Isolation and Propagation in Vitro of Peritoneal Mesothelial Cells

1989 ◽  
Vol 9 (4) ◽  
pp. 341-347 ◽  
Author(s):  
J. Thomas Hjelle ◽  
Barbara T. Golinska ◽  
Diane C. Waters ◽  
Kevin R. Steidley ◽  
David R. McCarroll ◽  
...  
Keyword(s):  
Peritoneal Dialysis ◽  
Cell Biology ◽  
Life Support ◽  
Cultured Cells ◽  
Mesothelial Cell ◽  
Experimental System ◽  
Mesothelial Cells ◽  
Growth Media ◽  
Peritoneal Mesothelial Cells

Mesothelial cells lining the peritoneal cavity are the primary site of molecular exchange during peritoneal dialysis, a life support system for over 50 000 patients worldwide. In this study, techniques are described for the isolation and propagation in culture of peritoneal mesothelial cells from rats and rabbits. For comparison, mesothelial cells were also obtained from the serosal surface of human colonic tissue. By electron microscopy the cultured cells were found to exhibit microvilli, a well developed endoplasmic reticulum and golgi apparatus, micropinocytotic vesicles, and lipid-filled intracellular vesicles. Immunochemical probes revealed the expression by these cells in vitro of cytokeratin, fibronectin, vimentin, and keratin, but not von Willebrand factor. Mesothelial cells from rat, rabbit, and human exhibited contact inhibition, but differences in growth rates and dependence on supplements to the growth media. This work provides a multispecies comparison of the behavior of mesothelial cells in vitro for the purpose of developing an experimental system for the study of mesothelial cell biology and the role of these cells in peritoneal dialysis.

Mesothelial Cells

2007 ◽  
Vol 27 (2_suppl) ◽  
pp. 110-115 ◽  
Author(s):  
Susan Yung ◽  
Chan Tak Mao
Keyword(s):  
Mesothelial Cell ◽  
In Vitro Models ◽  
Mesothelial Cells ◽  
Dynamic Membrane ◽  
Peritoneal Mesothelial Cells ◽  
Immunohistochemical Markers ◽  
Vitro Model ◽  
And Function

♦ Background The introduction of peritoneal dialysis (PD) as a modality of renal replacement therapy has provoked much interest in the biology of the peritoneal mesothelial cell. Mesothelial cells isolated from omental tissue have immunohistochemical markers that are identical to those of mesothelial stem cells, and omental mesothelial cells can be cultivated in vitro to study changes to their biologic functions in the setting of PD. ♦ Method The present article describes the structure and function of mesothelial cells in the normal peritoneum and details the morphologic changes that occur after the introduction of PD. Furthermore, this article reviews the literature of mesothelial cell culture and the limitations of in vitro studies. ♦ Results The mesothelium is now considered to be a dynamic membrane that plays a pivotal role in the homeostasis of the peritoneal cavity, contributing to the control of fluid and solute transport, inflammation, and wound healing. These functional properties of the mesothelium are compromised in the setting of PD. Cultures of peritoneal mesothelial cells from omental tissue provide a relevant in vitro model that allows researchers to assess specific molecular pathways of disease in a distinct population of cells. Structural and functional attributes of mesothelial cells are discussed in relation to long-term culture, proliferation potential, age of tissue donor, use of human or animal in vitro models, and how the foregoing factors may influence in vitro data. ♦ Conclusions The ability to propagate mesothelial cells in culture has resulted, over the past two decades, in an explosion of mesothelial cell research pertaining to PD and peritoneal disorders. Independent researchers have highlighted the potential use of mesothelial cells as targets for gene therapy or transplantation in the search to provide therapeutic strategies for the preservation of the mesothelium during chemical or bacterial injury.

Cancer Antigen 1251S Locally Produced in the Peritoneal Cavity during Continuous Ambulatory Peritoneal Dialysis

1994 ◽  
Vol 14 (2) ◽  
pp. 132-136 ◽  
Author(s):  
Ger C.M. Koomen ◽  
Michiel G.H. Betjes ◽  
Oésirée Zemel ◽  
Raymond T. Krediet ◽  
Frans J. Hoek
Keyword(s):  
Peritoneal Dialysis ◽  
Continuous Ambulatory Peritoneal Dialysis ◽  
Peritoneal Cavity ◽  
Mesothelial Cell ◽  
Linear Increase ◽  
Mesothelial Cells ◽  
Ca 125 ◽  
Cancer Antigen ◽  
The Relationship

The local production of cancer antigen (CA) 125 in the peritoneal cavity of 14 continuous ambulatory peritoneal dialysis patients was studied. In addition, the relationship between the concentration of mesothelial cells and CA 125 in the peritoneal dialysate effluent was examined. The median results and ranges were as follows: plasma CA 125 14 U/mL (range 10 23), dialysate CA 125 18 U/mL (range 5.2 76), dialysate/plasma ratio 1. 9 (range 0.61 -5.4), and number of mesothelial cells 400/mL (range 10 5000). Peritoneal concentrations of mesothelial cellsand CA 125 were positively correlated (r = 0.50, p < 0.01). Using a monoclonal antibody, CA 125-positive cells were found in the cytospin preparations of the cells of dialysis effluents. All these CA 125 positive cells were also positive for cytokeratin used as a mesothelial cell marker. In vitro experiments using mesothelial cells in monolayers showed a linear increase in CA 125 concentration both in time and in relation to the number of mesothelial cells. From these experiments a production rate of 24 U/hour/1 06 cells could be calculated. It is therefore concluded that CA 125 is locally produced in the peritoneal cavity during CAPD and that the mesothelial cells are the major source of this CA 125.

Mesothelial Cell Adherence to Vascular Prostheses and Their Subsequent Growth In Vitro

1994 ◽  
Vol 3 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Apollo Pronk ◽  
Arthur A.G.M. Hoynck Van Papendrecht ◽  
Piet Leguit ◽  
Henri A. Verbrugh ◽  
Roel P.A.J. Verkooyen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Attachment ◽  
Mesothelial Cell ◽  
Mesothelial Cells ◽  
Subsequent Growth ◽  
Vascular Prostheses ◽  
Peritoneal Mesothelial Cells ◽  
Viable Cells ◽  
Human Peritoneal Mesothelial Cells

Cell seeding may decrease the thrombogenicity of implanted vascular grafts, but its application is hampered by the limited availability of autologous endothelial cells. Human peritoneal mesothelial cells have blood flow supporting qualities and are readily available. This study investigated the adherence of mesothelial cells to vascular prostheses and their subsequent growth in vitro. Circular pieces of various vascular prosthetic materials were seeded with 51Chromium-labeled mesothelial and endothelial cells and left for either 5, 15, 30, 60, and 120 minutes. The unattached cells were removed and the degree of cell attachment was measured. The number of mesothelial cells to Dacron increased during the first 60 min up to 35.2 % of the seeded inoculum whereafter a plateau was reached. Scanning electron microscopy showed spreaded mesothelial cells adherent to the Dacron fibers. A significant increase in adherence was observed after preincubation of Dacron with 10 μg/mL fibronectin, but no improvement was found after preincubation with human serum albumin or gelatin. Mesothelial cells adhered better to Gelcoated than to Gelsealed or plain Dacron. The adherence of mesothelial cells to ePTFE (Teflon) was significantly poorer. No significant differences in adherence were found between mesothelial and endothelial cells. Mesothelial cell growth on Dacron resulted in a modest increase in the number of viable cells during 27 days, which implies biocompatibility of Dacron and mesothelial cells in vitro.

Peritoneal Mesothelial Cell Culture and Biology

2006 ◽  
Vol 26 (2) ◽  
pp. 162-193 ◽  
Author(s):  
Susan Yung ◽  
Fu Keung Li ◽  
Tak Mao Chan
Keyword(s):  
Cell Culture ◽  
Cell Biology ◽  
Mesothelial Cell ◽  
Mesothelial Cells ◽  
Peritoneal Mesothelial Cells ◽  
Immunohistochemical Markers ◽  
Tumor Dissemination ◽  
Structural And Functional Properties ◽  
Peritoneal Mesothelium ◽  
Peritoneal Mesothelial Cell

The peritoneal mesothelium is composed of an extensive monolayer of mesothelial cells that lines the body's serous cavity and internal organs and was previously thought to act principally as a protective nonadhesive lubricating surface to facilitate intracoelomic movement. With the introduction of peritoneal dialysis over three decades ago, there has been much interest in the cell biology of peritoneal mesothelial cells. Independent studies have highlighted specific properties of the peritoneal mesothelial cell, including antigen presentation, regenerative properties, clearance of fibrin; synthesis of cytokines, growth factors, and matrix proteins; and secretion of lubricants to protect the tissue from abrasion, adhesion, infection, and tumor dissemination. It is now evident that the mesothelium is not merely a passive membrane but, rather, a dynamic membrane that contributes substantially to the structural, functional, and homeostatic properties of the peritoneum. Since peritoneal mesothelial cells in culture possess immunohistochemical markers identical to mesothelial stem cells, the culture of mesothelial cells offers researchers an essential tool to assess their morphologic, structural, and functional properties. This review will discuss current procedures to isolate peritoneal mesothelial cells from human omental specimens, animal sources, and spent dialysate. Furthermore, the functional and morphologic properties of mesothelial cells are discussed, together with the potential use of mesothelial cell culture in research and clinical applications.

Improved In Vitro Biocompatibility of Bicarbonate-Buffered Peritoneal Dialysis Fluid

2006 ◽  
Vol 26 (6) ◽  
pp. 664-670 ◽  
Author(s):  
Nicolas Grossin ◽  
Marie-Paule Wautier ◽  
Jean-Luc Wautier ◽  
Pierre Gane ◽  
Redouane Taamma ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Peritoneal Dialysis ◽  
Growth Factor ◽  
Cell Biology ◽  
Transforming Growth Factor ◽  
Mesothelial Cell ◽  
Long Term Treatment ◽  
Peritoneal Dialysis Fluid ◽  
New Generation

Background Conventional peritoneal dialysis fluids (PDFs) have been shown to damage the mesothelial layer and are associated with the development of peritoneal fibrosis and neoangiogenesis. New-generation PDFs have therefore been developed with physiological pH and reduced levels of glucose degradation products (GDPs), precursors of advanced glycation end products (AGEs). In this work, we evaluated and compared the improved biocompatibility of two new-generation PDFs (Balance and bicaVera) using mesothelial cell biology; we also compared them to a standard PDF (stay·safe) (all PDFs by Fresenius Medical Care, Fresnes, France). Methods stay·safe, Balance, and bicaVera were tested for their effect on human peritoneal mesothelial cell (HPMC) viability by measuring cell proliferation and apoptosis, and oncosis induction. The formation of AGEs was evaluated by immunoassay. Transforming growth factor beta-1 and vascular endothelial growth factor (VEGF) were immunoassayed in HPMC supernatants exposed to the above PDFs. Results At 15 g/L glucose concentration, HPMC exposure to bicaVera resulted in higher cell proliferation compared to Balance ( p < 0.001) and stay·safe ( p < 0.001). Compared to the lactate-buffered PDFs (Balance and stay·safe), oncosis was significantly lower in cells exposed to bicaVera ( p < 0.05). bicaVera, containing lower amounts of GDPs, generated less AGE formation ( p < 0.05) and VEGF production ( p < 0.05) than either Balance or stay·safe. Conclusions New-generation PDFs with physiological pH and lower GDP levels, especially if bicarbonate-buffered (bicaVera), have fewer in vitro toxic effects on mesothelial cells and may contribute to peritoneal preservation, thus improving long-term treatment of PD patients.

scholarly journals miRNA589 Regulates Epithelial-Mesenchymal Transition in Human Peritoneal Mesothelial Cells

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Ke Zhang ◽  
Hao Zhang ◽  
Xun Zhou ◽  
Wen-bin Tang ◽  
Li Xiao ◽  
...  
Keyword(s):  
Epithelial Mesenchymal Transition ◽  
Mesothelial Cell ◽  
Mesothelial Cells ◽  
Control Group ◽  
Peritoneal Fibrosis ◽  
Mesenchymal Transition ◽  
Peritoneal Mesothelial Cells ◽  
Human Peritoneal Mesothelial Cells ◽  
E Cadherin

Background. microRNA (miRNA, miR) are thought to interact with multiple mRNAs which are involved in the EMT process. But the role of miRNAs in peritoneal fibrosis has remained unknown.Objective. To determine if miRNA589 regulates the EMT induced by TGFβ1 in human peritoneal mesothelial cell line (HMrSV5 cells).Methods. 1. Level of miR589 was detected in both human peritoneal mesothelial cells (HPMCs) isolated from continuous ambulatory peritoneal dialysis (CAPD) patients’ effluent and HMrSV5 cells treated with or without TGFβ1. 2. HMrSV5 cells were divided into three groups: control group, TGFβ1 group, and pre-miR-589+TGFβ1 group. The level of miRNA589 was determined by realtime PCR. The expressions of ZO-1, vimentin, and E-cadherin in HPMCs were detected, respectively.Results. Decreased level of miRNA589 was obtained in either HPMCs of long-term CAPD patients or HMrSV5 cells treated with TGFβ1. In vitro, TGFβ1 led to upregulation of vimentin and downregulation of ZO-1 as well as E-cadherin in HMrSV5 cells, which suggested EMT, was induced. The changes were accompanied with notably decreased level of miRNA589 in HMrSV5 cells treated with TGFβ1. Overexpression of miRNA589 by transfection with pre-miRNA589 partially reversed these EMT changes.Conclusion. miRNA589 mediates TGFβ1 induced EMT in human peritoneal mesothelial cells.

Glycerol Toxicity for Human Peritoneal Mesothelial Cells in Culture: Comparison with Glucose

1994 ◽  
Vol 17 (5) ◽  
pp. 252-260 ◽  
Author(s):  
J. Witowski ◽  
J. Knapowski
Keyword(s):  
Mesothelial Cell ◽  
Cellular Protein ◽  
Mesothelial Cells ◽  
Dependent Manner ◽  
Peritoneal Mesothelial Cells ◽  
Proline Incorporation ◽  
Human Peritoneal Mesothelial Cells ◽  
Dose Dependent Decrease ◽  
Dose Dependent

Glycerol has been proposed as a substitute osmotic agent for glucose in peritoneal dialysis fluids. We have compared the effect of glycerol and glucose on the function of human peritoneal mesothelial cells (HPMC) in vitro. The viability of HPMC was not affected by glycerol (up to 250 mM), whereas it was reduced by glucose in a time- and dose-dependent manner, as assessed by the LDH release. Although the incubation of HPMC with glycerol induced a dose-dependent decrease in HPMC proliferation, the effect was significantly less inhibitory than that produced by glucose. In HPMC treated with 90 mM of glycerol or glucose the incorporation of [3H]-thymidine had reached 79.0±19.3% and 55.3+4.0% of the control (p<0.05 and p<0.01), respectively. As measured by the [methyl-14C]-choline incorporation, the intracellular amount of newly synthesized phospholipids was reduced from (cpm/μg cellular protein) 147±58 in control HPMC to 59+15 in cells exposed to 90 mM of glucose (p<0.01), but not affected by glycerol (163±65). On the other hand, both glycerol and glucose (90 mM) decreased the synthesis of proteins (as assessed by the [3H]-proline incorporation) and interfered with potassium (86Rb) transport mechanisms in HPMC. Our data suggest that there exist some possibly advantageous aspects of glycerol as far as mesothelial cell biocompatibility profile is concerned.

scholarly journals Hyperbranched Polyglycerol is an Efficacious and Biocompatible Novel Osmotic Agent in a Rodent Model of Peritoneal Dialysis

2013 ◽  
Vol 33 (1) ◽  
pp. 15-27 ◽  
Author(s):  
Asher A. Mendelson ◽  
Qiunong Guan ◽  
Irina Chafeeva ◽  
Gerald A. da Roza ◽  
Jayachandran N. Kizhakkedathu ◽  
...  
Keyword(s):  
Peritoneal Dialysis ◽  
Flow Cytometric Analysis ◽  
Mesothelial Cell ◽  
Mesothelial Cells ◽  
Rodent Model ◽  
Water Soluble ◽  
Control Group ◽  
Sprague Dawley ◽  
Hyperbranched Polyglycerol

♦ObjectivesTo enhance the effectiveness of peritoneal dialysis (PD), new biocompatible PD solutions may be needed. The present study was designed to test the efficacy and biocompatibility of hyperbranched polyglycerol (HPG)—a nontoxic, nonimmunogenic water-soluble polyether polymer—in PD.♦MethodsAdult Sprague–Dawley rats were instilled with 30 mL HPG solution (molecular weight 3 kDa; 2.5% – 15%) or control glucose PD solution (2.5% Dianeal: Baxter Healthcare Corporation, Deerfield, IL, USA), and intraperitoneal fluid was recovered after 4 hours. Peritoneal injury and cellular infiltration were determined by histologic and flow cytometric analysis. Human peritoneal mesothelial cells were assessed for viability in vitro after 3 hours of PD fluid exposure.♦ResultsThe 15% HPG solution achieved a 4-hour dose-related ultrafiltration up to 43.33 ± 5.24 mL and a dose-related urea clearance up to 39.17 ± 5.21 mL, results that were superior to those with control PD solution ( p < 0.05). The dialysate-to-plasma (D/P) ratios of urea with 7.5% and 15% HPG solution were not statistically different from those with control PD solution. Compared with fluid recovered from the control group, fluid recovered from the HPG group contained proportionally fewer neutrophils (3.63% ± 0.87% vs 9.31% ± 2.89%, p < 0.0001). Detachment of mesothelial cells positive for human bone marrow endothelial protein 1 did not increase in the HPG group compared with the stain control ( p = 0.1832), but it was elevated in the control PD solution group (1.62% ± 0.68% vs 0.41% ± 0.31%, p = 0.0031). Peritoneal biopsies from animals in the HPG PD group, compared with those from control PD animals, demonstrated less neutrophilic infiltration and reduced thickness. Human peritoneal mesothelial cell survival after HPG exposure was superior in vitro ( p < 0.0001, 7.5% HPG vs control; p < 0.01, 15% HPG vs control). Exposure to glucose PD solution induced cytoplasmic vacuolation and caspase 3–independent necrotic cell death that was not seen with HPG solution.♦ConclusionsOur novel HPG PD solution demonstrated effective ultrafiltration and waste removal with reduced peritoneal injury in a rodent model of PD.

scholarly journals FC 105LITHIUM PRESERVES PERITONEAL MEMBRANE INTEGRITY BY REDUCING MESOTHELIAL CELL ΑB-CRYSTALLIN

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Rebecca Herzog ◽  
Guadalupe González ◽  
Maria Bartosova ◽  
Juan Manuel Sacnun ◽  
Lisa Daniel-Fischer ◽  
...  
Keyword(s):  
Cell Injury ◽  
Membrane Integrity ◽  
Mesothelial Cell ◽  
Mesothelial Cells ◽  
Peritoneal Membrane ◽  
Peritoneal Fibrosis ◽  
Peritoneal Mesothelial Cells ◽  
Αb Crystallin

Abstract Background and Aims Renal replacement therapy by peritoneal dialysis (PD) is limited in use and duration by progressive impairment of peritoneal membrane integrity and homeostasis. Preservation of peritoneal membrane integrity during chronic PD remains an urgent but long-unmet medical need. PD therapy failure results from peritoneal fibrosis and angiogenesis caused by hypertonic PD fluid (PDF)-induced mesothelial cytotoxicity. The incompletely defined pathophysiological mechanisms involved confound informed selection of therapeutic targets. Addition of cytoprotective agents to PDF have been shown to counteract pathophysiological mechanisms induced by current PDF. Lithium is a well described inhibitor of glycogen synthase kinase 3β and has recently been shown to also have nephroprotective effects in low doses. Here, we aim to characterize icodextrin-based, PDF-induced cellular injury with a combined omics approach and to investigate the effects of LiCl on the PD-induced observed molecular perturbations. Method To investigate mechanisms of acute cellular damage by PDF we chose an in vitro model of primary omental-derived peritoneal mesothelial cells with direct exposure to icodextrin-based PDF, followed by short-term or extended recovery for detection of short-term and long-term changes in transcriptome, proteome, and cell injury. 0, 2.5 or 10 mM LiCl were added to the PDF. In-vitro findings were validated in peritoneal biopsies (n=41) from pediatric PD and CDK5 patients or healthy controls and peritoneal effluents from adult and pediatric PD patients (n=27) or ascites samples (n=4) as control. For in-vivo experiments, healthy and uremic mice (C57/Bl6, female) were chronically exposed to PD-fluid without or with the addition of 5 mM LiCl via an implanted catheter. In-vivo overexpression of CRYAB was induced by i.p. injection of an adenoviral vector. All animal experiments and use of patient samples were approved by the local ethics committees and performed according to animal protection laws or the Declaration of Helsinki, respectively. Results LiCl significantly improved mesothelial cell survival in a dose-dependent manner. Combined transcriptomic and proteomic characterization of icodextrin-based PDF-induced mesothelial cell injury identified αB-crystallin as the mesothelial cell protein most significantly and consistently counter-regulated by LiCl. In-vitro and in-vivo overexpression of αB-crystallin triggered a fibrotic phenotype and PDF-like upregulation of vascular endothelial growth factor (VEGF), CD31-positive cells, and TGFβ-independent activation of TGFβ-regulated targets. In contrast, αB-crystallin knock-down decreased VEGF expression and early mesothelial-to-mesenchymal transition (MMT). LiCl reduced VEGF release and counteracted fibrosis- and angiogenesis-associated processes. αB-crystallin in patient-derived mesothelial cells was specifically upregulated in response to PDF and increased in peritoneal mesothelial cells from pediatric PD patient biopsies, correlating with markers of angiogenesis and fibrosis. Conclusion The cytoprotective effects of LiCl-supplemented PDF may be explained by counter-regulation of PD-induced angiogenesis via the novel target αB-crystallin. Reduction of mesothelial cell damage, peritoneal fibrosis and VEGF suggests therapeutic potential of this intervention. Repurposing LiCl as a cytoprotective PDF additive may offer a translatable therapeutic strategy to combat peritoneal membrane deterioration during PD therapy. Further study of LiCl-supplemented PDF is merited as a realistic approach to improving treatment longevity and patient outcomes during PD treatment.

HSP-Mediated Cytoprotection of Mesothelial Cells in Experimental Acute Peritoneal Dialysis

2010 ◽  
Vol 30 (3) ◽  
pp. 294-299 ◽  
Author(s):  
Thorsten O. Bender ◽  
Michael Böhm ◽  
Klaus Kratochwill ◽  
Hans Lederhuber ◽  
Michaela Endemann ◽  
...  
Keyword(s):  
Peritoneal Dialysis ◽  
Stress Response ◽  
Mesothelial Cell ◽  
Lactic Dehydrogenase ◽  
Mesothelial Cells ◽  
Cellular Stress Response ◽  
Protein Loss ◽  
Promising Therapeutic Approach

♦ BackgroundLow biocompatibility of peritoneal dialysis solution (PDS) injures mesothelial cells but also induces heat shock proteins (HSP), the main effectors of the cellular stress response. This study investigated whether overexpression of HSP upon pharmacologic induction results in cytoprotection of mesothelial cells in experimental PD.♦ MethodsStress response of mesothelial cells upon exposure to PDS was pharmacologically manipulated using glutamine as a co-inducer. In vitro, HSP-mediated cytoprotection was assessed by simultaneous measurements of HSP expression using Western blot analysis and viability testing using release of lactic dehydrogenase in cultured human mesothelial cells. In vivo, detachment of mesothelial cells from their peritoneal monolayer was assessed following exposure to PDS with and without the addition of glutamine in the acute rat model of PD.♦ ResultsIn vitro, mesothelial cell viability following exposure to PDS was significantly improved upon pharmacologic co-induction of HSP expression by glutamine (226% ± 29% vs 190% ± 19%, p = 0.001). In vivo, mesothelial cell detachment during exposure to PDS was reduced upon pharmacologic induction of HSP expression by glutamine (93 ± 39 vs 38 ± 38 cells, p = 0.044), resulting in reduced peritoneal protein loss (75 ± 7 vs 65 ± 4 mg, p = 0.045).♦ ConclusionOur results represent the first study of pharmacologic manipulation of HSP expression for cytoprotection of mesothelial cells following acute in vitro and in vivo exposure to PDS. PDS with added glutamine might represent a promising therapeutic approach against low biocompatibility of PDS but needs validation in a chronic PD model.

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