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 immunoglobulinfibronectin 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 morphological 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 disorder, 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 fibrils with both anti-K and anti-X light chain antisera and complement component (C3).14 We now report the detection and the immunohistochemiFrom 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 676
American
Journal
cal and biochemical characterization of proteins comprising the fibrils in a serum cryoprecipitate, which correspond to the proteins detected by immunoelectron microscopy, in the deposits of glomerular 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 evidence 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 vomiting, 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-glomerular basement membrane antibody, negative; C3 and C4 levels, normal; anti-streptolysin 0 antibody, negative; antihepatitis 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
Immunohistological
AND
METHODS
Studies of the Renal Tissue
The renal biopsy specimen was examined by standard optical, immunofluorescence, electron, and immunoelectron miof Kidney
Diseases,
Vol 28, No 5 (November), 1996: pp 676-684
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croscopy as previously described.14 For immunofluorescence fluorescein-labeled rabbit antibodies against human chainspecific Igs y, k, 01,K, A, C3, and fibrinogen (DAKO, Carpenteria, CA) were used. Immunoelectron microscopy of Eponembedded biopsy tissue was performed using unlabeled rabbit antibodies against human Ig -y, p, K, X chains, albumin, amyloid P component (DAKO), fibronectin (Fn) (Calbiothem, 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 centrifuged 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, Mallinckrodt), and anti-human fibrinogen 3 11 (American Diagnostica, Greenwich, CT) followed by gold-labeled goat antimouse IgG(Fc), (Amersham International, Buckinhamshire, England). Western blot analysis. The washed precipitate was solubilized in Laemmli sample buffer, and the proteins, separated on a gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (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% methanol and subjected to Western blot” and amino acid sequence analysis.‘8 For immunoblot analysis of the cryoprecipitate, the following 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 phosphatase-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% Tween20 (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 secondary antibodies conjugated to alkaline phosphatase. The biotinylated anti-Fn was detected with alkaline phosphatase-
labeled streptavidin; in all casesthe immunoblots were developed 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 Biosystems). Binding of biotinylated Fn to the serum precipitate by dotblot analysis. Human Fn was isolated from pooled normal plasma by sequential affinity chromatography on lysine-sepharose and gelatin-sepharose (Pharmacia Biotech Inc, Upsala, Sweden) as previously reported” and biotinylated as described. An aliquot of the washed serum precipitate was solubilized in 0.1 mol/L Tris-HCl buffer, pH 7.4, containing 4 mol/L urea and applied to a nitrocellulose membrane in several dots (2 &20 PL each). The membrane, with dried samples, was blocked with 3% nonfat milk in TBS-T. Binding of Fn to the immobilized protein was assessedby 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 activity (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 incubated 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 Westem 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 obliterated by sclerosis. All but two of the remaining 15 glomeruli had circumferential cellular crescents that compressed capillary tufts (Fig IA). Congo red-stained sections examined under polarization microscopy, showed no green birefringence typical of amyloid deposits. There was moderate interstitial edema and leukocytic infiltration. The vessels appeared normal. Immunojluorescence microscopy. Frozen sections containing four glomeruli incubated with fluoresceinated anti-human Ig antibodies showed smooth staining of compressed glomerular basement membranes, but not other structures, predominantly for IgG (Fig lB), both K and X light chains (not shown) and C3 (Fig 1C). Staining for
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Fig 1, (A) Glomerulus with circumferential cellular crescent (arrow) surrounding compressed capillary tufts (arrowheads). Periodic acidsilver methanamine, counterstained with hematoxylin and eosin. (Original magnification x280). lmmunofluorescence of frozen sections show glomerular smooth staining of compressed capillary tufts for IgG (6) and C3 (C). (Original magnification x250). (D) Electronmicrograph of a glometulus demonstrates randomly oriented fibrils in thickened glomerular basement membranes. En, endotheliil cell. Uranyl acetate and lead citrate. (Original magnification x15,ooo.)
fibrin was positive in crescents, but not in the capillary tufts, of all glomeruli. There was irregular 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 immunofluorescence. Electronmicroscopy and immunoelectron microscopy. The thickened convoluted glomerular capillary walls and increased mesangial matrix both contained granular densities intermingled with randomly oriented fibrils measuring 15 to 20 nm in diameter (Fig 1D). Immunoelectron 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-
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 microscopy 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 glomeruli or cryoprecipitate with antibodies to laminin or albumin (not shown). Biochemical Characterization of the Serum Precipitate Components Amino acid sequence analysis. Several protein 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-
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Fig 2. lmmunoelectron micrographs of a glometulus after incubation with anti-h (A), anti-K (B) and anti-Fn (C) antlbodiis followed by Protein A gold (15 nm). (A) Goldlabeled 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 citrate. (Original magnification x31,250) (B,C) Fibrils in GBM are decorated with gold patticles. (Original magnification X50,ooo.)
kd band yielded the sequence DIVMTQSPLSL, identified as an Ig K light chain, subgroup II.2o No sequence was obtained for the 50-kd band, which could represent an N-terminal blocked protein. The 75-kd band yielded the sequence
EVQLVEXGGGL, identified as Ig heavy chain subgroup VH III.*’ The sequence XXGMG/LIE was obtained from the 95-kd band. Homology search using the PIR database (National Biomedical Research Foundation, Georgetown VA) did
Fig 3. Electron microscopy of Epon-embedded serum cryopmcipitate demonstrates fibrils (between arrowheads) with periodic banding (small arrows), arranged in bundles. (Original magnification x3Cl,ooO.)
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Fig 4. lmmunoektron micrographs of LR Whiteembedded sections of the washed setum ctyoprecipitate. Bundles of fibrils are labeled with 15 nm gold probe after incubation with (A) antiK; (B) anti-A; (C) polyclonal anti-Fn; (D) monoclonal antiFn N-terminus. There is no labeling of fibrils after incubation with monoclonal anti-fibrinogen (E) or normal rabbit serum (F). (Original magnificatlorl x50,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). Because there was no immunoreactivity of the fibrils 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 (discussed further). Western blot analysis. The transferred proteins constituting the cryoprecipitated fib&, immunoreacted 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
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 identified in the amino acid sequences of the 28-kd band that corresponds to its described electrophoretie 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-specific antiserum; a number of other immunoreactive 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 immunofluorescence data, indicate that the serum precipitate is composed of a polyclonal IgG-IgM complex. 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 cryoglobulins and immune complexes in a variety of
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Blocked
N-terminus
?
XXGMYLIE
EVQLVEXGGGL Blocked
N-tcnainur
?
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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.19,29,30Less likely is the possibility of self-binding between the biotinylated Fn and the Fn in the serum fibrils attributable to the known ability of Fn to autopolymerizeS3’ DISCUSSION
Fibrillar-tubular accumulations in glomeruli occur in several different diseases. In some cases
DlVMTQ!SPLSL
Fig 5. Biochemical identification of the cryoprecipitated fibril components by amino acid sequence analysis. The fibril components were separated on 5% to 29% gradient SDS-PAGE, transferred onto lmmobilon P stained with Coomasie blue, and subjected to automated amino acid sequence analysis. The transferred bands and their corresponding N-terminal sequences are indicated by single letter code.= x denotes unidentified amino acid. Heterogeneity found at position 5 of the 95-kd component is indicated in small letters.
diseases.2’-28To test this, the transferred proteins were reacted with a biotinylated polyclonal antiFn antiserum. As demonstrated in Fig 6, lane 5, there was strong immunoreactivity of the 240kd band with the anti-Fn antibody, confirming the association of plasma Fn with the serum precipitated 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 antialbumin (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 dotblot analysis. The specificity of the Fn-binding affinity for the fibril components was further assessed by incubation of the immobilized precipitate with biotinylated Fn in a dot-blot assay. Figure 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-
14-
B r
1234
1
Fig 6. (A) Identification of the cryoprecipitated components by immunoblot analysis. The fibril components, separated by 5% to 29% gradient SDS-PAGE and electrotransferred on lmmobilon P were immunoreacted 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-fibronectin. After incubation with the corresponding alkaline phosphatase-labeled second antibodies or alkaline 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 components immobilized on a nitrocellulose membrane were incubated with: Dot 1: biotinylated Fn; Dot 2: rabbit anti-y heavy chain specific antibody followed by alkaline phosphatase-labeled goat anti-rabbi Ig (positive control); Dot 3: alkaline phosphatase labeled-streptavidin (negative control). Dot 4: represents purified plasma Fn immobilized on nitrocellulose and incubated with biotinylated Fn under identical conditions (negative control).
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fibrils form in the glomerular matrix in response to injury.32 In different types of glomerulonephritis, fibrils may form in deposits that tend to have characteristic ultrastructural and immunohistological features. For example, “finger print” crystalline configurations in polyclonal Ig deposits are distinctive of immune complexes in lupus GN.33 Fibrillar-crystalline deposits are sometimes seen in type II cryoglobulinemia.34 Randomly oriented fibrils with immunohistological reactivity according to the specific chemical type of fibril protein are characteristic of amyloid deposits. 35 In each of these glomerular diseases, immune complexes or precursor proteins are deposited 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 immunoglobulins or only fibronectin (Fn) have been found.36-38 Until the current study, the demonstration of serum 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 IgFn complexes in the serum. This conclusion is supported by both immunohistological and immunochemical evidence. First, we demonstrated immunoreactivity of the fibrils in both the serum precipitate and glomerular deposits, with chainspecific 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 terminal amino acid sequence analysis of the separated major cryofibril proteins, K light chain and a variable region type III of heavy chain were identified. 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 differentiated. 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
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light chain classes were detected both in the glomeruli 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 detected in the serum.4o Further studies of the cryoprecipitate 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 affected 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 precipitation and in this case for its resistance to solubilization. Tissue-specific factors may also play a role in deposition. Fn is a plasma and extracellular matrix glycoprotein that frequently has been associated with cryoglobulins, immune complexes, and Ig aggregates in myeloproliferative disorders, IgA nephropathy, and autoimmune diseases.2’-28 It is composed of discrete globular domains that bear affinities for numerous biological macromolecules, 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 interaction with integrin receptors in different cell types Fn is involved in chemotaxis,45,46 phagocytosis,47 neutrophil activation,48 and differentiation and terminal maturation of Ig secreting cells.49 It is known that Fn is a constituent of cryoglobulins 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 described 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 fibrils and the binding of Fn to the immobilized fibril protein in the affinity dot-blot assay described herein is most likely another manifestation of this specific binding interaction.
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immune complex-mediated disease? A pathologic study of two cases in the same family. Hum Path01 23:63-68, 1992 38. Assmann KJ, Koene RA, Wetzels JF: Familial glomerulonephritis characterized by massive deposits of fibronectin. Am J Kidney Dis 25:781-791, 1995 39. Brouet JC, Clauvel JP, Danon F, Klein M, Seligman M: Biological and clinical significance of cryoglobulins: A report of 86 cases. Am J Med 571775-779, 1974 40. D’ Amico G, Fomasieri A: Cryoglobulinemic glomerulonephritis: A membranoproliferative glomerulonephritis induced by hepatitis C virus. Am J Kidney Dis 25:361-369, 1995 41. Hynes RO: Fibronectins. New York, NY, SpringerVerlag, 1990 42. Mosher DF: Fibronectin. New York, NY, Academic Press, 1989 43. Homandberg GA, Kramer-Bjerke J: Thrombospondin binds to amino-terminal fragments of plasma fibronectin. Thromb Res 48:329-335, 1987 44. Rostagno A, Frangione B, Pearlstein E, Garcia-Pardo A: Fibronectin binds to amyloid P-component: Localization of the binding site to the 31,000 dalton C-terminal domain. Biochem Biophys Res Commun 140:12-20, 1986 45. Norris D, Clark RA, Swigart LM, Huff JC, Weston WL, Howell SE: Fibronectin fragment(s) are chemotactic for
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