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STUDY OF MAJOR HLA ANTIGENS IN AORTIC AND PULMONARY HOMOGRAFTS
SUSAN VERGHESE*, KM CHERIAN**
*Consultant Microbiologist, Department of Microbiology; **Director, Institute of Cardiovascular Diseases, Madras Medical Mission, Mogappair, Chennai 600 050.
Eight aortic and one pulmonary homografts were studied for the presence of major HLA Class I and Class II antigens. Cadaveric hearts were procured from the mortuary and kept in Hank's Balanced Salt Solution with antibiotics at 4oC. Bits were taken from the conduits and valves every 24 hours for 14 days during storage and snap frozen using liquid nitrogen. A total of 1368 sections were made using a cryostat. These sections were stained using 4 monoclonal antibodies. HLA Class I (M0736), Class II HLA DR (M0746), CD45 (M0701), and endothelial stain (MO616) were used. All monoclonals were procured from DAKO.
Class I antigen could be demonstrated on the endothelial surface of vessel wall from day 1 to day 4 to 5 of storage. It stained weaker and could not be demonstrated after day 10 of storage. Class I antigen was positive in very fresh valves and by day 5 to 6 could not be seen on the valve surface. Class II (HLA DR) antigen was present in the sub-endothelial layer from day 1 to day 12 to 14 of storage. They could also be demonstrated in valves and conduits released after cryopreservation. These Class II staining cells were also stained by CD45 antigen and hence they could be macrophages, histiocytes or leucocytes. Endothelium was very well demonstrated in vessel walls from day 1 to day 12 to 14 of storage. Endothelium could only be seen in very fresh valves. Storage in the liquid medium and sterilization procedures led to loss of endothelial lining of the valves. After cryopreservation and thawing Class I antigens could not be demonstrated on the valves and conduits. Class II antigens and CD45 stained cells continued to be demonstrated in the sub-endothelial layer and in the valve matrix. Endothelium was intact in the vessel wall after cryopreservation and thawing, but could not be seen in the released valves.
REVIEW OF LITERATURE
The first known successful implantation of a homologous cardiac valve, namely a canine valve into another dog's descending thoracic aorta was carried by Lamb and colleagues in 1952.[1] The first true orthotopic transplant of a valvular homograft, a canine tricuspid valve was completed in Hungary by Franscis Robiesek in 1953.[2] Gordon Murray completed a series of clinical insertions of aortic homografts into the descending thoracic aorta for relief of aortic insufficiency.[3] From 1960 onwards mechanical and biological valves were developed simultaneously. In 1962 Starr[4] described and implanted his first ball valve and Ross[5] and Barrat Boyes[6] first implanted homologous aortic valves in England and New Zealand respectively with full clinical success. It is estimated that more than 15000 homografts have been implanted world wide, half of them between 1962 and 1972, approximately 40% since 1985 and probably not more than 1500 between 1972 and 1985.[7] The reason for this gross disproportionate distribution could have been the following.
*The lack of competition from other biological valves as they were not available during the first period.
*The success of bovine prosthesis from mid 1970's to mid 1980's.
*The commencement of Cryolife Inc. in 1985 which provided good quality homografts mainly for surgeons in the United States of America.
Majority of the homograft valves in the current world experience are inserted as valved conduits in the heart. However a number of aortic homografts are used also for aortic valve replacement and aortic root replacement.
The early series of homografts inserted between 1962 and 1969 were sterilized by beta propiolactone, ethylene oxide[8] or gamma radiation.[9] Sterilization by radiation was proposed by Malm et al in 1967[9] and denounced by Beach and Malm in 1972.[10] However such methods led to nonviability of valve fibroblasts and post operative degeneration of elastic and collagen fibres. Valves harvested after death are contaminated with bacteria and fungi and it is essential to sterilise the harvested valves prior to use.
Being dissatisfied with the medium term clinical results using chemically sterilized homografts, Brain Barrat Boyes introduced antibiotic sterilization with high dose antibiotics in 1968.[11] Subsequently he reduced the concentration of antibiotic cocktail to decrease cytotoxicity.
To achieve a higher viability of heart valve allografts during long term preservation in nutrient medium, cryopreservation techniques were adopted, whereby 10% DMSO was added to the nutrient medium and the valves were finally stored in the vapour phase of liquid nitrogen (-170oC to -130oC). Refinement of cryopreservation techniques maintain cellular and tissue viability and therefore eliminates the possibility of structural damage as a factor for allograft degeneration. On the other hand it creates an immunological problem.[12] With cryopreserved heart valves a higher rate of viable endothelial cells and presence of surface antigens were detected, which could cause immunological reaction. Rejection of foreign antigens is largely dependent on alloantigens that are coded for by genes of the major histocompatibility locus; which are Class I (HLA ABC) and Class II (HLA DR, DP, DQ) antigens. Besides expression of antigens on the endothelial surface, dendritic cells have also been demonstrated to carry HLA antigens in a few studies.[12] The significance of endothelial cells and fibroblasts and the immunological basis of valve degeneration has not been extensively studied.[13] Valve cellularity includes endothelial cells (EC), fibroblasts and smooth muscle cells, each of which will be conserved some what differently by preservation techniques. Preservation of EC should enhance resistance to thrombosis, but because EC's can express major histocompatibility complex (MHC) Class I and Class II molecules, this may also promote a more immunogenic surface. Preservation of fibroblasts and smooth muscle cells may have practical importance because these are responsible for the maintenance of the amorphous and fibrillar extracellular matrix and are less immunogenic. However the smooth muscle cells present in the normal valves can also be potent modulators of an immune response. Finally variable amounts of myocardium a potent source of immunogenic material, are also incorporated into valve allografts.[14]
AIMS AND OBJECTIVES OF THE STUDY
Homograft valve use does not require tissue typing. Homograft valves are one of the earliest type of tissue valves to be used clinically and the factors which influence their durability whether patient of graft related have not been adequately studied. Rejection of foreign grafts is largely dependent on alloantigens that are coded for by genes of major histocompatibility locus. These antigens can be divided into Class I and (HLA ABC) and Class II (HLA DR, DP, DQ) which are necessary for antigen presentation. This study was undertaken to determine the localization of the major HLA antigens on different components on the human aortic and pulmonary homograft valves and conduits and to see the effect of sterilization and cryopreservation procedures on them. This study was undertaken to demonstrate HLA antigens on cardiac homograft conduits and valves to determine the effect of sterilization procedures, storage and cryopreservation on antigen expression.
MATERIAL AND METHODS
Eight aortic valves and conduits and one pulmonary valve and conduit were examined. The cadaveric hearts were collected from the mortuary within 24 hours after death. They were transported in Hanks Balanced Salt Solution (HBSS) with antibiotics. The age group of the donors ranged from 27 to 45 years. There were 7 males and 2 females. All the valves and conduits appeared normal. Two bits were taken from the valve and the conduits every day starting from 24 hours after death. The specimens were placed in a tissue paper with a drop of tissue embedding solution (Jung Tissue Freezing medium) and were snap frozen by immersion in liquid nitrogen. They were then stored and in the vapour phase of liquid nitrogen until sections were made using a Cryostat (Leica CM 1100). The procedure was repeated at intervals of 24 hours till the end of the 14th day of storage. During this period the homograft was stored in HBSS with antibiotics at 4oC. The antibiotics/antifungal used in the medium were the same as that which are used to sterilize the homografts in our Homograft Valve Bank.
10 tricuspid valve bits of previously cryo-preserved valves which had been frozen in M199 with 10% DMSO, upon thawing, were washed in MI99 with 5% DMSO and then twice with plain MI99. These bits were snap frozen in a similar manner as described above the sections of the tricuspid valve bits were also taken using a Cryostat.
5 aortic conduits and aortic valves which had been frozen in MI99 with 10% DMSO were thawed and rinsed in a similar manner and bits and sections were taken from the aortic walls and valve bits.
All sections were air dried for 24 hours at room temperature and wrapped in a silver foil and stored at -20oC till they were stained. The sections were fixed in acetone for 10 minutes prior to staining. Antigen expression was evaluated using monoclonal antibodies using the Alkaline Phosphatase Anti-alkaline Phosphatase (APAAP) method. The procedure was as described by Yacoub et al (1986).[15] Briefly, the sections were incubated with mouse monoclonal antibody (MAb). They were then washed with Tris buffered saline (TBS) and the section was incubated with goat anti-mouse antibody, washed again with Tris buffered saline followed by incubation with APAAP complex. After a further incubation and wash the substrate was finally added (Fast Red). The substrate was incubated on the section for 20 minutes, washed with Tris buffered saline, tap water and the slide was finally mounted using aqueous mounting fluid. The stained sections were studied immediately. The staining was graded as (+) to (++++) and recorded.
The monoclonal antibodies used in the study were:
*Mouse monoclonal anti-Human HLA Class I antigen (M0736) at a concentration of 1/50 in Tris buffered saline.
*Mouse monoclonal Anti-human Class II, HLA DR antigen (M0746) at a concentration of 1/50 in Tri buffered saline.
*Mouse monoclonal anti leucocyte common antigen (CD 45) at a concentration of 1/50 in Tris buffered saline.
*Mouse monoclonal anti human von Willebrand Factor (M0616) at a concentration of 1/50 in TBS, which was used to stain the endothelium.
Method of staining
Tissue Ag X
Mouse anti X monoclonal AB (M0736, M0746, CD45, M0616) at a dilution of 1/50 in TBS
Rabbit anti mouse immunoglobulin (Z0259), at a dilution of 1/25 in TBS
APAAP (D0651) at a dilution of 1/25 in TBS.
Alkaline phosphatase substrate (Fast Red)
Aqueous mount
RESULTS
Total number of fresh conduits and valves studied were 9 in number. The number of sections of conduits and valves that were sectioned and stained from each homograft were 152 (4x14 for conduits and 4x14 for valve). The total number of sections stained from fresh valves was 1368. Total number of tricuspid bits studied after cryopreservation and thawing were 10x4=40. Total number of aortic conduits(5) and valves(5) studied after cryopreservation and thawing were 10x4=40.
HLA Class I Ag : Aortic conduits from autopsy material showed positive staining of the endothelial surface ranging from very strong (++++) (Fig. 1) to strong (++) strain from day 1 to day 4 to 5 in HBSS with antibiotic (AB). The Class I antigen becomes weaker from day 4 to 5 but could be demonstrated upto day 8 to 9 (+) in most vessel walls.
by HLA ABC monoclonal antibody x 400.
by day 14 x 100.
well (++++) with HLA DR antibody. X 400.
Valves showed strong staining on day 1 to 2 on the endothelial surface (+++). Class I antigen could not be demonstrated after day 5 to 6 in all valves.
Class I antigen could also be demonstrated in the micro-vasculature of the adventitia of the aortic vessel walls, which also stained weaker after day 5 to 6.
Class I antigen could not be demonstrated in the vessel walls or adventitia or valve surface after day 10 (Fig. 2).
Class II antigens : Cells stained by HLA DR were present in the subendothelial layer in the aortic wall from day 1 to day 12 to 14. The staining was very strong (++++) (Fig. 3) to strong (++) by day 12 to 14 (Fig. 4). Class II antigen stained cells could also be found in the valve matrix (in the centre of the valve cusp) (Fig. 5) and just below the endothelium of the valve lining (Fig. 6). These valve matrix cells could be seen stained upto day 8 to 10 with Class II antigen.
CD45 Antigen : Cells stained by CD45 antigen in the sub-endothelial layer could be demonstrated upto day 12 to 14 of storage. The cells in the sub-endothelial lining which had stained by the Class II antigen also stained by CD45 antigen (Common leucocyte antigen (Fig. 7). The cells in the valve matrix also stained by the CD45 antigen. The HLA DR stained cells are probably leucocytes, macrophages or histiocytes as the CD45 antigen stains these cells.
Endothelial Stain : The aortic wall lining of the endothelium was intact up to day 14 (Figs. 8 and 9) of storage in HBSS with AB, and could be well stained by the endothelial stain. Thus Class I antigen was independent of the endothelial lining.
after 14 days with HLA DR (+) x 400.
just below the endothelium (++) x 400.
stained with HLA DR (+++) x 100.
day 3 of storage x 100.
below the endothelium (++++) x 400.
day 14 of storage x 100.
Valves : The endothelium is present in fresh valves. But with continued storage in HBSS with AB the endothelium could not be seen after day 4 to 5 of storage (Tables 1 and 2).
TABLE 1
Staining of Class I and Class II HLA antigens in conduits and valves during storage at 40C in HBBS with antibiotics1 2 3 4 5 6 7 8 9 10 11 12 13 14 Valve HLA ABC ++++ +++ ++ ++ + + - - - - - - - - HLA DR ++++ +++ +++ +++ ++ ++ ++ + + + - - - - CD45 +++ +++ ++ ++ ++ ++ + + + + - - - - Endothelium +++ ++ ++ + + - - - - - - - - - Aorta HLA ABC ++++ ++++ +++ ++ ++ + + + + + - - - - HLA DR ++++ ++++ ++++ ++++ ++++ ++++ ++++ +++ +++ +++ +++ ++ ++ + CD45 ++++ ++++ ++++ ++++ ++++ ++++ +++ +++ +++ +++ ++ ++ ++ + Endothelium ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ +++ +++ +++ +++ +++ ++++
TABLE 2
Staining of Class I and Class II HLA antigens in conduits and valves after cryopreservation and thawingHLA ABC - Aorta After Cryo Valve After Cryo
After Cryopreservation and thawing
Class I antigen could not be demonstrated on the vessel walls or on the valves.
Class II antigen could be demonstrated in the aorta which had been cryopreserved and thawed, in the sub-endothelial layer.
Class II antigen stained cells could also be seen to be weakly staining (+) in the valve matrix after cryopreservation and thawing.
CD45 stained cells could be seen in the sub-endothelial layer of the vessel wall and in the valve matrix. These were similar to the cells stained by Class II antigen. Endothelium in the vessel walls stained positive even after cryo preservation and thawing.
In the valves the endothelium could not be demonstrated after thawing. The procedure of sterilization and freezing and thawing probably destroyed the thin one celled layer of the endothelium on the valves.
DISCUSSION
Indian context
Homografts have been used in India only from the last 10 years in very few centres. Most of these homografts are procured from mortuaries during autopsy from sudden death victims, such as those dying from road traffic accidents or hanging cases. India being a tropical country, the rate of degeneration of the valves are probably higher. Also most mortuaries do not have adequate facilities of refrigeration and the bodies are not maintained at the desired low temperature. The antibiotics used and also the media used for sterilization and cryopreservation are probably different. There are very few reports of homograft valve use in the Indian literature. This study was undertaken to demonstrate the HLA allo-antigens on cardiac homografts in the Indian context.
Recent Work
Yacob et al (1986)[15] have demonstrated the presence of major HLA Class I and Class II antigens in the valves and on the vessel walls. According to their study Class I antigens were strongly expressed by both endothelial and interstitial cells only in very fresh valves. Both Class I and Class II antigens could not be detected after sterilization or after storage for 48 hours.[15] Besides Class I and Class II antigens dendritic cells have also been demonstrated in allograft valves.[16] Although ABO blood group antigens are also found on the endothelial surface, it has been studied and well established that there is no significant difference between ABO compatible and incompatible valves with regard to durability in the absence of immuno-suppression.[17] Some studies have demonstrated that degeneration and calcification of homografts to be more in aortic homografts compared to pulmonary valves especially in the younger age group.[18] This could be because of abnormal metabolism of structures and high calcium content of aortic homografts. Smith et al[19] have demonstrated that humoral antibodies are formed after implantation of homovital valves (implanted within 6 to 24 hours after retrieval) which is evident within the first year of life. This response is stronger when compared to antibodies produced after implantation of antibiotic treated valves. Recipients of homovital aortic valve homografts are known to produce specific antibodies to HLA determinants present on the cellular components of the valve tissue.[20] The HLA antibodies detected were all IgG and were able to fix complement suggesting that it was a long term secondary response. These antibodies were produced due to mismatched donor HLA antigens and not due to peri-operative blood transfusion. It has been demonstrated that fresh homovital valves with intact endothelium elicits a strong humoral response than an antibiotic treated valve.[20] It would follow that homovital valves would have a stronger immunological response and hence have more deleterious effect on the long term function of the valve. This does not happen. In fact, homovital and viable cryopreserved cardiac valve allografts have been shown to have a better long term function with lower incidence of re-operation for valve degeneration than fresh antibiotic sterilized valves which are maintained at 4oC before implantation.[21] This contradiction whereby allografts that elicit a more aggressive immune response have a better long term function, remains to be fully explained. One possible explanation for the different effects of antibodies after valve and organ transplantation is the likelihood of a much slower process of damage in the avascular homograft.[19] In addition viable endothelial cells are not obligatory for relatively long term valve function.
The presence of dendritic cells in the valve leaflet has not been confirmed in literature but Bobry shev and colleagues[16] have reported the presence of dendritic cells in aortic vessel walls. And the vessel wall represents an important component of the valve allograft. We have been able to show the presence of cells stained by common leucocyte antigen (CD45) in the aortic wall as well as in the matrix of the valve for up to 12 to 14 days duration during preservation at 4oC in HBSS with antibiotics.
Initiation of allogenic response requires intimate and specific contact between recipient T cells and viable donor cells expressing HLA Class II antigens. Only "professional antigen presenting cells" which display unique activation receptors and high density membrane HLA Class II are capable of unleashing the cascade of T cell activation in response to mismatched HLA antigen. In cryopreserved valve allografts no cell has so far been identified for this pivotal role. Valve leaflet endothelial cells can do so but they are not present in sufficient numbers in cryopreserved valves to mediate this function. Similarly valve fibroblasts and myocytes lack the critical numbers and antigen presenting capacity. Hoekstra and coworkers propose that vascular dendritic cells as the most likely initiator as only few of these extremely potent cells would be needed to trigger the anti-VA response.[22]
The endothelial cells have not been demonstrated consistently on the cryopreserved valve leaflets. Lupinittie and coworkers[23] showed that cryopreservation of human donor valves is associated with loss of endothelial cells. This has also been the experience of Hoekstra et al (1998) where a few endothelial cells could be demonstrated on the valve leaflets and vascular structures of cryopreserved valves before implantation. Our study differs in that endothelial lining of the aortic wall continues to be demonstrated up to the 12 to 14 day of preservation at 4oC and also can be demonstrated in most of the cryopreserved vessel walls (which have been thawed after cryopreservation). But we have not been able to demonstrate endothelial cells on the valve leaflets after the 4 to 5 day of preservation at 4oC in HBSS with AB. We have also not been able to demonstrate endothelial lining on the valve leaflets which have been cryopreserved and thawed. Hoekstra et al[22] have concluded that viable cells capable of expression of HLA Class I and Class II antigens are present in human valve allografts and antibodies induced by these grafts could damage the transplanted tissue and therefore contribute to graft dysfunction. This antibody production according to Hoekstra et al(1998) started approximately 4 weeks after valve transplantation. The delay could be because of the late presence of the Class I and Class II antigens (This could be because of the recovery phase of the cryopreserved cells after thawing and implantation).
Allograft preparation methods used in various centres differ in antibiotics used, nutrient medium used, varying ischaemic times before the valve is collected at autopsy and varying duration of antibiotic sterilization. This could lead to different antigenic load on the valves which are implanted. We were able to demonstrate HLA Class II antigen positive cells to be present in the sub-endothelial layer of the conduits upto day 12 to 14 of preservation in the antibiotic medium and in the valve matrix for up to day 10 of preservation in the antibiotic medium. These Class II antigen staining cells were also stained by CD45 (Common leucocyte antigen) monoclonal antibody. We have not used the specific monoclonal CD 1a to see if these cells belong to the dendritic cells. Class I antigens which are on the endothelial surface have been seen to disappear approximately around day 4 to 5 of preservation in the nutrient medium on the valves and approximately day 8 to 9 on the vessel wall.
We have not done studies in the homograft recipients to demonstrate the antibodies against Class I and Class II antigens. Lupinette et al[24] quote that allgoraft aortic valves are immunogenic and the immunogenicity is not altered by cryopreservation techniques. However prolonged storage in the nutrient medium at 4oC does reduce immunogenicity, but does not eliminate it. An association between immunological difference and calcification of the aortic valve allografts is present, but limited.[24] Immunogenicity could act along with other factors such as cellular viability, antibiotic exposure, duration of ischaemia, storage, media used and temperature and be a determinant for calcification of the valve. There are research workers who hold the opposite view. They quote that changes such as calcification and structural degeneration occurring in the homograft conduits and valves are not associated with immunological injury.[25] Rather the loss of viability according to him is more likely attributable to ischaemia, freezing and thawing as is used in cryopreservation. The valve degeneration that is seen in homografts is because there are no viable cells capable of synthesizing the new cellular matrix. Also according to Mitchell,[25] absence of significant neutrophils and mononuclear infiltrates in explanted cryopreserved valves even at time points where clear cut architectural changes and loss of cellular staining occurs leads to the conclusion that immunological phenomenon cannot be causally implicated in most homograft degenerations. Moreover in heart transplant patients where immunological phenomenon unequivocally caused cardiac failure (Either overwhelming rejection or homograft arteriopathy), evidence of immunological injury to the valves was not seen.[26] The hearts from the heart transplant group, even from the heart that have suffered rejection show near normal numbers of fibroblasts, the endothelium is intact and there is no hyalinization of the valve matrix or calcification seen.[27]
There are also reports that valves do not express many of the cell markers including ABO blood group antigens, which are present on other parts of the cardiovascular tree.[17] These authors have concluded that absence of blood group carbohydrate antigens on valvular endothelium suggest that blood group incompatibility does not play a significant role in homograft degeneration and hence blood group matching that is being performed in some of the centres does not have a role to play in preventing the degeneration of the homografts.
CONCLUSION
Homograft valves are one of the earliest types of tissue valves to be used clinically. They have been in use from 1962. No HLA typing or even blood group matching is done prior to insertion of homografts. The durability of the valves may be graft or patient related. One of the important variables is the antigenicity of different components of the graft at the time of insertion. Allograft aortic and pulmonary conduits and valves are immunogenic. We have demonstrated HLA ABC (Class I) and HLA DR (Class II) antigens on different components of the vessel wall and valve leaflets. They are strongly expressed on the endothelial surface (Class I) and in the sub-endothelial layer (Class II) of the vessel wall. Class I antigens disappear early at day 4 to 5 of storage in liquid medium at 4oC. Class II antigen continues to persist even upto day 12 to 14 of storage. Class II antigen staining cells are also present in the matrix of the valve leaflet. These Class II antigen staining cells can also be stained by CD45 monoclonal antibody hence they could be leucocytes, macrophages or histiocytes. Class I antigen is independent of the presence of the endothelial lining. Endothelium is well preserved in the vessel wall but in the valve leaflet endothelium could not be demonstrated.
ACKNOWLEDGEMENTS
The authors are deeply grateful to Rameshwardas Birla Smarak Kosh, Mumbai for their financial assistance.
We acknowledge the technical help of Microbiologists Mathew T and Maria CF.
REFERENCES
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16.Bobryshew YV, Lord SA. S-100 positive cells in the arterial intima and in atherosclerotic lesions. Cardiovascular 1995; 29 : 689-96.
17.Kadner A, Chen RH, Mitchell RN, Adams DH. Homograft cross matching is unnecessary due to absence of blood group antigens. Ann Thorac Surg 2001; 71 : S349-52.
18.Yankah AC, Alexi-Meskhishvili V, Weng A, Schorn K, Lange PE, Hetzer. Accelerated degeneration of allografts in the first two years of life. Ann Thorac Surg 1995; 60 : S71-7.
19.Smith JD, Hornick PI, Rasmi N, Rose MI, Yacoub MH. Effect of HLA mismatching and antibody status on "homovital" aortic valve homograft performance. Ann Thorac Surg 1998; 66 : S212-15.
20.Smith JD, Ogino H, Hunt D, Layor RM, Rose ML, Yacoub MH. Humoral Immune response to human aortic valve homografts. Ann Thorac Surg 1995; 60 : S127-30.
21.O'Brien MF, McGiffin DC, Stafford EG, et al. Allograft aortic valve replacement: long term comparative clinical analysis of viable cryopreserved and antibiotic 4oC stored valves. J Cardiac Surg 1991; 6 (suppl) : 534-43.
22.Hoekstra FME, Witvliet M, Knoop CY, Wasssenar C, Bogers JJC, Weimar W, Class FHJ. Immunogenic human leucocyte antigen Class II antigens on human cardiac valves induced specific alloantibodies. Ann Thorac Surg 1998; 66 : 2023-26.
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