A FUNCTIONAL CLASSIFICATION OF GRANULOMATOUS INFLAMMATION

A FUNCTIONAL CLASSIFICATION OF GRANULOMATOUS INFLAMMATION

Kenneth S. Warren

Division of Geographic Medicine Department of Medicine

Case Western Reserve University and University Hospitals Cleveland. Ohio 44106

Since the title of this international conference is Sarcoidosis and Other Granulo- matous Disorders, it might be worthwhile to begin this dissertation with a definition of the granuloma. Peter Ward, however, has said that “granulomatous inflammation is almost impossible to define.”’ The problem began in the 19th century with Virchow who described a granuloma as “a tumor or neoplasm made up of granulation tissue.”* As recently as 1974, in the Pathologic Basis of Disease, Stanley L. Robbins started his definition as follows: “The term granuloma implies a tumor-like mass of granulation tissue (actively growing fibroblasts and capillary bud^)."^ That the relationship between granuloma and granulation tissue is a puzzling one is exemplified by the fact that a t a Symposium on Inflammation, Mechanisms, and Control in 1972 Guido Majno asked me if the terms could be differentiated? My reply was that “the granuloma is an injurious reaction whether initiated by immunologic mechanisms, activation of chemical mediators of inflammation, or directly by toxic substances. In all cases, normal tissue is destroyed and replaced with inflammatory cells. When the immunologic reactivity ceases, the physical object is walled off, or the toxin is exhausted, tissue repair ensues. This is the point a t which granulation tissue is seen, as exemplified by the ap- pearance of fibroblasts and formation of new blood v e ~ s e l s . ” ~

Fortunately, Robbins went beyond granulation tissue in his definition of the granuloma adding, “However, the term should be restricted to a small 1-2 mm collection of modified macrophages or histiocytes almost invariably surrounded by a rim of mononuclear cells, principally lymphocyte^."^ Peter Ward states that “at present, granulomatous inflammation usually refers to an inflammatory response characterized by the presence of lymphocytes, monocytes and plasma cells.”’ These definitions of the granuloma, therefore, take both size and cellular composition into consideration. It must, however, be considered that accumula- tions of cells less than 1 mm or somewhat greater than 2 mm that also contain varying numbers of polymorphonuclear cells might be considered granulomas. Whereas localized collections of neutrophils may be called abscesses,’ many granulomas contain small numbers of neutrophils and, in some cases, relatively large numbers of eosinophils.6 Thus, I prefer to define the granuloma as a focal, chronic inflammatory reaction characterized by the accumulation and prolifera- tion of leukocytes, principally of the mononuclear type. Epstein has defined granulomatous inflammation in the broadest sense as any focal chronic inflammatory reaction with a variable complement of cell types, necrosis, and stromal pro~iferat ion.~

The basic etiological factor in the case of all granulomas is probably the presence of a nidus of insoluble material which, if small enough, is ingested by phagocytic cells or, if too large, remains extracellular. Examples of intracellular particles are tubercle bacilli, streptococcal cell walls, and silica; examples of

7

8 A n n a l s N e w York Academy of Sciences

TABLE I

INVOLVING THE GASTROINTESTINAL TRACT A CLASSIFICATION OF GRANULOMATOUS INFLAMMATIONS

Foreign-body Granulomas of Granulomas Unknown Etiology Infectious Granulomas

~~~~ ~~

Tuberculosis Starch, Talc Regional enteritis Syphilis Sutures Sarcoidosis Actinomycosis Lipid Granulomatous gastritis South American blastomycosis Barium Eosinophilic granuloma Histoplasmosis Mercury Allergic granulomatosis Amebiasis Primary biliary cirrhosis Schistosomiasis Lymphogranuloma venereum Bacterial infections

extracellular particles are schistosome eggs and plastic beads. In granulomatous diseases of unknown etiology such as sarcoidosis or regional enteritis, the putative insoluble material has not as yet been identified.

Granulomatous inflammations have been classified clini$ally into infectious and foreign-body lesions and those of unknown etiology. TABLE 1 shows this sort of classification as used for granulomatous inflammaiion of the gastrointestinal tract.’ For the lung, the various pneumoconioses such as silicosis, as- bestosis, and berylliosis would be added.

Pathologists and dermatologists generally define two types of granulomatous lesions variously called the infectious and foreign-body types,5 the hypersensitivity and colloidal types,’ or the immunologic and nonimmunologic, undifferentiated types’ with the major distinction between the two being the presence of either epithelioid cells or undifferentiated macrophages. A major problem, however, is that it is often not possible to achieve agreement among different observers as t o the presence or absence of epithelioid cells.

Spector is not happy with the terms “undifferentiated” and “epithelioid” used in this context, and suggests a classification of granulomas based on kinetic differences rather than m i c r o s c ~ p y . ~ * ’ ~ These differences are the so-called high and low turnover granulomas. “The former are characterized by high levels of recruitment, division, death and migration of cells, and are associated with irritants toxic to macrophages. Low-turnover lesions are characterized by longevity of the macrophage with infrequent division and a low rate of recruitment and are associated with irritants of low toxicity and which are difficult to digest. Each type is characterized by macrophages of typical ultrastructure.”’O The problem with this conceptualization is that high turnover granulomas are associated with stimuli as diverse as the injection of B. pertussis vaccine or BCG (B. Calmette-Gueiin), and the subcutaneous insertion of glass or plastic coverslips. Low turnover granulomas are evoked by carrageenan, a n unusual substance that has been shown to depress immunologic reactivity.” Furthermore, as Spector has pointed out, “in many situations the high turnover granuloma is merely a stage on the road to a low turnover reaction.””

In lieu of the above means of differentiating granulomas, a functional classification of granulomatous inflammation based on experimental evidence is now suggested (TABLE 2). The fundamental criterion for immunologic granulomas is altered reactivity following second and subsequent exposure to an antigenic sub-

Warren: A Functional Classification 9

stance, which is characterized by the accelerated evolution of a larger lesion! Once this anamnestic reactivity is established, the specificity of the reaction and its transfer with either cells, serum, or both can be determined. Further evidence for the role of immunologic mechanisms is the temporal association of granuloma formation with both in vivo and in vitro correlates of immediate or delayed hypersensitivity and the inhibition of granuloma formation by immunosuppressive measures. I t is worthy of note that the cellular composition of the lesions and the presence of antibody or cell-mediated reactivity provides helpful but only indirect evidence for the immunologic etiology of a particular lesion. To paraphrase Coombs and Ge11,'2 the mere demonstration of the presence of antibody or cell- mediated reactivity related to a clinical syndrome, often detectable only by sensi- tive tests, is little contribution to knowledge of pathogenesis, however useful in diagnosis; there must be some demonstration that such a reaction can in fact contribute to the actual pathological lesions found.

The primary characteristic of the immunologic granuloma, anamnestic reactivity, is not present in the nonimmunologic lesion.4 No matter how many times the inducing substance is injected into the tissues, the reactions are the same. Lesions of the inactive type develop rapidly to peak size and quickly diminish thereafter, a rim of cells walling off the foreign body as long as it remains in the tissues. Active lesions are characterized by the continuing turnover of phagocytic cells due to the toxic nature of ingested insoluble material. In certain cases, particles can initially elicit nonimmunological reactions, but following a sensitization process immunologic granulomas may result. Many of these different types of reactions, both immunologic and nonimmunologic, may go on simultaneously, although in most instances one type is predominant.

Immunologic Granulomas

Although tuberculosis is the prototype both of granulomatous diseases and of delayed hypersensitivity, a group of major authorities from Rich in 1951,'' to L u r k in 1964,14 and Raffel in 1971 I s have all considered the tubercle to be a reaction to the irritating effect of lipids. It is only recently that some evidence has been gathered suggesting anamnestic reactivity in the tubercle lesions. Dannenberg et al. showed that the BCG lesions of reinfection enlarged more rapidly than primary lesions and that macrophage turnover was more rapid.16 As far as 1 know, the specificity and passive transfer of this enhanced reactivity has not been demonstrated. The effect of immunosuppressive measures on the pathologic reactions induced by living bacteria would be difficult to evaluate because suppression of

TABLE 2 A FUNCTIONAL CLASSIFICATION OF GRANULOMATOUS INFLAMMATION

Granulomatous Inflammation Examples

immunologic granulomas Cell-mediated schistosomiasis mansoni Antibody-mediated schistosomiasis japonica (?)

Inactive plastic beads, bentonite Active silica, streptococcal cell walls

Nonimmunologic granulomas

10 Annals New York Academy of Sciences

cellular reactivity should lead t o multiplication and dissemination of the or- ganisms.

The first complete demonstration of the immunologic etiology of granulomatous inflammation was reported in 1967 with the Schistosoma mansoni egg granuloma.” S. mansoni, the so-called blood fluke, lives in the mesenteric or vesical venules of its mammalian host producing large numbers of eggs daily. These particles, measuring from 50 to 160 p in diameter, have a tanned, protein eggshell that protects the living organism within. The eggs embryonate within one week and the embryo or miracidium begins to secrete enzymes through ultra- microscopic pores in the eggshell. These enzymes enable the eggs t o pass through

60

X mE 50 E

. . 1 4 8 16

D A Y S 32

FIGURE I . Mean granuloma volumes I , 4, 8, 16, and 32 days after intravenous injection of S. mansoni eggs into the pulmonary microvasculature of unsensitized mice (Schisto), and mice injected intraperitoneally one month previously with Ascuris mum eggs (Asc-Schisto) or S. rnansoni eggs (Schisto-Schisto). *Volume calculated from measured diameters of the lesions.

the blood vessels and tissues into the lumen of the excretory organ. Large numbers of eggs are trapped in the tissues, however, where they live for approximately 2 weeks after embryonation. I n contrast to the tubercle bacillus these living or- ganisms neither multiply nor disseminate.

In the early 1960s von Lichtenberg developed a model for studying the S. mansoni egg granuloma by isolating viable eggs from the livers of infected animals and injecting them intravenously into the pulmonary microvasculature of mice.” In 1967, using this model we were able to demonstrate markedly accelerated, aug- mented granuloma formation on secondary injection of eggs one month following a primary intraperitoneal injection of eggs (FIGURE I ) . ” This reaction was shown

Warren: A Functional Classification 1 1

t o be specific in cross-injection studies with Ascaris suum eggs (FIGURE I), and i t was passively transferred with lymph node and spleen cells, but not with serum, The S. mansoni egg granuloma was then shown to be markedly inhibited by a variety of agents and conditions that are particularly immunosuppressive for cell-mediated reactions. These included neonatal thymectomy in both mice’’ and chickens;20 adult thymectomy followed by irradiation and bone marrow reconsti- tution;2’ antilymphocyte serum;22 a Hodgkin’s-like lesion;23 and a drug, niridazole, which suppresses cell-mediated reactions,24 but has little effect on antibody formation.25 By contrast, inhibitors of antibody formation such as chronic x-irradiation?6 Friend virus leukemia,23 and bursectomy in chickens” had no effect on granuloma formation.

When S. mansoni eggs were injected primarily into the lung arterioles of either mice or guinea pigs, the first inflammatory cells appeared around the eggs at 4 days, coincident with the development of delayed footpad swelling in the mice2’ and delayed dermal reactivity in the guinea pig.28 In the latter animal the onset of lymphocyte transformation to soluble S. mansoni egg antigens and the secretion of macrophage migration inhibitory factor by lymphocytes occurred 4 and 3 days, respectively, after the injection of the eggsz8 During the period of the full development of the granuloma, hemagglutinating antibodies were not detectable in mice2’ and y , and y2 antibodies were not found in the guinea pigs.28 Thus the S. mansoni egg granuloma appears to be largely an immunologic reaction of the cell-mediated type. Further evidence supporting the immunologic etiology of this lesion is the induction of tolerance to the egg granuloma in the offspring of pregnant female mice injected intravenously with soluble egg antigen^.^' Tolerance also followed injections of soluble egg antigens into neonatal mice.30 A modulation of granuloma formation has been found in chronic infection^,^' which is associated with the occurrence of rising antibody titers and with the development of unresponsiveness to antigen by splenic lymphocyte^.^^

Using a technique first described by Pellegrino and Brener,33 it has been possible to isolate intact S. mansoni egg granulomas from the lungs or liver of egg- injected or infected mice and study them in vitro. Recently, the cellular composition of these lesions has been determined by separating the cells with proteolytic enzymes and doing total and differential counts! Initially, the cells in highest con- centration in the lesions of both unsensitized and sensitized animals were lymphocytes. These declined in number, whereas macrophages continued to be present, and eosinophils increased. Studies have not been performed as yet to determine the relative numbers of B and T lymphocytes in the lesions, but functional investigations suggest that the latter are the major cells involved. Thus, when intact granulomas were maintained in vitro they emitted the lymphokines migration inhibitory factor and eosinophil stimulation promoter, particularly when soluble egg antigens were added. In the presence of I4C-labeled leucine, proteins were syn- thesized by the granulomas, but coprecipitation analysis with rabbit anti-mouse immunoglobulin serum revealed that they were not i m m u n ~ g l o b u l i n s . ~ ~

Allison has postulated the development of antibody-mediated granulomas, noting that in some “chronic inflammatory lesions B lymphocytes and their antibody-forming derivatives are prominent. These could locally produce antibody combining with antigen t o form c o m p l e x e ~ . ” ~ ~ Although formation of soluble immune complexes combined with complement has been associated primarily with polymorphonuclear infiltrates in the so-called Arthus reactions, Allison notes that these complexes may also be chemotactic for macrophages. Recent studies in our laboratories suggest the possibility that the S. japonicum egg granuloma may, a t least in the initial phases, be largely an antigen-antibody-complex-mediated


lesion?6 The lesions tended to form largely around aggregates of eggs in the tissues, suggesting the need for high local antigen concentrations. The initial lesions were essentially polymorphonuclear abscesses made up largely of eosinophils. This was succeeded by an influx of mononuclear cells including many plasma cells, although eosinophils still remained prominent. In contrast to S. mansoni infections, when soluble egg antigens were injected into the footpads of mice in different stages of schistosomiasis japonica, large immediate reactions developed and the swelling disappeared completely by 24 hours.“8

It is possible that both immediate and delayed hypersensitivity contribute simultaneously to the development of some granulomatous lesions, but evidence for this remains to be gathered. With respect to the S. mansoni egg granuloma, however, it is possible that antibody or immune complexes may be the key factor in the modulation of this largely cell-mediated lesion in a manner similar to the immunological blockade phenomenon of tumor immunology.”

As noted above, Dannenberg et al.I6 reported a sensitization phenomenon in the BCG lesions of reinfection. Using a different system, Boros and Warren in 197 1 had shown the development of immunologic-type granulomas around bentonite particles containing adsorbed mycobacterial culture filtrate in mice pre- sensitized with this same antigen.37 Similar studies were performed with S. mansoni egg antigens and the reactions were shown t o be specific. In a subsequent study this model was extended to Histoplasma capsulatum and was performed not only in antigen-injected mice, but in animals infected with the three organism^.^' Furthermore, passive transfer studies were successfully performed in each case with cells but not with serum. Thus, using this artificial model of the granuloma it was shown that antigens isolated from tubercle bacilli, histoplasma fungi, and schistosome helminth eggs are capable of eliciting specific cell-mediated-type granuloma formation.

Nonimmunologic Granulomas

When Franz von Lichtenberg developed the technique for studying the S . mansoni egg granuloma in vivo, he used divinyl-benzene copolymer beads as “foreign- body” granuloma controls.” In contrast to the prolonged lack of inflammatory activity (96 hours) following primary egg injections, von Lichtenberg showed that the lesion around the plastic beads began as early as 6 hours and reached its peak at 48 hours.” In-depth studies of this reaction subsequently reported by Keller- meyer and Warren revealed many differences between the bead and the schistosome egg reactions.39 Of particular importance was the fact that the granulomas in animals previously exposed to the beads were identical in both timing and cellular composition to those in animals that had never been exposed (FIGURE 2). Thus sensitization and anamnestic reactivity did not occur, specificity was not a rele- vant question, and there was nothing to transfer passively. In the acute formative stages the lesions consisted mainly of neutrophils which were succeeded by macrophages. In our studies of immunosuppressive conditions we have almost always used plastic-bead granulomas as controls; neither neonatal thymectomy,19 antilymphocyte serum,22 nor n i r i d a ~ o l e ~ ~ had any effect on the plastic-bead reaction.

In 1966 Ratnoff suggested that talc and silica might induce inflammatory responses in tissues by activating Hageman factor and initiating the formation of k i n i w m Based on this postulate, Kellermeyer and Warren performed a series of experiments indicating that the plastic-bead granuloma might also be dependent


on the activation of chemical mediators of i n f l a r n m a t i ~ n . ~ ~ I n vitro studies revealed that the plastic beads activated Hageman factor, generated kinin-like activity in normal human and mouse plasma, and induced vascular permeability- enhancing activity in guinea pig skin. In addition, beads injected intravenously into the pulmonary arterioles of pigeons, which are deficient in Hageman factor, elicited no inflammatory response. Finally, ellagic acid, an agent that activates Hageman factor in vivo and reportedly diminishes kininogen by consumptive de- pletion, markedly suppressed the inflammatory response t o beads in the pulmonary microvasculature of mice. Thus it was suggested that the nonim-

- a - a ~ 20 301 PRIOR T P IN.IFCTION /:( + S F

4 3 z (I W

a

LL 0

W a

I10

100

90

60

,*c=, - - . -

0 12 2 4 48 T I M E f h o u r s )

FIGURE 2. Mean granuloma diameters 0, 12, 24, and 48 hr after intravenous injection of divinyl-benzene copolymer beads into the pulmonary microvasculature o f control mice and those injected intraperitoneally with beads 2 weeks previously.

munologic granuloma was the result of the activation of chemical mediators of i n f l a m r n a t i ~ n . ~ ~

In ou r studies of granuloma formation using bentonite particles with adsorbed antigens, we observed that the particles themselves induced nonimmunologic-type granulomas, both when they were bare and when they contained antigen and were injected either into unsensitized animals or those sensitized to other antigen^.^'.^^ We have also observed that the lesions around silica particles intro- duced transtracheally or talc particles injected into the lungs of mice were identical to those of the plastic-bead granulomas with respect to time course, lack of


anamnestic reactivity, cellular composition, and suppression of the inflammation by ellagic acid.39

Small phagocytizable silica particles have a property not shared by the in- ducers of nonimmunologic granulomas described above in that they cause a chronic active lesion that is accompanied by considerable fibrosis. Allison has demonstrated that on ingestion by mononuclear phagocytes both silica and as- bestos particles interact with the membranes of secondary lysosomes making them permeable t o lysosomal enzymes and leading to death of the cells. The particles are then phagocytized again, resulting in a high turnover of cells. The extensive studies of the granuloma in streptococcal inflammation by Ginsberg et a[. revealed that phagocytic cells could not degrade the cell walls of group A streptococci!’ These cell walls are toxic to the phagocytes and when the cells die the particles are taken up by other cells with the development of chronic persistent lesions. Gins- berg has no evidence for anamnestic reactivity in his experiments. Thus when nonimmunologic, granuloma-inducing particles are nontoxic to macrophages, inactive lesions result, but if they are toxic to the phagocytes, active granulomas occur.

Although the categories of immunologic and nonimmunologic granulomas are quite distinct, they may under certain circumstances become blurred. It may seem that all foreign particles, whether they are antigen-containing and -secreting or not, should elicit a nonimmunologic granuloma prior to the onset of sensitization. It is interesting to note, however, that schistosome eggs, which d o not elicit inflammatory reactions for 4 days after primary injection, have an anticoagulant effect, prolonging the clotting time of Hageman-deficient plasma.39 They should not, therefore, activate the initial step in the pathway leading to the formation of chemical mediators of inflammation.

In addition, particles that usually elicit nonimmunologic inflammatory lesions may, on repeated exposure, sensitize the occasional individual, resulting in the development of immunologic granulomas. A good example of this type of response is the zirconium granuloma of the axilla related to the use of stick de- o d o r a n t ~ . ~ This was revealed to be a specific allergic reaction characterized histo- logically by epithelioid cells, which occurred in only about 3 percent of exposed individuals. A similar lesion is seen in berylliosis, a granulomatous lung disease due to inhalation of beryllium oxide. Only a small number of exposed individuals develop chronic berylliosis, and a delay of several months to years precedes the initial manifestations of disease. This granulomatous reactivity is also specific as shown by skin testing.43 It is important to realize, therefore, that idiosyncratic allergic reactions may occur not only to the above substances, but also to a wide variety of agents that usually cause nonimmunologic lesions.

In conclusion, it is proposed that the traditional classification of granulomas into infectious and foreign-body lesions, based largely on the morphological criterion of the presence or absence of the epithelioid cell, be superseded by a functional classification based on the mechanism of these reactions. Although the turnover rate of mononuclear cells in granulomas has provided information of great importance, it does not constitute a reasonable means of classifying the lesions. At the simplest level the functional concept of immunologic and nonimmunologic granulomas is based on the presence or absence of secondary anamnestic reactivity. This is a verifiable phenomenon and if found can be followed by a series of further investigations to elucidate the particular mechanism of each type of reaction. Thus this system is not merely a means of classifying complex pathological reactions to be filed away in textbooks for students to memorize. It


is a dynamic concept that could provide a means of solving the enigma of the granuloma, leading to an eventual understanding of granulomatous diseases of unknown etiology such as sarcoidosis.

Addendum: A Tentative Theory on Sarcoidosis

Dr. D. Geraint James sent me a copy of his recent review on the immunology of ~ a r c o i d o s i s ~ ~ just prior t o this Conference and asked me t o see if I could link what is going on in his field with investigations in our sphere. Carefully reading that interesting paper and thinking about work now in progress in our laboratory, I would like to present a tentative theory.

On the basis of the old and important criterion of morphology I believe that the sarcoid granuloma probably falls into the category of immunologic lesions. The nidus for the lesion may be related to the presence of laminated concretions composed of calcium and protein (Schaumann bodies), in the granulomas, or “asteroid bodies” in the giant cells of the lesion^,^ but the etiology of sarcoidosis remains unknown. The immunologic mechanism of the granulomas is probably cell-mediated, but then the question arises: How can cell-mediated granulomas exist in the presence of a generalized suppression of delayed hypersensitivity? Strangely enough, an answer has been suggested by our studies of schistosomiasis.

In 1964 we observed that during the course of a chronic S. mansoni infection granulomas around eggs newly arrived in the liver become smaller and ~rnal ler .4~ This was confirmed in subsequent investigations in which eggs were injected into the lungs of mice at different times in the course of natural i n f e ~ t i o n . ~ ’ We have been studying the mechanism of this modulation of granulomatous inflammation and have recently reported that coincident with the suppression of the granuloma there is an exponential rise in hemagglutinating antibodies to soluble S. mansoni egg antigens and an increase in the number of bands observed on immunodiffusion analysis.32 Accompanying these changes there was a gradual alteration in the response of lymphocytes to antigen as measured by their output of migration inhibitory factor, to the point where the cells became completely unresp~nsive.’~ These observations have been confirmed by Colley using related Furthermore, more recent studies have revealed that in the chronic stages of murine schistosomiasis mansoni a generalized unresponsiveness of lymphocytes to the T-cell mitogens phytohemagglutinin and concanavalin A develops.47

Thus it is possible that the chronic antigenic stimulus responsible for the “cell- mediated immunologic” sarcoid granuloma (probably a poorly biodegradable, antigen-releasing infectious agent) induces modulation phenomena in which the granuloma may be reduced somewhat in size, accompanied by a considerable increase in antibody levels and the development of lymphocyte unresponsiveness to both specific antigen and nonspecific mitogens.

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150-156. 47.

48. WARREN, K. S. Work in progress.

DISCUSSION

DR. M. F. KAGNOFF: I would like t o know if any work has been done to characterize the nature of the material inducing the granulomas in schistosomiasis japonicum. Perhaps Dr. Warren might make a few comments concerning antibody- induced granuloma formation?

DR. WARREN: There has been no clear-cut evidence of any antibody-induced granulomas, but Allison has often suggested that such a phenomenon could exist. Pathologically, japonicum lesions d o differ and they could possibly be the result of an antigen-antibody-complex-mediated reaction. I showed you some morphological reasons why they are different. With respect to the antigen, we have isolated soluble antigens from the S . juponicurn eggs and they induce immediate footpad swelling. With Dr. Boros and now with Dr. Pelley we have been working on the structure and purification of S. rnunsoni antigen. We have one antigen in pure form, MSA, which we think is the major granuloma-inducing antigen. Dr. Pelley has developed a radioimmunoassay for this antigen and when we add crude S . japonicum antigen there is less than 1% crossreactivity with the MSA antigen. So there are important differences between S. juponicum and S. rnansoni antigens.

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