- Email: [email protected]
δ-Heavy chain disease
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
a-Heavy
17, 584-594 (1980)
Chain
Disease
A Study of a Case
J. A. V1LPo,* K. IRJALA,* M. K. VILJANEN,t P. KLEMI,~ I. KOIJVONEN,$ AND T. RONNEMAA* *Departments University Central Turku, Turku;
of Clinical Hematology, Hospital: tDepartments and SMinerva Foundation
Clinical Chemistry, and Internal Medicine, Turku of Medical Microbiology and Pathology, University Institute for Medical Research, Helsinki, Finland
of
Received March 26, 1980 In this report we describe a new case of heavy chain disease, in which the serum M-component protein reacted with anti-&D (6 monospecific) but not with other antisera of heavy or light chain specificity. Due to kidney lesion, the clinical course of this heavy chain disease was rapidly fatal. The main histopathological finding in the renal biopsy was a thickening of the glomerular basement membrane, presumably caused by depositions of the paraprotein. There were no pathological proteins in the urine. The clinical picture resembled that often seen in multiple myeloma, that is plasmocytoid morphology of the malignant cells and osteolytic lesions. The real heavy chain nature of the serum M component was ascertained in the present work by immunoelectrophoresis, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with an immunological detection of d-heavy chain units, and by immunoselection. The molecular weight of the 6 complex appeared to be about 260,000 daltons. Owing to the molecular weight of about 65,000 daltons obtained for the major subunit of the paraprotein molecule in SDS-PAGE, a tetrameric structure of the intact serum S-chain paraprotein is suggested.
INTRODUCTION
Heavy chain diseases (HCD) are characterized by the presence of paraprotein molecules which consist of complete or incomplete heavy chains and are devoid of light chains. These abnormal proteins are present in serum, and in many cases, also in the urine. Since the first case described by Franklin in 1964 (1) more than one hundred cases of HCD have been published (2-6). The purpose of our present paper is to describe an elderly male patient (M.A.L.) with &HCD. The chemical analyses of the M-component protein, the electron microscopic characterization of the malignant plasma cells, as well as the ultrastructure of the kidney lesion resulting in anuria and death, are included in this report. CASE REPORT
Mr. M.A.L., aged 70 years, was referred to the Turku University Central Hospital in October, 1977, with a 2 months’ history of anorexia, dryness of the mouth, and frequent palpitations. His physician had noted elevated serum creatinine values and suspected kidney lesion. 584 0090-1229/80/120584-11$01.00/O Copyright @ 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
&HEAVY
CHAIN
DISEASE
585
The patient’s past history included paroxysmal tachycardia attacks for 10 years prior to admission, for which quinidine had been used. The family history was unremarkable. Physical examination on admission to our hospital did not reveal any significant pathological findings. Laboratory data are expressed by symbols: f (fasting), B (blood), S (serum), and P (plasma). Reference values for our laboratories are given in parentheses. Blood counts on admission were: B hemoglobin, 12.6 g/d1 (13.0- 16.5); B-packed cell volume, 0.40 (0.30-0.50); B platelets, 280 x log/liter (150-400); B leukocytes, 8.8 x 10s/liter (3.0-10.0); segmented neutrophils, 82%; bands, 2%; lymphocytes, 12%; monocytes, 4%; B reticulocytes, 0.4% (0.2-2). Electrolytes and acid-base balance values were; P-pH 7.34 (7.35-7.43); B-&O* 4.5 kPa (4.5-6.0); P-HCO, 18.5 mmol/liter (22-26); B-BE -7 mmol/liter (0 ? 2.5); P-potassium, 5.3 mmoVliter (3.3-4.8); P-sodium, 139 mmol/liter (135-145); fS-creatinine, 504 /*mol/liter (below 120); S-urate, 561 PmoVliter (190-390); fS-urea, 31.3 mmoVliter (3.0-8.5). Routine urinalysis was normal except some oxalate crystals were found; fS-cholesterol was 5.6 mmol/ liter (below 8.5) and fS-triglycerides 2.4 mmol/liter (below 2.0). The coagulation profile (APTT, PT, fibrinogen) was normal. The serum complement (C,; P,,-cglobulin) was 0.9 g/liter (0.6- 1.5) and the ESR was 67 mmhr. The serum total protein was 75 g/liter (62-85). Serum protein electrophoresis revealed a paraprotein with a mobility intermediate between the p2 and y globulins (Fig. lb). Through our routine immunotixation procedure (IO) applied to the patient’s serum, a clear precipitate was obtained with anti-a, but the paraprotein did not precipitate with aI’Iti-K or -A (Figs. lc-e). Therefore a tentative diagnosis of &HCD was made. Repeated quantitative serum immunoglobulin analyses (6) gave the following results (two different bleedings): IgG, 4.6-4.8 g/liter (7.0-17.0); IgA, 0.1-0.1 g/liter (0.7-3.5); and IgM, 0.3-0.4 g/liter (0.7-3.0). The result for the IgD (6 chains) was 8300 IUlml (below 100). According to the densitometry of the serum protein electrophoresis strip, the paraprotein fraction represented 9.5% of the total serum protein or 7.1 g/liter. No corresponding protein was detected in the urine by electrophoresis even after 200-fold concentration with an ultrafiltration system (MW cutoff being 15,000 daltons, Minicon type B-15, Amicon) (see Fig. If). Rectal biopsy was normal and no sign of amyloidosis was evident by Congo Red staining. From the sternal marrow aspiration biopsy a diagnosis of multiple myeloma was suggested; many pathological plasmocytoid cells with features of myeloma cells were observed in light and electron microscopic studies (for methods, see Ref. (7)) (Fig. 2). On the otherwise normal chest X ray, there was pleural thickening on the left side. A urinary tract X ray was normal. X rays of the skull showed two osteolytic lesions (typical of myeloma) in the temporal region. No lytic lesions were observed in the vertebral column, pelvis, or extremital bones. The structure of glomeruli, tubules, and interstitial tissue of the kidney was normal when a kidney biopsy specimen was examined by light microscopy. The specimen prepared for the immunofluorescence analysis of tissue immunoglobulins did not contain glomeruli. By this indirect immunofluorescence method no IgA, IgG, IgM, G, fibrinogen, or C,, could be detected in the renal medulla.
586
VILPO ET AL. a
b
C
d
e
f
FIG. 1. Serum and urine protein electrophoresis on cellulose acetate membranes (Microzone apparatus, gelatinized cellulose acetate membranes, barbital buffer, pH 8.6, voltage 200 V, time 50 min). In each pattern albumin is below and -y globulins are above. (a) Normal serum. (b-e) Serum of the patient with &HCD. In (b), note the M component (arrow) between & and y. Polyclonal -yglobulins are reduced in (b). In (c), (d), and (e) the routine serum protein electrophoresis was followed by an immunofixation procedure: after the usual run the specific proteins were identified by covering the membranes with cellulose acetate strips wetted in different monospecific antibody solutions (anti-IgG, IgA, IgM, K, A). After incubation the unprecipitated (unreacted) proteins were washed away. Coomassie brilliant blue stain. Anti-A (c) and anti-K (d) reacted only with polyclonal y globulins. Only anti-IgD (6 monospecific) reacted with the M component (e). (f) The urine protein electrophoresis of patient M.A.L. after 200-fold concentration. No M components are seen.
In electron microscopy (7) the basement membrane of glomeruli showed focal thickenings, which were due to subendothelial deposits of a material which was slightly coarser and more granular than the lamina densa of the basement membrane (Fig. 3). In other respects, the glomeruli, tubules, and the interstitial tissue appeared normal. The clinical course of the disease was rapidly fatal (Fig. 4). The patient’s general condition was reasonably good on admission, but the situation deteriorated rapidly, especially after Day 7 postadmission, when uremic symptoms including vomiting, headache, and general weakness appeared. Parenteral nutrition was necessary thereafter. In order to maintain diuresis, daily doses of 120 to 750 mg of furosemide were used. Diuresis was satisfactory (about 2000 ml daily) up to Day 16, but thereafter irreversible oliguria and anuria developed, and death occurred on Day 20 after admission. Consent for autopsy could not be obtained from the family. MATERIALS
AND METHODS
Patient’s set-a. Blood was drawn a few days prior to the patient’s death using citrate-phosphate-dextrose as anticoagulant. The plasma was separated by centrifugation and stored at -40°C. The serum was obtained by the recalcification of plasma in the usual manner. In order to prevent the “spontaneous” degradation of the M-component protein (8) 10 m&Z l -aminocaproic acid (EACA) was added to some lots of plasma as well as to the appropriate working solutions. Immunoelectrophoresis. This was performed in 1% agar gel in 0.07 M Verona1 buffer, pH 8.6 (9).
FIG 2. Top: Light microscopy of the bone marrow aspirates of S-HCD. Immature plasmoc ytoid cells a re seen (about 30% of the bone marrow cells). MGG stain, x 1200. Bottom: An electron n ticroplasmocytoid cell. The cytoplasm of the cell contains rough endoplr tsmic graph of a bone marrow reticul a in abundance, concentrated about the cell periphery. The Golgi complex (G) with small secret{ ory vesicles is prominent. Chromatin is scattered throughout the nucleus (N). Stained with uranyl acetate and lead citrate, x 11,000.
587
588
VILPO ET AL.
FIG. 3. An electron micrograph of a renal biopsy specimen from the patient with S-HCD. The capillary loop of a glomerulus is seen. There are subendothelial deposits (arrows) of material which is slightly coarser than that of the lamina densa of the basement membrane. The endothelial cell of the capillary (E); the foot processes (F) of the epithelial cells are without fusion. Stained with uranyl acetate and lead citrate, ~23,400.
S-HEAVY
CHAIN
Prednisone 20 mglday
.r 1 EZ .- c E zs
589
DISEASE
50
DAYS
IN
mg/day
TUCH
FIG. 4. The clinical course of patient M.A.L. with 6-HCD and worsening renal insufficiency. Serum urate (normal: 190-390 PmoL’liter), urea (3.0-8.5 mmol/liter), and creatinine (below 120 pmolkter) are indicated. The clinical situation did not improve during cytostatic or glucocorticoid therapy. The patient died of renal failure and anuria on Day 20 postadmission.
The immunoJixation procedure. Immunofixation (10) was done with the five available IgD-myeloma sera from different patients in order to control the capacity of our method to reveal the M-component light chains in myeloma cases. A sample of the M.A.L. serum was reduced with 0.2 M mercaptoethanol (2 hr at +37”C) and alkylated with 20% molar excess of iodoacetamide. Electrophoresis combined with immunofixation was done with the reduced + alkylated serum using anti-heavy and anti-light chain antisera. Purification of the paraprotein. Gel filtration chromatography on Sephadex G-200 was used. Other conditions were 0.05 M Tris-HCl buffer, pH 8.0, and a column measuring 83 x 7 cm2. Individual 5-ml fractions of the filtrate from the column were analyzed immunologically for the presence of a-heavy chains. Fractions containing S chains were pooled and concentrated by dialysis. Thereafter, the IgD-enriched protein pool was chromatographed on three Sepharose 4B columns containing anti-heavy chain antibodies (a, y, p., respectively) according to a method previously described (11). The molecular size determination. Size determination of 6 paraprotein was made using gel filtration on Sephadex G-200 as described above as well as on Sepharose 6B with an 83 x 7-cm2 column (0.05 M Tris-HCl buffer, pH 8.0). Serum or plasma proteins were “localized” using an enzyme immuno assay system as described below. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SDS-PAGE was performed in 5% gel after a full reduction (8 M urea-2% (w/v) SDS-2% (w/v) mercaptoethanol) as described by Lehtinen and co-workers (12). Enzyme immunoassay (EZA). The detection of different serum proteins in the fractions produced by gel filtration or SDS-PAGE was carried out by EIA based on the ability of polystyrene to bind proteins and polypeptides to form monolayers on them. After an incubation of 3 hr at +37”C in the cuvettes surplus proteins were washed and the proteins bound to the solid phase (cuvettes) were detected by using rabbit antisera against the below-mentioned serum proteins. The amount of
590
VILPO
ET
AL.
protein-specific rabbit antibodies was quantified by using alkaline phosphataseconjugated swine anti-rabbit IgG antibodies (Orion Diagnostica, Helsinki, Finland) (13). Zmmunosefection. This was performed in 1% agarose gel containing anti-K and anti-X antisera in appropriate amounts for precipitating light chain-containing immunoglobulins during the electrophoretic run. For other conditions, see Immunoelectrophoresis. Antibodies. The following monospecilic rabbit antibodies from Behringwerke AG (Frankfurt am Main, Germany) were used: anti-albumin, (II, p, 6, E, fibrinogen, transferrin. Anti-light chain antisera against K and h from Dakopatts (Copenhagen, Denmark) were used. RESULTS Zmmunoelectrophoresis and immunofixation. M.A.L. paraprotein was precipitated only with anti-6 but not with anti-K or anti-A in the routine immunoelectrophoresis and in the immunofixation (several dilutions of serum tested) (see Fig. 1). By contrast, all our five IgD-myeloma sera gave a monoclonal precipitation reaction with anti-6 and anti-A (immunofixation performed). The reduction plus alkylation did not reveal any monoclonal light chain protein in the original serum when analyzed by the immunofixation method. Purification of paraprotein. The purification procedure resulted in a highly purified fraction; according to the usual serum protein electrophoresis, Oughterlony radial immunodiffusion, and EIA. However l-2% contamination of IgG and corresponding light chains (K, h) was noted. Gel filtration chromatography on Sephadex G-200. The filtration pattern of M.A.L. serum and that of a normal human serum is illustrated in Fig. 5. In addition, the distribution of 6 and y heavy chains and K and A light chains in the effluent are illustrated. M.A.L.‘s serum differed from the normal human serum regarding the first peak of absorbance at 280 nm. The peak in M.A.L.‘s serum was relatively higher and consisted of two maxima. The last or “peptide” peak in M.A.L.‘s serum was a little higher than that of normal serum. The last peak in M.A.L.‘s serum did not react immunologically with anti-a, -y, -K, -A. EACA in a concentration of 10 mM did not prevent the appearance of this last peak. Molecular size ofparaprorein. The molecular size of the paraprotein was about 260,000 daltons as determined using gel filtration chromatography either on Sephadex G-200 or Sepharose 6B and M.A.L.‘s plasma proteins as molecular weight markers (albumin 69,000, transferrin 80,000, IgG 160,000, and fibrinogen 340,000 daltons). SDS -PAGE. The result of the SDS -PAGE with the purified paraprotein is illustrated in Fig. 6. The molecular weights of the two main protein bands were: 89,000 and 75,000 daltons; 33,000 was obtained for a weaker band. The 75,000daltons band was dominant. The two bands having the larger molecular size reacted immunologically with anti-8 antisera, but the third band (33,000 daltons) was immunologically nonreactive when analyzed by EIA or double radial immunodiffusion (anti-y, -6, -K, and -A tested). In a subsequent run the two protein fractions with larger molecular sizes had the molecular weights of 85,000 and
&HEAVY
CHAIN
591
DISEASE
00 280
20
10
0
300
500
400
::::‘.:::: ... : :. 1.0 : : -2 : 1 :i: :::., .... lJIL100. 200300
500
400
;[
,o& Y
’
’
’
’
-
-
400
’
’
500
’
FRACTIONS(ml) FIG. 5. Gel filtration chromatography of M.A.L. serum on Sephadex G-200. Above total protein concentration in the effluent (M.A.L.). NHS represents normal human serum for comparison. Below, the distribution of heavy and light chains between fractions as determined using EIA.
68,000 daltons; 32,000 daltons was obtained for the weaker fraction on that occasion. Zmmunosefcction. This procedure effectively selected other immunoglobulin molecules; only the a-heavy chain paraprotein was able to move in the gel as shown in Fig. 7. No precipitation arc was seen in immunoselection of the five IgD-A myeloma sera (results not shown). DISCUSSION
In the present paper we describe a new case (M.A.L.) of HCD on which the M-component protein gives a positive precipitation with anti-S, but not with any other anti-heavy (y, (Y, p, E) or anti-light chain (K, A) antisera. The presence of a light chain moiety in M.A.L.‘s serum paraprotein complex is excluded by several factors. Furthermore, a separate monoclonal light chain M component did not occur in our case. The clinical manifestations of HCD vary considerably depending on the heavy
592
VILF’O ET AL.
FIG. 6. SDS-PAGE of the purified M.A.L. nent and the third weaker fraction are given.
paraprotein.
The molecular weights of the two promi-
chain class (14). Usually the clinical presentation of HCD is closer to the presentation of malignant lymphomas than to that on multiple myeloma. Unlike most cases of HCD, the clinical picture of M.A.L. was indistinguishable from that usually seen in myeloma. This includes a typical myeloma-cell morphology of the pathological cell clone by light and electron microscopy. Osteolytic lesions seen in the present case are typical of myeloma as well. These findings are highly sugges-
FIG. 7. Immunoselection of M.A.L. serum. All the immunoglobulins which contain light chains are precipitated around the application well. Only the 6 paraprotein has been moved in the gel as seen from the anodic precipitin arc caused by anti-8 (arrow).
&HEAVY
CHAIN
DISEASE
593
tive for the neoplastic nature of the disease. In myeloma, serum levels of normal polyclonal IgG, IgA, and IgM are often reduced (15, 16). Especially, a marked decrease in IgG, IgA, and IgM is seen in IgD myeloma (17), and that was the situation in M.A.L., too. The clinical course of S-HCD of M.A.L. was very rapid. This was not due to hematological manifestations of the malignancy, since the hematological status of M.A.L. was quite good on admission. Instead, renal failure was fatal to this patient, The earliest sign recorded was a pathological serum creatinine value 46 days prior to hospitalization. The worsening azotemia was accompanied by hyperuricemia. The basic renal lesion seen by electron microscopy consisted of subendothelial deposits on the basement membrane of the glomeruli. Similar deposits have been found previously in multiple myeloma (18), and in cryoglobulinemia (19). At the time of the renal biopsy, there were no immunoglobulin light or heavy chains in the urine. This applies to the absence of other characteristic changes often seen in myeloma kidney (see Ref. (20)). In our patient the primary lesion, i.e., the subendothelial deposits, may be due to the high molecular weight protein complex, which cannot penetrate the basement membrane, due evidently to the S-heavy chain paraprotein. The lesion resulted in renal insufflciency and death. M.A.L. is the only case of IgD paraproteinemia so far, where the role of immunoglobulin light chains in the pathogenesis of the renal lesion can be excluded. The frequency of azotemia in IgD myeloma is definitely higher than in IgG and IgA myelomas but lower than in light-chain myelomas (17). The role of the S-heavy chain in the pathogenesis of the renal lesion should be emphasized. The presence of heavy chains in the urine is a typical finding in y-HCD. Bence Jones proteinuria is also found in many cases of p-HCD (5, 14,21), but unusual in cu-HCD (4). Moreover, in almost all patients of IgD myeloma the Bence Jones proteinuria appears (17). The urine of M.A.L. was clearly devoid of immunoglobulins. A natural explanation for this could be the large molecular size of M.A.L. paraprotein, its constant structure, and the absence of monoclonal light chains of the M-component protein. Structural studies of a number of y-, (Y-, and p-HCD have shown that most of the proteins are deleted heavy chains and that the deletions appear not to be random but involve certain specific regions of the molecule (22). Although the use of fibrinogen may make the molecular weight determination a little inaccurate, the large molecular size (260,000 daltons) obtained for M.A.L. S paraprotein is unique among the cases of HCD reported so far. The only natural explanation for the large molecular size is that the paraproteins consist of several protein subunits, probably S-chain origin. An analogical situation is seldom seen in HCD, but the largest heavy chain paraprotein molecules consisted only of two deleted heavy chains linked by normal interheavy chain disulfide bonds or by noncovalent links (23). Whether M.A.L. 260,000-daltons S complex comprises nonimmunoglobulin parts is open to question, but it does contain only immunoglobulin S chains as shown. The most likely structure of M.A.L. paraprotein is a S-heavy chain tetramer. The dominant subunit as determined using SDS-PAGE had a molecular size of 75,000 daltons (68,000). On the other hand, it has been clearly demonstrated previously that the obvious molecular weight of S chains is greatly dependent on the concentration of polyacrylamide gel (24, 25). For instance, a molecular weight of 78,000 daltons in
594
VILPO ET AL.
5% gel corresponded to that of 63,700 in 12.5% gel (24). Accordingly, the “true” molecular weight of the dominant SDS-PAGE subunit of M.A.L. paraprotein seems to be in the molecular weight range estimated for normal human S chains, i.e., from 60,000 (26) to 69,000 daltons (27). SDS-PAGE may be hampered by errors inherent in the technique and the absolute exclusion of a deletion in the S chain in our case is impossible. In SDS-PAGE two dominant and a weaker protein fraction were recorded when newly prepared protein was assayed. This type of behavior of IgD-myeloma protein S chains has been thoroughly studied by Goyert and co-workers (8). They noted two major bands (63,900 and 60,550 daltons) of S chain. The susceptibility of S chains to cleavage has been demonstrated (8). A (selective) destruction of the paraprotein light chain in IgD myeloma might mimic the picture seen in M.A.L. On the other hand, no reports are available concerning the susceptibility of IgD-immunoglobulin light chains to proteolysis or to other degradation, and the possibility that the paraprotein detected in this patient represents a degradation product seems unlikely. The clinical presentation of this patient is evidently unique, since no cases of this category have been described previously. More detailed chemical characterization of the paraprotein is in progress, although only a small amount of protein is available. REFERENCES 1. Franklin, E. C., Lowenstein, J., Bigelow, B., and Meltzer, M., Amer. J. Med. 37, 332, 1964. 2. Franklin, E. C., and Frangione, B., In “Contemporary Topics in Molecular Immunology,” (F. P. Inman and W. J. Mandy, Eds.), Vol. 4, p. 89, Plenum, New York, 1975. 3. Seligmann, M., Arch. Intern. Med. 135, 78, 1975. 4. Rambaud, J. C., and Seligmann, M., C/in. Gastroenterol. 5, 341, 1976. 5. Jonsson, V., Videbaek, A., Axelsen, N. H., and Harboe, M., Stand. J. Haematol. 16,209, 1976. 6. Mancini, G., Carbonara, A. O., and Heremans, J. F., Immunochemistry 2, 235, 1965. 7. Vilpo, J. A., Klemi, P., Lassila, O., Fraki, J., and Salmi, T. T., Cancer 46, in press, 1980. 8. Goyert, S. M., Hugh, T. E., and Spiegelberg, H. L., J. Immunol. 118, 2138, 1977. 9. Scheidegger, J. J., In?. Arch. Allergy 7, 103, 1955. 10. Irjala, K., and Rajamlki, A., &and. J. C/in. Lab. Invest. 39, 277, 1979. 11. Wofsy, L., and Burr, B., J. ImmunoL 103, 380, 1%9. 12. Lehtinen, P., Vuorio, E., and Kulonen, E., Biochem. J. 146, 565, 1975. 13. Viljanen, M. K., Acta Pathol. Microbial. Stand. C, in press, 1980. 14. Frangione, B., and Franklin, E. C., Semin. Hematol. 10, 53, 1973. 15. Cwynarski, M. T., and Cohen, S., Clin. Exp. Immunol. 8, 237, 1971. 16. Meyers, B. R., Hirschman, S. Z., and Axelrod, J. A., Amer. .I. Med. 52, 87, 1972. 17. Jancelewicz, Z., Takatsuki, K., Sugai, S., and Prudanski, W., Arch. Intern. Med. 135,87, 1975. 18. Abrahams, C., Pirani, C. L., and Pollack, V. E., J. Parhol. Bacterial. 92, 220, 1966. 19. Porush, J. G., Grishman, E., Alter, A. A., Mandelbaum, H., and Churg, J., Amer. J. Med. 47, 957, 1969. 20. Heptinstall, R. H., In “Pathology of the Kidney”, (R. H. Hepinstall, Ed.), 2nd ed., p. 498, Little, Brown, Boston, 1976. 21. Axelsen, N. H., Harboe, M., Jonsson, V., and Videbaek, A., Stand. .I. Haematol. 16,218,1976. 22. Franklin, E. C., “Plenary Sessions, XVII Congress of the International Society of Hematology,” p. 105, Librarie Arrette, Paris, 1978. 23. Faguet, G. B., Barton, B. P., Smith, L. L., and Garver, F. A., Blood 49, 495, 1977. 24. Jefferis, R., and Matthews, J. B., Immunol. Rev. 37, 25, 1977. 25. Spiegelberg, H. L., Immunol. Rev. 37, 3, 1977. 26. Spiegelberg, H. L., Prahl, J. W., and Grey, H. M., Biochemistry 9, 2115, 1970. 27. Leslie, G. A., Clem, L. W., and Rowe, D. S., Immunochemistry 8, 565, 1971.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
a-Heavy
17, 584-594 (1980)
Chain
Disease
A Study of a Case
J. A. V1LPo,* K. IRJALA,* M. K. VILJANEN,t P. KLEMI,~ I. KOIJVONEN,$ AND T. RONNEMAA* *Departments University Central Turku, Turku;
of Clinical Hematology, Hospital: tDepartments and SMinerva Foundation
Clinical Chemistry, and Internal Medicine, Turku of Medical Microbiology and Pathology, University Institute for Medical Research, Helsinki, Finland
of
Received March 26, 1980 In this report we describe a new case of heavy chain disease, in which the serum M-component protein reacted with anti-&D (6 monospecific) but not with other antisera of heavy or light chain specificity. Due to kidney lesion, the clinical course of this heavy chain disease was rapidly fatal. The main histopathological finding in the renal biopsy was a thickening of the glomerular basement membrane, presumably caused by depositions of the paraprotein. There were no pathological proteins in the urine. The clinical picture resembled that often seen in multiple myeloma, that is plasmocytoid morphology of the malignant cells and osteolytic lesions. The real heavy chain nature of the serum M component was ascertained in the present work by immunoelectrophoresis, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with an immunological detection of d-heavy chain units, and by immunoselection. The molecular weight of the 6 complex appeared to be about 260,000 daltons. Owing to the molecular weight of about 65,000 daltons obtained for the major subunit of the paraprotein molecule in SDS-PAGE, a tetrameric structure of the intact serum S-chain paraprotein is suggested.
INTRODUCTION
Heavy chain diseases (HCD) are characterized by the presence of paraprotein molecules which consist of complete or incomplete heavy chains and are devoid of light chains. These abnormal proteins are present in serum, and in many cases, also in the urine. Since the first case described by Franklin in 1964 (1) more than one hundred cases of HCD have been published (2-6). The purpose of our present paper is to describe an elderly male patient (M.A.L.) with &HCD. The chemical analyses of the M-component protein, the electron microscopic characterization of the malignant plasma cells, as well as the ultrastructure of the kidney lesion resulting in anuria and death, are included in this report. CASE REPORT
Mr. M.A.L., aged 70 years, was referred to the Turku University Central Hospital in October, 1977, with a 2 months’ history of anorexia, dryness of the mouth, and frequent palpitations. His physician had noted elevated serum creatinine values and suspected kidney lesion. 584 0090-1229/80/120584-11$01.00/O Copyright @ 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
&HEAVY
CHAIN
DISEASE
585
The patient’s past history included paroxysmal tachycardia attacks for 10 years prior to admission, for which quinidine had been used. The family history was unremarkable. Physical examination on admission to our hospital did not reveal any significant pathological findings. Laboratory data are expressed by symbols: f (fasting), B (blood), S (serum), and P (plasma). Reference values for our laboratories are given in parentheses. Blood counts on admission were: B hemoglobin, 12.6 g/d1 (13.0- 16.5); B-packed cell volume, 0.40 (0.30-0.50); B platelets, 280 x log/liter (150-400); B leukocytes, 8.8 x 10s/liter (3.0-10.0); segmented neutrophils, 82%; bands, 2%; lymphocytes, 12%; monocytes, 4%; B reticulocytes, 0.4% (0.2-2). Electrolytes and acid-base balance values were; P-pH 7.34 (7.35-7.43); B-&O* 4.5 kPa (4.5-6.0); P-HCO, 18.5 mmol/liter (22-26); B-BE -7 mmol/liter (0 ? 2.5); P-potassium, 5.3 mmoVliter (3.3-4.8); P-sodium, 139 mmol/liter (135-145); fS-creatinine, 504 /*mol/liter (below 120); S-urate, 561 PmoVliter (190-390); fS-urea, 31.3 mmoVliter (3.0-8.5). Routine urinalysis was normal except some oxalate crystals were found; fS-cholesterol was 5.6 mmol/ liter (below 8.5) and fS-triglycerides 2.4 mmol/liter (below 2.0). The coagulation profile (APTT, PT, fibrinogen) was normal. The serum complement (C,; P,,-cglobulin) was 0.9 g/liter (0.6- 1.5) and the ESR was 67 mmhr. The serum total protein was 75 g/liter (62-85). Serum protein electrophoresis revealed a paraprotein with a mobility intermediate between the p2 and y globulins (Fig. lb). Through our routine immunotixation procedure (IO) applied to the patient’s serum, a clear precipitate was obtained with anti-a, but the paraprotein did not precipitate with aI’Iti-K or -A (Figs. lc-e). Therefore a tentative diagnosis of &HCD was made. Repeated quantitative serum immunoglobulin analyses (6) gave the following results (two different bleedings): IgG, 4.6-4.8 g/liter (7.0-17.0); IgA, 0.1-0.1 g/liter (0.7-3.5); and IgM, 0.3-0.4 g/liter (0.7-3.0). The result for the IgD (6 chains) was 8300 IUlml (below 100). According to the densitometry of the serum protein electrophoresis strip, the paraprotein fraction represented 9.5% of the total serum protein or 7.1 g/liter. No corresponding protein was detected in the urine by electrophoresis even after 200-fold concentration with an ultrafiltration system (MW cutoff being 15,000 daltons, Minicon type B-15, Amicon) (see Fig. If). Rectal biopsy was normal and no sign of amyloidosis was evident by Congo Red staining. From the sternal marrow aspiration biopsy a diagnosis of multiple myeloma was suggested; many pathological plasmocytoid cells with features of myeloma cells were observed in light and electron microscopic studies (for methods, see Ref. (7)) (Fig. 2). On the otherwise normal chest X ray, there was pleural thickening on the left side. A urinary tract X ray was normal. X rays of the skull showed two osteolytic lesions (typical of myeloma) in the temporal region. No lytic lesions were observed in the vertebral column, pelvis, or extremital bones. The structure of glomeruli, tubules, and interstitial tissue of the kidney was normal when a kidney biopsy specimen was examined by light microscopy. The specimen prepared for the immunofluorescence analysis of tissue immunoglobulins did not contain glomeruli. By this indirect immunofluorescence method no IgA, IgG, IgM, G, fibrinogen, or C,, could be detected in the renal medulla.
586
VILPO ET AL. a
b
C
d
e
f
FIG. 1. Serum and urine protein electrophoresis on cellulose acetate membranes (Microzone apparatus, gelatinized cellulose acetate membranes, barbital buffer, pH 8.6, voltage 200 V, time 50 min). In each pattern albumin is below and -y globulins are above. (a) Normal serum. (b-e) Serum of the patient with &HCD. In (b), note the M component (arrow) between & and y. Polyclonal -yglobulins are reduced in (b). In (c), (d), and (e) the routine serum protein electrophoresis was followed by an immunofixation procedure: after the usual run the specific proteins were identified by covering the membranes with cellulose acetate strips wetted in different monospecific antibody solutions (anti-IgG, IgA, IgM, K, A). After incubation the unprecipitated (unreacted) proteins were washed away. Coomassie brilliant blue stain. Anti-A (c) and anti-K (d) reacted only with polyclonal y globulins. Only anti-IgD (6 monospecific) reacted with the M component (e). (f) The urine protein electrophoresis of patient M.A.L. after 200-fold concentration. No M components are seen.
In electron microscopy (7) the basement membrane of glomeruli showed focal thickenings, which were due to subendothelial deposits of a material which was slightly coarser and more granular than the lamina densa of the basement membrane (Fig. 3). In other respects, the glomeruli, tubules, and the interstitial tissue appeared normal. The clinical course of the disease was rapidly fatal (Fig. 4). The patient’s general condition was reasonably good on admission, but the situation deteriorated rapidly, especially after Day 7 postadmission, when uremic symptoms including vomiting, headache, and general weakness appeared. Parenteral nutrition was necessary thereafter. In order to maintain diuresis, daily doses of 120 to 750 mg of furosemide were used. Diuresis was satisfactory (about 2000 ml daily) up to Day 16, but thereafter irreversible oliguria and anuria developed, and death occurred on Day 20 after admission. Consent for autopsy could not be obtained from the family. MATERIALS
AND METHODS
Patient’s set-a. Blood was drawn a few days prior to the patient’s death using citrate-phosphate-dextrose as anticoagulant. The plasma was separated by centrifugation and stored at -40°C. The serum was obtained by the recalcification of plasma in the usual manner. In order to prevent the “spontaneous” degradation of the M-component protein (8) 10 m&Z l -aminocaproic acid (EACA) was added to some lots of plasma as well as to the appropriate working solutions. Immunoelectrophoresis. This was performed in 1% agar gel in 0.07 M Verona1 buffer, pH 8.6 (9).
FIG 2. Top: Light microscopy of the bone marrow aspirates of S-HCD. Immature plasmoc ytoid cells a re seen (about 30% of the bone marrow cells). MGG stain, x 1200. Bottom: An electron n ticroplasmocytoid cell. The cytoplasm of the cell contains rough endoplr tsmic graph of a bone marrow reticul a in abundance, concentrated about the cell periphery. The Golgi complex (G) with small secret{ ory vesicles is prominent. Chromatin is scattered throughout the nucleus (N). Stained with uranyl acetate and lead citrate, x 11,000.
587
588
VILPO ET AL.
FIG. 3. An electron micrograph of a renal biopsy specimen from the patient with S-HCD. The capillary loop of a glomerulus is seen. There are subendothelial deposits (arrows) of material which is slightly coarser than that of the lamina densa of the basement membrane. The endothelial cell of the capillary (E); the foot processes (F) of the epithelial cells are without fusion. Stained with uranyl acetate and lead citrate, ~23,400.
S-HEAVY
CHAIN
Prednisone 20 mglday
.r 1 EZ .- c E zs
589
DISEASE
50
DAYS
IN
mg/day
TUCH
FIG. 4. The clinical course of patient M.A.L. with 6-HCD and worsening renal insufficiency. Serum urate (normal: 190-390 PmoL’liter), urea (3.0-8.5 mmol/liter), and creatinine (below 120 pmolkter) are indicated. The clinical situation did not improve during cytostatic or glucocorticoid therapy. The patient died of renal failure and anuria on Day 20 postadmission.
The immunoJixation procedure. Immunofixation (10) was done with the five available IgD-myeloma sera from different patients in order to control the capacity of our method to reveal the M-component light chains in myeloma cases. A sample of the M.A.L. serum was reduced with 0.2 M mercaptoethanol (2 hr at +37”C) and alkylated with 20% molar excess of iodoacetamide. Electrophoresis combined with immunofixation was done with the reduced + alkylated serum using anti-heavy and anti-light chain antisera. Purification of the paraprotein. Gel filtration chromatography on Sephadex G-200 was used. Other conditions were 0.05 M Tris-HCl buffer, pH 8.0, and a column measuring 83 x 7 cm2. Individual 5-ml fractions of the filtrate from the column were analyzed immunologically for the presence of a-heavy chains. Fractions containing S chains were pooled and concentrated by dialysis. Thereafter, the IgD-enriched protein pool was chromatographed on three Sepharose 4B columns containing anti-heavy chain antibodies (a, y, p., respectively) according to a method previously described (11). The molecular size determination. Size determination of 6 paraprotein was made using gel filtration on Sephadex G-200 as described above as well as on Sepharose 6B with an 83 x 7-cm2 column (0.05 M Tris-HCl buffer, pH 8.0). Serum or plasma proteins were “localized” using an enzyme immuno assay system as described below. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SDS-PAGE was performed in 5% gel after a full reduction (8 M urea-2% (w/v) SDS-2% (w/v) mercaptoethanol) as described by Lehtinen and co-workers (12). Enzyme immunoassay (EZA). The detection of different serum proteins in the fractions produced by gel filtration or SDS-PAGE was carried out by EIA based on the ability of polystyrene to bind proteins and polypeptides to form monolayers on them. After an incubation of 3 hr at +37”C in the cuvettes surplus proteins were washed and the proteins bound to the solid phase (cuvettes) were detected by using rabbit antisera against the below-mentioned serum proteins. The amount of
590
VILPO
ET
AL.
protein-specific rabbit antibodies was quantified by using alkaline phosphataseconjugated swine anti-rabbit IgG antibodies (Orion Diagnostica, Helsinki, Finland) (13). Zmmunosefection. This was performed in 1% agarose gel containing anti-K and anti-X antisera in appropriate amounts for precipitating light chain-containing immunoglobulins during the electrophoretic run. For other conditions, see Immunoelectrophoresis. Antibodies. The following monospecilic rabbit antibodies from Behringwerke AG (Frankfurt am Main, Germany) were used: anti-albumin, (II, p, 6, E, fibrinogen, transferrin. Anti-light chain antisera against K and h from Dakopatts (Copenhagen, Denmark) were used. RESULTS Zmmunoelectrophoresis and immunofixation. M.A.L. paraprotein was precipitated only with anti-6 but not with anti-K or anti-A in the routine immunoelectrophoresis and in the immunofixation (several dilutions of serum tested) (see Fig. 1). By contrast, all our five IgD-myeloma sera gave a monoclonal precipitation reaction with anti-6 and anti-A (immunofixation performed). The reduction plus alkylation did not reveal any monoclonal light chain protein in the original serum when analyzed by the immunofixation method. Purification of paraprotein. The purification procedure resulted in a highly purified fraction; according to the usual serum protein electrophoresis, Oughterlony radial immunodiffusion, and EIA. However l-2% contamination of IgG and corresponding light chains (K, h) was noted. Gel filtration chromatography on Sephadex G-200. The filtration pattern of M.A.L. serum and that of a normal human serum is illustrated in Fig. 5. In addition, the distribution of 6 and y heavy chains and K and A light chains in the effluent are illustrated. M.A.L.‘s serum differed from the normal human serum regarding the first peak of absorbance at 280 nm. The peak in M.A.L.‘s serum was relatively higher and consisted of two maxima. The last or “peptide” peak in M.A.L.‘s serum was a little higher than that of normal serum. The last peak in M.A.L.‘s serum did not react immunologically with anti-a, -y, -K, -A. EACA in a concentration of 10 mM did not prevent the appearance of this last peak. Molecular size ofparaprorein. The molecular size of the paraprotein was about 260,000 daltons as determined using gel filtration chromatography either on Sephadex G-200 or Sepharose 6B and M.A.L.‘s plasma proteins as molecular weight markers (albumin 69,000, transferrin 80,000, IgG 160,000, and fibrinogen 340,000 daltons). SDS -PAGE. The result of the SDS -PAGE with the purified paraprotein is illustrated in Fig. 6. The molecular weights of the two main protein bands were: 89,000 and 75,000 daltons; 33,000 was obtained for a weaker band. The 75,000daltons band was dominant. The two bands having the larger molecular size reacted immunologically with anti-8 antisera, but the third band (33,000 daltons) was immunologically nonreactive when analyzed by EIA or double radial immunodiffusion (anti-y, -6, -K, and -A tested). In a subsequent run the two protein fractions with larger molecular sizes had the molecular weights of 85,000 and
&HEAVY
CHAIN
591
DISEASE
00 280
20
10
0
300
500
400
::::‘.:::: ... : :. 1.0 : : -2 : 1 :i: :::., .... lJIL100. 200300
500
400
;[
,o& Y
’
’
’
’
-
-
400
’
’
500
’
FRACTIONS(ml) FIG. 5. Gel filtration chromatography of M.A.L. serum on Sephadex G-200. Above total protein concentration in the effluent (M.A.L.). NHS represents normal human serum for comparison. Below, the distribution of heavy and light chains between fractions as determined using EIA.
68,000 daltons; 32,000 daltons was obtained for the weaker fraction on that occasion. Zmmunosefcction. This procedure effectively selected other immunoglobulin molecules; only the a-heavy chain paraprotein was able to move in the gel as shown in Fig. 7. No precipitation arc was seen in immunoselection of the five IgD-A myeloma sera (results not shown). DISCUSSION
In the present paper we describe a new case (M.A.L.) of HCD on which the M-component protein gives a positive precipitation with anti-S, but not with any other anti-heavy (y, (Y, p, E) or anti-light chain (K, A) antisera. The presence of a light chain moiety in M.A.L.‘s serum paraprotein complex is excluded by several factors. Furthermore, a separate monoclonal light chain M component did not occur in our case. The clinical manifestations of HCD vary considerably depending on the heavy
592
VILF’O ET AL.
FIG. 6. SDS-PAGE of the purified M.A.L. nent and the third weaker fraction are given.
paraprotein.
The molecular weights of the two promi-
chain class (14). Usually the clinical presentation of HCD is closer to the presentation of malignant lymphomas than to that on multiple myeloma. Unlike most cases of HCD, the clinical picture of M.A.L. was indistinguishable from that usually seen in myeloma. This includes a typical myeloma-cell morphology of the pathological cell clone by light and electron microscopy. Osteolytic lesions seen in the present case are typical of myeloma as well. These findings are highly sugges-
FIG. 7. Immunoselection of M.A.L. serum. All the immunoglobulins which contain light chains are precipitated around the application well. Only the 6 paraprotein has been moved in the gel as seen from the anodic precipitin arc caused by anti-8 (arrow).
&HEAVY
CHAIN
DISEASE
593
tive for the neoplastic nature of the disease. In myeloma, serum levels of normal polyclonal IgG, IgA, and IgM are often reduced (15, 16). Especially, a marked decrease in IgG, IgA, and IgM is seen in IgD myeloma (17), and that was the situation in M.A.L., too. The clinical course of S-HCD of M.A.L. was very rapid. This was not due to hematological manifestations of the malignancy, since the hematological status of M.A.L. was quite good on admission. Instead, renal failure was fatal to this patient, The earliest sign recorded was a pathological serum creatinine value 46 days prior to hospitalization. The worsening azotemia was accompanied by hyperuricemia. The basic renal lesion seen by electron microscopy consisted of subendothelial deposits on the basement membrane of the glomeruli. Similar deposits have been found previously in multiple myeloma (18), and in cryoglobulinemia (19). At the time of the renal biopsy, there were no immunoglobulin light or heavy chains in the urine. This applies to the absence of other characteristic changes often seen in myeloma kidney (see Ref. (20)). In our patient the primary lesion, i.e., the subendothelial deposits, may be due to the high molecular weight protein complex, which cannot penetrate the basement membrane, due evidently to the S-heavy chain paraprotein. The lesion resulted in renal insufflciency and death. M.A.L. is the only case of IgD paraproteinemia so far, where the role of immunoglobulin light chains in the pathogenesis of the renal lesion can be excluded. The frequency of azotemia in IgD myeloma is definitely higher than in IgG and IgA myelomas but lower than in light-chain myelomas (17). The role of the S-heavy chain in the pathogenesis of the renal lesion should be emphasized. The presence of heavy chains in the urine is a typical finding in y-HCD. Bence Jones proteinuria is also found in many cases of p-HCD (5, 14,21), but unusual in cu-HCD (4). Moreover, in almost all patients of IgD myeloma the Bence Jones proteinuria appears (17). The urine of M.A.L. was clearly devoid of immunoglobulins. A natural explanation for this could be the large molecular size of M.A.L. paraprotein, its constant structure, and the absence of monoclonal light chains of the M-component protein. Structural studies of a number of y-, (Y-, and p-HCD have shown that most of the proteins are deleted heavy chains and that the deletions appear not to be random but involve certain specific regions of the molecule (22). Although the use of fibrinogen may make the molecular weight determination a little inaccurate, the large molecular size (260,000 daltons) obtained for M.A.L. S paraprotein is unique among the cases of HCD reported so far. The only natural explanation for the large molecular size is that the paraproteins consist of several protein subunits, probably S-chain origin. An analogical situation is seldom seen in HCD, but the largest heavy chain paraprotein molecules consisted only of two deleted heavy chains linked by normal interheavy chain disulfide bonds or by noncovalent links (23). Whether M.A.L. 260,000-daltons S complex comprises nonimmunoglobulin parts is open to question, but it does contain only immunoglobulin S chains as shown. The most likely structure of M.A.L. paraprotein is a S-heavy chain tetramer. The dominant subunit as determined using SDS-PAGE had a molecular size of 75,000 daltons (68,000). On the other hand, it has been clearly demonstrated previously that the obvious molecular weight of S chains is greatly dependent on the concentration of polyacrylamide gel (24, 25). For instance, a molecular weight of 78,000 daltons in
594
VILPO ET AL.
5% gel corresponded to that of 63,700 in 12.5% gel (24). Accordingly, the “true” molecular weight of the dominant SDS-PAGE subunit of M.A.L. paraprotein seems to be in the molecular weight range estimated for normal human S chains, i.e., from 60,000 (26) to 69,000 daltons (27). SDS-PAGE may be hampered by errors inherent in the technique and the absolute exclusion of a deletion in the S chain in our case is impossible. In SDS-PAGE two dominant and a weaker protein fraction were recorded when newly prepared protein was assayed. This type of behavior of IgD-myeloma protein S chains has been thoroughly studied by Goyert and co-workers (8). They noted two major bands (63,900 and 60,550 daltons) of S chain. The susceptibility of S chains to cleavage has been demonstrated (8). A (selective) destruction of the paraprotein light chain in IgD myeloma might mimic the picture seen in M.A.L. On the other hand, no reports are available concerning the susceptibility of IgD-immunoglobulin light chains to proteolysis or to other degradation, and the possibility that the paraprotein detected in this patient represents a degradation product seems unlikely. The clinical presentation of this patient is evidently unique, since no cases of this category have been described previously. More detailed chemical characterization of the paraprotein is in progress, although only a small amount of protein is available. REFERENCES 1. Franklin, E. C., Lowenstein, J., Bigelow, B., and Meltzer, M., Amer. J. Med. 37, 332, 1964. 2. Franklin, E. C., and Frangione, B., In “Contemporary Topics in Molecular Immunology,” (F. P. Inman and W. J. Mandy, Eds.), Vol. 4, p. 89, Plenum, New York, 1975. 3. Seligmann, M., Arch. Intern. Med. 135, 78, 1975. 4. Rambaud, J. C., and Seligmann, M., C/in. Gastroenterol. 5, 341, 1976. 5. Jonsson, V., Videbaek, A., Axelsen, N. H., and Harboe, M., Stand. J. Haematol. 16,209, 1976. 6. Mancini, G., Carbonara, A. O., and Heremans, J. F., Immunochemistry 2, 235, 1965. 7. Vilpo, J. A., Klemi, P., Lassila, O., Fraki, J., and Salmi, T. T., Cancer 46, in press, 1980. 8. Goyert, S. M., Hugh, T. E., and Spiegelberg, H. L., J. Immunol. 118, 2138, 1977. 9. Scheidegger, J. J., In?. Arch. Allergy 7, 103, 1955. 10. Irjala, K., and Rajamlki, A., &and. J. C/in. Lab. Invest. 39, 277, 1979. 11. Wofsy, L., and Burr, B., J. ImmunoL 103, 380, 1%9. 12. Lehtinen, P., Vuorio, E., and Kulonen, E., Biochem. J. 146, 565, 1975. 13. Viljanen, M. K., Acta Pathol. Microbial. Stand. C, in press, 1980. 14. Frangione, B., and Franklin, E. C., Semin. Hematol. 10, 53, 1973. 15. Cwynarski, M. T., and Cohen, S., Clin. Exp. Immunol. 8, 237, 1971. 16. Meyers, B. R., Hirschman, S. Z., and Axelrod, J. A., Amer. .I. Med. 52, 87, 1972. 17. Jancelewicz, Z., Takatsuki, K., Sugai, S., and Prudanski, W., Arch. Intern. Med. 135,87, 1975. 18. Abrahams, C., Pirani, C. L., and Pollack, V. E., J. Parhol. Bacterial. 92, 220, 1966. 19. Porush, J. G., Grishman, E., Alter, A. A., Mandelbaum, H., and Churg, J., Amer. J. Med. 47, 957, 1969. 20. Heptinstall, R. H., In “Pathology of the Kidney”, (R. H. Hepinstall, Ed.), 2nd ed., p. 498, Little, Brown, Boston, 1976. 21. Axelsen, N. H., Harboe, M., Jonsson, V., and Videbaek, A., Stand. .I. Haematol. 16,218,1976. 22. Franklin, E. C., “Plenary Sessions, XVII Congress of the International Society of Hematology,” p. 105, Librarie Arrette, Paris, 1978. 23. Faguet, G. B., Barton, B. P., Smith, L. L., and Garver, F. A., Blood 49, 495, 1977. 24. Jefferis, R., and Matthews, J. B., Immunol. Rev. 37, 25, 1977. 25. Spiegelberg, H. L., Immunol. Rev. 37, 3, 1977. 26. Spiegelberg, H. L., Prahl, J. W., and Grey, H. M., Biochemistry 9, 2115, 1970. 27. Leslie, G. A., Clem, L. W., and Rowe, D. S., Immunochemistry 8, 565, 1971.