RU2616243C1 - Method for atherosclerotic plaques cellular composition determination - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method comprises surgical material taking, plaque grinding and enzymatic treatment, filtration, cell suspension staining to determine live/dead cells, followed by staining with monoclonal antibodies, flow cytometry conducting, for cell suspension preparation, a nylon filter is used with a pore size of 40 microns.

EFFECT: increased separation efficiency and accuracy of determination of the live cells population required for the study of composition of the immune cells from atherosclerotic plaques at different models of the flow cytometer.

1 dwg, 2 tbl, 1 ex

Description

The invention relates to medicine, in particular to cellular and molecular technology, and can be used in medical research in cardiology to study the cellular composition in atherosclerotic plaques in order to identify the immunological mechanisms of their maturation and rupture.

The complexity of the structure of atherosclerotic plaques has been known since the days of Virchow [R. Virkhov. Cellular pathology, as applied to the microscopic anatomy of normal and abnormal: trans. with him. Th 16. - M, in the printing house of Katkov and K., 1859. - P. 480], when it was found that they consist of various cellular components, including a large number of immune and smooth muscle cells. At the same time, the generally accepted theory that the development of atherosclerotic plaque occurs when foreign substance, modified lipoproteins, is deposited in the vascular wall, could not explain the cellular heterogeneity of the plaques. In this regard, an alternative hypothesis was formulated by R. Ross and L. Harker [Faggiotto A, Ross R, Harker L. Studies of hypercholesterolemia in the nonhuman primate. I. Changes that lead to fatty streak formation. Arteriosclerosis. - 1984. - No. 4. - P. 323-340; Ross R, Harker L. Hyperlipidemia and atherosclerosis. Science. - 1976. - No. 193. - P. 1094-1100] that the trigger factor in atherosclerosis is endothelial damage, accompanied by adhesion of leukocytes and platelets to its surface. Monocytes pass through the endothelial lining and turn into macrophages. At the same time, smooth muscle cells migrate from the media to the subendothelium and change their phenotype (dedifferentiation). It is believed that initially the immune system attempts to destroy a foreign object, in particular by phagocytosis by macrophages [Kruth HS Sequestration of aggregated low density lipoproteins by macrophages. Curr Opin Lipidol. - 2002. - No. 13. - P. 483-488]. Macrophages absorb lipoproteins, but this leads to the death of the phagocytes themselves and the formation of a focus of necrosis [Geng YJ, Libby P Progression of atheroma: A struggle between death and procreation. Arterioscler Thromb Vase Biol. - 2002. - No. 22. - P. 1370-1380], followed by capsule formation. Further development of atherosclerotic plaque is determined by the activity of the immune response, in particular inflammation.

The contribution of inflammation to the development of atherosclerosis has been discussed since the time of Rudolf Virchow, however, its decisive role in the growth and destruction of plaques has been recognized relatively recently. Inflammation is a complex phenomenon, including the migration of reactive cells, in particular lymphocytes and monocytes, and their multi-stage activation followed by the release of various cytokines.

The content of many pro-inflammatory cytokines in the blood is known to correlate with mortality in patients with coronary heart disease [Libby P, Okamoto Y, Rocha VZ, Folco E. Inflammation in atherosclerosis: transition from theory to practice. Circ J. - 2010. - No. 74. - P. 213-220; Stefanadi E, Tousoulis D, Papageorgiou N, Briasoulis A, Stefanadis C. Inflammatory biomarkers predicting events in atherosclerosis. Curr Med Chem. - 2010. - No. 17. - P. 1690-1707]. Thus, it was shown that an increase in the content of a certain group of lymphocytes in the blood is associated with an increase in the frequency of cardiovascular events in patients with coronary heart disease, rheumatoid arthritis, diabetes mellitus and chronic renal failure [Libby P. Role of inflammation in atherosclerosis associated with rheumatoid arthritis. Am J Med. - 2008. - No. 121. - P. S21-S31; Liuzzo G, Biasucci LM, Trotta G, Brugaletta S, Pinnelli M, Digianuario G, Rizzello V, Rebuzzi AG, Rumi C, Maseri A, Crea F. Unusual CD4_CD28null T lymphocytes and recurrence of acute coronary events. J Am Coll Cardiol. - 2007. - No. 50. - P. 1450-1458; Giubilato S, Liuzzo G, Brugaletta S, Pitocco D, Graziani F, Smaldone C, Montone RA, Pazzano V, Pedicino D, Biasucci LM, Ghirlanda G, Crea F. Expansion of CD4 + CD28null T-lymphocytes in diabetic patients: exploring new pathogenetic mechanisms of increased cardiovascular risk in diabetes mellitus. Eur Heart J. - 2011. - No. 32. - P. 1214-1226]. He not only systemic inflammation, but also a local remote inflammatory process affects the state of atherosclerotic plaques. For example, the association of incidence of periodontitis and coronary atherosclerosis has been identified [Humphrey LL, Fu R, Buckley DI, Freeman M, Helfand M. Periodontal disease and coronary heart disease incidence: a systematic review and meta-analysis. J Gen Intern Med. - 2008. - No. 23. - P. 2079-2086]. Even more important is the fact that inflammation in the plaque can cause its growth and rupture of the tire, leading to such clinical manifestations as unstable angina pectoris or acute myocardial infarction.

Like inflammation of any other localization, plaque inflammation is characterized by the infiltration and activation of immune cells [Naruko T, Ueda M, Haze K, van der Wal AC, van der Loos CM, Itoh A, Komatsu R, Ikura Y, Ogami M, Shimada Y, Ehara S, Yoshiyama M, Takeuchi K, Yoshikawa J, Becker AE. Neutrophil infiltration of culprit lesions in acute coronary syndromes. Circulation. - 2002. - No. 106. - P. 2894-2900; Taleb S, Tedgui A, Mallat Z. Regulatory T-cell immunity and its relevance to atherosclerosis. J Intern Med. - 2008. - No. 263. - R. 489-199]. However, a detailed analysis of the state of individual cells in plaques has not yet been performed, with the exception of traditional immunohistochemical analysis. Histochemical analysis revealed that complicated plaques obtained during carotid endarterectomy contain more lymphocytes [Rossmann A, Henderson B, Heidecker B, Seiler R, Fraedrich G, Singh M, Parson W, Keller M, Grubeck-Loebenstein B, Wick G T-cells from advanced atherosclerotic lesions recognize hHSP60 and have a restricted T-cell receptor repertoire. Exp Gerontol. - 2008. - No. 43. - P. 229-237] and dendritic cells [Erbel C, Sato K, Meyer FB, Kopecky SL, Frye RL, Goronzy JJ, Weyand CM. Functional profile of activated dendritic cells in unstable atherosclerotic plaque. Basic Res Cardiol. - 2007. - No. 102. - P. 123-132] than in control samples of arteries.

Despite the large amount of clinical and experimental data on the formation, maturation and rupture of atherosclerotic plaques, the mechanisms of these phenomena remain largely unknown. It has been shown that systemic inflammation plays an important role in this process [Tahara N, Imaizumi T, Virmani R, Narula J. Clinical feasibility of molecular imaging of plaque inflammation in atherosclerosis. J Nucl Med. - 2009. - No. 50. - P. 331-334].

Histochemical analysis [Gewaltig J, Kummer M, Koella C, Cathomas G, Biedermann BC. Requirements for CD8 T-cell migration into the human arterial wall. Hum Pathol. - 2008. - No. 39. - P. 1756-1762] and polymerase chain reaction with DNA isolated from atherosclerotic plaques [De Palma R, Del Galdo F, Abbate G, Chiariello M, Calabro R, Forte L, Cimmino G, Papa MF, Russo MG, Ambrosio G , Giombolini C, Tritto I, Notaristefano S, Berrino L, Rossi F, Golino P. Patients with acute coronary syndrome show oligoclonal T-cell recruitment within unstable plaque: evidence for a local, intracoronary immunologic mechanism. Circulation. - 2006. - No. 113. - P. 640-646], revealed the activation of T cells in an unstable plaque, which greatly expanded our knowledge of the factors associated with the instability of plaques.

However, this knowledge is not enough, since the polymerase chain reaction makes it possible to identify only the general characteristics of T-lymphocytes, and using immunohistochemistry, only one or two cellular antigens can be determined.

For a more complete description of lymphocytes and their role in the immunological mechanisms of plaque maturation and rupture, it is necessary to analyze lymphocytic phenotypes and their distribution in individual plaques. The only modern technology to accomplish these tasks is multicolor flow cytometry. One of the main obstacles to using modern multicolor flow cytometry technology to comprehensively study the parameters of cells contained in a plaque is the difficulty of isolating cells from a plaque without damaging the surface cell markers that determine the cell phenotype.

A known method for the isolation of lymphocytes from the tissue of atherosclerotic plaques of the carotid arteries. The method used the method of separation of tissue into small blocks, which were then placed in a cultivation medium for 4-6 hours with various activators of lymphocyte migration. Then, individual lymphocytes that migrated from tissue to medium during cultivation were collected, stained for surface markers CD4 and CD8, and examined using flow cytometry [Curry MP, Norris S, Golden-Mason L, Doherty DG, Deignan T, Collins C, Traynor O, McEntee GP, Hegarty JE, O'Farrelly C. Isolation of lymphocytes from normal adult human liver suitable for phenotypic and functional characterization. J Immunol Methods. - 2000. - No. 242 (1-2). - R. 21-31]. In this method, enzymatic hydrolysis of tissues and cell filtration were not carried out, only cells migrated into the culture medium were analyzed. This made it possible not to work out the technique for isolating living cells and separating their tissue debris, since only cells from the liquid medium were analyzed, and not from the sample tissue itself. In addition, the method considered surface markers of only two of these types. Therefore, this method does not make it possible to evaluate the full cellular composition of atherosclerotic plaques, since only individual CD4 and CD8 lymphocytes that independently migrated to the cultivation medium were studied.

In addition, there is a method of isolating lymphocytes from the tissue of the tonsils and cervical-vaginal tract [Saba E, Grivel JC, Vanpouille C, Brichacek B, Fitzgerald W, Margolis L, Lisco A. HIV-1 sexual transmission: early events of HIV-1 infection of human cervico-vaginal tissue in an optimized ex vivo model. Mucosal Immunol. - 2010. - No. 3 (3). - P. 280-290], where sections of lymphoid tissue, prepared into small blocks, were treated with type IV collagenase at a concentration of 5 mg / ml for 30-90 minutes. Next, the cell suspension was stained with various combinations of fluorescently-labeled antibodies to determine all types of cells isolated from lymphoid tissue, after which the cell composition was analyzed by flow cytometry. However, in this method, cells were isolated from tissue samples of solid immune organs, which had a low density and low heterogeneity, and therefore the researchers did not need additional treatment with highly concentrated enzymes and filtering the samples. However, such a method will not allow the selection of all types of cells from highly heterogeneous and densely structured tissue of atherosclerotic plaques.

Also described is a method for determining the composition of immune cells that were released from the tissue of the small intestine of pigs. In the method, enzymatic treatment with collagenase of large sections of the small intestine was carried out without its additional preparation. Subsequently, analysis was carried out using flow cytometry of lymphocytes released only from the internal homogeneous layer of intestinal cells [Solano-Aguilar GI, Vengroski KG, Beshah E, Lunney JK. Isolation and purification of lymphocyte subsets from gut-associated lymphoid tissue in neonatal swine. - 2000. - No. 241 (1-2). - P. 185-199]. As in the previous method, the isolation of cells in this study was carried out from the internal homogeneous layer of the soft tissue structure of the intestine, which allowed the use of a mixture of enzymes in low concentrations and not to carry out additional filtering of the samples. However, like the previous one, the described method will not allow the selection of all types of cells from highly heterogeneous and densely structured tissue of atherosclerotic plaques.

Thus, according to the literature, the existing methods for analyzing the cellular composition of tissues using flow cytometry allow either to analyze a small number of cells that were not isolated from tissues, but from the culture medium during short-term tissue cultivation; either isolate using low-activity enzymes without additional filtration and analyze cells from soft-tissue homogeneous structures, such as lymphoid organs of the intestine, lymph nodes. However, it is important to note that the structure of atherosclerotic plaques is extremely heterogeneous, plaques contain not only various cellular components, but also a large number of extracellular structures (crystals of calcium salts, cholesteric acids, necrotic masses). In this regard, the isolation of all cells from these structures requires testing the stages of filtering samples to increase the accuracy of the analysis of all plaque cells using flow cytometry.

A method for determining the cellular composition of atherosclerotic plaques of human carotid arteries using flow cytometry [Bonanno E, Mauriello A, Partenzi A, Anemona L, Spagnoli LG. Flow cytometry analysis of atherosclerotic plaque cells from human carotids: a validation study. Cytometry - 2000. - No. 39 (2). - R. 158-165]. In this method, as the material for determining the cellular composition of atherosclerotic plaques, the surgical material obtained during the carotid endarterectomy operation (atherosclerotic plaques) was used. Plaques were cut into pieces and then subjected to enzymatic treatment with collagenase I (250 u / ml) overnight at 37 ° C. The cell suspension was filtered through a 150 μm nylon filter and stained for flow cytometry with monoclonal antibodies to antigens associated with the cytoplasmic membrane for 30 minutes at 4 ° C, after which flow cytometry was performed. The disadvantage of this method is that after preparation of the tissue of atherosclerotic plaques and enzymatic treatment, the cell suspension was filtered using a 150 μm nylon filter, which will not allow reliable separation of individual types of immune cells in the analysis with high efficiency, as well as the use of modern flow cytometers with a restriction diameter of the sorting nozzle.

A known method of determining immune cells from atherosclerotic plaques of a person using flow cytometry [A new method for analyzing the cellular composition of atherosclerotic plaques / Grivel J.-S. [and others] // Creative Cardiology - 2012. - No. 1. - S. 26-40], which consists in the fact that as a material for determining the cellular composition of atherosclerotic plaques, the surgical material obtained during carotid endarterectomy (atherosclerotic plaques) is used, which is transported in RPMI 1640 culture medium at room temperature for not more than 2 hours. A 2 mm fragment is separated from the atherosclerotic plaque, which is fixed in 2% formalin. The remainder of the tissue is cut into blocks of 2 mm in size and subjected to enzymatic treatment. Pieces of the plaque are lysed with collagenase IV (1.25 mg / ml) in the presence of 0.2 mg / ml DNase I for 1 hour at 37 ° C with constant stirring. The cell suspension is filtered through a 100 μm nylon filter and washed in a PBS solution.

From the resulting cell suspension, 100 μl was taken to set up a negative control. To separate living cells from dead cells, the remaining cells are resuspended in a PBS solution and stained with a special dye for 15 minutes in the dark at room temperature, then washed with a PBS solution with 2% normal mouse serum. Then, the cell suspension is divided into the required number of tubes, leaving a part for analysis on living / dead cells, and stained monoclonal for determination of CD4 and CD8 T-lymphocytes, B-lymphocytes, NK-cells, as well as the degree of differentiation and activation of T-lymphocytes, for 15 minutes in the dark at room temperature. After staining, the cells are washed with PBS, fixed in a 1% solution of formaldehyde in PBS, and analyzed on a flow cytometer (type FACS Canto II, BD Biosciences, San Jose, CA). Monochromatic compensation reactions are set for each color in each experiment using spherical microparticles. As a control, the same antibody combinations are used as in the experiment to set the analysis area and confirm the specificity of the color, but minus one of the antibodies. The final analysis uses overlapping spectra of various fluorophores, taking into account the autofluorescence of the signals. This method was chosen for the prototype.

The disadvantage of this method is the reduced accuracy of separating the fraction of living cells from dead cells, leading to a decrease in the efficiency of determining the cellular composition of atherosclerotic plaques. The use of a nylon filter with a pore size of 100 μm when filtering does not provide sufficiently efficient removal of fragments and aggregates of dead cells, as well as aggregates of lipid cells and calcium salts, which leads to a very high intensity of autofluorescence of the signal from most lasers in the samples, which interferes with the analysis of data from using flow cytometry and reduces the accuracy of assessing the cellular composition of atherosclerotic plaques.

In addition, the use of a nylon filter with a pore size of 100 μm in the preparation of a cell suspension leads to the formation of large cell aggregates, and therefore the use of this suspension for analysis on flow cytometers of modern generation having a sorting nozzle diameter for focusing the fluid flow is less than 100 μm becomes not possible.

The objective of the invention is to increase the efficiency of assessing the composition of immune cells of atherosclerotic plaques.

The technical result consists in increasing the efficiency of isolation and the accuracy of determining the population of living cells necessary for studying the composition of immune cells from atherosclerotic plaques on various flow cytometer models.

This is achieved due to the fact that in the preparation of the cell suspension using a nylon filter with a pore size of 40 microns.

When passing through the pores of a nylon filter with a pore size of 40 μm, cell aggregates are broken down into smaller structures than when using a filter with a pore size of 100 μm, while aggregates of fragments of dead cells, as well as calcium salts or adventitia adipose tissue do not pass through the pore 40 atherosclerotic plaques. Reducing the size of structures in the suspension makes it possible to use the latest generation of flow cytometers-sorters with a sorting nozzle with a diameter of 70-100 μm (Fig. 1, a - image of the fluid flow of a flow cytometer-sorter when analyzing a cell suspension filtered using a 100 μm nylon filter ; Fig. 1, b - image of the fluid flow of a flow cytometer sorter when analyzing a cell suspension filtered with a 40 μm nylon filter). As can be seen in fig. 1a, when analyzing a cell suspension filtered through a 100 μm filter, moments of blocking the flow of fluid from the flow cytometer by cell aggregates arise. When analyzing a cell suspension filtered through a 40 μm filter, a fragment of a possible flow stop was identified, however, with a higher resolution, it was determined that blocking the fluid flow does not occur (Fig. 1, b).

In addition, the removal of these structures from cell samples leads to a significant decrease in the intensity of autofluorescence in the spectrum of fluorochromes coupled to the analyzed antibodies and their isotypes. At the same time, the fluorescence level of isotypic controls is reduced to 5% of the positive signal fluorescence (Table 1).

As can be seen from the table. 1, the level of autofluorescence decreases when using a 40 μm filter over all fluorescence channels of the flow cytometer. Thus, the accuracy of separating negative events from positive events by the intensity of the fluorescent signal and a more accurate estimate of the number of living cells carrying the surface markers under study becomes much higher (Table 2).

As can be seen from the table. 2, the number of cell aggregates, dead cells, and doublet events becomes significantly lower when using a 40 μm filter compared to a 100 μm filter.

Figures explaining the invention:

fig. 1, a and b - the image of the moments of stopping the flow of fluid flow cytometer sorter in the analysis of cell suspension.

The method is as follows:

As a material for the isolation of immune cells from atherosclerotic plaques, surgical material obtained during an endarterectomy (atherosclerotic plaques) is used, which is transported in RPMI 1640 medium at room temperature for no more than 2 hours. A 2 mm fragment is separated from the atherosclerotic plaque, which is fixed in 2% formalin. The remainder of the tissue is cut into blocks of 2 mm in size and then subjected to enzymatic treatment. Pieces of the plaque are lysed with collagenase IV (1.25 mg / ml) in the presence of 0.2 mg / ml DNase I for 1 hour at 37 ° C with constant stirring. The cell suspension is filtered through a 100 μm nylon filter and washed in a PBS solution. From the resulting cell suspension, 100 μl was taken to set up a negative control. To separate living cells from dead cells, the remaining cells are resuspended in a PBS solution and stained with a special dye for 15 minutes in the dark at room temperature, then washed with a PBS solution with 2% normal mouse serum. Then, the cell suspension is divided into the required number of tubes, leaving a part for analysis on living / dead cells, and stained monoclonal for determination of CD4 and CD8 T-lymphocytes, B-lymphocytes, NK-cells, as well as the degree of differentiation and activation of T-lymphocytes, for 15 minutes in the dark at room temperature. After staining, the cells are washed with PBS solution, fixed in a 1% solution of formaldehyde in PBS, and analyzed on any type of flow cytometer (for example, FACS Aria II, BD Biosciences, San Jose, CA). Monochromatic compensation reactions are set for each color in each experiment using spherical microparticles. As a control, the same antibody combinations are used as in the experiment to set the analysis area and confirm the specificity of the color, but minus one of the antibodies. The final analysis uses overlapping spectra of various fluorophores, taking into account the autofluorescence of the signals.

Example

Atherosclerotic plaque from the right internal carotid artery during endarterectomy in a 69-year-old male patient was taken as the source of the test material. The plaque was placed in an RPMI 1640 transport medium. Samples were delivered to the laboratory within 1.5 hours. In the laboratory, a plaque sample was placed in a Petri dish, examined and described macroscopically, photographed and, under sterile conditions, cut into 2 mm blocks. 1 piece was filled with a solution of 2% formalin for further histological examination. The remaining pieces of the plaque were placed in eppendorf tubes containing collagenase IV solution (1.25 mg / ml) in the presence of 0.2 mg / ml DNase I, incubated for 1 hour at 37 ° C with constant stirring. After that, the cells were filtered through a 40 μm nylon filter and washed in PBS solution. 100 μl was taken from the resulting cell suspension for negative control. The remaining cells were resuspended in a PBS solution and stained with a Pacific Orange dye with a reactive amino group (Invitrogen, Carlsbad, CA) for 15 minutes in the dark, washed with a PBS solution with 2% normal mouse serum. The cells were divided in 50 μl for staining with combinations of antibodies and for analysis of living / dead cells. The following combinations of monoclonal antibodies diluted to the optimal titer were used for staining: (1) CD45 Cy7-PE, CD3 Cy5.5-PerCp, CD4 eFluor780-APC, CD28 PE, CD27 FITC, CD197 APC and CD45RA eFluor 450 to identify T- populations memory cells and naive T cells; (2) CD45 Cy7-PE, CD3 Cy5.5-PerCp, CD4 eFluor780-APC, CD25 PE, CD146 FITC, CD38 APC and Pacific blue HLA-DR for determining T-cell activation; and (3) CD45 Cy7-PE, CD3 Cy5.5-PerCp, CD4 eFluor780-APC, CD 19 PE, CD16 FITC, CD56 APC, CD8 eFluor450 for analysis of T cells, B cells and natural killers (NK) . Staining was carried out in 5 ml tubes for 15 minutes in the dark, then the cells were washed with PBS solution and fixed with 1% formalin solution. One-color compensation reactions were also set for each color in each experiment using spherical CompBeads microparticles (Becton Dickinson). As a control, the same antibody combinations were used as in the experiment, but minus one of the antibodies, to set the analysis area and confirm the specificity of the color. The final analysis used overlapping spectra of various fluorophores, taking into account the autofluorescence of the signals.

When filtering with 40 μm pores, the total autofluorescence background of the isotypic controls was less than 5% of the fluorescence of positive events.

In the analysis using flow cytometry of living cells isolated from their plaques, the following results were obtained:

The number of leukocytes (CD45 + cells): 457500 cells per 1 mg of plaque tissue: granulocytes - 17.10%, monocytes - 2.63%, lymphocytes - 31.50%.

The number of lymphocytes: 144 113 cells per 1 mg of plaque tissue:

T-lymphocytes - 76.40% (110 102 cells);

B-lymphocytes - 2.00% (288 cells);

Natural killers - 17.80% (25 652 cells): activated - 10.10%, mature - 0.62%, immature - 0.00%);

NKT cells - 0.25% (358 cells): CD4 +% - 0.00%, CD8dim - 55.60%, CD8 +% - 11.10%, NK-like T cells - 33.30%).

When analyzing the differentiation of T-lymphocytes:

naive cells: CD8 Naive - 0.89%, CD4 Naive - 2.15%;

memory cells:

central memory cells: CD8 Tcm - 25.90%, CD4 Tcm - 36.20%;

effector memory cells:

CD8 Tem - 60.90% (Early differentiated - 33.80%, Terminally differentiated - 37.50%, Intermediate differentiated - 28.70%);

CD4 Tem - 30.30% (Early differentiated - 29.60%, Terminally differentiated - 7.99%, Intermediate differentiated - 62.41%).

When analyzing the activation of T-lymphocytes:

CD8 CD25 - 16.00%, CD8 CD38 - 47.30% and CD8 HLA-DR - 66.00%;

CD4 CD25 - 30.10%, CD4 CD38 - 33.00% and CD4 HLA-DR - 52.20%.

Thus, we have successfully studied the degree of differentiation and activation of the entire population of immune cells isolated from atherosclerotic plaque tissue.

Claims (1)

The method of determining the cellular composition of atherosclerotic plaques, which consists in collecting surgical material, grinding the plaque, its enzymatic processing, filtering, staining the cell suspension to determine live / dead cells, then staining with monoclonal antibodies and flow cytometry, characterized in that when preparing the cell suspension use a nylon filter with a pore size of 40 microns.

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