Journal of
J. Neurol.,216, 51--56 (1977)
Neurology ~) by Springer-Verlag 1977
A Heat Stable Serum Inhibitor of an Antigen Antibody Reaction of Subacute Sclerosing Panencephalitis* D. Karcher, M. Noppe, and A. Lowenthal The Department of Neurochemistry, Born-Bunge Foundation, Universitaire lnstelling Antwerpen, 59, Filip Williotstraat, 2600 Berchem, Antwerpen, Belgium
Summary. A factor which inhibits the antigen antibody reaction was isolated from the sera of patients affected with subacute sclerosing panencephalitis (SSPE), multiple sclerosis (MS) and also of control subjects. This factor inhibits the immune reaction between SSPE serum IgGs and measles virus. This inhibiting factor was detected in the alpha globulin region after agar gel electrophoresis, and is heat stable at 100°C for ten minutes. Key words: Subacute sclerosing panencephalitis - Immunoglobulins - Hyperimmunization - Inhibiting factor.
Zusammenfassung. Von Patienten mit subakuter sklerosierender Panencephalitis, mit multipler Sklerose und auch von Kontrollpersonen wurde ein Faktor im Serum isoliert, welcher die Antigen-Antik6rper-Reaktion hemmt. Dieser Faktor hemmt die Immunreaktion zwischen SSPE-Serum IgGs und Masernvirus. Dieser hemmende Faktor befindet sich in der Agargel-Elektrophorese in der Region der Alphaglobuline und ist bei 100°C w~ihrend 10min hitzestabil.
Subacute sclerosing panencephalitis (SSPE) can be classified as a hyperimmune disease. The virus is persistent and can be isolated in biopsies from brain or lymph nodes at every stage of the disease [1]. This persistent antigen triggers the formation ofautologous antibodies during the whole evolution of the disease, except perhaps during the terminal stage. High measles antibody titers have been measured in the serum, cerebrospinal fluid (CSF), and tissue extracts, and restricted antibody heterogeneity is observed on agar gel electrophoresis. These immunoglobulins (IgGs) are partially specific, and they can be absorbed with measles virus in the CSF of those patients [2]. In a study of experimental hyperimmunization with chitine in rabbits, serum IgGs have been absorbed specifically by incubating the chitine with the immuno*
This investigation was supported by F.G.W.O., M.N.O., U.I.A., Belgium
52
D. Karcher et al,
globulins with restricted h e t e r o g e n e i t y [3). I g G a b s o r p t i o n with chitine was a c c o m p a n i e d b y the loss o f some oligoclones. Using measles virus, we have p r e v i o u s l y a p p l i e d this technic to the C S F o f patients with SSPE a n d multiple sclerosis (MS) [2], a n d this o b s e r v a t i o n has been c o n f i r m e d recently b y B o o e [4]. But we have been u n a b l e to achieve the s a m e results with whole serum. T h e a n t i b o d y antigen r e a c t i o n was a t t a i n e d with isolated I g G s , a n d the I g G s were a b s o r b e d with measles virus. This difference in b e h a v i o r between whole serum a n d purified I g G s f r o m an identical serum specimen was investigated. I n h i b i t i n g factors have been d e t e c t e d in the s e r u m o f patients with t u b e r culosis, chronic c u t a n e o u s candidiasis, hepatitis, r h e u m a t o i d arthritis, a t a x i a telangiectasia a n d cancer, all diseases a s s o c i a t e d with intense antigenic stimul a t i o n [5]. Since there is also a n intense antigenic s t i m u l a t i o n in SSPE, it seems likely t h a t the i n h i b i t o r y f a c t o r r e p o r t e d here is similar to the " h u m o r a l i m m u n o suppressive f a c t o r " d e s c r i b e d b y C o o p e r b a n d et al. [5].
Material and Methods Serum and CSF samples were collected from seven patients affected with SSPE and two with MS. The diagnosis of SSPE was made clinically, electroencephalographically,and sometimes at autopsy; it was always confirmed by agar gel electrophoresis and measurement of measles antibody titers. The diagnosis of MS was made clinically. The disease progressed characteristically with bouts, remissions, and widespread neurological manifestations, and it was confirmed by agar gel electrophoresis of the CSF.
Strains of Measles Virus A commercially available measles antigen (Behringwerke, RAO) and two strains of measles virus (Woodfolk and Edmonston) were used; in addition a strain of measles-like virus (LEC) isolated from a patient affected with SSPE, was kindly provided by Prof. V. ter Meulen, of Wiirzburg.
Electrophoresis A sample of 0.3 mg purified SSPE serum lgG dissolved in 20 ~tl of physiological saline was used as a control and compared with a sample of 0.3 mg of the purified SSPE serum IgG dissolved in 201xl of viral suspension. They were incubated overnight at room temperature, and then contrifuged. Both supernatants were run electrophoretically on the same plate. The plates were scanned and the total surface areas compared. To detect the inhibiting factor the following procedure was applied in all case: samples of 0.6 mg of the purified SSPE serum IgG were dissolved in 20 Ixl viral suspension and incubated with 20 ~tl of a 1 mg per ml solution of the various fractions and compared by electrophoresis with SSPE serum IgG incubated, either with the viral suspension alone, or with physiological saline.
Radioimmunoassay IgGs isolated from SSPE were labelled with I125by the method of Greenwood et al. [6]. Labelled IgGs (20 000 cpm) were rotated overnight at room temperature with specific rabbit antihuman IgG antibodies coupled to cyanogen bromide powdered cellulose as immunoabsorbent. The working concentration for the immunoabsorbent was chosen to yield approximately 37% of bound labelled IgG.
A Heat Stable Serum Inhibitor of an Antigen Antibody Reaction
53
The same technic was repeated under identical conditions, except for the addition of the various preparations of inhibiting factor in serial dilutions of similar concentrations. The different fractions for radioimmunoassay in initial concentrations of 0.1 mg/100~tl were serially diluted to 1/32. The decrease in counts per minute reflected the inhibiting effect on the reaction.
Detection of the Inhibiting Factor The factor inhibiting the antigen antibody reaction in the SSPE serum was detected after application of the following methods: i) preparative electrophoresis on agar gel carried out on glass plates 15.5 x 13.0 cm using barbital buffer pH 8.6. A strip of gel was cut parallel to the electrophoretic migration at the end of the run, and served as reference. Five different classes of proteins were isolated: albumin and c~1, ~2, fl and y globulins. The gel was deep frozen, thawed several times, and then centrifuged. The supernatant was drawn off, dialyzed and lyophilized. ii) serum precipitation with 0.126 M sodium sulfate. The supernatant was dialyzed and lyophilized. iii) the lyophilized supernatant was dissolved and precipitated with 2.8 M ammonium sulfate. The precipitate was dissolved in physiological saline, dialyzed and lyophilized. iv) the supernatant from the procedure (ii) or the precipitate from (iii) was eluted from a D E A E Sephadex A50 column with a 0.05 M triethanolamine--0.005 M EDTA buffer pH 7.5, using a stepwise 1M NaC1 gradient mixer, for radioimmunoassay. The eluates were checked by Mancini plates for the presence of IgGs. The preparations were passed on a Sepharose CNBr immunoabsorbent column coupled to antihuman IgGs serum to eliminate IgGs.
Results 1. A f t e r electrophoresis o f a s a m p l e o f 0.3 m g o f t h e p u r i f i e d S S P E s e r u m I g G diss o l v e d in 20~tl o f p h y s i o l o g i c a l saline, the c o n c e n t r a t i o n was c a l c u l a t e d b y d e n s i t o m e t r y . T h e a v e r a g e v a l u e was 248 c o n v e n t i o n a l unit areas f o r n i n e
!3
4! 2
O
t
3 4
Fig. 1. Electrophoretic pattern of serum IgGs of patient affected with subacute sclerosing panencephalitis. 1 and 3: IgGs taken as control and dissolved in physiological saline. 2: IgGs incubated with LEC, a strain of measles-like virus. Note the marked decrease. 4: IgGs incubated with Woodfolk strain of measles virus. Note the marked decrease
54
D. Karcher et al.
determinations. After incubation of the IgGs with measles (Woodfolk) or LEC virus, it dropped to 53 (Fig. 1). Thus at least 79% of the IgGs shown by electrophoresis were precipitated when purified IgGs were incubated with virus, while there was no measurable decrease when lyophilized serum was used. The antigen antibody reaction was inhibited by the following fractions (and no decrease of the I g G concentration was measured): (i) only the fraction migrating in the ~ region after preparative electrophoresis in agar gel; (ii) the supernatant after 0.125 M sodium sulfate precipitation; (iii) the precipitate of this same supernatant after adding 2.8 M a m m o n i u m sulfate; (iv) the second, the third and the fifth fraction after elution from D E A E Sephadex A50 column with a NaC1 gradient. ~ globulins are present in all those fractions; (v) serum diluted 1 : 10 and heated at 100°C for 10min. Equal quantities (3 mg/40 2) of each fraction were added either before incubation of the antigen with its analogous antibody, or to the mixture prepared for radioimmunoassay (2 mg/ml). 2. As large amounts of purified IgGs and virus would be needed to check the effect of each fraction on the antigen antibody reaction by agar gel electrophoresis, radioimmunoassay was used to test the blocking effect on the antigen antibody
Fractions of SSPE serum supernatant (0.126M sodium sulfate precipitation) collected after passage on DEAE Sephadex A50 column IgG I t25 bound (Bo)
9021
Inhibiting effect (in %)
Fraction Fraction Fraction Fraction Fraction Fraction Fraction
5087 2700 3007 7787 3300 3879 7795
44 71 67 14 64 57 14
1 2 3 4 5 6 7
IgG I ~25bound (B~) 8208 4445 6206
Inhibiting effect (in %) 640 698 1274
92 84 80
Table 1. Inhibiting effect expressed in % decrease in counts per minute (RIA)
Table 2. Control sera heated at 100°C for 10 min
A Heat Stable Serum Inhibitor of an Antigen Antibody Reaction
55
immune reaction. When working with labelled IgGs, the supernatant after 0.126 M sodium sulfate precipitation or its precipitate formed after treatment with 2.8 M ammonium sulfate was not considered to be reliable for RIA since the presence of IgGs in the preparations, competing with labelled IgGs, may give false positive readings. The fractions collected from the column (DEAE Sephadex A50: fractions 2, 3 and 5) gave very satisfactory results (Table 1). All those fractions were located in the ~ region after agar gel electrophoresis and inhibited the antigen antibody reaction. The heated control serum also inhibited the reaction (Table 2). The inhibitions varied from 64% to 92% (Tables 1 and 2).
Discussion
Previously the CSF immunoglobulins were found to be absorbed with different strains of measles virus [2,4]: a decrease of the CSF oligoclonal IgGs was clearly observed. Samples of CSF are available in too small quantities to permit widespread investigation. Serum presents many practical advantages, but the serum antibodies from patients with SSPE with restricted antibody heterogeneity are not precipitated by measles virus. However, IgGs isolated from sera precipitated readily with different strains of measles virus. It appears that the total serum antibody precipitation is inhibited and there may be a factor which blocks the immune reaction. Swich et al. [7] described a heat stable serum factor which impairs the cell mediated immune response in SSPE sera. In all these experiments serum and IgGs from the same specimen of serum were compared. Cooperband et al. [5] have reviewed extensively the work done during the last seven years on humoral immunosuppressive factors, and have concluded that there is a non-specific peptide suppressor that may be isolated with plasma alpha globulins. This substance is probably identical with the inhibiting factor we have observed. They discuss its immunosuppressive effect in relation to transplantation, the immune response and the action of phytohemagglutinin, among other agents. They did not report inhibiting activity associated with antigen antibody binding. In the present experiments, the substance acting as blocking or inhibiting factor which prevented the antigen antibody reaction was detected in the globulin region after agar gel electrophoresis. This inhibiting factor was present in all the sera investigated from seven patients with SSPE, two with MS, two control subjects and samples of pooled sera, each collected from about 20 patients in polyclinics of local hospitals. The serum of the patients affected with SSPE have either a more potent or higher concentration of the inhibiting factor in their sera. This will be determined after purification of the inhibiting factor. The present inhibiting factor is reminiscent of the non-specific inhibitor described ten years ago by Rovnova [8]. The role, function, and underlying mechanisms of the inhibiting factor in diseases where hyperimmunization occurs during its natural history underlines the interest of this investigation. It is hoped that this investigation will shed some light on the genesis of an immune disease such as SSPE.
56
D. Karcher et al.
References 1. Horta-Barbosa, L., Hamilton, R., Wittig, B., Fuccilo, D. A., Sever, J.: Subacute sclerosing panencephalitis: isolation of suppressed measles virus from lymph node biopsies. Science 173, 840--841 (1971) 2. Lowenthal, A., Karcher, D., Bollengier, F.: M-components in Neurology. Neue Forschungsergebnisse des Hirnstoffwechsels und der Entmarkungsenzephalomyelitis, ,pp. 295--297 (R. M. Schmidt, ed.). Halle (Saale): Martin-Luther-Universit/it 1974 3. Janssens, J.: Homogene konijnen antilichamen tegen micrococcus lysodeikticus. Thesis: Faculteit der Wetenschappen, Vrije Universiteit, Brussel, 1972 4. Booe, I. Ma., Tourtellotte, W. W., Brandes, D. W.: Brain and measles specific immunoglobulin G in the cerebrospinal fluid of patients with SSPE. American Academy of Neurology 28th annual meeting. Neurology (Minneap.) 26, 377 (1976) 5. Cooperband, S. R., Nimberg, R., Schmid, K., Mannick, J. A.: Humoral immunosuppressive factors. Transpl. Proc. 3, 225--242 (1976) 6. Greenwood, F. C., Hunter, W. M., Glover, J. S.: The preparation of 131-I-labelled human growth hormone of high specific radioactivity. Biochem. J. 89, 114--123 (1963) 7. Swick, H. M., Brooks, W. H., Roszman, T. L., Caldwell, D.: A heat stable blocking factor in the plasma of patients with subacute sclerosing panencephalitis. Neurology (Minneap.) 26, 84--88 (1976) 8. Rovnova, Z. I., Kosyakov, P. N.: Inhibitors and antibodies in the course of immunization of animals with viral preparations. Acta Virol. 11, 69--77 (1967) Received January 25, 1977
J. Neurol.,216, 51--56 (1977)
Neurology ~) by Springer-Verlag 1977
A Heat Stable Serum Inhibitor of an Antigen Antibody Reaction of Subacute Sclerosing Panencephalitis* D. Karcher, M. Noppe, and A. Lowenthal The Department of Neurochemistry, Born-Bunge Foundation, Universitaire lnstelling Antwerpen, 59, Filip Williotstraat, 2600 Berchem, Antwerpen, Belgium
Summary. A factor which inhibits the antigen antibody reaction was isolated from the sera of patients affected with subacute sclerosing panencephalitis (SSPE), multiple sclerosis (MS) and also of control subjects. This factor inhibits the immune reaction between SSPE serum IgGs and measles virus. This inhibiting factor was detected in the alpha globulin region after agar gel electrophoresis, and is heat stable at 100°C for ten minutes. Key words: Subacute sclerosing panencephalitis - Immunoglobulins - Hyperimmunization - Inhibiting factor.
Zusammenfassung. Von Patienten mit subakuter sklerosierender Panencephalitis, mit multipler Sklerose und auch von Kontrollpersonen wurde ein Faktor im Serum isoliert, welcher die Antigen-Antik6rper-Reaktion hemmt. Dieser Faktor hemmt die Immunreaktion zwischen SSPE-Serum IgGs und Masernvirus. Dieser hemmende Faktor befindet sich in der Agargel-Elektrophorese in der Region der Alphaglobuline und ist bei 100°C w~ihrend 10min hitzestabil.
Subacute sclerosing panencephalitis (SSPE) can be classified as a hyperimmune disease. The virus is persistent and can be isolated in biopsies from brain or lymph nodes at every stage of the disease [1]. This persistent antigen triggers the formation ofautologous antibodies during the whole evolution of the disease, except perhaps during the terminal stage. High measles antibody titers have been measured in the serum, cerebrospinal fluid (CSF), and tissue extracts, and restricted antibody heterogeneity is observed on agar gel electrophoresis. These immunoglobulins (IgGs) are partially specific, and they can be absorbed with measles virus in the CSF of those patients [2]. In a study of experimental hyperimmunization with chitine in rabbits, serum IgGs have been absorbed specifically by incubating the chitine with the immuno*
This investigation was supported by F.G.W.O., M.N.O., U.I.A., Belgium
52
D. Karcher et al,
globulins with restricted h e t e r o g e n e i t y [3). I g G a b s o r p t i o n with chitine was a c c o m p a n i e d b y the loss o f some oligoclones. Using measles virus, we have p r e v i o u s l y a p p l i e d this technic to the C S F o f patients with SSPE a n d multiple sclerosis (MS) [2], a n d this o b s e r v a t i o n has been c o n f i r m e d recently b y B o o e [4]. But we have been u n a b l e to achieve the s a m e results with whole serum. T h e a n t i b o d y antigen r e a c t i o n was a t t a i n e d with isolated I g G s , a n d the I g G s were a b s o r b e d with measles virus. This difference in b e h a v i o r between whole serum a n d purified I g G s f r o m an identical serum specimen was investigated. I n h i b i t i n g factors have been d e t e c t e d in the s e r u m o f patients with t u b e r culosis, chronic c u t a n e o u s candidiasis, hepatitis, r h e u m a t o i d arthritis, a t a x i a telangiectasia a n d cancer, all diseases a s s o c i a t e d with intense antigenic stimul a t i o n [5]. Since there is also a n intense antigenic s t i m u l a t i o n in SSPE, it seems likely t h a t the i n h i b i t o r y f a c t o r r e p o r t e d here is similar to the " h u m o r a l i m m u n o suppressive f a c t o r " d e s c r i b e d b y C o o p e r b a n d et al. [5].
Material and Methods Serum and CSF samples were collected from seven patients affected with SSPE and two with MS. The diagnosis of SSPE was made clinically, electroencephalographically,and sometimes at autopsy; it was always confirmed by agar gel electrophoresis and measurement of measles antibody titers. The diagnosis of MS was made clinically. The disease progressed characteristically with bouts, remissions, and widespread neurological manifestations, and it was confirmed by agar gel electrophoresis of the CSF.
Strains of Measles Virus A commercially available measles antigen (Behringwerke, RAO) and two strains of measles virus (Woodfolk and Edmonston) were used; in addition a strain of measles-like virus (LEC) isolated from a patient affected with SSPE, was kindly provided by Prof. V. ter Meulen, of Wiirzburg.
Electrophoresis A sample of 0.3 mg purified SSPE serum lgG dissolved in 20 ~tl of physiological saline was used as a control and compared with a sample of 0.3 mg of the purified SSPE serum IgG dissolved in 201xl of viral suspension. They were incubated overnight at room temperature, and then contrifuged. Both supernatants were run electrophoretically on the same plate. The plates were scanned and the total surface areas compared. To detect the inhibiting factor the following procedure was applied in all case: samples of 0.6 mg of the purified SSPE serum IgG were dissolved in 20 Ixl viral suspension and incubated with 20 ~tl of a 1 mg per ml solution of the various fractions and compared by electrophoresis with SSPE serum IgG incubated, either with the viral suspension alone, or with physiological saline.
Radioimmunoassay IgGs isolated from SSPE were labelled with I125by the method of Greenwood et al. [6]. Labelled IgGs (20 000 cpm) were rotated overnight at room temperature with specific rabbit antihuman IgG antibodies coupled to cyanogen bromide powdered cellulose as immunoabsorbent. The working concentration for the immunoabsorbent was chosen to yield approximately 37% of bound labelled IgG.
A Heat Stable Serum Inhibitor of an Antigen Antibody Reaction
53
The same technic was repeated under identical conditions, except for the addition of the various preparations of inhibiting factor in serial dilutions of similar concentrations. The different fractions for radioimmunoassay in initial concentrations of 0.1 mg/100~tl were serially diluted to 1/32. The decrease in counts per minute reflected the inhibiting effect on the reaction.
Detection of the Inhibiting Factor The factor inhibiting the antigen antibody reaction in the SSPE serum was detected after application of the following methods: i) preparative electrophoresis on agar gel carried out on glass plates 15.5 x 13.0 cm using barbital buffer pH 8.6. A strip of gel was cut parallel to the electrophoretic migration at the end of the run, and served as reference. Five different classes of proteins were isolated: albumin and c~1, ~2, fl and y globulins. The gel was deep frozen, thawed several times, and then centrifuged. The supernatant was drawn off, dialyzed and lyophilized. ii) serum precipitation with 0.126 M sodium sulfate. The supernatant was dialyzed and lyophilized. iii) the lyophilized supernatant was dissolved and precipitated with 2.8 M ammonium sulfate. The precipitate was dissolved in physiological saline, dialyzed and lyophilized. iv) the supernatant from the procedure (ii) or the precipitate from (iii) was eluted from a D E A E Sephadex A50 column with a 0.05 M triethanolamine--0.005 M EDTA buffer pH 7.5, using a stepwise 1M NaC1 gradient mixer, for radioimmunoassay. The eluates were checked by Mancini plates for the presence of IgGs. The preparations were passed on a Sepharose CNBr immunoabsorbent column coupled to antihuman IgGs serum to eliminate IgGs.
Results 1. A f t e r electrophoresis o f a s a m p l e o f 0.3 m g o f t h e p u r i f i e d S S P E s e r u m I g G diss o l v e d in 20~tl o f p h y s i o l o g i c a l saline, the c o n c e n t r a t i o n was c a l c u l a t e d b y d e n s i t o m e t r y . T h e a v e r a g e v a l u e was 248 c o n v e n t i o n a l unit areas f o r n i n e
!3
4! 2
O
t
3 4
Fig. 1. Electrophoretic pattern of serum IgGs of patient affected with subacute sclerosing panencephalitis. 1 and 3: IgGs taken as control and dissolved in physiological saline. 2: IgGs incubated with LEC, a strain of measles-like virus. Note the marked decrease. 4: IgGs incubated with Woodfolk strain of measles virus. Note the marked decrease
54
D. Karcher et al.
determinations. After incubation of the IgGs with measles (Woodfolk) or LEC virus, it dropped to 53 (Fig. 1). Thus at least 79% of the IgGs shown by electrophoresis were precipitated when purified IgGs were incubated with virus, while there was no measurable decrease when lyophilized serum was used. The antigen antibody reaction was inhibited by the following fractions (and no decrease of the I g G concentration was measured): (i) only the fraction migrating in the ~ region after preparative electrophoresis in agar gel; (ii) the supernatant after 0.125 M sodium sulfate precipitation; (iii) the precipitate of this same supernatant after adding 2.8 M a m m o n i u m sulfate; (iv) the second, the third and the fifth fraction after elution from D E A E Sephadex A50 column with a NaC1 gradient. ~ globulins are present in all those fractions; (v) serum diluted 1 : 10 and heated at 100°C for 10min. Equal quantities (3 mg/40 2) of each fraction were added either before incubation of the antigen with its analogous antibody, or to the mixture prepared for radioimmunoassay (2 mg/ml). 2. As large amounts of purified IgGs and virus would be needed to check the effect of each fraction on the antigen antibody reaction by agar gel electrophoresis, radioimmunoassay was used to test the blocking effect on the antigen antibody
Fractions of SSPE serum supernatant (0.126M sodium sulfate precipitation) collected after passage on DEAE Sephadex A50 column IgG I t25 bound (Bo)
9021
Inhibiting effect (in %)
Fraction Fraction Fraction Fraction Fraction Fraction Fraction
5087 2700 3007 7787 3300 3879 7795
44 71 67 14 64 57 14
1 2 3 4 5 6 7
IgG I ~25bound (B~) 8208 4445 6206
Inhibiting effect (in %) 640 698 1274
92 84 80
Table 1. Inhibiting effect expressed in % decrease in counts per minute (RIA)
Table 2. Control sera heated at 100°C for 10 min
A Heat Stable Serum Inhibitor of an Antigen Antibody Reaction
55
immune reaction. When working with labelled IgGs, the supernatant after 0.126 M sodium sulfate precipitation or its precipitate formed after treatment with 2.8 M ammonium sulfate was not considered to be reliable for RIA since the presence of IgGs in the preparations, competing with labelled IgGs, may give false positive readings. The fractions collected from the column (DEAE Sephadex A50: fractions 2, 3 and 5) gave very satisfactory results (Table 1). All those fractions were located in the ~ region after agar gel electrophoresis and inhibited the antigen antibody reaction. The heated control serum also inhibited the reaction (Table 2). The inhibitions varied from 64% to 92% (Tables 1 and 2).
Discussion
Previously the CSF immunoglobulins were found to be absorbed with different strains of measles virus [2,4]: a decrease of the CSF oligoclonal IgGs was clearly observed. Samples of CSF are available in too small quantities to permit widespread investigation. Serum presents many practical advantages, but the serum antibodies from patients with SSPE with restricted antibody heterogeneity are not precipitated by measles virus. However, IgGs isolated from sera precipitated readily with different strains of measles virus. It appears that the total serum antibody precipitation is inhibited and there may be a factor which blocks the immune reaction. Swich et al. [7] described a heat stable serum factor which impairs the cell mediated immune response in SSPE sera. In all these experiments serum and IgGs from the same specimen of serum were compared. Cooperband et al. [5] have reviewed extensively the work done during the last seven years on humoral immunosuppressive factors, and have concluded that there is a non-specific peptide suppressor that may be isolated with plasma alpha globulins. This substance is probably identical with the inhibiting factor we have observed. They discuss its immunosuppressive effect in relation to transplantation, the immune response and the action of phytohemagglutinin, among other agents. They did not report inhibiting activity associated with antigen antibody binding. In the present experiments, the substance acting as blocking or inhibiting factor which prevented the antigen antibody reaction was detected in the globulin region after agar gel electrophoresis. This inhibiting factor was present in all the sera investigated from seven patients with SSPE, two with MS, two control subjects and samples of pooled sera, each collected from about 20 patients in polyclinics of local hospitals. The serum of the patients affected with SSPE have either a more potent or higher concentration of the inhibiting factor in their sera. This will be determined after purification of the inhibiting factor. The present inhibiting factor is reminiscent of the non-specific inhibitor described ten years ago by Rovnova [8]. The role, function, and underlying mechanisms of the inhibiting factor in diseases where hyperimmunization occurs during its natural history underlines the interest of this investigation. It is hoped that this investigation will shed some light on the genesis of an immune disease such as SSPE.
56
D. Karcher et al.
References 1. Horta-Barbosa, L., Hamilton, R., Wittig, B., Fuccilo, D. A., Sever, J.: Subacute sclerosing panencephalitis: isolation of suppressed measles virus from lymph node biopsies. Science 173, 840--841 (1971) 2. Lowenthal, A., Karcher, D., Bollengier, F.: M-components in Neurology. Neue Forschungsergebnisse des Hirnstoffwechsels und der Entmarkungsenzephalomyelitis, ,pp. 295--297 (R. M. Schmidt, ed.). Halle (Saale): Martin-Luther-Universit/it 1974 3. Janssens, J.: Homogene konijnen antilichamen tegen micrococcus lysodeikticus. Thesis: Faculteit der Wetenschappen, Vrije Universiteit, Brussel, 1972 4. Booe, I. Ma., Tourtellotte, W. W., Brandes, D. W.: Brain and measles specific immunoglobulin G in the cerebrospinal fluid of patients with SSPE. American Academy of Neurology 28th annual meeting. Neurology (Minneap.) 26, 377 (1976) 5. Cooperband, S. R., Nimberg, R., Schmid, K., Mannick, J. A.: Humoral immunosuppressive factors. Transpl. Proc. 3, 225--242 (1976) 6. Greenwood, F. C., Hunter, W. M., Glover, J. S.: The preparation of 131-I-labelled human growth hormone of high specific radioactivity. Biochem. J. 89, 114--123 (1963) 7. Swick, H. M., Brooks, W. H., Roszman, T. L., Caldwell, D.: A heat stable blocking factor in the plasma of patients with subacute sclerosing panencephalitis. Neurology (Minneap.) 26, 84--88 (1976) 8. Rovnova, Z. I., Kosyakov, P. N.: Inhibitors and antibodies in the course of immunization of animals with viral preparations. Acta Virol. 11, 69--77 (1967) Received January 25, 1977
