Isolation and characterization of circulating immune complexes in patients with hepatitis B systemic vasculitis

Isolation Complexes

A method ylene glycol

and Characterization of Circulating Immune in Patients with Hepatitis B Systemic Vasculitis

is described for the (PEG) precipitation

(Staph

A).

IC of elevated

BSAianti-PSA. Studies levels of IC in serum,

Analysis detectable to immune

Techniques

for

isolation of immune and competitive

recovery

of IC from in patients but levels

of IC in serum revealed in whole serum. The complex

disease%

complexes (IC) employing binding to staphylococcal

Staph

A were

with hepatitis did not correlate

established

B systemic closely

both HBsAg and anti-HBs. methodology described may

in which

the

inciting

antigen

using with

polyethprotein

vasculitis disease

revealed activity.

whereas anti-HBs was have important application is not

A

preformed

not

known.

INTRODUCTION

The report in 1970 by Gocke of (11. ( 1) of four patients with polyarteritis nodosa in association with the Australia antigen was described as “the first recognition in man of a systemic vasculitis mediated by an immunologic reaction to a virus.” Sergent et nl. (2) reported 14 additional cases of systemic vasculitis, of whom five (31%) were positive for hepatitis B surface antigen (HBsAg). Subsequent series have reported the incidence of HBsAg in systemic vasculitis to be between 23 and 55% (3-5). A recent survey reported three cases of systemic vasculitis among 266 chronic HBsAg carriers on hemodialysis, a 1.2% prevalence of systemic vasculitis in this select patient population. As a model for an immune complex disease in man, hepatitis B vasculitis provides a circumstance in which the inciting antigen is known and in which sensitive immunoassays are available for the detection of antigens and antibodies. The immunofluorescent study of inflamed vessels in these patients has revealed HBsAg, BlC globulin Clq. IgG and IgM, thus implicating immune complexes (IC) as pathogenic mediators of this disease ( 1). Recent reviews on the subject of IC (6,7) have outlined the wide range of assays for detecting IC in human serum. Of the multiple assays most are not antigen specific but rather rely upon changes in immunoglobulin (Ig) properties or reactivities subsequent to formation of IC with the relevant antigen. The recently described staphylococcal A binding assay (SBA) (8) employs an insoluble, relatively inert immunoabsorbent which binds, via staphylococcal protein A (Staph A), the Fc part of IgG molecules. In the present study, this technique is applied to ’ Postdoctoral Fellow of ‘The 2 Present address: Immunology :j The 10021.

Lindsley

F. Kimball

Arthritis Division. Research

Foundation. Center Institute

for of The

364 0090-1229/81/120364-I

1%01.00/O

Disease New

Control. York

Atlanta. Blood

Center,

GA

30333. New

York,

N.Y.

I’MMUNE

COMPLEXES

the isolation and characterization systemic vasculitis.

IN

HEPATITIS

of circulating

MATERIALS

B VASCULITIS

IC in patients with hepatitis

365 B

AND METHODS

Reagents und buffers. Phosphate-buffered saline with sodium azide (PBS-AZ) was 0.01 M phosphate, 0.15 M NaCl, 0.1% AZ, pH 7.2-7.4. Bovine serum albumin (BSA) was obtained from Pentex Biochemicals, Kankakee, Illinois. Human IgG (Cohn fraction II, Pentex, Kankakee, Ill.) was further purified by DEAE-cellulose chromatography. Aggregation of IgG by heat (63°C for 20 min) was followed by chromatography on Sephadex G-200 column (Pharmacia Fine Chemicals, Piscataway, N.J.). Sucrose density gradient ultracentrifugation indicated that the aggregated IgG preparation contained < 10% monomeric (7 S) IgG (8). Rabbit antiserum to BSA was prepared by toepad immunization with complete Freund’s adjuvant. The IgG fraction of the antisera was obtained by DEAEcellulose chromatography (DE-52, Whatman Ltd., Maidenstone, Kent, England). The BSA and the IgG fraction of the rabbit anti-BSA antiserum were radiolabeled by the lactoperoxidase method (9). Serum. Blood was allowed to clot at room temperature for 2 hr. Serum was removed by centrifugation and stored at -70°C in 0.4-ml aliquots. Samples were thawed just before use and were used only once. Prqjiwtned IC. Quantitative precipitation curves were obtained for B&$/antiBSA IC in which the antigen moiety was radiolabeled and in which the antibody moiety was radiolabeled. Soluble IC were formed at slight antigen excess. Staph A hitding nssuy (MA). The methods for this assay have been described in detail (8). Briefly, the SBA is performed by precipitation of serum in 5% polyethylene glycol (PEG). When incubated with 5% PEG, 5% of monomeric IgG is precipitated whereas 82% of aggregated IgG is precipitated. Subsequent incubation of the dissolved PEG precipitate with heat-killed formalin-fixed Staphylococci possessing protein A. The incubation is done under conditions in which complexed IgG is preferentially bound and monomeric IgG is competitively eluted by monomeric rabbit IgG. Residual IgG present on the Staph A is then quantitated by incubation with ‘““I-labeled rabbit anti-human IgG antiserum, and after washing, the bound radioactivity is compared to a standard curve constructed with known amounts of heat-aggregated IgG. Experiments to isolate the IC employed a variety of eluants (see Table 1). Material was eluted from the Staph A by incubating the staph-bound IC with 200 ~1 of the selected eluant for 30 min at 37°C with gentle agitation. This was then centrifuged at 1OOOgfor 20 min. The supernatant was removed, and dialyzed against PBS. These experiments included normal human serum (NHS) and buffer controls, as well AS control incubations with Wood 46, a strain of Staphylococci lacking protein A. Coluttln chrotnutogruphy c$ IC. Following elution from the Staph A with glycine--HCl, pH 2.8, the eluate was applied to Sephadex G-200 column (Pharmacia Fine Chemicals, Piscataway, N.J.) which was poured in the same buffer. Column bed volume was 100 ml and l.O-ml fractions were collected, neutralized, then assayed for immune reactivity.

366

INMAN

E-T Al..

SDS-pol~crcr?llunlide gel electropharesis (SDS -PAGE). For these studies, IC were eluted from the Staph A by incubation with 2% SDS for 30 min at 37°C. The eluate was combined with an equal volume of the following: 0.1 M Tris, 0.0015 .W EDTA, 20% glycerol. 2% SDS, and Bromphenol blue. 0.1 mgiml, pH 7.5. This mixture was placed in boiling water for 2 min, then applied to a 5% polyacrylamide gel. and run at 100 V for 90 min in a buffer of 0.1 M phosphate, 0.2% SDS, pH 7.2. The IO-cm gels were sliced into l-mm fractions in a Gilson gel slicer, and fractions counted in a gamma counter. Sucrose density grudient ultrat.elltr~lcagtiorI (SDS-UC). This was performed in sucrose density gradients at pH 7.4 (15-45%’ sucrose in PBS) and at pH 3.2 (15-45s sucrose in 0.2 M glycine-HCI). The gradients were allowed to diffuse overnight at 4°C before sample application. Ultracentrifugation was performed in a Beckman Model L2 ultracentrifuge using a SW56 rotor at 40,000 rpm for 16 hr at 4°C. The gradient tubes were punctured at the bottom and 0.2-m] fractions collected. Hepatitis B reuctant,s. Radioimmunoassays for the hepatitis B surface antigen (HBsAg) and antibody (anti-HBs), and for the antibody to hepatitis B core antigen (anti-HBc) were performed with the Abbott kits (Aus-RIA, Aus-Ab, Cor-Ab: Abbott Diagnostics, North Chicago, Ill.). E antigen (e Ag) and anti-e antibody were measured by solid-phase RIA (10) through the courtesy of Dr. A. R. Neurath at the New York Blood Center. Other LISSU~S. The Raji cell radioimmunoassay for IC was performed as described by Theofilopoulos et rrl. ( 11). The modified ‘““I-Clq binding assay (ClqBA) was done as described in detail by Zubler et al. (12). Quantitative immunoglobulin concentrations were determined by radialimmunodiffusion (Hyland Immunoplates, Travenol Lab. Costa Mesa, Calif.). Total hemolytic complement was determined by the method of Kent and Fife (13). The IgA fraction of serum was isolated by agarose block electrophoresis (14). The fractions containing IgA were then further purified by adsorption and elution from a column containing anti-IgA antibodies covalently linked to cyanogen bromide-activated Sepharose 4B ( 15). Purity of the preparation was confirmed by immunoelectrophoresis and Ouchterlony double diffusion. C‘ompetitil’r RIA for serum unti-HBs. lzSI-HBsAg was incubated with test serum or with the resolubilized 5% PEG precipitate of the serum for 1 hr at room temperature (100 ~1 of each reactant used). This was then transferred to a well containing solid-phase HBsAg on a polystyrene bead. This was incubated for 1 hr at room temperature and then the bead was washed with PBS three times, and was counted in a gamma counter. Control incubations with normal human serum (NHS) were performed concurrently.

RESULTS

Soluble

IC of izjI-BSA/anti-BSA, 100 ~1, were combined with 100 ~1 0.8% at 37°C for 2 hr. This was then centrifuged for 20 min at 1OOOg. and the pellet washed twice with PBS-AZ. One milliliter of rabbit

Staphylococci and incubated

IMMUNE

COMPLEXES

IN HEPATITIS

367

B VASCULITIS

monomeric IgG, 5 mg/ml, was added to the pellet, and incubated at 37°C for 2 hr. Repeat centrifugation and washing was performed as above. To the pellet, 200 ~1 of the specific eluant was added, and this was incubated at 37°C for 30 min. This was centrifuged at 1OOOg for 20 min, then the supernatant was decanted and counted in a gamma counter. Results (Table 1) show that selected eluants yielded a high percentage recovery of the radiolabeled antigen from the Staph A, with 2% SDS giving the highest yield. 12”1-BSA alone did not bind to the Staph A. To be assured that the eluants cleaved the immune complex from the Staph A, and not merely the antigen-antibody bond within the complex, these experiments were repeated using IC in which the antibody moiety was radiolabeled. The comparable recovery rates (Table 1) for these complexes indicate that these eluants effect a cleavage of the Fc-protein A bond.

To demonstrate directly the recovery of both antigen and antibody from the Staph A, complexes with different moieties radiolabeled were bound to SA as described above. IC were eluted from Staph A with 2% SDS and analyzed by SDS-PAGE. Figure 1 illustrates the pattern of the eluted components from the complexes. Control samples of radiolabeled BSA and rabbit IgG appeared in identical fractions to the respective peaks.

Figure 2 illustrates the course of a patient with hepatitis B vasculitis who presented with arthralgias, weakness, and a foot drop. Increasing abdominal pain, accompanied by a progressive rise in white blood cell count (WBC) and serum amylase heralded intestinal infarction, and the patient died shortly after a diagnostic laparotomy. Autopsy revealed disseminated necrotizing vasculitis with active arteritis of the pancreatic artery. Serial IC levels were elevated throughout the patient’s course until his moribund postoperative state. By the SBA, IC levels ranged from 300 to 600 &ml (normal range < 10 &ml), and by the Raji cell assay

TABLE I ELUTION OF PREFORMED COMPLEXES FROM STAPHYL.OCOCCI Immune complex

Eluant

Percentage elution”

““I-BStVanti-BSA

3 M NaSCN 3.5 M M&I, 0.1 M LIS 2% SDS

81.0 13.5 76.5 85.6

1 + -c c

1.1 4.6 I.5 0.1

BSAi”;‘l-anti-BSA

3 M NaSCN 3.5 .M M&I, 0.1 .I4 LIS” 2% SDS

85.8 f 1.1 14.9 t 1.7 83.3 f 1.9 87.5

’ Elution calculated by percentage of bound radioactivity that is eluted from .Stc~ph~/oc~~~c~i.Mean i SEM. ’ Lithium diiodosalicylate.

36X

INMAN

FIG. I. 5% after absorption in which to positions

BSA

SDS-polyacrylamide to and elution

is radiolabeled of uncomplexed

from (---) ““I-BSA

ET

Al..

gel electrophoresis. Strrph~ktc,r,t.ci with and in which and ““1-1gG

Analysis of preformed immune 2% SDS. Patterns of BSA/anti-BSA

anti-BSA controls.

is radiolabeled respectively,

(- - -t. electrophoresed

Peaks

complexes complexes correspond in a separate

experiment.

00 PREDNISONE (MGIDAY)

40

1 OL

D.$ATH

3ooor AMYLASE (MG/dLl e-0

‘500 i

~;~~:j

E?y*

j;

“B sL\s

e

+

+

4

HBs Ab HEc Ab ’ A9 l Ab

+ +

+ +

+ +

+ +

0

2

4

6

0

IO

12

14

I6

18 20

with

acute

22

24

26

HOSPITAL FIG.

2.

Serological

studies

in a patient

hepatitis

26

sATsplgpAHy;A

I 30 DAY

B systemic

vasculitis

IMMUNE

COMPLEXES

IN HEPATITIS

369

B VASCULITIS

levels of 600-900 pg/ml were detected (normal range ~20 pg/ml). The Clq binding assay was negative throughout. Serum hepatitis B reactants were measured by solid-phase radioimmunoassay. These profiles did not change qualitatively during the course and were as follows: serum was positive for HBsAg, e Ag, and antiHBc, and was negative for anti-HBs and anti-e antibodies. Serum HB Reactants

in HB Vasculitis

Comparable serum HB profiles on four other patients with HB vasculitis are recorded in Table 2. The presence of HBsAg, e Ag, and anti-HBc were constant findings for all patients. Anti-HBs and anti-e antibodies were not detected in whole serum. Studies oj’ IC in HB Vasculitis To characterize the IC in these patients, elution studies were performed on two of the patients and in two patients with uncomplicated acute hepatitis type B as controls (Table 3). Low levels of IC, 6 and 55 &ml, were detected in the two hepatitis patients as opposed to the higher levels found in the patients with vasculitis, 275 and 260 pglml. NaSCN was used to elute the IC from the Staph A, and the Staph A eluates were dialyzed against PBS prior to radioimmunoassay for HB reactants. HBsAg was demonstrable in the eluate of the vasculitis patients, but anti-HBs was not detected in the intact eluate. Although both patients with hepatitis had HBsAg present in serum, the failure to detect the antigen in the Staph A eluates of either patient ruled against nonspecific binding of the HBsAg to the Staph A. These studies were expanded to include additional HB vasculitis patients (Table 4). Elevated IC values were detected for all patients but bore no relationship to activity or severity of disease. Patients 1 and 2 (IC 400 and 175 Fg/mll were studied during the acute phase of vasculitis. Patients 3 and 4 (IC 150 and 95 &ml) were studied one year following the acute phase of the disease, at which time hypertension was the dominant residual manifestation. Patient 5 (IC 245 kg/ml) was studied 10 years after acute vasculitis, at which time she appeared as an asymptomatic HBsAg carrier. In each case IC were isolated by NaSCN elution from the Staph A and analyzed by RIA for HB reactants. All patients had

SERUM

HEPATITIS

HBsAg

TABLE B REACTANTS

2 IN HEPATITIS

Anti-HBs

(cpm)

(cm)

2 3 4 5

4,924 12,053 6.437 6.524 19.793

120 89 114 136 I18

+ +

NHS

233

105

0

Patient I

B VASCULITIS

Anti-HBc + + t

e Ag + + + +

Anti-e

t

0 0 0 0 0

0

0

370

lNMAN

IMMUNE

COMPLEXES

ET

AI,

TABLE 3 B VASCUI.IIIS

IN HEPATITIS

AND

HEPANTIS

R H~PA~I

I IS -..

HB Patient Immune HBsAg

complexes in Staph

(&ml)” A eluate

fl Normal ’ Staph

human A binding

serum. assay.

” RIA

for

HBsAg

on

eluted

from

HB Patient

I

2

Patient

260

175 19.54

(cpm)’

material

vasculitis

A with

of Anti-HBs

Patient

3

NHS”

6

159

0

56

I94

3 izf NaSCN.

HBsAg demonstrable in the eluate, but neither anti-HBs anti-HBc was detected in the NaSCN eluate. Demonstration

I

55

4924

Staph

hepatitis

nor e Ag. In a single case,

in IC

A competition radioimmunoassay employing radiolabeled antigen provided indirect evidence that anti-HBs was present in IC. Solid-phase HBsAg (bound to polystyrene bead) bound no free lz51-HBsAg. When lZ51-HBsAg was incubated with serum of two patients, slight increments in binding, compared to NHS, was observed (Fig. 3). However, when the PEG precipitate was studied, an enhanced binding was observed. This effect was not observed in NHS, nor in serum containing free anti-HBs but no IC. This phenomenon whereby exogenous antigen exchanged with endogenous antigen within the IC implicated the presence of antiHBs in the IC. Direct evidence for the antibody was obtained by eluting the IC from the Staph A with 0.2 M glycine-HCl, pH 2.8. The isolated complexes were applied to a Sephadex G-200 column in the same acidic buffer to allow chromatography in a dissociative medium. After neutralization of the fractions, each was tested for anti-HBs activity by RIA (Fig. 4). A discrete peak of activity at that fraction with a rise in optical density indicated anti-HBs reactivity. Chromatography of lz51-IgG

CIRCuLA~rINc

IMMUNE

TABLE 4 COMPLEXES (CIC)

IN HEPA HB

CIC (&ml aggregate equivalents)”

Patient

reactants

B VAX~&.~

I IS

in CIC

HBsAg (cpm)

Anti-HBs lcpm)

Anti-HBc

140 147 156

0 0 0

0 0 0

e Ag

I 2

400 17s

4024 3515

3 4

I50 9.5

2101 1175

5

245

7909

163 137

0

0 0

331

130

0

0

NHS Ii Staph

I’ITIS

.s A binding

assay.

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COMPLEXES

IN

HEPATITIS

B VASCIJLITIS

371

14 13 12 CT8 11 25 E 10 9 8 7 A

A

B

Patient

R

Patient

1

A

2

B NHS

FIG. 3. Competitive RIA for anti-HBs. ““I-HBsAg incubated with serum (Al or 5% PEG precipitate (Bl, and radioactivity bound to insoluble HBs Ag is measured (cpm). Patients I and 2 with HB vasculitis. NHS, Normal human serum.

confirmed position of IgG. As mentioned serum of this patient was negative. Other Serological

above, RIA for anti-HBs

on the whole

Studies

Serum total hemolytic complement (CH,,) was depressed in two of the acute cases (42 and 70 CH,, units, respectively, normal > 150). In case 1 quantitative Ig determination by radial immunodiffusion showed an increased level of serum IgA (920 mg/dl, normal ~600 mg/dl). IgA was purified from this serum by agarose block electrophoresis and affinity chromatography, then assayed for antibody reactivity. The serum IgA was negative for anti-HBs reactivity by RIA, and was positive for anti-HBc by RIA. The IgA had no anti-e reactivity. SDG ultracentrifugation analyses were performed at neutral and acidic pH to address the

-7.

1251-lgG 4 4

20

26

30

34

38

42

46

50

54

Fraction

FIG. 4. Sephadex G-200 chromatography in glycine-HCI. same buffer. After neutralization of fractions. Anti-HBs patient 5 with HB vasculitis.

pH 2.8. of material eluted from SA with assayed by RIA. Serum studied was from

373

INMAN

E’l-

AI

possibility that antigenic systems contributed to the CIC. The distribution ofe Ag by RIA was predominantly in the 7 S fraction on both gradients. Anti-HBc activity was present in the 119 S fractions, but failed to show a significant shift after acidification. DISCUSSION In order to develop the methodology for isolation and characterization of IC in human serum, we have employed the principles of differential solubility in PEG, and competitive binding to the protein A molecule on certain strains of S. (II(WIIS. For studies of binding and recovery of IC, preformed complexes of BSA/anti-BSA were selected since protein A binds rabbit IgG as well as human IgG, and both moieties can be easily radiolabeled. Previous investigators have used similar complexes in this regard (16, 17). Although uncomplexed antigen did not bind to Staph A, IC were bound and could be recovered from the SA with a variety of eluants. Variable labeling experiments demonstrated that the protein A-IgG bond was cleaved as well as the antigen-antibody bond. Recovery of both the antigen and antibody moieties of the IC was confirmed by SDS-PAGE. It was concluded that this technique could be applied to the isolation of IC in immune complex disease in man. Since the association of systemic vasculitis with hepatitis B virus infection was first reported, it has been hypothesized that circulating IC are mediators of vessel injury, by analogy with experimental serum sickness. IC assays have had variable results in previous studies. In two series comprising 12 patients, none of the patients had cryoglobulins demonstrable (2, 18). The monoclonal rheumatoid factor assay, and immunodiffusion for Clq precipitins have been negative in the 10 patients studies (2). Ultracentrifugation analysis was negative in one study (1) while a later report found C3 and IgG as well as HBsAg in the heavy fractions of the gradient (19). The Raji cell assay was positive in one patient studied, and the level of IC so detected bore an inverse relationship to inhibition of antibodydependent, cell-mediated cytotoxicity (19). In a recent report of three patients, two had both a positive PEG precipitation test and a positive solid-phase Clq binding assay (20). All patients in the present study had detectable circulating IC by the SBA, a technique with high sensitivity for IC that are heterogeneous in size and variable in antigen:antibody content (8). Differential solubility of hepatitis B reactants in PEG was studied by Neurath cf crl. (21). It was observed that in 6% PEG the Dane particles and tubule forms of the virus were precipitable, while the spheres were present in the supernatant. Lambert r/ rrl. (22) employed the principle that in 10% PEG all detectable HBsAg is precipitated by 105%PEG as the basis for detecting Ig binding to HBsAg. Our use of 5% PEG in the SBA might result in precipitation of uncomplexed highmolecular-weight forms of HBsAg, such as the Dane particle, but the subsequent binding to protein A would depend upon Ig bound to HBsAg. The specificity of this procedure for antigen in IC form is supported by the absence of HBsAg in the Staph A eluate from the uncomplicated hepatitis patients in whom HBsAg was present in serum but in whom very low levels of circulating IC were detected. To meet strict criteria for IC formation, the antibody reactivity of the Ig moiety

IMMUNE

COMPLEXES

IN

HEPATITIS

B VASCULITIS

373

of the 1C should be demonstrable. It is an unusual finding to observe coincident HBsAg and anti-HBs in whole serum. This phenomenon was observed in 5 of 30 patients with HB vasculitis (5). and in 3 of 10 patients in Duffy’s series (18). Usually this finding reflects prior infection with two different subtypes of HB virus (23-25). In general it has required more refined techniques to demonstrate the anti-HBs in the serum of these patients. The “heavy” IgG in SDG-UC analyses (1, 19) have not been assayed for anti-HBs reactivity. If the patient is in marked antigen excess, such that any anti-HBs is in complex form, a negative result could be obtained in the solid-phase, sandwich-method radioimmunoassay for the antibody. Separation of antigen and antibody accomplished under dissociative conditions must preclude reassociation after the medium is neutralized to allow for the radioimmunoassay. The present study employed Sephadex G-200 chromatography in an acidic medium, with subsequent neutralization of the fractions to accomplish this. From the demonstration of circulating IC of HBsAg/anti-HBs in hepatitis B vasculitis patients one cannot conclude that this is the only, or even the major, antigen-antibody system contributing to complex formation. The example of hepatitis B membranous nephropathy illustrates the point that circulating antigen may not reflect deposited antigen in injured tissue (26). We found by ultracentrifugation no convincing evidence implicating either HBcAg/anti-HBc or HBeAgianti-e in contributing to formation of IC in these patients, but other antigenic systems, such as the S antigen (27), could be playing such a role. The pathogenic role of the HBsAg/anti-HBs in hepatitis B vasculitis cannot be addressed by the present data. We observed no correlation of IC levels with the activity or duration of the vasculitis. Similar discordance with disease activity has been previously observed with SDG-UC (28) and electron microscopy (29). Furthermore, it is clear that circulating IC can occur in a variety of different liver diseases, both with and without extrahepatic manifestations (6, 30-35). It may be that impaired hepatic reticuloendothelial system function during acute and chronic hepatitis may result in impaired clearance of circulating IC. Quantitation of such clearance capabilities as well as immunochemical differentiation of pathogenic from nonpathogenic IC in this disease awaits further study. REFERENCES I. Gocke. D. J.. Morgan, C.. Lockshin, M. D., Hsu. K.. Bombardierie, S.. and Christian, C. L.. f.(/tll~rf 2, 1149. 1970. 2. Sergent. J. S., Lockshin, M. D.. Christian, C. L.. and Gocke, D. J.. &fe~lic~i,re 55, 1, 1976. 3. Travers. R. L.. Allison. D. J.. Brettle, R. P., and Hughes. (;. R. V.. Sr/ri;,t. Artliriris Rhe~~wr. 8, 184. 1979. 4. Leib. E. S., Restivo. C., and Paulus, H. E., Amer. J. ,Ilc>d. 61, 93. 1979. 5. Trepo. C. G.. Zuckerman, A. J., Bird, R. C., and Prince. A. M., ./. C/j/i. Parho/. 27, 863. 1974. 6. Theofilopoulos, A. N., and Dixon, F. J., A~VNII. fm~~~rrmd. 28, 69. 1980. 7. Inman. R. D.. and Day, N. K., Amrr. J. Med. 70, 1097, 1981. 8. McDougal. J. S.. Redecha, P. B.. Inman, R. D.. and Christian. C. L., J. C/i/r. Invest. 63, 627. 1979. 9. Marchalonis, J. J., Biocherrl. J. 113, 299, 1969. 10. Neurath, A. R., Strick. N.. Smuzness, W.. Stevens, C. E.. and Harley, E. J.,J. Goi. Viro/. 42, 493. 1979).

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22%.

Etrpl.

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(Lr~&~~t~)

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and Pellegrino. Sharp. J. T.,

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S.. Nott{r(,

232.

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