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Quantification of de novo central nervous system igg measles antibody synthesis in sspe.

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Quantification of de Novo
Central Nervous System IgG Measles
Antibody Synthesis in SSPE
Wallace W. Tourtellotte, MD, PhD,"t Booe I. Ma, PhD,"? D. B. Brandes, MS, MD,"?
M. J. Walsh, MD,"? and Alfred R. Potvin, PE, PhDS
De novo central nervous system (CNS) IgG measles antibody synthesis rate was determined in two patients with
subacute sclerosing panencephalitis (SSPE). Cerebrospinal fluid and sera were incubated with different concentrations of measles virus to determine maximum absorption of IgG. The difference in IgG concentration before and
after incubation was considered the amount of measles-specific IgG antibody. Various tests were done to assess the
immunological specificity of the absorption test. Using our empirical formula, the de novo CNS IgG measles antibody synthesis rate for one patient with SSPE was 57 mg/day, or 59% of the total CNS IgG synthesis rate; for the
other it was 145 mg/day, or 82%. Daily determination of CNS measles antibody by empirical formula provides a
quantification of humoral immunity in SSPE that corrects for both passage of measles antibody across a damaged
blood-brain barrier and the effect of an elevated blood antibody level.
Tourtellotte WW, Ma BI, Brandes DB, Walsh MJ, Potvin AR: Quantification of de novo central nervous
system IgG measles antibody synthesis in SSPE. Ann Neurol 9:551-556, 1981
Subacute sclerosing panencephalitis (SSPE) is a rare,
slowly progressive human encephalopathy caused by
persistent measles infection [ l , 141. A remarkable
feature of this illness is the presence of increased
immunoglobulin G (IgG) in the cerebrospinal fluid
(CSF) and of oligoclonal bands on CSF electrophoresis. Oligoclonal IgG banding is frequently
demonstrable in the sera of SSPE patients as well.
Early radioactive exchange studies with radiolabeled
IgG and albumin showed considerable quantities of
IgG synthesized within the brains of patients with
SSPE [3]. Extracts from brains of affected individuals
also demonstrated high IgG concentration and
measles antibody activity as well as IgG oligoclonal
banding [ 5 , 151. Subsequently, Ma and associates [6],
Vandvik et a1 [16], and Mehta and co-workers [7]
extended these observations and confirmed that from
10 to 90% of total IgG from CSF and brain extracts
of SSPE patients showed antimeasles activity. The
reason for this pattern of humoral immune response
is unknown, but it may be a consequence of continued stimulation by antigen of the measles virus.
An unusual feature of this disorder is the presence of
considerable cross-idiotypy among the immunoglobulins produced by patients in response to per-
sistent measles virus infection. Strosberg et a1 [lo]
were able to demonstrate cross-idiotypy for IgG in 7
of 18 patients. This is evidence for homogeneity in
the antibody response of SSPE patients to viral
antigen.
Conventionally, the host humoral response to
virus infection is expressed qualitatively as titer.
Comparison of titers of antiviral antibody in CSF and
serum has provided evidence for synthesis of antiviral antibody inside the central nervous system (CNS)
in certain viral and demyelinating disorders [8]. Precise quantitation of the antiviral specificity of this
synthesized immunoglobulin of CSF and serum IgG
and of its fluctuations over time would be valuable in
interpreting the immune response to antigen within
the CNS and in following the stages of disease over
time in response t o putative therapeutic interventions. In this report, we further characterize the
immunological reaction in SSPE by quantifying the
de novo CNS IgG measles antibody synthesis rate.
From the 'Neurology and Research Services, VA Wadsworth
Medical Center, Los Angeles, CA 90073, the +Department of
UCLA School of Medicine, Los A n g e b CA 90024,
and the $Biomedicaland Electrical Engineering Departments, The
University of Texas at Arlington, Arlington, TX 76019.
Received Aug 11, 1980, and in revised form Oct 5. Accepted for
publication Nov 11, 1980.
Materials and Methods
CSF and serum were obtained from two patients with
SSPE. The diagnosis was confirmed at autopsy.
Electroimmunodiffusion (EID) was used as previously
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551
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described [ 131 to quantify albumin and IgG simultaneously
in sera diluted 1 : 200 and in CSF, either 5 p1 neat or diluted
as necessary. In brief, the supporting medium is a combination of agarose and Noble agar containing hyperimmune
serum from rabbits immunized with human IgG and albumin. Standards and unknown specimens are placed in the
wells of the matrix, and an electric field is applied across the
EID plate. Albumin migrates toward the anode and IgG
toward the cathode; a precipitation line forms at the zone
of antigen-antibody equivalence. Comparison of the length
of the “rockets” enables calculation of IgG and albumin in
the unknown specimens. All plates contain a multiple sclerosis (MS) CSF pool standard, which should read 11.2 ?
0.9 mgldl (mean -+ 2 SD) for IgG and 34.8 2 1.6 mgldl for
albumin. If the MS CSF pool is not within the known
range, the EID plate is discarded.
Measles virus (Edmonston strain, from Flow Laboratories, Rockville, M D ) with a hemagglutination antigen
titer of 1: 64, was concentrated 100 times under negative
pressure in a collodion bag [4] (Schleicker and Schull, Inc.,
Keene, NH). In the measles absorption procedure, various
concentrations of measles were incubated with equal volumes (50 pl) of CSF or serum diluted 1 : 100 at 37°C for 2
hours and 4°C for 16 hours. To serve as controls, CSF was
incubated with equal volumes of 0.9% sodium chloride o r
the culture medium containing the cells in which thc
measles virus was grown. This was then centrifuged at
125,000 rcf to sediment immune complexes. EID to determine absorbed IgG was repeated o n the supernatant
fluid. The difference in IgG concentration before and after
incubation was considered the amount of measles antibody.
The de novo CNS IgG synthesis rate, in milligrams per
day, was computed using the empirical formula:
De novo CNS IgGsvN= [(IgGcsv
-
(AlbrsF -
-
IgGs
369
230
where IgGcsF is the concentration of IgG found in the pais the serum IgG concentration, AlbrsF is
tient’s CSF,
the albumin concentration in CSF and Alb, is the albumin
concentration in serum. The basis for the formula and its
validation are found elsewhere [ 1I]. De novo C N S measles
antibody synthesis rate was determined similarly except
that serum and CSF antimeasles IgG obtained from the
measles absorption test were used in lieu of IgG.
In order to obtain additional evidence that an immunological reaction had occurred in the absorption procedure,
counterimmunoelectrophoresis (CIE) was used [9]. CIE
permits IgG, a cathodic protein, to migrate toward anodic
measles virus. If antibody to measles were present in the
CSF, a precipitation line should develop. If the concentrate
of IgG is diluted, the precipitation line should develop
closer to the CSF well. O n agarose plates (Bioware Inc.,
Wichita, KS), 50 pl of CSF (neat and diluted 1 : 1) before
and after absorption was placed in the anodic hole and 50
pl of measles preparation concentrated 100 times was
placed in the cathodic hole. After electrophoresis, the
plates were washed and stained with Coomassie Blue, a
sensitive, nonspecific protein stain.
552 Annals of Neurology
Vol 9
No 6 June 1981
Immunofixation was carried out to identify that oligoclonal bands on electrophoretograms were formed by IgG.
Oligoclonal bands were reacted with rabbit antihuman IgG
on Panagel agarose plates (Millipore Co., Bedford, MA) as
previously described [2] to demonstrate oligoclonal IgG
before and after the measles absorption procedure. In addition, immunofixation of CSF oligoclonal IgG with horse
antiserum against lambda IgG or goat antiserum against
kappa IgG (Hyland Laboratories, Costa Mesa, CA) was also
carried out to determine kappa and lambda light chain
specificity of the oligoclonal bands absorbed by measles.
Results
An EID plate showing the result of an SSPE CSF
measles absorption procedure is shown in Figure 1.
The first four wells (numbers run right to left) contain solutions with known concentrations of IgG and
albumin. The fifth well is the standard CSF pool from
MS patients with known IgG and albumin. The SSPE
CSF that was not absorbed was placed in wells 6 and
12. The remaining wells contained the supernatant
fluid after CSF from the same SSPE patient had been
incubated with various concentrations of measles
virus. The absorption of CSF IgG reached a maximum of 60% at a measles concentration of x25.
Doubling this measles concentration produced no
further absorption. Accordingly, this specimen contained 5.8 mgidl of measles IgG antibody (7.7 - 3.7
mg/dl before and after incubation, respectively). In
contrast to results for IgG, albumin was not absorbed, and in a separate experiment a panel of other
viruses (rubella, herpes simplex, adenovirus, parainfluenza type I and 11, and mumps) showed no IgG
absorption from CSF o r serum. Although we have no
explanation f o r the symmetrical lateral lines seen in
holes 7 through 10, they must be related to constituents in the media or cells containing the virus
since their intensity increases with measles concentration. The lines never interfered with measurement
of rocket length. When media and cells without virus
were used, rockets did not form.
Values for CSF and serum albumin and IgG obtained by EID as well as de novo CNS IgG synthesis
rate and IgG measles synthesis rate for the two SSPE
patients are shown in the Table. For each patient,
determinations of the same specimens done on two
occasions showed good agreement. IgG synthesis
rates for the two patients averaged 96 and 178 mg
per day (normal, 3.3) while IgG measles synthesis
rates averaged 57 and 145 mg per day, or 59 and
82%, respectively, of the total IgG synthesized.
To obtain additional evidence that an immunological reaction rather than nonspecific precipitation
had occurred in the absorption procedure, CIE was
performed using SSPE CSF before and after measles
absorption. Typical results from an SSPE patient are
F i g I . (Patient 1i Typical electroimmunodi~usionplate
showing an SSPE CSF measles absorption test. All information
relating to albumin lies above the midline of the wellr, that related to lgG, below the midline.
Rates of de Novo CNS IgG Synthesis and 1gG Measles
Synthesis Utilizing Our Empirical Formula in Two
Cases of SSPE
shown in Figure 2. All bottom wells contained measles virus concentrated 100 times. Upper wells l and 4
held CSF before absorption. A precipitation line was
formed after electrophoresis, as indicated by the arrows. Since CSF in well 4 was diluted 1 : 1 with 0.9%
sodium chloride, the lines of precipitation are less
intense and closer to the CSF well. Upper wells 2 and
3 contained CSF that was previously incubated with
measles virus concentrated 50 and 100 times, respectively. No precipitation line was seen. Similar results for CSF and sera were found for both patients
with SSPE. Accordingly, the results support a specific
measles antibody reaction and the presence of
measles antibody in the SSPE CSF.
Figure 3 shows changes in oligoclonal IgG bands
with agarose gel electrophoresis of CSF stained with
Coomassie Blue for one patient with SSPE before
and after incubation with measles. Two of the five
bands were absorbed out and the other three were
reduced in intensity. Results of immunofixation
showed that another band was absorbed out. A similar result was found for the other SSPE patient.
These results indicate the presence of specific
measles antibody in some oligoclonal bands and not
Determination
CSF IgG (rngldl)
Total IgG
IgG measles
CSF albumin (mgldl)
Before measles absorption
After measles absorption
Serum IgG (mgldl)
Total IgG
IgG measles
Serum albumin
(mgldl)
Before measles absorption
After measles absorption
De novo C N S IgG
synthesis rate
(mglday)
Total IgG
IgG measles
Percent d e novo C N S
IgG synthesis
rate due to
measles virus
Patient 1:
Determination
Patient 2:
Determination
1
1
2
2
12
24
13
39
28
43
30
18
22
20
20
22
24
20
20
1,720
400
1,600
360
1,920
0
1,880
80
4,900
5,100
4,000
4,400
4,900
5,300
4,000
3,800
94
54
57
98
60
61
166
189
149
79
23
140
84
Tourtellotte et al: C N S Measles Ab Synthesis in SSPE
553
F i g 2. (Patient 21 Counterimmunoelectrophoresisof an SSPE
CSF before and after absorption with measles virus (upper
wells) versus measles virus concentrated I00 times (lower wells).
I
SSPE-CSF + 0,9% SODIUM CHLORIDE
SSPE-CSF + MEASLES
MEASLES + 0,9% SODIUM
CHLORIDE
SSPE-CSF + 0,9% SODIUM CHLORIDE
SSPE-CSF
+
MEASLES
1
I
IMMUNOF
Ix
MEASLES + 0.9% SODIUM CHLORIDE
I
Fig 3. (Patient 21 Agarose gel (Panagel) electrophoresis with
and without immunoflxation (rabbit antihuman IgGi of CSF
from a patient with SSPE before and after incubation with
measles, and of the measles virus preparation.
554 Annals of Neurology Vol 9 No 6 June 1981
others, and further validate the absorption procedure.
T o determine if oligoclonal IgG absorbed by
measles was specific for kappa or lambda light chain
IgG, bands separated by agarose electrophoresis
were immunofixed with antiserum against kappa or
lambda IgG. Comparison of bands before and after
incubation with measles indicated that primarily
kappa light chain oligoclonal IgG bands were absorbed by measles, further supporting the immunological specificity of the measles absorption procedure.
Discussion
As part of the measles absorption procedure using
SSPE CSF and sera, maximum absorption was determined by titration. Validation that the absorption
procedure was due to measles IgG antibodies was
obtained by utilizing CIE and by agarose electrophoresis followed by immunofixation with rabbit
antihuman IgG and antiserum against kappa and
lambda light chains.
We used a live virus that should contain all viral
components. Accordingly, CSF antibody against
certain fractions such as nucleocapsid may have been
inadequately removed. Hence, our estimate of CNS
IgG measles antibody synthesis might be a minimal
value. Alternatively, if the antigen preparation was
highly contaminated by polypeptides from the cells
in which cross-reactions of the human IgG with such
proteins were generated, then the amount of specific
antimeasles IgG present in serum and CSF specimens
might be overestimated. Additional studies are required to resolve these issues.
Absorption experiments with other viruses also
were performed on these SSPE CSF samples utilizing our absorption method to quantitate antibodies. Five myxoviruses (herpes simplex type 1, mumps,
rubella, cytomegalovirus, and parainfluenza virus,
obtained from Flow Laboratories) and their tissue
culture, which served as a control, were concentrated
25 o r 50 times. N o absorption was obtained. Accordingly, the specificity of the measles antibody
synthesis for SSPE in these patients was documented.
Although our empirical formula has been validated
and used [ 111 to calculate d e novo C N S IgG synthesis rate, the formula was utilized for the first time in
this study to measure CNS antibody production. The
same physiological principles that govern CNS IgG
production in MS (filtration of IgG from serum into
CSF across the intact blood-brain barrier and filtration of albumin on a 1: 1 molecular equivalence with
IgG as a measure of the health of the blood-brain
barrier) should be applicable to de novo CNS IgG
measles antibody synthesis rate. Confirmation could
be obtained from a radioactive exchange test using
labeled IgG measles antibody. Cutler and Tourtellotte [3] described a test of this general concept. IgG
from the CSF of one SSPE patient was labeled with radioactive iodine 131 and injected intravenously, and
IgG specific activity in serum and CSF was measured
serially. Fractional transfer rates and equilibrium
specific activities of labeled serum and CSF IgG were
nearly identical. The results did not support the possibility of selective transport of antibody from blood
to CSF in SSPE.
De novo CNS IgG synthesis rate was calculated in
the two SSPE cases studied. From the measles absorption procedure, CSF and serum antibody per
deciliter were measured and substituted into our
empirical formula to calculate measles IgG antibody
synthesized in the CNS per day. We found that 59
and 82% of the total IgG synthesized were antibodies to measles antigen; thus, in the two patients, 41
and 18% were synthesized for other reasons. Additional SSPE cases should be studied to confirm this
result.
One explanation for this finding, which is similar to
that hypothesized for MS [ 121, may be recruitment of
committed lymphocytes to the SSPE central nervous
system, resulting in antibody synthesis that recapitulates the body’s antigenic exposure. This proposal
was not supported by our experiments, in which we
used viruses to which most individuals are exposed.
Another possibility is that a mutant measles virus
or a neoantigen developed in the CNS during the
course of the disease. Accordingly, this resulted in
synthesis of IgG antibody that did not cross-react
with the measles virus used in the absorption test. To
test this proposal in part [6], CSF from an SSPE
patient containing 96 pglml of IgG was incubated
with measles. The resulting IgG concentration was
47 pglml. The matched brain preparation (20%
homogenate in 0.9% sodium chloride) containing 5 1
pglml of IgG was also incubated with measles, and
the resulting IgG concentration was 28 pglml. When
CSF and brain, presumably free of measles antibody,
were reacted with each other, the resulting concentration was 57 pglml, which was 18 pg/ml less than
expected (47 28 - 57 = 18).Thus, 18% (18/96 x
100) of CSF I& reacted with something in SSPE
brain. When the experiment was replicated with
normal and MS brain and CSF, measured values
agreed with calculated values. Thus evidence from
this study suggests that an antibody may exist in SSPE
CSF to antigen other than the measles virus preparation used, e.g., a mutant measles virus in the SSPE
brain or a neoantigen that may be induced by the persistent measles virus. Additional SSPE cases should
be studied to confirm this result.
We have extended these results with nine patients
who had clinically definite MS and found a mono-
+
Tourtellotte et al: CNS Measles Ab Synthesis in SSPE
555
tonically increasing relationship between the percentage of IgG absorbed and de novo C N S IgG synthesis rate. In three control patients (two with stroke
and one with cardiovascular disease), no IgG absorption was found (unpublished data).
Quantitative estimation of measles antibody
specificity, in milligrams of antibody per milliliter of
CSF or serum, should be superior to antibody titer
measurements. With the method described in this
report to obtain antibody values in serum and CSF as
well as albumin values to assess health of the bloodbrain barrier, the de novo CNS IgG antibody synthesis rate can be calculated from our empirical formula.
The formula automatically combines blood antibody
levels and blood-brain barrier health. Determination
of de novo C N S antibody synthesis rates in consecutive specimens might offer higher accuracy than measurements of antibody titers in CSF and blood.
For other diseases in which the antigen is known
and available, it should be possible to develop an absorption procedure similar to that described here for
SSPE. In particular, this approach may have value in
herpes simplex encephalitis. Since the antigen is
available for absorption studies and effective therapy
exists, our procedure may be helpful in assessing
therapeutic regimens.
Supported in part by VA Medical Research Funds; the National
Neurological Research Bank funded by the National Multiple
Sclerosis Society, the National Institute of Neurological and
Communicative Disorders and Strokes, The National Institute of
Mental Health, and the Hereditary Disease Foundation; and Organized Research Funds from The University of Texas at Arlington.
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