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ELECTRON MICROSCOPICAL AND IMMUNOLOGICAL OBSERVATIONS ON THE SERUM-HEPATITIS (S.H.) ANTIGEN IN PRIMARY BILIARY CIRRHOSIS
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ELECTRON MICROSCOPICAL AND IMMUNOLOGICAL OBSERVATIONS ON THE SERUM-HEPATITIS (S.H.) ANTIGEN IN PRIMARY BILIARY CIRRHOSIS N. D. C. FINLAYSON
KAI KROHN P. T.
JOKELAINEN
K. E. ANDERSON
22
August
1970
particles already described in association with s.H. antigen. 6-8 In addition, high-voltage immuno-electroosmophoretic studies suggest that in P.B.c. the particles may contain either s.H. antigen or a related antigen. Previous negative findings of immunodiffusion studies may have been due to the occurrence of the particles as immune complexes.6 Patients and Methods
A. M. PRINCE
Patients
Division of Gastroenterology, Department of Medicine, and Department of Pathology, Cornell University Medical College, Department of Anatomy, New York Medical College, and Laboratory of Virology, New York Blood Center
with P.B.C. were studied. The relevant clinical shown in tables i and 11. In addition, we findings studied 3 healthy blood-donors who were chronic s.H.antigen carriers and 6 healthy laboratory personnel.
Particles identical to those associated with S.H. antigen were found by electron microscopy in the sera of 11 out of 12 patients with primary biliary cirrhosis. Antigen and/or antibody to S.H. antigen or antiserum were found in 9 out of 10 cases by immunological methods. It is suggested that immune complexes involving an antigen similar to or identical with S.H. antigen may be present in primary biliary cirrhosis, and that it may be important in the pathogenesis of the disease.
Sera One
Sum ary
Introduction
SERUM-HEPATITIS
(S.H.) antigen(Australia [Au] antigen,2hepatitis antigen,3 hepatitis-associated antigen 4) has been reported in acute viral hepatitis and in various chronic idiopathic liver diseases.5 It has not, however, been identified in primary biliary cirrhosis (P.B.C.) by routine immunodiffusion tests.4.5 We describe here the finding, by electron microscopy, of virus-like particles in the sera of 12 patients with P.B.C. These included particles identical with the three
12
women are
or more serum-samples were taken from each subject. Specimens were stored either for a few days at - 20°C or for up to a year at —70°C. In some cases only a small amount of serum was available for analysis. Immunological Methods Sera were tested for the presence of s.H. antigen by a
modified
immunodiffusion method9 and
immunoIn the latter method, 31/2 x 4 inch lantern slides were covered with 10 ml. of molten 1-0% agarose suspended in ’Veronal’ buffer (0-05 M, pH 8-6). Wells 3 mm. in diameter and 1 cm. apart were punched (fig. 1). The s.H. antiserum was placed in the anodal wells, and the test and standard s.H. sera were put in the cathodal wells. Electrophoresis was carried out for 1-2 hours at 12-15 V per cm. the agarose temperature being kept at 23°C. To increase the sensitivity of the tests, serum samples were concentrated 5 times with acrylamide gel (’ Lyophogel ’, Gelman Instruments). The antiserum used was 4-fold concentrated serum " S ", derived from a patient with haemophilia.1 0-5 ml. concentrated serum from 3 of the patients with P.B.C., the 3 patients with chronic S.H. antigenannia, and 1 s.H.-negative control were each
electro-osmophoresis (I.E.O.P.) technique."
TABLE I-CLINICAL CHARACTERISTICS OF PATIENTS WITH PRIMARY BILIARY CIRRHOSIS
*Acute non-specific illness month before onset. ’ Present - Absent. N.V. Not visualised. K.D. Not done.
7669
an
380 TABLE II-LABORATORY FINDINGS IN PATIENTS WITH PRIMARY BILIARY CIRRHOSIS
*Bodansky units (normal:
<
4-0)
tinternational units (normal: <85)
placed in a central trough in an agarose plate and electrophoresed for 2 hours, as described above. The anodal and cathodal areas of the plate were then removed separately, leaving a 5 mm. margin around the trough. The agarose blocks were then placed in test-tubes, frozen and thawed four times, and centrifuged at 15,000g for 20 minutes, after which the supernatants, 2-3 ml. in volume, were retained. 0-5 ml. of each was prepared for electron microscopy as described below, and the rest of the sample was concentrated 5 times and tested for s.H. antigen and antibody by immunodiffusion. Antimitochondrial, antinuclear, and smooth-muscle antibodies were detected by the indirectfluorescence method of Coons 11 using rat tissue as antigen with serum dilutions of 1/5 and 1/25.
Fig. 1-High-voltage immunoelectro-osmophoresis, showing identity reactions between P.B.C. serum and reference S.H. antigen (a) or antibody (b).
Isopycnic Density-gradient Fractionation Sera from 3 patients with P.B.C. (cases 1, 3, and 7) and the 3 chronic s.H.-antigen carriers were fractionated.on a cxsium-chloride gradient. 0-4 ml. of serum, or a mixture of 0-2 ml. of carrier serum and 0-2 ml. of serum S held for 24 hours at 4°C, was placed on a 4-2 ml. CsCI gradient with
TABLE III-OCCURRENCE OF S.H. ANTIGEN OR ANTIBODY AS DEMONSTRATED BY HIGH-VOLTAGE IMMUNOELECTRO-OSMOPHORESIS AND ELECTRON MICROSCOPY IN PATIENTS WITH PRIMARY BILIARY CIRRHOSIS, S.H. ANTIGEN CARRIERS, AND HEALTHY CONTROLS
381 concentration range of 20-32-8% (w/v). Sodium-chloride/ citrate buffer (NaCl 0-1 M, Na-citrate 0.01 M, 02 l4s) pH 7-5 was used. The samples were centrifuged for 24 hours at 145,000g in a Spinco model L2-65b ultracentrifuge, using an SW 65L Ti rotor. Fractions (10 drops each) were screened for s.H. antigen by i.E.o.P. The density of alternate fractions was determined by measurement of refraction index. Fractions with a
a
glycine
Na-glycine
density between 1-20 and 1-23 were pooled, dialysed against sodium-chloride/glycine/citrate buffer and concentrated 5 times with lyophogel. The concentrated fractions were tested for s.H. antigen by I.E.O.P. and studied by electron microscopy. Electron Microscopy Serum pellets were studied by the method of Almeida and Waterson. Negative staining of the specimens was done with phosphotungstic acid (pH 7-2). Results (table HI) Electron Microscopy Particles 160-250 A in diameter were seen in the serum of all the P.B.c. patients, the majority of particles being between 180 A and 220 A. These were granular, rounded spheres without a central structure (fig. 2a). In all cases, most of the particles appeared in clumps, but some occasional single particles were
Fig. 3-Frequency distribution of particle diameter in 7 cases of primary biliary cirrhosis (-) and 3 cases of chronic S.H. antigenaemia (- --). In every case at least 200 particles were measured and the percentage in each size range was calculated. The means and standard deviation of these percentages are plotted. seen. Within clumps interparticulate filamentous bridges, 50-150 A long, were often seen (fig. 2a). In 11 of the 12 P.B.C. cases larger particles up to 450A in diameter were observed, either singly or within the clumps of smaller particles. Generally, the larger
also
particles resembled the smaller ones morphologically, in that they did not show any inner structure (fig. 2b). In 3 cases the larger particles had clearly visible outer and inner envelopes, as well as a central core (fig. 2c). Rod-shaped particles, 200 A in diameter, were seen in small but varying numbers in 4 cases (figs. 2a and 2c). Clumps of particles were frequently associated with or surrounded by lipid-like material (fig. 2d). The
Fig. 2-Electron micrograph
of
negatively stained particles in
sera
from the 3 carriers were found to contain similar to those described above; treatment of these sera with s.H. antibody caused clumping of the particles. In 7 cases of P.B.C. (cases 1, 4, 7, 8, 9, 10, 12), where clumps were loose enough for individual
sera
particles
from primary biliary cirrhosis
(phosphotungstic acid, pH 7-2).
a Clumped spherical particles of 180-220 A diameter. Arrow indicates a rod-shaped particle with the same diameter. ( x 180,000). Inset 360,000 showsinterparticulate bridges. c Clumr of smaller particles, and a larger particle of 420 A diameter (reduced to two-thirds from ’.: 180,000). Particles of 400-450 A diameter showing varving degrees of internal structure, clumped with spherical and rod-shaped particles 10,000 . d Spherical panicles in association with lipid-like material (x 180,000). _
382
particles to be seen, and in the 3 symptom-free s.H.-antigen carriers, the frequency distribution of particle diameters was determined. Particle sizes did not differ significantly between the two groups (fig. 3). In the other P.B.C. cases dense clumping or surrounding lipid material prevented accurate measurement. Particles
were not
found in the
serum
of any of the 6
healthy control subjects.
Immunological Studies S.H. antigen could
not be demonstrated with gel diffusion in any of the sera from P.B.C. patients or healthy controls, whereas the 3 carriers had a positive reaction. Because of these negative results, highvoltage I.E.O.P. was carried out. In 9 of 10 cases of P.B.C. the patient’s serum formed a precipitation line with the antiserum to s.H. antigen, and in 5 cases it gave a similar reaction with s.H.-antigen-positive serum. In most cases it was not possible to demonstrate identity of these lines with known precipitates of s.H. antigen or antibody because the precipitation lines in P.B.C. sera took 2 hours to develop by which time the control lines, appearing between 30 and 60
minutes, were broadening. Occasionally, however, identity with s.H. antigen or antibody could be shown (fig. 1). The concentrated serum-samples from the P.B.C. patients and negative controls obtained by elution from agarose did not react with s.H. antigen or antibody in immunodiffusion, whereas those from the s.H.-antigenaEmia subjects reacted with s.H. antiserum. Electron microscopy revealed particles in the anodal eluates in the P.B.c. and s.H.-positive subjects, but not in the controls: the majority of the particles in P.B.c. sera were
still in
clumps, whereas mainly single.
those from
s.H.-
positive Density-gradient Fractionation sera were
In order to compare the density of the particles observed in cases of P.B.C. with those known to contain the s.H. antigen, fractionation by CsCI density gradient was done. S.H. antigen could be detected by I.E.O.P. in unconcentrated fractions (densities 1-211-24) derived from carrier sera but not from P.B.C. sera or from a mixture of carrier sera and serum S. However, when fractions with densities between 1-21 and 1-24 were pooled and concentrated 5 times, i.E.o.p. revealed the presence of s.H. antigen in the 3 P.B.C. sera, in the 3 carrier sera, and in 1 of the mixtures of s.H. carrier serum and S.H. antibody. Electron microscopy of these pooled fractions revealed particles, mainly with diameters between 180-220 A, in the P.B.c. and carrier sera. Discussion
In primary biliary cirrhosis intrahepatic biliary obstruction of unknown cause occurs. Usually of gradual onset in women aged 40 to 60 years, it progresses slowly to a fatal endstage. Silent extrahepatic biliary obstruction can mimic P.B.c. and must be excluded radiologically or at operation: alternatively, the presence of antimitochondrial antibodies, which are seldom found in extrahepatic biliary obstruction,12 strongly supports a diagnosis of p.B.C.13 Our patients have the age, sex, and clinical features characteristic of P.B.c. 6 patients (cases 1-6) had no evidence of extra-
hepatic obstruction at laparotomy, and a further 4 (cases 7-10) had a radiologically normal extrahepatic biliary tree in addition to antimitochondrial antibodies. In 1 case (case 11) the extrahepatic biliary system was not investigated, but there was no clinical evidence of prolonged extrahepatic obstruction and antimitochondrial antibodies were present. The cause of P.B.C. is unknown, but the presence of autoimmune antibodies has led to the suggestion that it is associated with abnormal immune reactivity.14 Coyne et al. 15 have reported finding s.H. antigen in the nuclei of liver parenchymal cells of one patient with P.B.c. by immunofluorescence, but the serum of that patient, and all other patients with this disease reported in the literature, was negative for s.H. antigen by immunodiffusion.4.5 Similarly in our work, the immunodiffusion test did not reveal s.H. antigen in any of the P.B.C. cases; more sensitive methods gave evidence suggesting the presence of s.H. antigen in the serum of most P.B.C. cases studied. We found both small spherical particles and larger ones, in some cases with a definite substructure, which were morphologically identical to those already described as containing s.H. antigen. .6-8 These particles were found to have the same density as s.H. antigen, and the concentrated fractions of this density gave a precipitation reaction with S.H. antiserum in immunoelectro-osmophoresis. In addition, 9 out of 10 concentrated sera from P.B.C. cases gave a precipitation reaction with serum containing either s.H. antigen or antibody. In most instances, identity of the antigens and antibodies with s.H. antigen and antibody could not be demonstrated. This was related to the fact that in the I.E.O.P. system precipitates first appear and then broaden if electrophoresis is continued, so that by the time the precipitates given by P.B.c. serum appears, the control precipitates had become more diffuse. This difference may be the result of clumped particles migrating more
slowly. However, as identity was occasionally seen, we feel that, taking into account the morphological evidence, it is likely that the particles contain either s.H. antigen or a closely related antigen. The great majority of particles in the P.B.c. sera were in clumps. These clumps may represent antigenantibody complexes, since interparticulate bridges compatible with antibody were shown by electron microscopy. This would account for the negative results obtained by routine immunodiffusion studies of P.B.C. sera. There is evidence that I.E.O.P. is more sensitive than immunodiffusion for detecting both s.H. antigen and its antibody, and it has been postulated that some of the increase in sensitivity may depend on the ability of the technique to separate some antigen-antibody complexes.10 Our results suggest that the system causes the clumped particles to migrate into the agarose, and this could be another explanation for its greater sensitivity. This would also explain the negative results obtained in immunodiffusion experiments using the eluates from I.E.O.P. tests. The fact that both antigen and antibody could be found in the same serum specimen in 4 cases, and antibody as well as electron microscopic particles in 2 other cases, further suggests that complexes can be present. The frequency with which we found particles which
j .
!
; i ’
383
antigen in cases of P.B.C. suggests that be intimately related to the disease process. they could been It has suggested 16 that some diseases associated with immunological deficiency predispose to the development of a chronic antigen-carrier state. It might be that this applies to P.B.c. However, there is no clinical evidence to suggest that our patients were unusually susceptible to infectious diseases. Furthermore, the incidence of chronic s.H. antigenaemia in patients with major immunological deficiency is not as high as that observed here.5 ,16 Liver damage in P.B.c. could be the result either of continuing replication of a virus, or of the host’s immune response. Our observation of the simultaneous occurrence of antigen and antibody, and the presence of clumps of particles possibly joined by antibody, are compatible with the existence of immune complexes. The presence of immune complexes has been postulated both in acute hepatitis 17 and in active chronic hepatitis with S.H. antigenaemia.6 This latter condition and P.B.c. may not be wholly separate clinical syndromes,la and when these diseases are associated with S.H. antigen, the basic pathogenic mechanism may be broadly similar. We wish to thank Dr. W. Eisenmenger, Dr. M. A. Payne, Dr. E. H. Ahrens, and Dr. N. M. Luger for providing materials for these studies. We also thank our technicians, Miss Marthe Fauconnet, Miss Tellervo Huima, and Miss Kathleen Burke for their hard work. We appreciate the reading of this manuscript and the many helpful suggestions by Dr. Henry Kunkel and Dr. Norman B. Javitt. This work was supported in part by training grant T01-AM5430 from the National Institutes of Health. One of us (A. M. P.) is the recipient of a career scientist award from the Health Research Council of the City of New York under contract may contain s.H.
number 1-533. Requests for reprints should be addressed to K. K., Laboratory of Virology, New York Blood Center, 310 East 67th Street, New York, New York, 10021. REFERENCES 1. Prince, A. M. Proc. natn Acad. Sci. U.S.A. 1968, 60, 814. 2. Blumberg, B. S., Alter, H. J., Visnich, S. A. J. Am. med. Ass. 1965,
191, 541. Gocke, D. J., Kavey, N. B. Lancet, 1969, i, 1055. Fox, R. A., Niazi, S. P., Sherlock, S. ibid. 1969, ii, 609. Wright, R., McCollum, R. W., Klatskin, G. ibid. p. 117. Almeida, J. D., Waterson, A. P. ibid. p. 983. Bayer, M. E., Blumberg, B. S., Werner, B. Nature, 1968, 218, 1057. Dane, D. S., Cameron, C. H., Briggs, M. Lancet, 1970, i, 695. Prince, A. M. ibid. 1968, ii, 462. Prince, A. M., Burke, K. Science (in the press). Coons, A. H., Kaplan, M. H. J. exp. Med. 1950, 91, 1. Doniach, D., Roitt, I. M., Walker, J. G., Sherlock, S. Clin. exp. Immunol. 1966, 1, 237. 13. Sherlock, S. in Diseases of the Liver and Biliary System; chap. 11. Philadelphia, 1968. 14. Popper, H., Schaffner, F. in Popper and Schaffner’s Progress in Liver Diseases; chap. 21. New York and London, 1970. 15. Coyne, V. E., Millman, 1., Cerda, J., Gerstley, B. J. S., London, T., Sutnic, A., Blumberg, B. S. J. exp. Med. 1970, 131, 307. 16. Blumberg, B. S., Sutnick, A. I., London, W. T. Bull. N.Y. Acad. Med. 1968, 44, 1566. 17. Shulman, N. R., Hirschman, R. J., Barker, L. F. Ann. intern. Med. 1970, 72, 257. 18. Doniach, D., Walker, J. G., Roitt, I. M., Berg, P. A. New Engl. J. Med. 1970, 282, 86.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
" Has anyone ever asked the question-why does the of clinical medicine start in the middle, proceed to the end, before coming back to the beginning ? Has any medical school thought of the revolutionary step of starting the clinical curriculum with pxdiatrics ? "-Prof. CHARLOTTE AxDERSOX, Putting Paediatrics in its Place, an inaugural lecture delivered in the University of Birmingham on Feb. 5, 1970, p. 11.
study
COMBINED TREATMENT OF PARKINSONISM WITH L-DOPA AND AMANTADINE C. C. FREARS
R. B. GODWIN-AUSTEN S. BERGMANN National
J. D.
Hospital, London W.C.1
PARKES
King’s College Hospital,
R. P.
KNILL-JONES
London S.E.5
A double-blind crossover trial of combined treatment with amantadine and L-dopa was conducted in twenty-four patients with parkinsonism. Twelve patients were on longterm treatment with L-dopa, and amantadine was added to their treatment; while twelve patients had L-dopa added to long-term treatment with amantadine. Further benefit was shown in the group in whom amantadine treatment was supplemented by the addition of L-dopa, whereas no additional benefit was detected in patients on optimal L-dopa therapy when amantadine was added. Combined treatment with these drugs is only indicated when the maximum tolerated dose of L-dopa is very small.
Summary
Introduction AMANTADINE was first given to patients with Parkinson’s disease by Schwab et al.1 and the definite improvement demonstrated was confirmed in subsequent trials.2,3 The effect of amantadine is less than that of L-dopa,3but Schwaband Scotti5 have claimed that the two drugs have an additive therapeutic action. Since toxic effects frequently limit the use of optimal doses of L-dopa, a combination of L-dopa and amantadine might be of greater value than either drug alone. We have combined treatment with L-dopa and amantadine in patients with parkinsonism to see whether an additive effect of these two drugs could be demonstrated. Patients and Methods Twenty-four patients with parkinsonism were studied. These were divided into two groups; group i comprised twelve patients already taking L-dopa, and group 11 twelve patients already taking amantadine. Eighteen patients were male, six patients were female; their ages ranged from forty-seven to sixty-seven; and they included patients with idiopathic, postencephalitic, and arteriosclerotic parkinsonism. Group-I patients had been on continuous treatment with L-dopa (0-75-5 g. daily) for more than eight months. Most of these patients were also taking anticholinergic drugs and all had benefited from L-dopa. In two patients the improvement had been dramatic, in nine patients moderate, and in one patient slight; but all had residual subjective and
objective disability. Group-II patients
had all been on treatment with amantadine (200-500 mg. daily) for three to six months. A preliminary assessment established that all patients in this group could tolerate 3 g. of L-dopa daily. All these patients had shown a favourable response to amantadine, with moderate improvement in seven and slight improvement in five. The benefit from both L-dopa and amantadine had been maintained, but no continuing further improvement was occurring at the start of the present trial. Before either
ELECTRON MICROSCOPICAL AND IMMUNOLOGICAL OBSERVATIONS ON THE SERUM-HEPATITIS (S.H.) ANTIGEN IN PRIMARY BILIARY CIRRHOSIS N. D. C. FINLAYSON
KAI KROHN P. T.
JOKELAINEN
K. E. ANDERSON
22
August
1970
particles already described in association with s.H. antigen. 6-8 In addition, high-voltage immuno-electroosmophoretic studies suggest that in P.B.c. the particles may contain either s.H. antigen or a related antigen. Previous negative findings of immunodiffusion studies may have been due to the occurrence of the particles as immune complexes.6 Patients and Methods
A. M. PRINCE
Patients
Division of Gastroenterology, Department of Medicine, and Department of Pathology, Cornell University Medical College, Department of Anatomy, New York Medical College, and Laboratory of Virology, New York Blood Center
with P.B.C. were studied. The relevant clinical shown in tables i and 11. In addition, we findings studied 3 healthy blood-donors who were chronic s.H.antigen carriers and 6 healthy laboratory personnel.
Particles identical to those associated with S.H. antigen were found by electron microscopy in the sera of 11 out of 12 patients with primary biliary cirrhosis. Antigen and/or antibody to S.H. antigen or antiserum were found in 9 out of 10 cases by immunological methods. It is suggested that immune complexes involving an antigen similar to or identical with S.H. antigen may be present in primary biliary cirrhosis, and that it may be important in the pathogenesis of the disease.
Sera One
Sum ary
Introduction
SERUM-HEPATITIS
(S.H.) antigen(Australia [Au] antigen,2hepatitis antigen,3 hepatitis-associated antigen 4) has been reported in acute viral hepatitis and in various chronic idiopathic liver diseases.5 It has not, however, been identified in primary biliary cirrhosis (P.B.C.) by routine immunodiffusion tests.4.5 We describe here the finding, by electron microscopy, of virus-like particles in the sera of 12 patients with P.B.C. These included particles identical with the three
12
women are
or more serum-samples were taken from each subject. Specimens were stored either for a few days at - 20°C or for up to a year at —70°C. In some cases only a small amount of serum was available for analysis. Immunological Methods Sera were tested for the presence of s.H. antigen by a
modified
immunodiffusion method9 and
immunoIn the latter method, 31/2 x 4 inch lantern slides were covered with 10 ml. of molten 1-0% agarose suspended in ’Veronal’ buffer (0-05 M, pH 8-6). Wells 3 mm. in diameter and 1 cm. apart were punched (fig. 1). The s.H. antiserum was placed in the anodal wells, and the test and standard s.H. sera were put in the cathodal wells. Electrophoresis was carried out for 1-2 hours at 12-15 V per cm. the agarose temperature being kept at 23°C. To increase the sensitivity of the tests, serum samples were concentrated 5 times with acrylamide gel (’ Lyophogel ’, Gelman Instruments). The antiserum used was 4-fold concentrated serum " S ", derived from a patient with haemophilia.1 0-5 ml. concentrated serum from 3 of the patients with P.B.C., the 3 patients with chronic S.H. antigenannia, and 1 s.H.-negative control were each
electro-osmophoresis (I.E.O.P.) technique."
TABLE I-CLINICAL CHARACTERISTICS OF PATIENTS WITH PRIMARY BILIARY CIRRHOSIS
*Acute non-specific illness month before onset. ’ Present - Absent. N.V. Not visualised. K.D. Not done.
7669
an
380 TABLE II-LABORATORY FINDINGS IN PATIENTS WITH PRIMARY BILIARY CIRRHOSIS
*Bodansky units (normal:
<
4-0)
tinternational units (normal: <85)
placed in a central trough in an agarose plate and electrophoresed for 2 hours, as described above. The anodal and cathodal areas of the plate were then removed separately, leaving a 5 mm. margin around the trough. The agarose blocks were then placed in test-tubes, frozen and thawed four times, and centrifuged at 15,000g for 20 minutes, after which the supernatants, 2-3 ml. in volume, were retained. 0-5 ml. of each was prepared for electron microscopy as described below, and the rest of the sample was concentrated 5 times and tested for s.H. antigen and antibody by immunodiffusion. Antimitochondrial, antinuclear, and smooth-muscle antibodies were detected by the indirectfluorescence method of Coons 11 using rat tissue as antigen with serum dilutions of 1/5 and 1/25.
Fig. 1-High-voltage immunoelectro-osmophoresis, showing identity reactions between P.B.C. serum and reference S.H. antigen (a) or antibody (b).
Isopycnic Density-gradient Fractionation Sera from 3 patients with P.B.C. (cases 1, 3, and 7) and the 3 chronic s.H.-antigen carriers were fractionated.on a cxsium-chloride gradient. 0-4 ml. of serum, or a mixture of 0-2 ml. of carrier serum and 0-2 ml. of serum S held for 24 hours at 4°C, was placed on a 4-2 ml. CsCI gradient with
TABLE III-OCCURRENCE OF S.H. ANTIGEN OR ANTIBODY AS DEMONSTRATED BY HIGH-VOLTAGE IMMUNOELECTRO-OSMOPHORESIS AND ELECTRON MICROSCOPY IN PATIENTS WITH PRIMARY BILIARY CIRRHOSIS, S.H. ANTIGEN CARRIERS, AND HEALTHY CONTROLS
381 concentration range of 20-32-8% (w/v). Sodium-chloride/ citrate buffer (NaCl 0-1 M, Na-citrate 0.01 M, 02 l4s) pH 7-5 was used. The samples were centrifuged for 24 hours at 145,000g in a Spinco model L2-65b ultracentrifuge, using an SW 65L Ti rotor. Fractions (10 drops each) were screened for s.H. antigen by i.E.o.P. The density of alternate fractions was determined by measurement of refraction index. Fractions with a
a
glycine
Na-glycine
density between 1-20 and 1-23 were pooled, dialysed against sodium-chloride/glycine/citrate buffer and concentrated 5 times with lyophogel. The concentrated fractions were tested for s.H. antigen by I.E.O.P. and studied by electron microscopy. Electron Microscopy Serum pellets were studied by the method of Almeida and Waterson. Negative staining of the specimens was done with phosphotungstic acid (pH 7-2). Results (table HI) Electron Microscopy Particles 160-250 A in diameter were seen in the serum of all the P.B.c. patients, the majority of particles being between 180 A and 220 A. These were granular, rounded spheres without a central structure (fig. 2a). In all cases, most of the particles appeared in clumps, but some occasional single particles were
Fig. 3-Frequency distribution of particle diameter in 7 cases of primary biliary cirrhosis (-) and 3 cases of chronic S.H. antigenaemia (- --). In every case at least 200 particles were measured and the percentage in each size range was calculated. The means and standard deviation of these percentages are plotted. seen. Within clumps interparticulate filamentous bridges, 50-150 A long, were often seen (fig. 2a). In 11 of the 12 P.B.C. cases larger particles up to 450A in diameter were observed, either singly or within the clumps of smaller particles. Generally, the larger
also
particles resembled the smaller ones morphologically, in that they did not show any inner structure (fig. 2b). In 3 cases the larger particles had clearly visible outer and inner envelopes, as well as a central core (fig. 2c). Rod-shaped particles, 200 A in diameter, were seen in small but varying numbers in 4 cases (figs. 2a and 2c). Clumps of particles were frequently associated with or surrounded by lipid-like material (fig. 2d). The
Fig. 2-Electron micrograph
of
negatively stained particles in
sera
from the 3 carriers were found to contain similar to those described above; treatment of these sera with s.H. antibody caused clumping of the particles. In 7 cases of P.B.C. (cases 1, 4, 7, 8, 9, 10, 12), where clumps were loose enough for individual
sera
particles
from primary biliary cirrhosis
(phosphotungstic acid, pH 7-2).
a Clumped spherical particles of 180-220 A diameter. Arrow indicates a rod-shaped particle with the same diameter. ( x 180,000). Inset 360,000 showsinterparticulate bridges. c Clumr of smaller particles, and a larger particle of 420 A diameter (reduced to two-thirds from ’.: 180,000). Particles of 400-450 A diameter showing varving degrees of internal structure, clumped with spherical and rod-shaped particles 10,000 . d Spherical panicles in association with lipid-like material (x 180,000). _
382
particles to be seen, and in the 3 symptom-free s.H.-antigen carriers, the frequency distribution of particle diameters was determined. Particle sizes did not differ significantly between the two groups (fig. 3). In the other P.B.C. cases dense clumping or surrounding lipid material prevented accurate measurement. Particles
were not
found in the
serum
of any of the 6
healthy control subjects.
Immunological Studies S.H. antigen could
not be demonstrated with gel diffusion in any of the sera from P.B.C. patients or healthy controls, whereas the 3 carriers had a positive reaction. Because of these negative results, highvoltage I.E.O.P. was carried out. In 9 of 10 cases of P.B.C. the patient’s serum formed a precipitation line with the antiserum to s.H. antigen, and in 5 cases it gave a similar reaction with s.H.-antigen-positive serum. In most cases it was not possible to demonstrate identity of these lines with known precipitates of s.H. antigen or antibody because the precipitation lines in P.B.C. sera took 2 hours to develop by which time the control lines, appearing between 30 and 60
minutes, were broadening. Occasionally, however, identity with s.H. antigen or antibody could be shown (fig. 1). The concentrated serum-samples from the P.B.C. patients and negative controls obtained by elution from agarose did not react with s.H. antigen or antibody in immunodiffusion, whereas those from the s.H.-antigenaEmia subjects reacted with s.H. antiserum. Electron microscopy revealed particles in the anodal eluates in the P.B.c. and s.H.-positive subjects, but not in the controls: the majority of the particles in P.B.c. sera were
still in
clumps, whereas mainly single.
those from
s.H.-
positive Density-gradient Fractionation sera were
In order to compare the density of the particles observed in cases of P.B.C. with those known to contain the s.H. antigen, fractionation by CsCI density gradient was done. S.H. antigen could be detected by I.E.O.P. in unconcentrated fractions (densities 1-211-24) derived from carrier sera but not from P.B.C. sera or from a mixture of carrier sera and serum S. However, when fractions with densities between 1-21 and 1-24 were pooled and concentrated 5 times, i.E.o.p. revealed the presence of s.H. antigen in the 3 P.B.C. sera, in the 3 carrier sera, and in 1 of the mixtures of s.H. carrier serum and S.H. antibody. Electron microscopy of these pooled fractions revealed particles, mainly with diameters between 180-220 A, in the P.B.c. and carrier sera. Discussion
In primary biliary cirrhosis intrahepatic biliary obstruction of unknown cause occurs. Usually of gradual onset in women aged 40 to 60 years, it progresses slowly to a fatal endstage. Silent extrahepatic biliary obstruction can mimic P.B.c. and must be excluded radiologically or at operation: alternatively, the presence of antimitochondrial antibodies, which are seldom found in extrahepatic biliary obstruction,12 strongly supports a diagnosis of p.B.C.13 Our patients have the age, sex, and clinical features characteristic of P.B.c. 6 patients (cases 1-6) had no evidence of extra-
hepatic obstruction at laparotomy, and a further 4 (cases 7-10) had a radiologically normal extrahepatic biliary tree in addition to antimitochondrial antibodies. In 1 case (case 11) the extrahepatic biliary system was not investigated, but there was no clinical evidence of prolonged extrahepatic obstruction and antimitochondrial antibodies were present. The cause of P.B.C. is unknown, but the presence of autoimmune antibodies has led to the suggestion that it is associated with abnormal immune reactivity.14 Coyne et al. 15 have reported finding s.H. antigen in the nuclei of liver parenchymal cells of one patient with P.B.c. by immunofluorescence, but the serum of that patient, and all other patients with this disease reported in the literature, was negative for s.H. antigen by immunodiffusion.4.5 Similarly in our work, the immunodiffusion test did not reveal s.H. antigen in any of the P.B.C. cases; more sensitive methods gave evidence suggesting the presence of s.H. antigen in the serum of most P.B.C. cases studied. We found both small spherical particles and larger ones, in some cases with a definite substructure, which were morphologically identical to those already described as containing s.H. antigen. .6-8 These particles were found to have the same density as s.H. antigen, and the concentrated fractions of this density gave a precipitation reaction with S.H. antiserum in immunoelectro-osmophoresis. In addition, 9 out of 10 concentrated sera from P.B.C. cases gave a precipitation reaction with serum containing either s.H. antigen or antibody. In most instances, identity of the antigens and antibodies with s.H. antigen and antibody could not be demonstrated. This was related to the fact that in the I.E.O.P. system precipitates first appear and then broaden if electrophoresis is continued, so that by the time the precipitates given by P.B.c. serum appears, the control precipitates had become more diffuse. This difference may be the result of clumped particles migrating more
slowly. However, as identity was occasionally seen, we feel that, taking into account the morphological evidence, it is likely that the particles contain either s.H. antigen or a closely related antigen. The great majority of particles in the P.B.c. sera were in clumps. These clumps may represent antigenantibody complexes, since interparticulate bridges compatible with antibody were shown by electron microscopy. This would account for the negative results obtained by routine immunodiffusion studies of P.B.C. sera. There is evidence that I.E.O.P. is more sensitive than immunodiffusion for detecting both s.H. antigen and its antibody, and it has been postulated that some of the increase in sensitivity may depend on the ability of the technique to separate some antigen-antibody complexes.10 Our results suggest that the system causes the clumped particles to migrate into the agarose, and this could be another explanation for its greater sensitivity. This would also explain the negative results obtained in immunodiffusion experiments using the eluates from I.E.O.P. tests. The fact that both antigen and antibody could be found in the same serum specimen in 4 cases, and antibody as well as electron microscopic particles in 2 other cases, further suggests that complexes can be present. The frequency with which we found particles which
j .
!
; i ’
383
antigen in cases of P.B.C. suggests that be intimately related to the disease process. they could been It has suggested 16 that some diseases associated with immunological deficiency predispose to the development of a chronic antigen-carrier state. It might be that this applies to P.B.c. However, there is no clinical evidence to suggest that our patients were unusually susceptible to infectious diseases. Furthermore, the incidence of chronic s.H. antigenaemia in patients with major immunological deficiency is not as high as that observed here.5 ,16 Liver damage in P.B.c. could be the result either of continuing replication of a virus, or of the host’s immune response. Our observation of the simultaneous occurrence of antigen and antibody, and the presence of clumps of particles possibly joined by antibody, are compatible with the existence of immune complexes. The presence of immune complexes has been postulated both in acute hepatitis 17 and in active chronic hepatitis with S.H. antigenaemia.6 This latter condition and P.B.c. may not be wholly separate clinical syndromes,la and when these diseases are associated with S.H. antigen, the basic pathogenic mechanism may be broadly similar. We wish to thank Dr. W. Eisenmenger, Dr. M. A. Payne, Dr. E. H. Ahrens, and Dr. N. M. Luger for providing materials for these studies. We also thank our technicians, Miss Marthe Fauconnet, Miss Tellervo Huima, and Miss Kathleen Burke for their hard work. We appreciate the reading of this manuscript and the many helpful suggestions by Dr. Henry Kunkel and Dr. Norman B. Javitt. This work was supported in part by training grant T01-AM5430 from the National Institutes of Health. One of us (A. M. P.) is the recipient of a career scientist award from the Health Research Council of the City of New York under contract may contain s.H.
number 1-533. Requests for reprints should be addressed to K. K., Laboratory of Virology, New York Blood Center, 310 East 67th Street, New York, New York, 10021. REFERENCES 1. Prince, A. M. Proc. natn Acad. Sci. U.S.A. 1968, 60, 814. 2. Blumberg, B. S., Alter, H. J., Visnich, S. A. J. Am. med. Ass. 1965,
191, 541. Gocke, D. J., Kavey, N. B. Lancet, 1969, i, 1055. Fox, R. A., Niazi, S. P., Sherlock, S. ibid. 1969, ii, 609. Wright, R., McCollum, R. W., Klatskin, G. ibid. p. 117. Almeida, J. D., Waterson, A. P. ibid. p. 983. Bayer, M. E., Blumberg, B. S., Werner, B. Nature, 1968, 218, 1057. Dane, D. S., Cameron, C. H., Briggs, M. Lancet, 1970, i, 695. Prince, A. M. ibid. 1968, ii, 462. Prince, A. M., Burke, K. Science (in the press). Coons, A. H., Kaplan, M. H. J. exp. Med. 1950, 91, 1. Doniach, D., Roitt, I. M., Walker, J. G., Sherlock, S. Clin. exp. Immunol. 1966, 1, 237. 13. Sherlock, S. in Diseases of the Liver and Biliary System; chap. 11. Philadelphia, 1968. 14. Popper, H., Schaffner, F. in Popper and Schaffner’s Progress in Liver Diseases; chap. 21. New York and London, 1970. 15. Coyne, V. E., Millman, 1., Cerda, J., Gerstley, B. J. S., London, T., Sutnic, A., Blumberg, B. S. J. exp. Med. 1970, 131, 307. 16. Blumberg, B. S., Sutnick, A. I., London, W. T. Bull. N.Y. Acad. Med. 1968, 44, 1566. 17. Shulman, N. R., Hirschman, R. J., Barker, L. F. Ann. intern. Med. 1970, 72, 257. 18. Doniach, D., Walker, J. G., Roitt, I. M., Berg, P. A. New Engl. J. Med. 1970, 282, 86.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
" Has anyone ever asked the question-why does the of clinical medicine start in the middle, proceed to the end, before coming back to the beginning ? Has any medical school thought of the revolutionary step of starting the clinical curriculum with pxdiatrics ? "-Prof. CHARLOTTE AxDERSOX, Putting Paediatrics in its Place, an inaugural lecture delivered in the University of Birmingham on Feb. 5, 1970, p. 11.
study
COMBINED TREATMENT OF PARKINSONISM WITH L-DOPA AND AMANTADINE C. C. FREARS
R. B. GODWIN-AUSTEN S. BERGMANN National
J. D.
Hospital, London W.C.1
PARKES
King’s College Hospital,
R. P.
KNILL-JONES
London S.E.5
A double-blind crossover trial of combined treatment with amantadine and L-dopa was conducted in twenty-four patients with parkinsonism. Twelve patients were on longterm treatment with L-dopa, and amantadine was added to their treatment; while twelve patients had L-dopa added to long-term treatment with amantadine. Further benefit was shown in the group in whom amantadine treatment was supplemented by the addition of L-dopa, whereas no additional benefit was detected in patients on optimal L-dopa therapy when amantadine was added. Combined treatment with these drugs is only indicated when the maximum tolerated dose of L-dopa is very small.
Summary
Introduction AMANTADINE was first given to patients with Parkinson’s disease by Schwab et al.1 and the definite improvement demonstrated was confirmed in subsequent trials.2,3 The effect of amantadine is less than that of L-dopa,3but Schwaband Scotti5 have claimed that the two drugs have an additive therapeutic action. Since toxic effects frequently limit the use of optimal doses of L-dopa, a combination of L-dopa and amantadine might be of greater value than either drug alone. We have combined treatment with L-dopa and amantadine in patients with parkinsonism to see whether an additive effect of these two drugs could be demonstrated. Patients and Methods Twenty-four patients with parkinsonism were studied. These were divided into two groups; group i comprised twelve patients already taking L-dopa, and group 11 twelve patients already taking amantadine. Eighteen patients were male, six patients were female; their ages ranged from forty-seven to sixty-seven; and they included patients with idiopathic, postencephalitic, and arteriosclerotic parkinsonism. Group-I patients had been on continuous treatment with L-dopa (0-75-5 g. daily) for more than eight months. Most of these patients were also taking anticholinergic drugs and all had benefited from L-dopa. In two patients the improvement had been dramatic, in nine patients moderate, and in one patient slight; but all had residual subjective and
objective disability. Group-II patients
had all been on treatment with amantadine (200-500 mg. daily) for three to six months. A preliminary assessment established that all patients in this group could tolerate 3 g. of L-dopa daily. All these patients had shown a favourable response to amantadine, with moderate improvement in seven and slight improvement in five. The benefit from both L-dopa and amantadine had been maintained, but no continuing further improvement was occurring at the start of the present trial. Before either