Fibrillary Glomerulonephritis Related to Serum Fibrillar Immunoglobulin-Fibronectin Complexes

Agueda Rostagno, PhD, Ruben Vidal, PhD, Asok Kumar, PhD, Joseph Chuba, PhD, George Niederman, MD, Leslie Gold, PhD, Bias Frangione, MD, PhD, Jorge Ghiso, PhD,

and Gloria Gallo, MD

0 Fibrillary glomenrlonephritis is a disease of uncertain origin and pathogenesis characterized by nonamyloidotic fibrils in glomeruli. We report immunohistological, immunochemical, and biochemical studies of a serum fibrillar cryoprecipitate obtained from a patient with fibrillaty glomerulonephritis, that formed on prolonged storage at 4°C. By Western blot and amino acid sequence analysis, the cryoprecipitated fibril components consisted of immunoglobulins, heavy chains y and CL, light chains K and A, and fibronectin, similar to the proteins identified by immunofluorescence and immunoelectron microscopy in the glomerular fibrils. These findings support the hypothesis that serum precursors may be the source of the fibrillar deposits and suggest a role for immunoglobulin- fibronectin complexes in the pathogenesis of fibrillary glomerulonephritis. 0 1996 by the National Kidney Foundation, Inc.

INDEX WORDS: Fibrillary ON; cryoprotein; fibronectin; immune complexes.

F IBRILLARY glomerulonephritis (GN) as first described by Rosenmann and Eliakim’

and subsequently by others,2-‘5 is characterized by the presence of fibrils in glomeruli of patients who have no known associated disease. Although the fibrils in fibrillary GN exhibit some morpho- logical features similar to amyloid, they differ in several ways: the fibril diameter in fibrillary GN (16 to 24 nm) is larger than in amyloid (8 to 12 nm), and the fibrils are not twisted or congophilic as they are in amyloid deposits.14 Furthermore, unlike light chain amyloidosis and other diseases with fibrillar deposits, a B-cell proliferative dis- order, dysproteinemia, or serum precursors have not been identified in fibrillary GN.

Evidence suggesting that the fibrils in fibrillary GN are polymerized polyclonal immunoglobulin (Ig) deposits of immune complexes was obtained by an immunoelectron microscopic study of renal biopsy tissues from patients with fibrillary GN that showed specific immunoreactivity of the fi- brils with both anti-K and anti-X light chain anti- sera and complement component (C3).14 We now report the detection and the immunohistochemi-

From the Department of Pathology, New York University Medical Center, New York: and Maimonides Medical Center, Brooklyn, NY.

Received June 5, 1996; accepted July 17, 1996. Supported by grants from the National Institutes of Health

service (AR 02594, AI 32110), Bethesda, MD. Address reprint requests to Gloria Gallo, MD, Department

of Pathology, NYU Medical Center, 560 First Ave, New York, NY 10016.

0 1996 by the National Kidney Foundation, Inc. 0272-6386/96/2805-0003$3.00/O

cal and biochemical characterization of proteins comprising the fibrils in a serum cryoprecipitate, which correspond to the proteins detected by im- munoelectron microscopy, in the deposits of glo- merular fibrils from a patient with fibrillary GN.

CASE REPORT

A 54-year-old woman with a history of hypertension for 2 years presented 4 days before admission with progressive leg swelling for the preceding 3 weeks. There was no evi- dence of previous renal disease. Laboratory studies showed a serum creatinine of 5 mg/dL, which rose 3 days later to 7 mg/dL. The urine protein excretion was 5 g/24 hours.

On hospital admission, she gave a history of a 20-lb weight gain over the previous 2 to 3 months, nausea without vom- iting, and irregular sleep patterns. She denied shortness of breath, cough, hemoptysis, fever, rash, arthralgia, dysuria, or hematuria. The physical examination was significant for a blood pressure of 160/90 mm Hg and bilateral 3 to 4+ pitting edema of the lower extremities.

Laboratory findings showed blood urea nitrogen, 114 mg/ dL; serum creatinine, 9.5 mg/dL; antineutrophil cytoplasmic antibody, negative; antinuclear antibody, negative; anti-glo- merular basement membrane antibody, negative; C3 and C4 levels, normal; anti-streptolysin 0 antibody, negative; anti- hepatitis B surface antigen, negative; anti-hepatitis C virus, negative; and rheumatoid factor, negative. The microscopic examination of the urine showed red blood cells as well as red cell casts. Renal sonography was normal, and nuclear scan showed a severe decrease in renal function.

Hemodialysis was started 4 days after admission because of a rapidly increasing serum creatinine to 12.5 mg/dL. A renal biopsy was performed 1 week after admission and 4 weeks after apparent onset.

MATERIALS AND METHODS

Immunohistological Studies of the Renal Tissue The renal biopsy specimen was examined by standard opti-

cal, immunofluorescence, electron, and immunoelectron mi-

676 American Journal of Kidney Diseases, Vol 28, No 5 (November), 1996: pp 676-684

CRYOPROTEINS IN FIBRILLARY GLOMERULONEPHRITIS 677

croscopy as previously described.14 For immunofluorescence fluorescein-labeled rabbit antibodies against human chain- specific Igs y, k, 01, K, A, C3, and fibrinogen (DAKO, Carpen- teria, CA) were used. Immunoelectron microscopy of Epon- embedded biopsy tissue was performed using unlabeled rab- bit antibodies against human Ig -y, p, K, X chains, albumin, amyloid P component (DAKO), fibronectin (Fn) (Calbio- them, San Diego, CA), laminin (Telios Pharmaceuticals, Inc., San Diego, CA), followed by 15 nm gold-labeled protein A (Amersham, Rockford, IL).

Immunohistochemical and Biochemical Analysis of the Serum Precipitate

A small precipitate formed in the patient’s serum, first noted after 4 months of storage at 4°C. The precipitate, which did not redissolve on warming at 37°C to 4O”C, was centri- fuged and washed five times in cold Tris-buffered saline (TBS), pH 7.4.

Ultrathin sections of the washed precipitate fixed in 2.5% glutaraldehyde or 4% paraformaldehyde, embedded in Epon or LR White respectively, were immunoreacted with a panel of the unlabeled rabbit anti-human antibodies listed above, including K and A light chains, y and p heavy chains, albumin, laminin, amyloid P component, Fn, and normal rabbit serum followed by gold-labeled protein A as previously described.14 Also used were mouse monoclonal antibodies against the amino terminal domain of Fn (Fn a-NT, gift from Dr. Angeles Garcia-Pardo, Madrid, Spain); anti-cell binding domain of Fn, (Fn a-CBD, N-296, Mallinckrodt, Inc, St. Louis, MO); anti-carboxyl-terminal region of Fn, (Fn a-CT, N-296, Mal- linckrodt), and anti-human fibrinogen 3 11 (American Diag- nostica, Greenwich, CT) followed by gold-labeled goat anti- mouse IgG(Fc), (Amersham International, Buckinhamshire, England).

Western blot analysis. The washed precipitate was solu- bilized in Laemmli sample buffer, and the proteins, separated on a gradient sodium dodecyl sulfate polyacrylamide gel elec- trophoresis (5% to 20% of acrylamide monomer),‘6 were transferred to Immobilon P membranes (Millipore, Bedford, MA) using 10 mmol/L cyclohexylaminopropane sulfonic acid (Sigma, St Louis, MO) buffer, pH 11, containing 10% metha- nol and subjected to Western blot” and amino acid sequence analysis.‘8

For immunoblot analysis of the cryoprecipitate, the follow- ing antibodies were used: rabbit anti-human Fn (Calbiochem), biotinylated employing immunopure-NHS-LC-Biotin (Pierce, Rockford, IL) according to the manufacture’s instructions; affinity-purified goat anti-human Ig heavy chains y and p (Calbiochem); rabbit anti-human Ig light chain h (DAKO) and K (The Binding Site, San Diego, CA); and alkaline phos- phatase-labeled anti-rabbit and anti-mouse Igs (Tago Inc, Burlingame, CA).

The Immobilon membrane was blocked overnight with 3% nonfat dried milk in TBS, pH 7.4, containing 0.5% Tween- 20 (TBS-T), and incubated separately for 1 hour with either polyclonal anti-human y, CL, K, h chain-specific antibodies or biotin-labeled anti-Fn antisera. After washing with TBS-T, the membranes were incubated with the corresponding sec- ondary antibodies conjugated to alkaline phosphatase. The biotinylated anti-Fn was detected with alkaline phosphatase-

labeled streptavidin; in all cases the immunoblots were devel- oped with the Promega Protoblot system @omega Corp., Madison, WI).

Amino acid sequence analysis of the serum precipitate components. The immobilized protein bands, visualized by staining with Coomassie blue R-250 in 50% methanol, were excised from the membrane and the N-terminal amino acid sequence determined by automated Edman degradation on a 477A microsequencer (Applied Biosystems, Foster City, CA). The resulting phenylthiohydantoin derivatives were identified using an on-line 120 PTH analyzer (Applied Bio- systems).

Binding of biotinylated Fn to the serum precipitate by dot- blot analysis. Human Fn was isolated from pooled normal plasma by sequential affinity chromatography on lysine-seph- arose and gelatin-sepharose (Pharmacia Biotech Inc, Upsala, Sweden) as previously reported” and biotinylated as de- scribed. An aliquot of the washed serum precipitate was solu- bilized in 0.1 mol/L Tris-HCl buffer, pH 7.4, containing 4 mol/L urea and applied to a nitrocellulose membrane in sev- eral dots (2 &20 PL each). The membrane, with dried sam- ples, was blocked with 3% nonfat milk in TBS-T. Binding of Fn to the immobilized protein was assessed by incubation with biotinylated Fn (20 &500 PL TBS containing 0.1% bovine serum albumin and 0.1% Tween-20) for 3 hours at room temperature, followed by alkaline phosphatase-labeled streptavidin for 1 hour. As a control for self-Fn binding activ- ity (autopolymerization), a 2-pg sample of purified Fn was immobilized onto the membrane and reacted under identical conditions with biotinylated Fn. For additional controls, dots of identical alliquots of the solubilized precipitate were incu- bated with either alkaline phosphatase-labeled streptavidin alone (negative control) or with rabbit anti-human Ig-specific antisera, followed by anti-rabbit Ig as described for the West- em Blot analysis (positive control).

RESULTS

Pathological Features of the Renal Biopsy and Serum Precipitate

Light microscopy. There were 17 glomeruli in the biopsy, two of which were globally obliter- ated by sclerosis. All but two of the remaining 15 glomeruli had circumferential cellular cres- cents that compressed capillary tufts (Fig IA). Congo red-stained sections examined under po- larization microscopy, showed no green birefrin- gence typical of amyloid deposits. There was moderate interstitial edema and leukocytic infil- tration. The vessels appeared normal.

Immunojluorescence microscopy. Frozen sec- tions containing four glomeruli incubated with fluoresceinated anti-human Ig antibodies showed smooth staining of compressed glomerular base- ment membranes, but not other structures, pre- dominantly for IgG (Fig lB), both K and X light chains (not shown) and C3 (Fig 1C). Staining for

678 ROSTAGNO ET AL

fibrin was positive in crescents, but not in the capillary tufts, of all glomeruli. There was irregu- lar staining for IgM and negative staining for IgA. The patient’s serum failed to exhibit binding to glomerular basement membranes of normal kidney substrate examined by indirect immuno- fluorescence.

Electronmicroscopy and immunoelectron mi- croscopy. The thickened convoluted glomeru- lar capillary walls and increased mesangial matrix both contained granular densities inter- mingled with randomly oriented fibrils measur- ing 15 to 20 nm in diameter (Fig 1D). Immuno- electron microscopy of ultrathin sections of glomeruli demonstrated specific labeling of the fibrils with antibodies to X (Fig 2A) and K (Fig 2B) light chains, y and p heavy chains, amyloid P component (not shown), and Fn (Fig 2C), but not normal rabbit serum.

Immunoreactivity of the cryoprecipitate. The serum precipitate (Fig 3) was composed of bun-

Fig 1, (A) Glomerulus with circumferential cellular cres- cent (arrow) surrounding compressed capillary tufts (arrowheads). Periodic acid- silver methanamine, counter- stained with hematoxylin and eosin. (Original magnification x280). lmmunofluorescence of frozen sections show glo- merular smooth staining of compressed capillary tufts for IgG (6) and C3 (C). (Original magnification x250). (D) Elec- tronmicrograph of a glometu- lus demonstrates randomly oriented fibrils in thickened glomerular basement mem- branes. En, endotheliil cell. Uranyl acetate and lead ci- trate. (Original magnification x15,ooo.)

dles of parallel fibrils of indeterminate length measuring up to 440 ,um. The bundles were 280 nm in maximum width, consisting of two to four fib&, each measuring 70 nm and exhibiting 18.75-nm periodic banding. Immunoelectron mi- croscopy showed specific labeling of the fibrils with rabbit anti-human K and X light chains and Fn (Fig 4A to D), amyloid P component, y and p heavy chains (not shown), but not fibrinogen nor normal rabbit serum (Fig 4E F). There was no immunoreactivity of the fibrils in either glo- meruli or cryoprecipitate with antibodies to lami- nin or albumin (not shown).

Biochemical Characterization of the Serum Precipitate Components

Amino acid sequence analysis. Several pro- tein bands, whose apparent molecular masses corresponded to 28, 50, 75, 95, and 240 kd (Fig 5), were detected on the electrotransferred blots. Amino acid sequence analysis of the excised 28-

CRYOPROTEINS IN FBRILIARY GLOMERULONEPHRITIS 679

Fig 2. lmmunoelectron micrographs of a glometulus after incubation with anti-h (A), anti-K (B) and anti-Fn (C) antlbodiis followed by Pro- tein A gold (15 nm). (A) Gold- labeled fibrils in glomerular basement membranes (GBM) and at the luminal interface (arrows) in continuity with the GBM. Ep, epitheliil podocyte; En, endotheliil podocytes. Uranyl acetate and lead ci- trate. (Original magnification x31,250) (B,C) Fibrils in GBM are decorated with gold patti- cles. (Original magnification X50,ooo.)

kd band yielded the sequence DIVMTQSPLSL, EVQLVEXGGGL, identified as Ig heavy chain identified as an Ig K light chain, subgroup II.2o subgroup VH III.*’ The sequence XXGMG/LIE No sequence was obtained for the 50-kd band, was obtained from the 95-kd band. Homology which could represent an N-terminal blocked search using the PIR database (National Biomed- protein. The 75-kd band yielded the sequence ical Research Foundation, Georgetown VA) did

Fig 3. Electron micros- copy of Epon-embedded serum cryopmcipitate dem- onstrates fibrils (between arrowheads) with periodic banding (small arrows), ar- ranged in bundles. (Original magnification x3Cl,ooO.)

not provide unequivocal identification of this protein. However, there are identical sequences in laminin (amino acid residues 1007 to 1011, (Y chain; residues 130 to 134, p chain) and collagen V (residues 464 to 468 of the al(V) chain). Be- cause there was no immunoreactivity of the fi- brils in the serum precipitate or glomeruli with anti-laminin in the immunoelectron microscopy analysis, it is more likely that the 95kd protein corresponds to a fragment of a collagen molecule rather than laminin. No sequence information was obtained for the 240-kd band, which could represent an amino terminal blocked protein (dis- cussed further).

Western blot analysis. The transferred pro- teins constituting the cryoprecipitated fib&, im- munoreacted with a panel of antibodies in the Western blot analysis, are shown in Fig 6. The 28-kd band immunoreacted with both anti-h (lane 1) and anti-K (lane 2) antibodies, corroborating the immunofluorescence and immunoelectron microscopy findings of the renal biopsy and the fibrils in the cryoprecipitate. Because only a K

light chain sequence was obtained for this band, it is likely that the comigrated X light chain is either blocked at the N-terminus, as is the case

ROSTAGNO ET AL

Fig 4. lmmunoektron mi- crographs of LR White- embedded sections of the washed setum ctyoprecipi- tate. Bundles of fibrils are la- beled with 15 nm gold probe after incubation with (A) anti- K; (B) anti-A; (C) polyclonal anti-Fn; (D) monoclonal anti- Fn N-terminus. There is no labeling of fibrils after incuba- tion with monoclonal anti-fi- brinogen (E) or normal rabbit serum (F). (Original magnifi- catlorl x50,ooo.)

of some X I and II light chain subgroups, or the yield was insufficient for amino acid sequencing. Amyloid P component detected in the cryofibrils by immunoelectron microscopy was not identi- fied in the amino acid sequences of the 28-kd band that corresponds to its described electropho- retie mobility. The 50-kd band immunoreacted with anti-y chain-specific antiserum (lane 3). The 75kd band that was identified as Ig heavy chain subgroup VH III by amino acid sequence, immunoreacted with anti-p (lane 4) chain-spe- cific antiserum; a number of other immunoreac- tive fragments most likely represent minor Ig fragments containing the p chain epitope that are shown by the high sensitivity of the assay, but are not evident by Coomassie Blue stain in Fig 5. These results, in agreement with the immuno- fluorescence data, indicate that the serum precipi- tate is composed of a polyclonal IgG-IgM com- plex.

The molecular weight of the 240-kd protein, as well as the demonstrated blocked N-terminus, suggested the likelihood that this protein band corresponds to Fn, a multifunctional protein that has been found consistently associated with cryo- globulins and immune complexes in a variety of

CRYOPROTEINS IN FIBRILLARY GLOMERULONEPHRITIS

Blocked N-terminus ?

XXGMYLIE EVQLVEXGGGL

Blocked N-tcnainur ?

DlVMTQ!SPLSL

Fig 5. Biochemical identification of the cryoprecip- itated fibril components by amino acid sequence anal- ysis. The fibril components were separated on 5% to 29% gradient SDS-PAGE, transferred onto lmmobilon P stained with Coomasie blue, and subjected to auto- mated amino acid sequence analysis. The transferred bands and their corresponding N-terminal sequences are indicated by single letter code.= x denotes uniden- tified amino acid. Heterogeneity found at position 5 of the 95-kd component is indicated in small letters.

diseases.2’-28 To test this, the transferred proteins were reacted with a biotinylated polyclonal anti- Fn antiserum. As demonstrated in Fig 6, lane 5, there was strong immunoreactivity of the 240- kd band with the anti-Fn antibody, confirming the association of plasma Fn with the serum pre- cipitated fibrils. To exclude the possibility that the presence of Fn could represent nonspecific trapping of serum proteins within the precipitate, the electrotransferred proteins were additionally tested for human albumin that is present in the serum in a 17-fold excess (w:w) with respect to Fn. The negative reaction obtained with anti- albumin (data not shown) suggests that the Fn is not a serum contaminant, but more likely an integral constituent of the fibrillar aggregates.

Binding of Fn to cryojibril components by dot- blot analysis. The specificity of the Fn-binding affinity for the fibril components was further as- sessed by incubation of the immobilized precipi- tate with biotinylated Fn in a dot-blot assay. Fig- ure 6B, Dot 1, demonstrates that labeled Fn specifically bound to the fibril components, in contrast to the absence of binding to immobilized Fn, Dot 4, under the described experimental con-

681

ditions. This result favors the interpretation of specific binding of Fn to the Ig molecules in the cryoprecipitate, a mechanism that has been demonstrated in other studies.19,29,30 Less likely is the possibility of self-binding between the bio- tinylated Fn and the Fn in the serum fibrils attrib- utable to the known ability of Fn to autopoly- merizeS3’

DISCUSSION

Fibrillar-tubular accumulations in glomeruli occur in several different diseases. In some cases

14-

B 1234

r 1 Fig 6. (A) Identification of the cryoprecipitated

components by immunoblot analysis. The fibril com- ponents, separated by 5% to 29% gradient SDS-PAGE and electrotransferred on lmmobilon P were immu- noreacted with different antibodies. Lane 1: anti-h light chain; Lane 2: ant& light chain; Lane 3: affinity purified anti-y heavy chain; Lane 4: affinity-purified anti-p heavy chain; Lane 5: biotin-labeled anti-fibro- nectin. After incubation with the corresponding alka- line phosphatase-labeled second antibodies or alka- line phosphatase-labeled streptavidin, the reaction was developed using the Protoblot system. (B) Binding of biotin-labeled fibronectin to the isolated fibrils by affinity dot-blot assay. The solubilized fibrfl compo- nents immobilized on a nitrocellulose membrane were incubated with: Dot 1: biotinylated Fn; Dot 2: rabbit anti-y heavy chain specific antibody followed by alka- line phosphatase-labeled goat anti-rabbi Ig (positive control); Dot 3: alkaline phosphatase labeled-strep- tavidin (negative control). Dot 4: represents purified plasma Fn immobilized on nitrocellulose and incu- bated with biotinylated Fn under identical conditions (negative control).

682 ROSTAGNO ET AL

fibrils form in the glomerular matrix in response to injury.32 In different types of glomerulonephri- tis, fibrils may form in deposits that tend to have characteristic ultrastructural and immunohisto- logical features. For example, “finger print” crystalline configurations in polyclonal Ig depos- its are distinctive of immune complexes in lupus GN.33 Fibrillar-crystalline deposits are some- times seen in type II cryoglobulinemia.34 Ran- domly oriented fibrils with immunohistological reactivity according to the specific chemical type of fibril protein are characteristic of amyloid de- posits. 35 In each of these glomerular diseases, immune complexes or precursor proteins are de- posited from the circulation.

In fibrillary GN, the origin and nature of the fibrils has been less certain. In most cases, Igs and complement have been detected in glomeruli,‘~‘5 suggesting that the fibrils are immune complexes. But in a small number of cases, no immunoglobu- lins or only fibronectin (Fn) have been found.36-38 Until the current study, the demonstration of se- rum precursors as the source of fibrillar deposits in fibrillary GN has been lacking.

In the current case of fibrillary GN, we report the first evidence that the fibrils in the glomeruli are most likely deposits derived from mixed Ig- Fn complexes in the serum. This conclusion is supported by both immunohistological and im- munochemical evidence. First, we demonstrated immunoreactivity of the fibrils in both the serum precipitate and glomerular deposits, with chain- specific antibodies against Ig light chains, K and X, heavy chains, y and /.L, amyloid P component, and Fn. Second, we identified the same proteins, y, p, K, A, and Fn, in the isolated serum fibrils analyzed by immunoblot. Third, by amino termi- nal amino acid sequence analysis of the separated major cryofibril proteins, K light chain and a vari- able region type III of heavy chain were identi- fied.

Although the serum fibrils were composed of Igs that precipitated in the cold, a characteristic of cryoglobulins, the effects of temperature and time factors in the fibril formation cannot be dif- ferentiated. The prolonged time for occurrence of precipitation and the resistance of the fibrils to solubilization on warming argues against the precipitate being a true cryoglobulin. In any case, type I cryoglobulin, by definition composed of a monoclonal Ig,39 can be excluded because two

light chain classes were detected both in the glo- meruli and the cryoprecipitate. Also, type II mixed cryoglobulin is unlikely because the serum complement level was normal, and rheumatoid factor and hepatitis C virus antigen were not de- tected in the serum.4o Further studies of the cryo- precipitate would be necessary to determine whether hepatitis C RNA is present and if both the y and ,u Igs are polyclonal, as seen in type III cryoglobulin.39

The mechanism(s) involved in Ig precipitation, fibril formation, and tissue deposition are still uncertain. Different factors have been implicated, such as an aberrant Ig protein structure related to the primary amino acid sequence that is af- fected by temperature and pH. In addition, other proteins such as Fn, in this case identified as one of the major components of the precipitate, could affect solubility and account for the cold precipi- tation and in this case for its resistance to solubi- lization. Tissue-specific factors may also play a role in deposition.

Fn is a plasma and extracellular matrix glyco- protein that frequently has been associated with cryoglobulins, immune complexes, and Ig aggre- gates in myeloproliferative disorders, IgA ne- phropathy, and autoimmune diseases.2’-28 It is composed of discrete globular domains that bear affinities for numerous biological macromole- cules, including collagen, proteoglycans,4’342 thrombospondin,43 and amyloid P component.44 Because of its varied ligand binding interactions, Fn participates in many physiological activities related to the immune system. Through the inter- action with integrin receptors in different cell types Fn is involved in chemotaxis,45,46 phagocy- tosis,47 neutrophil activation,48 and differentiation and terminal maturation of Ig secreting cells.49

It is known that Fn is a constituent of cryo- globulins and circulating immune complexes both in the sera of patients with autoimmune diseases and in the synovium of patients with rheumatoid arthritis.2’-28.50 The previously de- scribed specific biochemical interaction between Fn and Igs can explain the association of Fn with immune complexes and Ig aggregates.‘9,29,30,5’ The presence of Fn in the precipitated serum fi- brils and the binding of Fn to the immobilized fibril protein in the affinity dot-blot assay de- scribed herein is most likely another manifesta- tion of this specific binding interaction.

CRYOPROTEINS IN FIBRILLARY GLOMERULONEPHRITIS 683

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