healthy tissues, physiologically relevant in vitro models need to be developed. The usage of 3D spheroids as targets for TCR-transduced T cells represents a powerful strategy to test killing capacity or possible safety issues of TCRs, which could fill the gap between 2D co-cultures and animal models. Compared to 2D cell layers, cells within a 3D spheroid structure more closely resemble the physiological in vivo situation regarding cell-cell-interactions, proliferation rates, hypoxia, and gene expression. Here we describe the use of 3D spheroids generated from tumor cell lines for assessment of TCR efficacy or healthy primary cells/iPSC-derived cells for evaluation of potential TCR-derived toxicity. HLA-A2-positive cells served as target cells in functional co-culture assays with CD8+ T cells transduced with an HLA-A2-restricted TCR. 3D spheroids have been generated using ULA (ultra-low attachment) plates or via the hanging-drop culture method. Spheroids were co-cultured with TCR-transduced T cells and killing of spheroids was analyzed in real-time using the IncuCyte live cell imaging system. For better visualization of spheroid killing, tumor cell lines were transduced using a lentivirus encoding a nuclear-restricted red fluorescent protein or by adding Annexin V red fluorescent reagent for detection of apoptotic cells. Thereby, spheroid killing could be quantified via decrease or increase in red fluorescence over time. Killing of 3D spheroids derived from healthy cells was analyzed via phase contrast and evaluation of spheroid morphology. TCR-transduced T cells showed efficient killing of tumor cell spheroids, confirming the results generated in a 2D system. Furthermore, 3D spheroids generated from healthy cells (e.g. normal human lung fibroblasts) or iPSC-derived cells (e.g. cardiomyocytes or neurons) were not killed via TCR-transduced T cells, whereas exogenous loading of the spheroids with the specific peptide led to efficient killing, showing the general susceptibility to TCR-mediated killing of healthy cell 3D spheroids.In summary, we showed that functional in vitro assays using 3D spheroid structures using tumor cell lines or healthy cells represent an elegant approach to assess both efficacy and potential toxicity of a given TCR in vitro. The analyzed TCR showed potent efficacy as well as a favorable safety pattern in 3D in vitro cultures. Citation Format: Maja Buerdek, Kathrin Mutze, Kai Pinkernell, Dolores J. Schendel. In vitro evaluation of TCR efficacy and toxicity using 3D spheroid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2303.

Arsenic trioxide and tetraarsenic oxide induce cytotoxicity and have a synergistic effect with cisplatin in paclitaxel-resistant ovarian cancer cells

Background: Arsenic compounds (As2O3 and As4O6) have demonstrated anticancer effects in various malignancies. In this study, the cytotoxicity of arsenic compounds on ovarian cancer cell lines and the anticancer activity of the combination of arsenic compounds and cisplatin IN chemoresistant ovarian cancer cells were investigated.Methods: We investigated the cytotoxicity of As2O3 and As4O6 and their combinations with cisplatin in the paclitaxel-sensitive ovarian cancer cell lines SKOV3ip1 and HeyA8 and paclitaxel-resistant ovarian cancer cell lines SKOV3TRip2 and HeyA8-MDR. Growth and apoptosis were evaluated by MTT assay and annexin V assay using flow cytometry, respectively. For detection of apoptotic cells, immunofluorescence was performed using a cleaved caspase-3 antibody. Cell-cycle distribution was determined by propidium iodide staining and flow cytometry.Results: Treatment of each cell line with As2O3 or As4O6 led to a marked dose-dependent inhibition of cell growth. As2O3 and As4O6 treatment induced caspase-3-dependent apoptosis in all cell lines compared to the respective control groups (p < .05). As2O3 and As4O6 induced apoptosis of paclitaxel-sensitive and -resistant cancer cell lines following G2/M cell cycle arrest (p < .05). A synergistic effect was achieved by combining cisplatin with As2O3 or As4O6 in the paclitaxel-resistant ovarian cancer cell lines.Conclusions: As2O3 and As4O6 can inhibit cell growth and induce apoptosis in paclitaxel-sensitive and -resistant ovarian cancer cell lines. Their combination with cisplatin resulted in a synergistic effect in paclitaxel-resistant cancer cell lines. These results suggest that arsenic compounds may be given in monotherapy or combination therapy with cisplatin for treating paclitaxel-resistant ovarian cancer.

THE EFFECT OF THE COLD STRESS ON MORPHOLOGICAL AND FUNCTIONAL PARAMETERS OF THE THYMUS IN THE EXPERIMENT

The thymus is the central organ of the immunogenesis, the functioning of which determines the protective reactions of the organism. Various stressors can influence the immune organs. One of these factors is exposure to low temperatures. The aim of this research was studying the effect of experimental cold stress on the morphological and functional state of rat thymus using morphometric analysis. The cold exposure was done by placing the rats in individual cages in a refrigeration chamber at a temperature of -10 ± 20 °C for 1 hour at the same time of day for 7, 14, 21, and 30 days. Histological examination of thymus preparations stained with hematoxylin and eosin; on the 7th day after exposure to cold, the area of the cortical substance is significantly reduced compared with the control group, while the area of the medulla tends to increase. Analysis of the cell composition showed an increase in the number of epithelia-reticular cells in the subcapsular zone and a slight decrease in the number of lymphocytes. On the 14th day of exposure, the areas of cortical and medulla and the cortical-cerebral index do not differ from the indicators of intact control. Morphometric counting of cellular elements showed an increase in the number of lymphocytes, epithelia-reticulocytes, and macrophages. After 21 days of the experiment, data on the relative areas of cortical and medulla shows a significant decrease in the rate compared with all the first groups. The counting of cellular elements during this period showed marked changes observed in all morphological and functional zones of the thymus. In the subcapsular zone, there is a sharp decrease in the number of dividing lymphocytes. The number of epithelia-reticular cells is increased relative to the norm. There is frequent detection of apoptotic bodies and fat cells. In the medulla, there is an increase in the number of epithelia-reticulocytes compared with the 7th and 14th day. On the 30th day after exposure, the areas of cortical, medulla, and cortical-cerebral index remained at the same level as on the 21st day. There is a significant decrease in epithelia-reticular cells in the subcapsular zone, a significant decrease in the cortical substance and the cortico-medullary zone compared with all groups, and a significant increase in their number in the medulla. Hassall’s bodies are characterized by pronounced degenerative changes in epithelia-reticulocytes with cellular debris. In the subcapsular zone, a reduced number of cells with mitosis figures is maintained. It was established that as a result of cold stress on days 7, 21 and 30, there is a redistribution of volume and cellular composition, various structural and functional zones, indicating a decrease in the functional activity of the thymus, an increase in the death of lymphocytes by apoptosis, a decrease in mitotic activity, and accumulation of macrophages. Morphological and functional data of the 14th day of the experiment indicate the development of compensatory-adaptive changes in the thymus on the cold effect, manifested in the form of activation of cell division in the cortical substance and the cortical-medullary zone.

Morphological study of aorta in patients with acute aortic syndromes

Purpose of the study is to investigate the morphology of the aortic wall in patients with acute aortic syndrome.Materials and methods. The results of morphological study of aneurysmatic fragments (n-52), which were collected intraoperatively, each fragment were prepared on the basis of the SI "V.T.Zaycev Institute of General and Urgent Surgery AMSU", which were coloured with hematoxilin eosin, elastic fibers/ were incubated with antibodies to IgG, and with Apo-Alert DNA Fragmentation to determine apoptosis.Results. Post-coarctation aneurysms had the following microscopic picture: a decrease in the medial layer with small fields of disorganization of elastic fibers and smooth muscle cells (SMC). The detection of apoptotic cells in the sections is minimal. In the group of acute aortic dissection aneurysms in 40 % of cases, the elastic fibers were fragmented, the SMC are chaotic with separate parts of the isiocytane medineocrine - a typical pattern for Marfan's syndrome. A microscopic study of thoracoabdominal aneurysms showed a significant decrease in the middle layer with the development of fibrosis. When counting the total number of cells, a decrease in smooth muscle cells was found on average by 54 %. The aneurysms of the abdominal aorta were characterized by major degenerative changes. Reducing the medial layer and fibrous changes were also the most significant. Reduction of the medial layer and fibrous changes were most significant in this groupConclusions. All types of complicated aneurysms are characterized by insufficiency of smooth muscle cells in aortic wall. The study found that a decrease in the number of smooth muscle cells can occur due to apoptosis. There is a clear correlation with inflammatory infiltration by cells that produce apoptosis induction

Neurotoxic Effects of Stanozolol on Male Rats' Hippocampi: Does Stanozolol cause apoptosis?

Stanozolol is an anabolic-androgenic steroid which is commonly abused by athletes for improved energy, appearance, and physical size. It has been previously shown to cause changes in behaviour and has various physical effects. Studies have previously been conducted on its neurotoxic effect on the central nervous system (CNS), which are typically psychological in nature. This study was performed to investigate the apoptotic effect of stanozolol on different parts of the rat hippocampus. Sixteen male Wistar rats were divided randomly into two groups (experimental and control). The experimental group received subcutaneous injections of stanozolol (5mg/kg/day) for consecutive 28 days, whereas the control group received saline using the same dosing schedule and administration route. After routine procedures, coronal sections of rat brain were stained with Toluidine blue and TUNEL for pre-apoptotic and apoptotic cell detection, respectively. In order to compare groups, the mean number of TUNEL-positive and pre-apoptotic neurons per unit area were calculated and analysed. Histopathological examination revealed that the mean number of pre-apoptotic and apoptotic neurons in the CA1, CA2, CA3 and DG areas of the hippocampus were significantly increased in the stanozolol treated group. In conclusion, stanozolol abuse may induce pre-apoptotic and apoptotic cell formation in different regions of the hippocampus.

Changqin NO. 1 inhibits neuronal apoptosis via suppressing GAS5 expression in a traumatic brain injury mice model.

The present study was designed to investigate the mechanism of the traditional Chinese medicine Changqin NO. 1 on the amelioration of traumatic brain injury (TBI). Adult male C57BL/6J mice and newborn mice were used to generate a mouse TBI model and harvest primary neurons, respectively. The localizations of specific neural markers neuropilin-1 (Nrp-1), growth-associated protein-43 (GAP-43) and microtubule-associated protein Tau (Tau) were examined in brain tissues by immunohistochemistry. Terminal deoxynucleotidyl transferase dUTP nick end labeling apoptotic cell detection in tissue sections and the CCK-8 cell viability assay were performed to examine neuronal apoptosis. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were also carried out in this study. The association between long non-coding RNA (lncRNA) growth-arrest specific 5 (GAS5), miR-335 and RAS p21 GTPase activating protein 1 (Rasa1) was disclosed using the dual-luciferase reporter assay. Changqin NO. 1 inhibited TBI-induced neuronal apoptosis in vivo and in vitro. GAS5 functioned as a competing endogenous RNA (ceRNA) by sponging miR-335 to upregulate Rasa1 expression in mouse neuronal cells. Further investigations demonstrated that GAS5 promoted neuronal apoptosis following TBI via the miR-335/Rasa1 axis. In vivo experiments indicated that Changqin NO. 1 exerted neuroprotection during TBI via the GAS5/miR-335/Rasa1 axis. Changqin NO. 1 promoted neuroprotective effects by inhibiting neuronal apoptosis via the GAS5/miR-335/Rasa1 axis in TBI.

Evaluation of low-level diode laser irradiation and various irrigant solutions on the biological response of stem cells from exfoliated deciduous teeth

This study aimed to evaluate cytotoxic effects and the apoptosis of Gallium-Aluminum-Arsenide (GaAlAs) diode laser irradiation, sodium hypochlorite (NaOCl), ozonated water and ethylene diamine tetraacetic acid (EDTA) on stem cells from human exfoliated deciduous teeth (SHEDs). Cells were exposed to EDTA (5%, 8.5%, 17%), NaOCl (1%, 2.5%, 5%) ozonated water (5, 10, 20 μg/ml) and GaAlAs diode laser irradiation (energy densities of 0.5, 1, 1.5 j/cm2). Culture medium included D-MEM, supplemented with 15% foetal bovine serum, 1% l-glutamine, 1% penicillin-streptomycin, 1% gentamycin, amphotericin-B and served as control group. The prepared irrigants were added to the relevant wells and incubated with the cells at 37 °C for 5, 10 and 15 min. The cells in the laser group were also incubated at 37 °C for 5, 10 and 15 min after the laser application. Cell viability and proliferation were analysed with the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. The percentage of cell viability showed a significant reduction in all concentrations of the EDTA and NaOCl groups when compared to the control group, diode laser irradiation and ozonated water groups at 5th, 10th and 15th minutes respectively but high cytotoxic effects of all EDTA and NaOCl groups with decreased over 50% of cell viability were observed at the 15th minute. Also EDTA group with 17% concentration (17%E) presented the lowest survival rate on SHEDs with mean of 21.67% ± 6.101 at this time interval. The lowest toxic effects were observed at the 5th minutes compared to other time periods at experimental groups. For detection of apoptotic cells, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) method was performed. According to the MTT results, doses showed the highest toxicity (cell survival decreased over 50%) in each group were selected for TUNEL assay (17% EDTA; 1% NaOCl; 10 μg/ml Ozonated water; 1.5 j/cm2 diode laser irradiation). The significantly lowest percentages of TUNEL-positive cells were detected in ozonated water (10.67% ± 2.93) and diode laser irradiation (13.24% ± 7.61) compared to EDTA (39.89% ± 11.54) and NaOCl (31.15% ± 10.64) respectively. Also the difference between percentage of TUNEL-positive cells in EDTA and NaOCl groups was not significant. Synergistic combination of ozonated water and diode laser irradiation may be used in the disinfection step of necrotic root canals.

Corrigendum: Bioluminescence Resonance Energy Transfer (BRET)-coupled Annexin V-functionalized Quantum Dots for Near-Infrared Optical Detection of Apoptotic Cells

Corrigendum: Bioluminescence Resonance Energy Transfer (BRET)-coupled Annexin V-functionalized Quantum Dots for Near-Infrared Optical Detection of Apoptotic Cells

Effect of hesperetin on inflammatory and oxidative status in trinitrobenzene sulfonic acid-induced experimental colitis model

In our study, the effect of hesperetin on inflammatory and oxidative status in trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis model was investigated through different methods. Eighteen Wistar albino male rats were divided in to three groups: Group I (Control, n = 8; 1 ml physiological saline), Group II (Colitis, n = 8; 1 ml TNBS), Group III (Hesperetin, n = 8; 1 ml TNBS and 100 mg/kg hesperetin). Macroscopic and microscopic scores were calculated to determine the damage to the colon at the end of the experiment. Serum tumor necrosis factor-α (TNF-α) and tissue interleukin-6 (IL-6) levels were determined using the ELISA method. Myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) levels were investigated spectrophotometrically. The TUNEL method was used for the detection of apoptotic cells in the colon tissue. Inducible nitric oxide synthase (iNOS) and nuclear factor-kappa-B (NF-ĸβ) expression in the colon were determined immunohistochemically. Hesperetin administration has shown to significantly reduce levels of MPO, MDA, and proinflammatory agents (TNF-α, IL-6, and NF-ĸβ). It has also been proven to inhibit mucosal apoptosis. This study indicates that hesperetin is protective against TNBS-induced colitis model via antiinflammatory, antioxidant and antiapoptotic effects.

Evaluation of 99mTc-rhAnnexin V-128 SPECT/CT as a diagnostic tool for early stages of interstitial lung disease associated with systemic sclerosis

BackgroundGiven the need for early detection of organ involvement in systemic sclerosis, we evaluated 99mTc-rhAnnexin V-128 for the detection of early stages of interstitial lung disease (ILD) in respective animal models using single photon emission computed tomography (SPECT/CT).MethodsIn bleomycin (BLM)-challenged mice, fos-related antigen 2 (Fra-2) transgenic (tg) mice and respective controls, lung injury was evaluated by analysis of hematoxylin and eosin (HE) and Sirius red staining, with semi-quantification of fibrosis by the Ashcroft score. Apoptotic cells were identified by TUNEL assay, cleaved caspase 3 staining and double staining with specific cell markers. To detect early stages of lung remodeling by visualization of apoptosis, mice were injected intravenously with 99mTc-rhAnnexin V-128 and imaged by small animal SPECT/CT. For confirmation, biodistribution and ex vivo autoradiography studies were performed.ResultsIn BLM-induced lung fibrosis, inflammatory infiltrates occurred as early as day 3 with peak at day 7, whereas pulmonary fibrosis developed from day 7 and was most pronounced at day 21. In accordance, the number of apoptotic cells was highest at day 3 compared with saline controls and then decreased over time. Epithelial cells (E-cadherin+) and inflammatory cells (CD45+) were the primary cells undergoing apoptosis in the earliest remodeling stages of experimental ILD. This was also true in the pathophysiologically different Fra-2 tg mice, where apoptosis of CD45+ cells occurred in the inflammatory stage. In accordance with the findings on tissue level, at day 3 in the BLM and at week 16 in the Fra-2 tg model, biodistribution and/or ex vivo autoradiography showed increased pulmonary uptake of 99mTc-rhAnnexin V-128 compared with controls. However, accumulation of the radiotracer and thus the signal intensity in lungs was too low to allow the differentiation of healthy and injured lungs in vivo.ConclusionAt the tissue level, 99mTc-rhAnnexin V-128 successfully demonstrated early stages of ILD in two animal models by detection of apoptotic epithelial and/or inflammatory cells. In vivo, however, we did not detect early lung injury. It remains to be investigated whether the same applies to human ILD.

Esculentoside A suppresses breast cancer stem cell growth through stemness attenuation and apoptosis induction by blocking IL-6/STAT3 signaling pathway.

Breast cancer stem cells (CSCs) survive in inflammatory microenvironment, their survival are regulated by inflammatory cytokines and signaling pathways. Esculentoside A (EsA), a triterpene saponin derived from the root of Phytolacca esculenta, possesses antiinflammation effects; but whether it has anticancer activity is unknown. The purpose of this study is to test the inhibitory effect of EsA on the growth of breast CSCs and to elucidate its probable mechanisms of action. The proliferation inhibitory effect of EsA on breast CSCs in vitro were determined by cytotoxicity, mammosphere formation inhibition, apoptotic cell detection assays, and in vivo tumor growth inhibition experiment. The possible molecular mechanisms elucidating the inhibitory effect of EsA on breast CSC growth were examined with western blotting. EsA caused proliferation and mammosphere formation inhibition of breast CSCs; induced breast CSCs apoptotic death; suppressed the growth of tumors generated from breast CSCs significantly; the expressions of stemness proteins including ALDH1A1, Sox2, and Oct4 were downregulated; proapoptotic proteins, Bax and cleaved caspase-3 were upregulated, whereas the antiapoptotic protein Bcl-2 was reduced; IL-6/STAT3 pathway proteins including IL-6, phosphorylated STAT3 (Tyr705), and STAT3 (Ser727) were downregulated significantly in EsA-treated breast CSCs and tumor tissues. EsA inhibited breast CSC growth in vitro and in vivo through stemness attenuation and apoptosis induction by blocking IL-6/STAT3 signaling pathway; it might serve as a novel candidate agent for human breast cancer treatment and/or prevention.

Restoring Ovarian Function With Human Placenta-Derived Mesenchymal Stem Cells in Autoimmune-Induced Premature Ovarian Failure Mice Mediated by Treg Cells and Associated Cytokines.

Regulatory T (Treg) cells play a key role in the regulation of autoimmunity and transplantation. Human placenta-derived mesenchymal stem cell (hPMSC) transplantation has a potential to restore ovarian dysfunction associated with premature ovarian failure (POF), while the exact function of the Treg cells in the transplantation still needs to be further investigated. In this study, hPMSCs were intravenously injected into POF mice following zona pellucida glycoprotein 3 (pZP3) treatment. Ovarian function was measured by analyzing estrous cycle, folliculogenesis, and hormone secretion, also, with the detection of apoptotic granular cells (GCs) in ovarian tissues. To determine whether immune response is involved in the regulation of ovarian function change, the population of Treg cell populations and expression of associated cytokines, for example, transforming growth factor β (TGF-β) and interferon γ (IFN-γ) were measured. After hPMSCs transplantation, the injured ovarian function is significantly improved. Also, the pZP3-treatment-induced apoptotic GCs were significantly decreased as compared with the POF mice. The transplantation of hPMSCs significantly increased the population of Treg cells which was inhibited by pZP3 treatment. The decrease in TGF-β and increase in IFN-γ in serum caused by pZP3 treatment have been reversed following hPMSCs transplantation. These findings strongly suggest that the recovery of ovarian function in POF mice is mediated via the regulation of Treg cells and production of associated cytokines following hPMSCs transplantation.