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THE JOURNAL OF INVESTIGATIVE DERMATOLOG Y, 67:129-135, 1976 Copyright © 1976 by The Williams & Wilkins Co.
Vol. 67, No . 1 Printed in U.S.A.
DIAGNOSTIC PROCEDURES IN IMMUNODERMATOLOGY
RUDI H. CORMANE, M.D ., AND SYED SHAFI ASGHAR, PH.D.
Department of Dermatology, Binnengasthuis, University of Amsterdam, Amsterdam, The Netherlands
Most immunologic diseases are caused by the derailment of the humoral or cellular pathways of the immunologic defense system. This derailment results from numerous factors such as the inability of the patient to remove the pathogen; the consumption, defect, or deficiency in any component of these pathways, and the overproduction of any of the components.
To diagnose these immunologic disorders one has to detect the pathogen and the reactions caused by it and to determine the cause of its nonclearance. The immunofluorescence technique has been invaluable in detecting both the antigen that causes the disease and the reactions initiated by the antigen, such as the production of antibodies and the activation of the complement system. The immunoperoxidase technique has also been used for these purposes in certain instances. For detecting the circulating immune complexes which occur as intermediates in the chain of reactions initiated by the antigen, various physicochemical and biologic techniques have been used. However, none of these tests seems to be totally reliable for determining whether circulating immune complexes are present.
The consumption of complement was detected by hemolytic estimations and radial immunodiffusion or rocket electrophoresis. These techniques were also useful in detecting the hereditary deficiencies in immunoglobulins and components of classical and alternative pathways of complement activation. Since these techniques cannot be used to estimate IgE, the radioallergosorbent test was used to measure such levels in atopic patients.
Cellular hypersensitivity was detected with skin tests together with methods which assess the ability of lymphocytes to produce mediators in response to antigen. Many of these mediator assays, however, are not suitable for this purpose. A satisfactory substitute appears to be to determine the factor in antigen-stimulated, lymphocyte culture supernatants which activates macrophages to take up radiolabeled colloidal gold or radiolabeled glucosamine. In contact allergic dermatitis, an increase in the IgD-bearing lymphocytes and granulocytes has also been correlated with cellular hypersensitivity. Lymphocytes and polymorphonuclear leukocytes coated with antibodies mainly directed against nuclear antigens of the basal layer cells of the noninvolved epidermis have invariably been encountered in psoriasis. The use of these findings for diagnostic purposes and for understanding the mechanisms of certain diseases is being explored.
The factors that cause the various immunologic diseases in man can be broadly divided into four categories: (1) self antigens; (2) foreign antigens such as viruses, bacteria, chemicals, and drugs which may form immunogens with body proteins; (3) foreign substances such as bacterial polysaccharides; and (4) immunodeficiencies and other
Reprint requests to: Dr. R. H. Cormane, Department of IJermatology, Binnengasthuis, University of Amsterdam, A. msterdam, The Netherlands.
Abbreviations: ECF-A: eosinophil chemotactic factor of anaphylaxis FTC: fluorescein thiocyanate LPF: lymphocyte proliferation factor MAF: macrophage activator factor MIF: migration inhibition factor MNLCTX: mononuclear chemotactic factor PG: prostaglandin PMN: polymorphonuclear RAST: radioallergosorbent test SRS-A: slow-reacting substance of anaphylaxis
genetic abnormalities. These factors can cause derailment of the humoral or cellular pathways of immunologic reactions. Figure 1 shows the main humoral immunologic reactions but not the side reactions, such as abnormalities in the mechanism of blood coagulation caused by the activation of alternative pathway of complement activation.
In normal individuals, these reactions appear to be inhibited by agents in the blood, but they could be activated by'factors such as endotoxins or by antigens if antibodies have already been produced. After the source of their activation is removed, they return to the unactivated state. In a disease state, some of these reactions are activated, depending on whether the activator is an antigen or an endotoxin, and remain so during the course of the disease either because the activator is continuously resupplied or because the patient's reticuloendothelial system or some other eliminating mechanism is unable to remove it. Other causes of disease
129
130 CORMANE AND ASGHAR
IgE-sensitized _ circulatiflg ... ----- Ag + Ab Basophil or Mast cell
baS01PhiiS immune complex I Clumping of platelet, release of vasoacti ve
IgE (Ab) IgE (Ab) '- .J i + allergen (Ag)
Release of chemical mediators
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Activating substance such as endotoxin polysaccharide
~ IF---IF
I I
Platelet-activating amines Immune complex (IC) C3+P ,. P (deposited) ---,-
I factor ---------..
increased permeability ~ C4 C2 : +C3PAse (Factor D)
C CI V C3 Mg+ + , 1- -t ): _________________ C3PA(FactorB)
C42 ------l.. ,/,:r "C3b inhibitor /' ~/
Attraction of PMN-leukocyte
Tissue injury ... -----and release of lysosomal constituents
chemotactic factors
C3a C3b- C3bi I
....... ------ I I I
1------- C5
_------ I \ C5a C5b
1 + C6-C9
C5b- C9 membrane attack
FIG. 1. Summary of main humoral immunologic reactions in clinical hypersensitivity and disease. Ag = antigen, Ab = antibody, CI-C9 = components of complement, IF = initiating factor, P = properdin, C3PA = C3 proactivator, C3PAase = C3 proactivator convertase.
states include a defective component of these pathways, e.g. , a deficient inhibitor of the first component of complement in hereditary angioneurotic edema and the overproduction of IgE in atopic patients.
First, we shall describe these pathways briefly. If antibodies have been produced by an antigen, addition of more antigen may lead to the formation of antigen-antibody complex. This complex may be fixed or circulating depending on whether the antibodies have been produced against a fixed antigen such as autoantigen or whether the antigen is circulating or is one of the proteins of the circulatory system. If the complex is circulating and large ( > 198), it will be deposited between the endothelial cells in such a way [1] that a plateletactivating factor is released from IgE-sensitized basophils by immune complexes and the vasoactive amines are released by the platelet-activating factor. Because of increased vascular permeability, larger complexes are trapped in and around the wall of the blood vessel.
Whatever the mechanism of deposition, the immune complexes either before or after deposition can activate the complement sequence that leads to the generation of chemotactic factors and is followed by the attraction of polymorphonuclear (PMN)-leukocytes, the lysosomal enzymes of which may cause tissue injury [2].
If the alternative pathway has been activated, say by endotoxin, complement from the C3 stage will be activated by a mechanism described by Muller-Eberhard [3], which involves an initiating factor, properdin, C3 proactivator, C3 proactivator convertase, C3, and probably unknown proteins of this pathway. After complement from C3 has been activated, the remaining steps of complement action leading to tissue injury and membrane attack are common to both pathways.
In addition to the mechanism that activates the complement system, there are other causes of
allergic disorder in atopic patients, i.e., IgE antibodies which combine with mast cells through Fc fragments. When allergen reacts with cell-bound IgE, the enzyme sequence is activated and such chemical mediators as histamine, the slow-reacting substance of anaphylaxis (8R8-A), prostaglandins (PGs), eosinophil chemotactic factor of anaphylaxis (ECF -A), and kallikrein are released.
The cellular pathways of self-defense involve various cell types. One of them, the B-Iymphocyte, is the immunoglobulin-bearing lymphocyte found in lymph nodes. The other cell, the T -lymphocyte, is the monocyte which originates in the bone marrow, is processed in the thymus, and is devoid of antibody. At least these two types are needed to express cellular hypersensitivity. When sensitized lymphocytes come in contact with antigen, they release several proteins, such as the migration inhibition factor (MIF), the skin reactive factor, and about 10 more which have a pronounced effect on other cells involved. These factors, perhaps by autocatalytic or positive feedback effect, help to produce biologic activities from nonsensitized lymphocytes as well. By virtue of their different properties, they lead to the localized accumulation of nonspecific inflammatory mononuclear cells and, in extreme reaction, to tissue necrosis (see detailed descriptions by Lawrence and Lardy [4), David [5), and Asghar and Cormane [6]) .
Both humoral and cellular immunologic pathways may be operative at the same time in a patient or an experimental animal. Besides their activation by certain pathogens, some genetic deficiency, defects or overproduction may eventually impair the function of the whole pathway and may cause the disease.
We have described these pathways briefly because our aim is not to discuss the mechanisms of immunologic reactions but to show that the method of diagnosing immunologic disorders should be based on either a recognition of the
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factors that are responsible for initiating the disease or the detection of reactions which have been activated by these factors. Thus, the diagnosis of immunologic diseases should be based on the criteria described in Table 1.
DETECTION OF ANTIGEN
In an immunologic reaction caused by an antigen, microprecipitation of antigen and antibodies may occur in and around the blood vessel walls or immune complexes may be found in the circulation before they have been deposited. So far, there is a scarcity of data showing antigen parts of immune complexes which are circulating, but in certain cases the immunofluorescence technique has been used to demonstrate the antigen in the deposited immune complexes. The rationale and the technical details of the immunofluorescence technique have been described by Beutner et al [7], and the requisites for achieving peak performance with immunofluorescence microscopy have already been discussed by Ploem [8].
Using this technique, Wemambu et al [9] found evidence that microbacterial antigen is the cause of the Arthus-like reaction in a form of leprosy characterized by neutrophil infiltrates. Parish [10] demonstrated Streptococcus pyogenes group A and D antigens in cutaneous vasculitis and erythema nodosum. Streptococcal, candidal, and microbacterial antigens were also detected in the lesions of vasculitis.
Simultaneous staining for the immunoglobulins
TABLE I. Basis of diagnosis various immunologic disorders
1. Detection of antigen
2. Detection of antibod-les
3. Detection of immune complexes
4. Involvement of complement by classical or alternate pathway
5. Immunodeficiencies and other genetic abnormalities
Detection of cellular hypersensitivity and studies of ancillary cells
Whether fixed, circulating, self or foreign. Identification if possible.
Whether locally fixed, circulating, or on the surface of ancillary cells.
Whether circulating or fixed.
Whether complement is activated locally or systemically. Whether complement levels are altered and fragments detectable.
Whether there is deficiency or genetic defect in immunoglobulins, complement components of classical or alternative pathway, or inhibitors of these systems.
Whether delayed hypersensitivity is operative. Whether antibodies directed against a particular antigen are present on ancillary cell surface.
IMMUNODERMATOLOGY 131
and bacterial antigens with rhodamine and fluorescein thiocyanate (FTC}-labeled antisera occasionally revealed them in combination. Parish [10] has also demonstrated the antibody specificity of immunoglobulins against the antigen in the lesion. The extracellular isolated protein antigen of Strep. pyogenes group A and antigen extracted from Staphylococcus aureus were unequivocally fixed on aggregated IgG in the lesions.
The immunofluorescence technique is now established as a diagnostic and investigative tool in immunodermatology. Another technique, which uses other markers, is the immunoperoxidase technique in which peroxidase-coupled antibody reacts with antigen and the colored enzyme product serves as a stain. Although this technique is not as sensitive as the immunofluorescent technique [11], it is useful for certain purposes in light and in electron microscopy [12].
DETECTION OF ANTIBODIES
Direct and indirect immunofluorescence techniques have been useful in detecting antibodies, both fixed and circulating. Several antibodies under different pathologic conditions have been detected by this technique, but only the most important are listed in Table II.
Antibodies may also occur on the surface of cells such as lymphocytes or PMN-Ieukocytes (see below).
DETECTION OF IMMUNE COMPLEXES
The mechanism of circulating immune complex disease is the same as that of fixed complexes except that active processes are involved in the deposition of immune complexes. The persistent presence of complexes can cause such pathologic effects as anaphylaxis, Arthus-like reaction, granulomata, and hyperviscosity. Immunofluorescence microscopy is now indispensable for detecting deposited immune complexes. The circulating complexes can be detected by various physicochemical and biologic methods (Tab. III).
Physicochemical Methods
The physicochemical methods depend on the separation of complexes from serum immunoglobulins because of size, charge, or solubility characteristics. Analytical ultracentrifugation has been used to demonstrate 22S IgG-IgM complexes in the serum of patients with rheumatoid arthritis
TABLE II. Antibodies detectable in the skin and/or serum by immunofluorescence technique in various diseases
Substrates for antibodies
Subcellular constituents Stratum corneum Intercellular space material Basal cell cytoplasm a and
membrane Basement membrane
Diseases
SLE Psoriasis vulgaris Pemphigus, burns Drug reaction
Pemphigoid
132 CORMANE AND ASGHAR
TABLE III . Methods employed for the detection of circulating immune complexes
Physicochemical methods
Analytical ultracentrifugation
Density gradient ultracentrifugation
Electron microscopy Cryoprecipitation
Differential solubility
Biologic methods
Reaction with Clq
Reaction with rheumatoid factor
Platelet aggregation Histamine release from
guinea-pig lung Increased antibody con
centration after destruction of antigen
Anticomplementary activity
and 11-13S IgG-IgG complexes in the serum of W aldenstrom 's benign hypergammaglobulinemic purpura [13]. This technique, however, is useful only for detecting large quantities of circulating complexes (>5% of total serum proteins). Other complexes can be detected by density gradient centrifugation. Immune complexes in the sediment have been visualized by electron microscopy [14].
Other methods of detecting immune complexes take advantage of the fact that the latter have decreased solubility at 4 ec, at low ionic strength, or in salting-out conditions. These approaches have, however, been unsatisfactory for several reasons, including the fact that under these conditions many unwanted proteins, such as antibodies with rheumatoid factor activity and Clq, tend to precipitate.
Biologic Methods
Several biologic methods have been used to detect circulating immune complexes. C1q has been used to detect immune complexes because it can precipitate with them in agarose gel. Asghar, Faber, and Cormane [15], however, have shown in the serum of a patient with allergic vasculitis that C1q gave a strong precipitation line with some unidentified material which could not be shown to be an immune complex. Similarly, precipitation of complexes by isolated monoclonal rheumatoid factors may give a false positive reaction.
A comparatively more sensitive and reproducible procedure for detecting soluble immune complexes has been reported [16]. Radiolabeled C1q is reacted with sera containing immune complexes. Both free and complex bound (1251) Clq are separated by selective precipitation with polyethylene glycol. Complex bound radioactivity is the measure of immune complexes. This method is more sensitive than agarose gel precipitation but may have similar drawbacks of reaction with nonimmune Clq reactants. In addition, the different Clq levels in serum samples might have some effect since higher C1q levels may cause more competition with radioactive C1q than lower levels. More
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work is needed to confirm the suitability of this method for immune complex detection.
Immune complexes were detected by virtue of their property to release histamine from guinea-pig lungs [17] and to aggregate platelets [18]. When antigen is known to be in complexes, an increase in free antibody concentration after enzymic degradation of antigen suggests the presence of immune complexes. DNA-anti-DNA complex has been detected by this method [19].
Decreased levels of total complement or a specific component of complement (C3) associated with the degradation products of complement (~lC - ~lA + a2D) have been used to measure circulating immune complexes. Recent data on the alternative pathway, however, suggest that these observations implicate a nephritic factor or the activation of the alternative pathway at any other step [3 ].
Thus, these biologic tests do not conclusively identify the immune complexes circulating at any one time. Several factors affect the accuracy of these tests: the degree of lattice formation required to initiate the biologic test reaction, the excess of antigen or antibody in the sera at the time of testing, pseudoimmune complexes which behave like them, and the immunoglobulin class of antibodies in complexes. No current technique can demonstrate the presence or absence of immune complexes. Perhaps some of these techniques, both physicochemical and biologic, should be combined but such a combination would be too cumbersome for routine diagnoses. Further research is obviously needed.
INVOLVEMENT OF COMPLEMENT BY CLASSICAL OR ALTERNATIVE PATHWAY
If fluorescent antibodies directed against the individual components of the classical or alternative pathway are used, the binding of these components at the site of the lesions can be followed. This technique, which does not differ from that used to detect antigen or antibody, can be used with FTC-labeled antisera to any of the complement components. Immunofluorescence has been especially useful with anti-C3 since C3 is present in the immunologic lesions in largest amounts. C4, C5, and Clq have also been detected in lesions. In all of the diseases listed in Table III concomitant deposition of complement has been observed. Deposition of properdin and C3 proactivator have been observed in kidney biopsies of systemic lupus erythematosus (SLE) nephritis, chronic membranoproliferative glomerulonephritis, and other diseases [20], but such data with these or other components of alternative pathway are scarce in skin diseases.
Evidence of in vivo complement activation by either of the pathways is the detection of fixed components, decreased levels of the components of alternative or classical pathway, and the formation in vivo of breakdown products of components of these pathways. These phenomena have been
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studied in detail only with regard to total hemolytic complement and C3. a 2D, in particular, is found in the circulation of patients with hypocomplementemic membranoproliferative glomerulonephritis. Occasionally in other immune disorders (e.g., hypocomplementemic SLE), C3 breakdown has been observed [21]. Additional research into other skin diseases will probably yield more information.
Total hemolytic activity in the serum can be measured by estimating the degree of hemolysis of sensitized sheep erythrocytes caused by various dilutions of test serum [22]. The individual components can also be estimated by hemolytic methods which have clearly been described by Lachmann, Hobard, and Aston [23]. Several hemolytic methods can also estimate the alternative pathway as well as its components. Alternatively, the levels of these components or their breakdown products can be determined antigenically [24,25]. A sample is introduced in the holes at the bottom of the gel which contains monospecific antiserum. The gel plate is allowed to stand, as in the Mancini technique, or electrophoresed. The area of ring or the height of the precipitate is the measure of antigen concentration or, in this case, of the concentration of component of these pathways.
IMMUNODEFICIENCIES AND OTHER GENETIC ABNORMALITIES
The term immunodeficiency encompasses a broad spectrum of abnormalities in the host defense that ranges from mild to severe and is paralleled by increasing susceptibility to different pathogens. Hereditary deficiencies in immunoglobulins, complement components, inhibitors of complement system and the constituents of PMNleukocytes, as well as B- and T-cell function, have been reported. These deficiencies can easily be tested by the techniques already. described. Deficiencies in B-cell and T-cell -function can be detected by assaying such functions as antibody and MIF production.
In those patients who are only functionally deficient in a particular protein, the protein may be defective or have impaired biologic activity; for example , the defective C1-esterase inhibitor in hereditary angioneurotic edema and the defective IgA in some cases of atopy. In such cases, the protein is estimated according to its activity as well as antigenically. These activities are too nUmerous and diverse to allow us to describe how the activities of the components of the defense system are determined. Some of the immunodeficiencies and genetic defects, together with the diseases with which they are associated, are listed i.n Table IV.
Finally, the' techniques described so far do not detect another defect, not in the structure, but in :he quantity of production. This is the higher levels )f IgE in atopic patients. Diagnosis in vitro based m IgE levels is becoming a routine analytical Jrocedure in many laboratories. The radio aller;osorbent test (RAST) [25] seems to be the most
IMMUNODERMATOLOGY 133
TABLE IV. Some examples of genetic immunologic deficiencies and the defects associated with disease states
Disease Deficiency
Wiskott-Aldrich Syndrome Deficiency in T-cell function
Some forms of chronic Deficiency in T-cell mucocutaneous candidiasis function
Some cases of atopy and Defect in or deficiency a range of disorders of IgA
Hereditary angioneurotic Defect in or deficiency edema of Cl-esterase inhibitor
Arthritis of sex-linked Gammaglobulin deficiency agammaglo bulinemia
Lupus-like illness C2 deficiency
suitable large-scale test for IgE antibody. The rationale of this technique is that when allergen bound to an insoluble matrix such as cellulose is incubated with test serum, allergen-polymer-IgE complex is formed. Radioactive anti-IgE can combine with this complex, and the radioactivity in the insoluble matrix gives the amount of IgE present in the serum.
DETECTION OF CELLULAR HYPERSENSITIVITY AND STUDIES OF ANCILLARY CELLS
Many authorities still regard skin tests as the only valid measure of cutaneous delayed hypersensitivity. Several studies, however, report that many assays of cellular hypersensitivity in vitro are equally good procedures. These assays are based on evaluating the ability of lymphocytes to produce mediators after antigen challenge or challenge with immunologic equivalent. They include the tests of release of (1) lymphocyte proliferation factor (LPF), (2) migration inhibition factor (MIF), (3) macrophage activator factor (MAF) , and (4) mononuclear chemotactic factor (MNLCTX).
Some of these methods have drawbacks. Since antigen-induced proliferation of lymphocytes can occur without delayed hypersensitivity or with Arthus reaction, measuring LPF is not necessarily an expression of delayed-type hypersensitivity . As far as using the ability of lymphocytes to produce MIF to test delayed hypersensitivity is concerned, despite various technical modifications, the prolonged multistep procedures are subjeCt to numerous technical problems; hence there is considerable variation within and between assays. In addition, competing molecular entities in the supernatant fractions which enhance macrophage migration, plus the fact that B-cell products such as antibodies can inhibit macrophage migration, make it hard to believe that the MIF assay really correlates with delayed hypersensitivity. Similarly, the technique which involves assaying MNLCTX can be complicated by the products of complement factors such as C5a. Furthermore, both T-cell and B-cell mitogens stimulate T -cells to produce MNLCTX. Even nonmitogenic substances which interact with B-cell membranes such as anti-
134 CORMANE AND ASGHAR
immunoglobulins, C3 sites, and antigen-antibody complexes are all potent activators of B-cell MNLCTX production [26].
Some investigators have advocated alternative mediator assays, namely, MAF [27]. Antigenstimulated lymphocyte culture supernatants contain a factor which causes macrophages to take up radiolabeled colloidal gold. This assay is quantitative, rapid, and sensitive, but gold particles must not be allowed to aggregate; otherwise phagocytosis occurs and gives a nonspecific result. Another assay for macrophage activation which uses the uptake of radiolabeled glucosamine has been described.
Direct immunofluorescence studies of biopsies taken from positive patch tests elicited by various antigens in our laboratory revealed lymphocytes with membrane-bound immunoglobulins throughout the dermis and epidermis, particularly in and around the blood vessel walls [28]. With the immunofluorescence technique, we found immunoglobulins and complement-bearing leukocytes and lymphocytes in the lesional skin of specimens of patients with various dermatoses [28,29]. In all the cases of allergic contact dermatitis examined, the percentage of IgD-bearing lymphocytes and PMN -leukocytes was increased. This altered ratio of circulating B-cells and T -cells may indicate an impaired cooperation of at least these cell populations. Like lymphocyte populations, the PMNleukocyte populations consist of cells with and without immunoglobulins on the cell membrane in the same ratio as in healthy controls.
Another finding observed by the immunofluorescence technique was the occurrence of antibodies in eluates of lymphocytes and PMN -leukocytes obtained from 5 untreated psoriasis patients [30]. These antibodies appeared to be directed mainly against the nuclei of the basal cell layer (Fig. 2); however, antibodies directed against the nuclei of epidermal and dermal cells have also been seen. Since the eluted antibodies are directed mainly against nuclear antigens of dividing epidermal cells and not against the nuclei of all cells, it may be that the nuclear antigens are the nonhistone proteins which regulate the growth of the dividing epidermal cells [31].
The lymphocyte eluates of 3 patients with discoid lupus erythematosus and 2 with pemphigoid contained IgG antibody reactive with normal human skin or guinea-pig basement membrane. Similarly, lymphocyte eluates from a patient with pemphigus contained IgG antibody reactive with intercellular substances of monkey esophagus, and those of a patient with a drug reaction contained antibody reactive with basal cells of stratified epithelium_ [32]. These results may prove especially useful in diagnosing diseases associated with cellular hypersensitivity.
INDICATIONS FOR THE FUTURE
Further research is needed to fill in the many gaps that exist in the field of diagnosis in dermatol-
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FIG. 2. Eluate from the peripheral lymphocytes of a patient with psoriasis vulgaris with FTC-labeled antihuman IgD to show positive immunofluorescence of the nuclei of the basal cell layer of the uninvolved skin of the same patient.
ogy. Some of the most obvious gaps are listed below.
1. Various approaches must be used to detect the antigen responsible for the disease state and to uncover the etiology of many diseases.
2. More accurate methods of detecting immune complexes must be found.
3. More studies are needed on the components of the alternative pathways of complement activation and their involvement in disease states. Production of antibodies against all of them will enable us to estimate them quantitatively.
4. Further studies must be done on the antibodies coated on ancillary cells.
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