2020 Oct 23;16:1023-1029. doi: 10.2147/TCRM.S268130. Regulation of Fas antibody induced neutrophil apoptosis is both caspase and mitochondrial dependent. Saudi J Biol Sci. Neutrophils are the type of white blood cell that attack bacteria in the blood. The supernatant or cytosolic fraction was stored at -70°C until 30 μL was run on Western blot. Neutrophils (1 × 106 cells/mL) were isolated from healthy male (n = 26) and female (n = 26) volunteers and incubated in vitro for 24 hours. The effects of estradiol and progesterone on spontaneous caspase 3 activation at 6 and 24 hours assessed by Western blot and densitometry. 20 Progesterone and estradiol levels rise throughout pregnancy and may peak at a 100-fold higher than normal nonpregnant values. This retrospective study included 1,468 patients with suspected bacteremia and sepsis. We also acknowledge the generous gift of anti-cytochrome c antibody from Prof Seamus Martin, Molecular Cell Biology Laboratory, Department of Genetics, Smurfit Institute, Trinity College, Dublin. Error bars indicate mean ± SD. Neutrophils incubated with progesterone or estradiol demonstrated a significant decrease in active caspase 3 protein expression at 6 hours that was confirmed by densitometry (Figure 7A). The expression of the (active) proteolytic cleavage product of caspase 3 protein was increased following 6 and 24 hours of neutrophil incubation in vitro. Effects of Neutrophil-to-Lymphocyte Ratio Combined With Interleukin-6 in Predicting 28-Day Mortality in Patients With Sepsis. An inhibitory effect may be seen at a lower dose of Fas antibody. Patients on chemotherapy can become neutropenic. Accessibility Neutrophils (1 × 106 cells/mL) isolated from male (▪; n = 10) and female (♦; n = 10) controls were incubated in vitro with increasing physiologic doses (10-12 to 10-7 g/mL) of estradiol (A), progesterone (B), or both in combination (C). A period of neutropenia may last a few days, followed by normal levels for the rest of the cycle. It is not measured directly, but calculated by multiplying the white blood cell count by the Neutrophils Percentage or the Relative Neutrophil Count in the sample. All experiments used both male and female subjects. This may explain the differences between the PMA-induced responses.48 Neutrophil CD11b expression in men and women was unaffected by physiologic doses of female sex hormones. A normal (absolute) neutrophil count is between 2500 and 7500 neutrophils per microliter of blood. Sacks GP, Studena K, Sargent K, Redman CW. Overview. Spontaneous apoptosis of neutrophils was quantified by flow cytometry as the percent of cells with hypodiploid DNA.20,21 Cells (1 × 106/mL) were centrifuged at 130g for 5 minutes and then gently resuspended in 400 μL hypotonic fluorochrome solution (200 mL phosphate-buffered saline [PBS], 10 mg propidium iodide, 3.4 mM sodium citrate, 1 mM Tris [tris(hydroxymethyl)aminomethane], 0.1 mM EDTA [ethylenediaminetetraacetic acid], 0.1% Triton X-100). Estradiol has previously been shown to induce an undefined inhibitor of active caspases through a receptor-mediated nongenomic pathway in neurons.49 Estrogen withdrawal stimulates the processing of caspase 3 to its active form in chick oviduct, culminating in the induction of apoptosis.50 Decreased apoptosis in MCF-7 breast cancer cell line51 and in B cells33 is believed to be due to increased Bcl-2 expression. "my doctor said i have small in size blood red cells i'm not sure what that means? All measurements were performed under the same instrument settings. (B) Densitometry was performed and the density of each band (percent of total pixels) expressed as the mean ± SD of all blots. (A) Men, n = 2; women, n = 3. This would be immunologically advantageous by enhancing the number of circulating neutrophils available to combat infection. These blots are representative of 1 of at least 3 separate experiments. Bacteremia and sepsis are common causes of morbidity and mortality worldwide, with incorrect or delayed diagnoses being associated with increased mortality. Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. For this number, a normal range of an absolute neutrophil count is 1.5 to 8.0. There was no significant additive effect when these steroid hormones were added in combination over the same physiologic range (Figure 2). RU486 (RU; 10-6 M) was added at time 0 hours and percentage apoptosis was assessed by flow cytometry at 24 hours (n = 11). Significance was assumed for P values less than .05. Each graph represents at least 5 separate experiments performed in duplicate (mean ± SEM). Vegeto E, Pollio G, Pellicari C, Maggi A. Estrogen and progesterone induction of survival of monoblastoid cells undergoing TNF-α-induced apoptosis. From conception to senescence, women have a significant survival advantage.1 Men are more susceptible to sepsis and subsequent morbidity and mortality than women of reproductive age.2-4 The incidence of sepsis in postmenopausal women increases to levels almost equal to those seen in age-matched men.5,6 The exact mechanism mediating this sexual dimorphism is unclear. : 168 Up to 17% of female human neutrophil nuclei has a drumstick-shaped appendage which contains the inactivated X chromosome. The technique used was dextran (3%) sedimentation and centrifugation through a discontinuous Ficoll gradient; red blood cells were lysed using E-Lyse.20 The neutrophil pellet was resuspended in DMEM culture medium supplemented with 10% FCS, 1% glutamine, 1% penicillin/streptomycin solution, and amphotericin B at a concentration of 1 × 106 cells/mL. The pellet containing the mitochondrial fraction was resuspended in 100 μL universal immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM ethylene glycol tetraacetic acid [EGTA], 0.2% Triton X-100, 0.3% NP-40, 1 mM PMSF, leupeptin, aprotinin) for 10 minutes on ice. We also examined the contribution of these hormones to the persistence and resolution of an inflammatory response. Altered caspase expression results in delayed neutrophil apoptosis in acute pancreatitis. Procalcitonin performance in detecting serious and invasive bacterial infections in children with fever without apparent source: a systematic review and meta-analysis. Mc Laughlin GJ, Anderson ID, Grant IS, Fearon KC. In addition to killing cancer cells, chemotherapy can also destroy neutrophils and other healthy cells. Institutional ethical committee approval from the Mater Misericordiae Hospital was received for the study period. Kindzelskii AL, Huang J-B, Chaiworapongsa T, et al. Watson RW, O'Neill A, Brannigen AE, et al. Both increased41,42 and decreased ROI43-46 production have been reported in response to female sex hormones. Upon activation, neutrophils can change their shape and size and move like an amoeba cell. Copyright © 2003 by The American Society of Hematology, Copyright ©2020 by American Society of Hematology, https://doi.org/10.1182/blood-2003-02-0649. The progesterone receptor antagonist RU486 (10-6 M) reversed progesterone-induced (10-7g/mL) delays in apoptosis. T cells from women have displayed aromatase activity and male-derived T cells have increased 5α-reductase activity.39 This, in turn, may mediate the cellular immunosuppression seen in men following trauma-hemorrhage. Early Blood Biomarkers to Improve Sepsis/Bacteremia Diagnostics in Pediatric Emergency Settings. Prognostic Value of Neutrophil-to-Lymphocyte Ratio in Predicting Death Risk in Patients with Severe Hand, Foot and Mouth Disease. Cancer and cancer treatments. Both cell proliferation and cell death have been described in the setting of breast cancer in response to sex steroids.31 Female sex hormones have been shown to delay apoptosis in the peripheral blood mononuclear cells of women with normal menstrual cycles,32 B cells,33 and endothelial cells.34 Tumor necrosis factor (TNF)-induced apoptosis in monoblastoid cells is also delayed by these hormones.35 Estrogen also has proinflammatory effects on the uterus and promotes neutrophil influx. eCollection 2018. Predictive value of procalcitonin for excluding bloodstream infection: results of a retrospective study and utility of a rapid, quantitative test for procalcitonin. Bekesi G, Kakucs R, Varbiro S, et al. The effect of pregnancy on polymorphonuclear leukocyte function. The difference between specific caspase activity at 0 and 1 hour was calculated and expressed as caspase activity per microgram protein.24. 2020 Oct;27(10):2847-2852. doi: 10.1016/j.sjbs.2020.07.005. The aim of this study was to clarify the cutoff value of the neutrophillymphocyte ratio (NLR) according to procalcitonin (PCT) level in the decision-making processes for bacteremia and sepsis. Smith JA, Wiedemann MJ. Privacy, Help Neutropenia can be classified as mild, moderate, or severe, depending on the number of neutrophils in a sample of blood. (B) ROI activity was assessed in neutrophils preincubated with sex steroids for 6 hours and then stimulated with DHR (baseline [BL]) alone or in addition to PMA (respiratory burst [RB]). ICI 182 780 (ICI; 10-8 M) was added at time 0 hours and percentage apoptosis was assessed by flow cytometry at 24 hours following propidium iodide staining (n = 8). 2017 Nov;15(11):1041-1057. doi: 10.1080/14787210.2017.1400907. Tsukimori K, Maeda H, Ishida K, Nagata H, Koyanagi T, Nakano H. The superoxide generation of neutrophils in normal and preeclamptic pregnancies. White blood cells in general, and neutrophils in particular, are important for infection control in the body. Prepublished online as Blood First Edition Paper, June 5, 2003; DOI 10.1182/blood-2003-02-0649. Neutrophils, along with eosinophils and basophils, constitute a group of Cell lysates were prepared from 10 × 106 cells using caspase isolation and incubation buffers. Fraser H, Davidge ST, Clanachan AS. In children, the count will vary with age. Spyridopoulos I, Sullivan AB, Kearney M, Isner JM, Losordo DW. (see video link in the sidebar for a real neutrophil in action). Another recent study provided additional evidence for the role of neutrophils and neutrophil extracellular traps (NETs) in antiviral host defense 36). This is in contrast to LPS and granulocyte-macrophage colony-stimulating factor (GM-CSF), which delay apoptosis and prevent the induction of Fas antibody-induced apoptosis.20 The delay in neutrophil apoptosis with estradiol and progesterone is relatively subtle and not as exaggerated as that seen with LPS and GM-CSF. Progesterone via its receptor antagonizes the pro-inflammatory activity of estrogen in the mouse uterus. To understand how these caspases are activated we examined the effects of neutrophil aging on spontaneous release of cytochrome c from the mitochondria. Depending on the total white cell count (WBC) an elevation in the percentage of neutrophils may reflect either true neutrophilia (high WBC) or lymphopenia (normal or low WBC). In this study, we would like to analyze peripheral blood neutrophil-lymphocyte ratio (NLR) in breast cancer patients and its correlation with disease staging. Dose-dependent effects of estradiol, progesterone, or both in combination on spontaneous neutrophil apoptosis. Total cellular protein was extracted at 6 and 24 hours from control neutrophils and cells incubated with estradiol or progesterone (10-8 g/mL). 2003;102:2653-2659). This report represents the first study to determine the limits of the use of NLR in the diagnosis of infection or sepsis using a cutoff value of <5 when sufficient exclusion criteria are used. Normal neutrophils (1 × 106 cells/mL) were incubated for 1 hour either with or without estradiol, progesterone, or both in combination (10-8 g/mL), before the addition of LPS (1 μg/mL). Testosterone is converted to dihydrotestosterone (DHT) in the cell by 5α-reductase. The apoptosome pathway to caspase activation in primary human neutrophils exhibits dramatically reduced requirements for cytochrome c. Bain BJ, England JM. Cytosolic cytochrome c release was assessed using Western blot analysis. Results are expressed as mean ± SD unless stated otherwise. Enhanced immune responses in females, as opposed to decreased responses in males following haemorrhagic shock and resuscitation. It's important to know what are considered normal neutrophil levels, to identify whether they are in fact low.Although blood tests can show these levels, they may vary slightly from one laboratory to another. Normal neutrophils (1 × 106 cells/mL) were incubated for 6 hours with estradiol, progesterone, or both in combination (10-8 g/mL). This demonstrates that the delay in apoptosis associated with female sex steroids is mediated through their respective steroid hormone receptors. A minimum of 5000 events were collected and analyzed. The delay in apoptosis was mediated at the level of the mitochondria with decreased release of cytochrome c, which may alter caspase cleavage and activity. *P < .05 versus control at 6 hours. Cells were stimulated with 1 μL (16 μM) phorbol 12-myrisate 13-acetate (PMA) for 20 minutes at 37°C. National Library of Medicine Variations in leukocyte count during the menstrual cycle. Schroder J, Kahlke V, Staubach KH, Zabel P, Stuber F. Gender differences in human sepsis. Modulating hormonal responses may be an effective therapeutic tool in combating inflammatory diseases. Divergent immune responses in male and female mice after trauma-haemorrhage: dimorphic alterations in T lymphocyte steroidogenic enzyme activities. Normal pregnancy and preeclampsia both produce inflammatory changes in peripheral blood leukocytes akin to those of sepsis. So, if there are high neutrophil counts in blood, it means something is going on in the body. (A) Fas antibody was added and the percentage apoptosis assessed at 24 hours by propidium iodide staining and flow cytometry. The density of each band was measured using the UN-SCAN-ITgel Version 5.1 program (Silk Scientific, Orem, UT). The specific receptor antagonists, ICI 182 780 and RU486, reversed the delays in apoptosis following coincubation with exogenous estradiol and progesterone, respectively. Relationship between oxidative burst activity and CD11b expression in neutrophils and monocytes from healthy individuals: effects of race and gender. Often these patients will revert to normal neutrophil levels should chemotherapy cease. Liu S, Wang X, She F, Zhang W, Liu H, Zhao X. Following a 5-minute incubation on ice, the cells were pelleted at 1000g for 5 minutes at 4°C. 2021 Mar 16;12:639735. doi: 10.3389/fimmu.2021.639735. Crouch SPM, Crocker IP, Fletcher J. DHT had no significant effect on male and female neutrophil apoptosis in physiologic doses. The solution was then pelleted at 10 000g for 10 minutes at 4°C and the supernatant or mitochondrial fraction stored at -70°C until 30 μL was run on a Western blot. Estrogens are also cited as having a protective role in neurodegenerative and cardiac diseases through a variety of mechanisms including blockade of oxidation, antagonism of nitric oxide synthase activity,15 and interference with the apoptotic process in a variety of cell systems. Tulchinsky D, Hobel CJ. Mathur S, Mathur RS, Goust JM, Williamson HO, Fudenburg HH. Search for other works by this author on: Bone RC. Supported by grants from the Health Research Board of Ireland (Clinical Research Fellowship, E.J.M. Adjusting Your Diet Eat fruits and vegetables high in vitamin C. This vitamin is a great way to boost … New tests or markers that allow a more rapid and less costly detection of bacteremia and sepsis have been investigated. 2021 Jan 22;16(1):e0245748. Remmers DE, Wang P, Cioffi WG, Bland KI, Chaudry IH. We studied DHT (10-9 M), which had no significant effect on male (control = 24.2% ± 0.5%; DHT = 23.0% ± 2.5%; n = 4) and female (control = 16.0% ± 4.1%; DHT = 12.2% ± 3.0%; n = 4) neutrophil apoptosis at physiologic doses. One important finding of this study, which will serve as a baseline to measure future progress, is the presence of many gaps in the information on pathogens that constitute a major health risk. Aliquots of the lysates (40 μL) were diluted in caspase incubation buffer (40 μL) and 20 mM (5 μL) Ac-LEHD-AMC (caspase 9) or 20 mM Ac-DEVD-AMC (caspase 3) and incubated for 1 hour at 3°C in a 96-well Costar plate (Corning, NY). This may indicate the ability of these hormones to delay but not completely inhibit apoptosis. Effects of estradiol and progesterone on the priming effect. doi: 10.1371/journal.pone.0249301. Typically, this represents a neutrophil count greater than 7700/microliter (µL).Note that a finding of neutrophilia should not be based on a differential count reported in percentages. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. Neutrophils (1 × 106 cells/mL) were incubated with estradiol, progesterone, or both (10-7 g/mL) for 24 hours. No statistically significant correlations were found for paclitaxel compliance and sensory neuropathy in the primary analysis. 17beta-Estradiol induces an inhibitor of active caspases. The neutrophil phenotype in this case is also different from the extreme immune activation seen in SIRS.18 Female sex hormones have been shown to induce a significant female survival advantage in a number of disease states including neurodegeneration, cardiac disease, and sepsis.5,9,10 These beneficial effects may be partially mediated by the effects of these hormones on the immune system. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. 2018 Jul 15;2018:3758068. doi: 10.1155/2018/3758068. Doyle BT, O'Neill AJ, Newsholme P, Fitzpatrick JM, Watson RW. Prevention and treatment information (HHS). Cytoplasmic fractions were also extracted at 18 hours from control neutrophils and cells incubated with LPS, estradiol, progesterone, or both (10-8 g/mL). also my neutrophil relative was a 73.8 which is out of range." Am J Ther. Variants in the drug transporter ABCB1 gene are possibly associated with the neutrophil suppressing effect of … eCollection 2021. McGowan JE, Barnes MW, Finland N. Bacteremia at Boston City Hospital: occurrence and mortality during 12 selected years (1935-1972) with special reference to hospital acquired cases. If … The loss of IAP expression during HL-60 cell differentiation is caspase-independent. Bacteremia and sepsis are common causes of morbidity and mortality worldwide, with incorrect or delayed diagnoses being associated with increased mortality. Despite the effects of these hormones on spontaneous apoptosis they had no effect on Fas antibody-induced apoptosis. The ability of these hormones to prime for an additive response to lipopolysaccharide (LPS) was also investigated. Thus, female sex hormones mediate delayed neutrophil apoptosis in both sexes and enhance female intracellular production of ROIs. Generation of ROIs was evaluated by flow cytometry using the technique of Smith and Wiedemann23 Neutrophils (500 μL [1 × 106/mL]) were incubated with DHR 123 (100 μM) at 37°C for 10 minutes. Mizushima Y, Wang P, Jarrar D, Cioffi WG, Bland KI, Chaudry IH. Furthermore, during human pregnancy when female sex hormones are elevated, neutrophil apoptosis is delayed. In this study, we demonstrated that neutrophil survival was significantly increased in women. 2019 Apr 10;55(4):99. doi: 10.3390/medicina55040099. Since neutrophils account for the majority of the circulating leukocytes (about 60%), in most instances, white blood cell (WBC) counts of over 11,000/μL represent neutrophilia. PMA-induced ROI release was enhanced by estradiol and progesterone in women. Neutrophil, type of white blood cell (leukocyte) that is characterized histologically by its ability to be stained by neutral dyes and functionally by its role in mediating immune responses against infectious microorganisms. The neutrophil count may be high with infe… Preincubation for 1 hour with estradiol or progesterone had no effect on Fas antibody-induced apoptosis (Figure 6A). Knoferl MW, Angele MK, Diodato MD, et al. Propidium iodide DNA-binding staining using flow cytometry assessed neutrophil apoptosis at 24 hours. The fact that the active protein is expressed but the activity is inhibited and apoptosis delayed would indicate that these hormones inhibit the functional activity of the caspase. Djordjevic D, Rondovic G, Surbatovic M, Stanojevic I, Udovicic I, Andjelic T, Zeba S, Milosavljevic S, Stankovic N, Abazovic D, Jevdjic J, Vojvodic D. Mediators Inflamm. Please enable it to take advantage of the complete set of features! These studies demonstrate a novel and interesting paradox whereby estradiol increases neutrophil survival without an increase in neutrophil activation. However, if phagocytosis fails, apoptotic cells undergo secondary necrosis. Neither aromatase nor 5α-reductase activities have been described in the mature neutrophil, although HL60 cells and progenitor hematopoietic CD34 cells have aromatase activity.40 Because testosterone can be converted to estradiol by aromatase and would therefore not give a true indication of androgenic effects, we studied DHT rather than testosterone. Neutrophil apoptosis is delayed in patients with inflammatory bowel disease. Testosterone is converted to DHT in the cell by 5α-reductase and by aromatase to estradiol. This response was not mediated by Fas expression on the surface of the neutrophil, because this was unchanged by these steroid hormones (Figure 6B). Toward an epidemiology and natural history of SIRS (systemic inflammatory response syndrome). Similarly, 500 μL neutrophils (1 × 106cells/mL) were stained with 5 μL Fas antibody (50 μg/mL) and centrifuged at 150g for 5 minutes. ), the Mater College (Dublin, Ireland), and the Children's Research Fund, Our Lady's Hospital for Sick Children. *P < .05 versus control; **P < .05 versus estradiol; ***P < 0.05 versus progesterone (mean ± SEM). There are two major causes for neutrophilia: • We are grateful to Dr Colm Morrissey, Mater Misericordiae Hospital, Conway Institute for his critical reading of this paper. From the Department of Surgery, Mater Misericordiae University Hospital; Conway Institute of Biomolecular and Biomedical Research, University College Dublin; Department of Paediatrics, Coombe Women's Hospital; and Department of Neurology, Our Lady's Hospital for Sick Children, University College Dublin, Ireland. (B) Densitometry of the 3 blots was performed and the density of each band (percent of total pixels) expressed as mean ± SEM. Harvey BJ, Doolan CM, Condliffe SB, Renard C, Alzamora R, Urbach V. Non-genomic convergent and divergent signalling of rapid responses to aldosterone and estradiol in mammalian colon. eCollection 2021. Estradiol administration after trauma-haemorrhage improves cardiovascular and hepatocellular functions in male animals. Cyclic variations in white cell sub-populations in the human menstrual cycle: correlations with progesterone and estradiol. Numerous factors may cause neutropenia through destruction, decreased production or abnormal storage of neutrophils. Our study used the intracellular dye DHR 123. □ indicates male (n = 6) and ▪ indicates female controls (n = 6). (A) Cytosolic cytochrome c release was assessed using Western blot analysis. Low neutrophil count. The effects of estradiol and progesterone on spontaneous cytochrome c release: Western blot and the expression of MnSOD in cytosolic and mitochondrial fractions. Neutrophil production takes place in the bone marrow from myeloid cells. Error bars indicate mean ± SD. This number is also written as 1,500 to 8,000/mm3. FOIA Patients were grouped according to the following PCT criteria: levels <0.05 ng/ml (healthy group), 0.05-0.5 ng/ml (local infection group), 0.5-2 ng/ml (systemic infection group), 2-10 ng/ml (sepsis group), and >10 ng/ml (sepsis shock group). Cancer chemotherapy is a common cause of neutropenia. Your doctor also may need to repeat the blood test to confirm your results or order additional tests … Neutrophil counts may be lower than normal for any number of reasons, including both diseases and treatments. Dys-regulated expression of neutrophil apoptosis in the systemic inflammatory response syndrome. For example, a 70% Relative Neutrophil Count may seem within normal limits. However, because similar effects are seen with caspase 9 activity, we investigated cytochrome c release and the mitochondria as a site for apoptotic inhibition. Would you like email updates of new search results? Estradiol and progesterone did not increase the ability of LPS to delay neutrophil apoptosis or to increase CD11b expression (Figure 4). We postulate that by altering the phenotype of the neutrophil these hormones prepare the female host for potential inflammatory insults. Testosterone-induced responses would therefore not give a true indication of androgenic effects. The supernatant was aspirated, the pellet resuspended in 400 μL PBS, and the mean channel fluorescence assessed by flow cytometry. DHT is the active metabolite mediating androgenic effects in the cell. The reaction was then halted by placing samples on ice. Normal haematological values: sex difference in neutrophil count. The mechanism and use of these hormones as an adjunctive treatment in sepsis or shock warrant further investigation. Clipboard, Search History, and several other advanced features are temporarily unavailable. Neutrophils were isolated from healthy male (n = 26) and female (n = 26) volunteers with an age range of 26 to 33 years; fully informed consent was given by all volunteers. Both estradiol and progesterone had an inhibitory effect on cytochrome c release indicating the ability of these hormones to regulate apoptosis at the level of the mitochondria and thus preventing caspase activation and apoptosis. Ştefanescu S, Cocoş R, Turcu-Stiolica A, Mahler B, Meca AD, Giura AMC, Bogdan M, Shelby ES, Zamfirescu G, Pisoschi CG. Neutrophils are short-lived with an … Tissue-protective effects of estrogen involve regulation of caspase gene expression. We further show that these differences are cell specific and likely driven by sex hormones. Brannigan AE, O'Connell PR, Hurley H, et al. Female neutrophils had an increased baseline ROI production on coincubation with estradiol and progesterone in combination and therefore the Δ difference between baseline and PMA-stimulated was reduced. Progesterone but not estradiol treatment did result in a decrease in active caspase 3 protein expression at 24 hours (Figure 7B).
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