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Protein composition of white matter myelin in subacute sclerosing panencephalitis
Journal of the neurological Sciences
15
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
Protein Composition of White Matter Myelin in Subacute Sclerosing Panencephalitis P. RIEKKINEN, J. PALO, A. ARSTILA, U. K. RINNE, H. FREY, H. SAVOLAINEN AND E. KIVALO Departments of Neurology and Electron Microscopy, University of Turku, Turku, and Department of Neurology, University of Helsinki, Helsinki (Finland) (Received 20 November, 1970)
INTRODUCTION
Results of some recent studies have related subacute sclerosing panencephalitis (SS PE) to measles virus infection in the central nervous system (CNS) (Horta-Barbosa et al. 1969 ; Meulen et al. 1969). The virus may at least be partially the causative agent of the disease although a recent report suggests that in many cases of SSPE reported in the literature measles may not be the only aetiological factor (Brody and Detels 1970). Increased measles titres in serum and cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients have been described (Panelius 1969; Panelius et al. 1971) although conflicting results have also been reported. Although demyelination is characteristic of both MS and SSPE, the clinical course and age distribution of patients is different. It is therefore possible that the tissue responses underlying demyelination are different despite the fact that the results of some studies on lipid and fatty acid composition of myelin and white matter have shown similarities (Gerstl et al. 1970; Svennerholm et aL 1970). The loss of basic or encephalitogenic protein from myelin during the early steps 6f demyelination has been observed in MS (Hallpike and Adams 1970; Einstein et al. 1970; Riekkinen et al. 1970), but according to our knowledge this has not been reported for SSPE. Antibodies against encephalitogenic protein do not seem to be associated only with MS (Field and Caspary 1970; Caspary and Field 1970) andthe whole problem about autoantibodies against myelin is unresolved (Lumsden and Jennings 1970). To further clarify the nature of demyelination in SSPE we analysed the protein composition of myelin in one SSPE autopsy brain. This paper will report data obtained during this study. Moreover findings are compared in SSPE and MS samples. This study was supported by the Finnish Research Council for Medical Sciences and the Sigrid Jus61ius Foundation.
J. neurol. Sci., 1971, 14:15-20
16
P. RIEKKINENeta]. MATERIALS AND METHODS
Case report An 18-year-old boy was admitted to the Department of Neurology, University of Turku, because of suspected SSPE. Symptoms characteristic of SSPE had lasted for 4 months and the clinical findings confirmed the diagnosis. Measles titres were 1 : 256 in serum and 1 : 32 in cerebrospinal fluid. Other virus titres were normal. A colloidal precipitation test showed a marked left-sided paralytic curve in active steps (10, 9, 7, 6, 4, 3, 2, 1) and cellulose acetate electrophoresis showed an increase of 7-globulins up to 6 2 ~ of the total CSF proteins from the same sample. Also agar gel electrophoresis revealed an increase of the 7-subfractions in the cathodal area. The electroencephalogram (EEG) showed changes characteristic of SS PE. The patient's condition deteriorated very rapidly and he died 6 months after the onset of the illness. An autopsy was performed within 24 hr of death. Histological findings confirmed the clinical diagnosis. Brain tissue was taken for microscopic and biochemical analyses. It was stored at - 25 ° C until it could be analysed. Preparation of myelin White matter was dissected from the periventricular areas in which there was profound histological demyelination. Myelin was separated according to the method of Riekkinen and Rumsby (1969). The purity of the myelin was analysed using enzymic markers for evaluating contamination by other membrane fractions (acid phosphatase for lysosomes, succinyl-dehydrogenase for mitochondria, lactate dehydrogenase for cell sap, DNA and RNA for nuclei and microsomes respectively). Quantitative morphometric (electron microscopic) analysis of purified myelin fractions was performed in order to check possible contamination by other membranes. For control purposes white matter from other autopsy brains was dissected from areas corresponding with the SSPE brain. These control brains did not show any sign of demyelination on the basis of clinical and autopsy findings. One MS white matter sample from autopsy material sent to us by Prof. W. W. Tourtellotte (U.S.A.) was also analysed. This sample was taken from an area just outside an MS plaque. Myelin separation from control and MS material was performed as for the SSPE sample. Protein electrophoresis The method of Mehl and Halaris (1970) was used without modifications. The proteins were solubilized in phenol-formic acid-water (14 : 3 : 3, w/v/v) solution. The details have been published elsewhere (Riekkinen et al. 1970). Cytochrome c (Boehringer) served as a marker during the run.
RESULTS
Purity of myelin Results for marker enzymes showed that there was barely detectable contamination
from lysosomes, mitochondria, cell sap, microsomes and nuclei. Electron microscopic analysis mainly revealed compact myelin sheaths and some smaller membrane fragments, which were probably derived from disrupted myelin lamellae during the homogenization and ultracentrifugation procedures (Fig. 1).
Protein composition of myelin The system of Mehl and Halaris (1970) allows separation of all protein material from membranes at the same time. This was of special importance in the present study because the proportions of different proteins could be compared directly. The characteristic pattern of human myelin with three main fractions was observed in the control samples in addition to some minor fractions (Fig. 2). The previously reported decrease of encephalitogenic (basic) protein (BP) in the MS sample was again confirmed (Fig. 2). The encephalitogenic protein fraction of the SSPE sample was, however, identical with that of the control sample (Fig. 2). Scanning of the gels with a microdensitometer (Canalco Model E) confirmed these findings and gave findings consistent with there being two different fractions separated from the same portion of SSPE brain white matter and in several electrophocetic runs from each sample (Fig. 3). J. neurol. Sci., 1971, 14:15--20
PROTEIN COMPOSITION OF WHITE MATTER MYELIN IN
SSPE
17
Fig. 1. Electronmicrograph of purified myelin fraction. Most of the fraction is composed of fragments of myelin sheath. The contamination by other organelles is negligible, x 15,000.
As can be seen in Fig. 3 the major proteolipid protein (PP) in SSPE myelin (in the middle zone of the electrophoretic run) appeared as a double band. This phenomenon was never observed in the controls and in the MS sample. The two bands moved further apart when electrophoresis was prolonged and they could also be recorded with the scanning system. Removal of the lipids from the samples with chloroformmethanol (2:1, v/v) extraction produced no change in the pattern.
DISCUSSION
Basic or encephalitogenic protein is known to play an important role in the structural integrity of adult human myelin. It appears to be an especially vulnerable part of the membrane and is detached from the membrane in a wide variety of conditions in which the membrane is damaged e.g. by physicochemical factors, lysosomal acid hydrolases or trypsin digestion (Hallpike and Adams 1970; Einstein et al. 1970). Furthermore we have recently demonstrated, as would be expected, that the amount of encephalitogenic protein is significantly decreased in samples taken from brains obJ. neurol. Sci., 1971, 14:15-20
P. RIEKKINEN e l al.
18
MS
Normal Fig. 2. Typical electrophoretic pattern of myelin from control, SSPE and MS samples. 15-20 pg of protein was applied for each run.
MS
___J~
SSPE
iI
NORMAL Fig. 3. Scanning pattern of eleetrophoretograms. The arrow shows the direction of the run. The fast-moving band contains basic (encephalitogenic) protein (BP) in the control and SSPE specimens but in the MS sample that protein was just detectable. Note that the proteolipid protein (PP) appears as a double band,
tained at autopsy from MS patients. However, at present it is not clear why and how this selective loss of the encephalitogenic protein occurs. For this reason our present results are of considerable interest since they clearly show that extensive demyelination may occur without any significant change in the quantity or quality of encephalitogenic protein, as in the brain of a patient with SSPE, when compared to control samples. This finding also suggests that the pathogenesis of demyeliJ. neurol. Sci., 1971, 14:15-20
PROTEIN COMPOSITION OF WHITE MATTER MYELIN IN
SSPE
19
nation in MS and SSPE may be quite different, in spite of the fact that there are some similarities in the chemical changes which occur in both diseases (Gerstl et al. 1970; Svennerholm et al. 1970). Although we have so far analysed material from only 1 case of SSPE it is difficult to conceive that this difference in the protein composition of MS and SSPE myelin is due to artefacts since the electrophoretic runs of the SSPE myelin were repeated several times, always with the same results. How this difference in the mechanism of demyelination could be explained, we do not know. One possibility could be that in SSPE myelin is peeled off and phagocytized in toto either by surrounding glial cells or by haematogenous cells, as is the case with the demyelination which occurs in experimental allergic encephalomyelitis (EAE) (Raine et al. 1969; Wisniewski et al. 1969; Miiller and Meulen 1969). In MS the situation is in this regard clearly different since we and others have previously demonstrated that in spite of the increase in the acid hydrolase activity which also occurs outside the plaques, myelin is not directly phagocytosed by surrounding cells (Rinne et al. 1969; Suzuki et al. 1970). Rather it appears that in MS the digestion of myelin by heterophagic lysosomes is a secondary phenomenon which is preceded by other degradative mechanisms. Also the difference in the encephalitogenic protein content of myelin in MS and in our SSPE sample may in some way reflect differences in the course of the two diseases. ACKNOWLEDGEMENT
The authors are grateful to Prof. W. W. Tourtellotte from the University of Michigan Medical Center, Ann Arbor, Mich., for supplying a sample of MS brain. SUMMARY
Myelin was separated from a sample of Cerebral white matter obtained at autopsy from a patient with subacute sclerosing panencephalitis (SSPE), using sucrose density gradient centrifugation techniques. The purity of the myelin was confirmed by electronmicroscopic and biochemical criteria. The protein composition of the myelin was evaluated on horizontal polyacrylamide gel electrophoresis. In contrast to the myelin from a case of multiple sclerosis (MS) where the amount of encephalitogenic protein was decreased, the SS PE myelin showed a normal band representing encephalitogenic factor.
REFERENCES BRODY, J. A. AND R. DETELS (1970) Subacute sclerosing panencephalitis. A zoonosis following aberrant measles, Lancet, ii: 500-501. CASPARY, E. A. AND E. J. FIELD (1970) Europ. Neurol., In the press. EINSTEIN, E. R., K. B. DALAI. AND J. CSEJTEY (1970) Increased protease activity and changes in basic proteins and lipids in multiple sclerosis plaques, d. neuroL Sci., 11: 109-121. FIELD, E. J. AND E. A. CASPARY (1970) Lymphocyte sensitisation : an in-vitro test for Cancer?, Lancet, i: 1337-1341.
J. neurol. Sci.,1971,14:15-20
20
P. RIEKKINEN et al.
GERSTL,B., L. F. ENG, M. TAVASTJERNA,J. K. SMITHANDS. L. KRUSE(1970) Lipids and proteins in multiple sclerosis white matter, J. Neurochem., 17: 677-689. HALLPIKE, J. F. AND C. W. M. ADAMS(1970) Proteolysis and local pathogenesis of the multiple sclerosis lesion. Histochemical and biochemical studies. In: Proceedings of the 6th International Conqress ~[ Neuropathology, Paris, September, pp. 487488. HORTA-BARBOSA,L., D. A. FUCCILLOANDJ. L. SEVER(1969) Subacute sclerosing panencephalitis. Isolation of a measles virus from a brain biopsy, Nature (Lond,), 22t: 974, LUMSDEN, C. E. AND M. JENNINGS(1970) Anti-myelin antibodies in multiple sclerosis. Significance for pathogenesis. In: Proceedings of the 6th International Congress of Neuropathology, Paris, September, p. 489. MAI, K. (1969) Measles antibodies in multiple sclerosis patients and controls (biostatistical evaluation). Pathogenesis and etiology of demyelinating diseases, Int. Arch. Allergy, 36:109-113. MEnL, E. ANt) A. HALARIS(1970) Stoichiometric relation of protein components in cerebral myelin from different species, J. Neurochem., 17: 659-668. MEULEN,V. T., G. ENDERS-RUCKLE,D. MOLLERANDG. JOPPICH(1969) Immunohistological, microscopical and neurochemical studies on encephalitides, Part 3 (Subacute progressive panencephalitis. Virological and immunohistological studies), Acta neuropath. (Berl.), 12: 244-259. MOLLER, D. AND V. T. MEULEN(1969) Immunohistological, fine structural and neurochemical studies on encephalitides, Part 2 (Subacute progressive panencephalitis. Neurohistological and histochemieal studies), Acta neuropath. (Berl.), 12: 227-243. PANELIUS, M. (1969) Studies on epidemiological, clinical and etiological aspects of multiple sclerosis, Acta neurol, scand., Suppl. 39: 1-82. PANELIUS,M., U. K. RINNEAND A. A. SALMI(1971) Measles infection in multiple sclerosis. In Progress in Research and Treatment of Multiple Sclerosis, In press. RAINE,C. S., H. WISNIEWSKIANDJ. PRINEAS(1969) An ultrastructural study of experimental demyelination and remyelination, Part 2 (Chronic experimental allergic encephalomyelitis in the peripheral nervous system), Lab. Invest., 2l: 316-327. RmKKINEN, P. J. AND M. G. RUraSBY (1969) Esterase activity in purified myelin preparations from beef brain, Brain Research, 14: 772-775. RIEKKINEN, P., J. P•Lo, A. U. ARSTtLA,H. S~,VOLAINEN,U. K. RINNE, E. KIVALOA~qDH. FRE¥ (1970) Protein composition of multiple sclerosis myelin, Arch. Neurol. (Chic.), In press. SUZUKI, K., J. M. ANDREWS,J. M. WALTZ AND R. D. TERRY (1970) Ultrastructural studies of multiple sclerosis, Lab. Invest., 20: 444-454. SVENNERHOLM, L., M. HALTIA AND P. SOURANDER(1970) Chronic sclerosing panencephalitis, Part 2 (A neurochemical study), Acta neuropath. (Berl.), 14: 293-303. W ISlqmWSKI,H., J. PRINEASAriDC. S. RAIN~(1969) An ultrastructural study of experimental demyelination and remyelination, Part 1 (Acute experimental allergic encephalomyelitis in the peripheral nervous system), Lab. lnvest., 21: 105-118.
J. neurol. Sci., 1971, 14:15-20
15
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
Protein Composition of White Matter Myelin in Subacute Sclerosing Panencephalitis P. RIEKKINEN, J. PALO, A. ARSTILA, U. K. RINNE, H. FREY, H. SAVOLAINEN AND E. KIVALO Departments of Neurology and Electron Microscopy, University of Turku, Turku, and Department of Neurology, University of Helsinki, Helsinki (Finland) (Received 20 November, 1970)
INTRODUCTION
Results of some recent studies have related subacute sclerosing panencephalitis (SS PE) to measles virus infection in the central nervous system (CNS) (Horta-Barbosa et al. 1969 ; Meulen et al. 1969). The virus may at least be partially the causative agent of the disease although a recent report suggests that in many cases of SSPE reported in the literature measles may not be the only aetiological factor (Brody and Detels 1970). Increased measles titres in serum and cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients have been described (Panelius 1969; Panelius et al. 1971) although conflicting results have also been reported. Although demyelination is characteristic of both MS and SSPE, the clinical course and age distribution of patients is different. It is therefore possible that the tissue responses underlying demyelination are different despite the fact that the results of some studies on lipid and fatty acid composition of myelin and white matter have shown similarities (Gerstl et al. 1970; Svennerholm et aL 1970). The loss of basic or encephalitogenic protein from myelin during the early steps 6f demyelination has been observed in MS (Hallpike and Adams 1970; Einstein et al. 1970; Riekkinen et al. 1970), but according to our knowledge this has not been reported for SSPE. Antibodies against encephalitogenic protein do not seem to be associated only with MS (Field and Caspary 1970; Caspary and Field 1970) andthe whole problem about autoantibodies against myelin is unresolved (Lumsden and Jennings 1970). To further clarify the nature of demyelination in SSPE we analysed the protein composition of myelin in one SSPE autopsy brain. This paper will report data obtained during this study. Moreover findings are compared in SSPE and MS samples. This study was supported by the Finnish Research Council for Medical Sciences and the Sigrid Jus61ius Foundation.
J. neurol. Sci., 1971, 14:15-20
16
P. RIEKKINENeta]. MATERIALS AND METHODS
Case report An 18-year-old boy was admitted to the Department of Neurology, University of Turku, because of suspected SSPE. Symptoms characteristic of SSPE had lasted for 4 months and the clinical findings confirmed the diagnosis. Measles titres were 1 : 256 in serum and 1 : 32 in cerebrospinal fluid. Other virus titres were normal. A colloidal precipitation test showed a marked left-sided paralytic curve in active steps (10, 9, 7, 6, 4, 3, 2, 1) and cellulose acetate electrophoresis showed an increase of 7-globulins up to 6 2 ~ of the total CSF proteins from the same sample. Also agar gel electrophoresis revealed an increase of the 7-subfractions in the cathodal area. The electroencephalogram (EEG) showed changes characteristic of SS PE. The patient's condition deteriorated very rapidly and he died 6 months after the onset of the illness. An autopsy was performed within 24 hr of death. Histological findings confirmed the clinical diagnosis. Brain tissue was taken for microscopic and biochemical analyses. It was stored at - 25 ° C until it could be analysed. Preparation of myelin White matter was dissected from the periventricular areas in which there was profound histological demyelination. Myelin was separated according to the method of Riekkinen and Rumsby (1969). The purity of the myelin was analysed using enzymic markers for evaluating contamination by other membrane fractions (acid phosphatase for lysosomes, succinyl-dehydrogenase for mitochondria, lactate dehydrogenase for cell sap, DNA and RNA for nuclei and microsomes respectively). Quantitative morphometric (electron microscopic) analysis of purified myelin fractions was performed in order to check possible contamination by other membranes. For control purposes white matter from other autopsy brains was dissected from areas corresponding with the SSPE brain. These control brains did not show any sign of demyelination on the basis of clinical and autopsy findings. One MS white matter sample from autopsy material sent to us by Prof. W. W. Tourtellotte (U.S.A.) was also analysed. This sample was taken from an area just outside an MS plaque. Myelin separation from control and MS material was performed as for the SSPE sample. Protein electrophoresis The method of Mehl and Halaris (1970) was used without modifications. The proteins were solubilized in phenol-formic acid-water (14 : 3 : 3, w/v/v) solution. The details have been published elsewhere (Riekkinen et al. 1970). Cytochrome c (Boehringer) served as a marker during the run.
RESULTS
Purity of myelin Results for marker enzymes showed that there was barely detectable contamination
from lysosomes, mitochondria, cell sap, microsomes and nuclei. Electron microscopic analysis mainly revealed compact myelin sheaths and some smaller membrane fragments, which were probably derived from disrupted myelin lamellae during the homogenization and ultracentrifugation procedures (Fig. 1).
Protein composition of myelin The system of Mehl and Halaris (1970) allows separation of all protein material from membranes at the same time. This was of special importance in the present study because the proportions of different proteins could be compared directly. The characteristic pattern of human myelin with three main fractions was observed in the control samples in addition to some minor fractions (Fig. 2). The previously reported decrease of encephalitogenic (basic) protein (BP) in the MS sample was again confirmed (Fig. 2). The encephalitogenic protein fraction of the SSPE sample was, however, identical with that of the control sample (Fig. 2). Scanning of the gels with a microdensitometer (Canalco Model E) confirmed these findings and gave findings consistent with there being two different fractions separated from the same portion of SSPE brain white matter and in several electrophocetic runs from each sample (Fig. 3). J. neurol. Sci., 1971, 14:15--20
PROTEIN COMPOSITION OF WHITE MATTER MYELIN IN
SSPE
17
Fig. 1. Electronmicrograph of purified myelin fraction. Most of the fraction is composed of fragments of myelin sheath. The contamination by other organelles is negligible, x 15,000.
As can be seen in Fig. 3 the major proteolipid protein (PP) in SSPE myelin (in the middle zone of the electrophoretic run) appeared as a double band. This phenomenon was never observed in the controls and in the MS sample. The two bands moved further apart when electrophoresis was prolonged and they could also be recorded with the scanning system. Removal of the lipids from the samples with chloroformmethanol (2:1, v/v) extraction produced no change in the pattern.
DISCUSSION
Basic or encephalitogenic protein is known to play an important role in the structural integrity of adult human myelin. It appears to be an especially vulnerable part of the membrane and is detached from the membrane in a wide variety of conditions in which the membrane is damaged e.g. by physicochemical factors, lysosomal acid hydrolases or trypsin digestion (Hallpike and Adams 1970; Einstein et al. 1970). Furthermore we have recently demonstrated, as would be expected, that the amount of encephalitogenic protein is significantly decreased in samples taken from brains obJ. neurol. Sci., 1971, 14:15-20
P. RIEKKINEN e l al.
18
MS
Normal Fig. 2. Typical electrophoretic pattern of myelin from control, SSPE and MS samples. 15-20 pg of protein was applied for each run.
MS
___J~
SSPE
iI
NORMAL Fig. 3. Scanning pattern of eleetrophoretograms. The arrow shows the direction of the run. The fast-moving band contains basic (encephalitogenic) protein (BP) in the control and SSPE specimens but in the MS sample that protein was just detectable. Note that the proteolipid protein (PP) appears as a double band,
tained at autopsy from MS patients. However, at present it is not clear why and how this selective loss of the encephalitogenic protein occurs. For this reason our present results are of considerable interest since they clearly show that extensive demyelination may occur without any significant change in the quantity or quality of encephalitogenic protein, as in the brain of a patient with SSPE, when compared to control samples. This finding also suggests that the pathogenesis of demyeliJ. neurol. Sci., 1971, 14:15-20
PROTEIN COMPOSITION OF WHITE MATTER MYELIN IN
SSPE
19
nation in MS and SSPE may be quite different, in spite of the fact that there are some similarities in the chemical changes which occur in both diseases (Gerstl et al. 1970; Svennerholm et al. 1970). Although we have so far analysed material from only 1 case of SSPE it is difficult to conceive that this difference in the protein composition of MS and SSPE myelin is due to artefacts since the electrophoretic runs of the SSPE myelin were repeated several times, always with the same results. How this difference in the mechanism of demyelination could be explained, we do not know. One possibility could be that in SSPE myelin is peeled off and phagocytized in toto either by surrounding glial cells or by haematogenous cells, as is the case with the demyelination which occurs in experimental allergic encephalomyelitis (EAE) (Raine et al. 1969; Wisniewski et al. 1969; Miiller and Meulen 1969). In MS the situation is in this regard clearly different since we and others have previously demonstrated that in spite of the increase in the acid hydrolase activity which also occurs outside the plaques, myelin is not directly phagocytosed by surrounding cells (Rinne et al. 1969; Suzuki et al. 1970). Rather it appears that in MS the digestion of myelin by heterophagic lysosomes is a secondary phenomenon which is preceded by other degradative mechanisms. Also the difference in the encephalitogenic protein content of myelin in MS and in our SSPE sample may in some way reflect differences in the course of the two diseases. ACKNOWLEDGEMENT
The authors are grateful to Prof. W. W. Tourtellotte from the University of Michigan Medical Center, Ann Arbor, Mich., for supplying a sample of MS brain. SUMMARY
Myelin was separated from a sample of Cerebral white matter obtained at autopsy from a patient with subacute sclerosing panencephalitis (SSPE), using sucrose density gradient centrifugation techniques. The purity of the myelin was confirmed by electronmicroscopic and biochemical criteria. The protein composition of the myelin was evaluated on horizontal polyacrylamide gel electrophoresis. In contrast to the myelin from a case of multiple sclerosis (MS) where the amount of encephalitogenic protein was decreased, the SS PE myelin showed a normal band representing encephalitogenic factor.
REFERENCES BRODY, J. A. AND R. DETELS (1970) Subacute sclerosing panencephalitis. A zoonosis following aberrant measles, Lancet, ii: 500-501. CASPARY, E. A. AND E. J. FIELD (1970) Europ. Neurol., In the press. EINSTEIN, E. R., K. B. DALAI. AND J. CSEJTEY (1970) Increased protease activity and changes in basic proteins and lipids in multiple sclerosis plaques, d. neuroL Sci., 11: 109-121. FIELD, E. J. AND E. A. CASPARY (1970) Lymphocyte sensitisation : an in-vitro test for Cancer?, Lancet, i: 1337-1341.
J. neurol. Sci.,1971,14:15-20
20
P. RIEKKINEN et al.
GERSTL,B., L. F. ENG, M. TAVASTJERNA,J. K. SMITHANDS. L. KRUSE(1970) Lipids and proteins in multiple sclerosis white matter, J. Neurochem., 17: 677-689. HALLPIKE, J. F. AND C. W. M. ADAMS(1970) Proteolysis and local pathogenesis of the multiple sclerosis lesion. Histochemical and biochemical studies. In: Proceedings of the 6th International Conqress ~[ Neuropathology, Paris, September, pp. 487488. HORTA-BARBOSA,L., D. A. FUCCILLOANDJ. L. SEVER(1969) Subacute sclerosing panencephalitis. Isolation of a measles virus from a brain biopsy, Nature (Lond,), 22t: 974, LUMSDEN, C. E. AND M. JENNINGS(1970) Anti-myelin antibodies in multiple sclerosis. Significance for pathogenesis. In: Proceedings of the 6th International Congress of Neuropathology, Paris, September, p. 489. MAI, K. (1969) Measles antibodies in multiple sclerosis patients and controls (biostatistical evaluation). Pathogenesis and etiology of demyelinating diseases, Int. Arch. Allergy, 36:109-113. MEnL, E. ANt) A. HALARIS(1970) Stoichiometric relation of protein components in cerebral myelin from different species, J. Neurochem., 17: 659-668. MEULEN,V. T., G. ENDERS-RUCKLE,D. MOLLERANDG. JOPPICH(1969) Immunohistological, microscopical and neurochemical studies on encephalitides, Part 3 (Subacute progressive panencephalitis. Virological and immunohistological studies), Acta neuropath. (Berl.), 12: 244-259. MOLLER, D. AND V. T. MEULEN(1969) Immunohistological, fine structural and neurochemical studies on encephalitides, Part 2 (Subacute progressive panencephalitis. Neurohistological and histochemieal studies), Acta neuropath. (Berl.), 12: 227-243. PANELIUS, M. (1969) Studies on epidemiological, clinical and etiological aspects of multiple sclerosis, Acta neurol, scand., Suppl. 39: 1-82. PANELIUS,M., U. K. RINNEAND A. A. SALMI(1971) Measles infection in multiple sclerosis. In Progress in Research and Treatment of Multiple Sclerosis, In press. RAINE,C. S., H. WISNIEWSKIANDJ. PRINEAS(1969) An ultrastructural study of experimental demyelination and remyelination, Part 2 (Chronic experimental allergic encephalomyelitis in the peripheral nervous system), Lab. Invest., 2l: 316-327. RmKKINEN, P. J. AND M. G. RUraSBY (1969) Esterase activity in purified myelin preparations from beef brain, Brain Research, 14: 772-775. RIEKKINEN, P., J. P•Lo, A. U. ARSTtLA,H. S~,VOLAINEN,U. K. RINNE, E. KIVALOA~qDH. FRE¥ (1970) Protein composition of multiple sclerosis myelin, Arch. Neurol. (Chic.), In press. SUZUKI, K., J. M. ANDREWS,J. M. WALTZ AND R. D. TERRY (1970) Ultrastructural studies of multiple sclerosis, Lab. Invest., 20: 444-454. SVENNERHOLM, L., M. HALTIA AND P. SOURANDER(1970) Chronic sclerosing panencephalitis, Part 2 (A neurochemical study), Acta neuropath. (Berl.), 14: 293-303. W ISlqmWSKI,H., J. PRINEASAriDC. S. RAIN~(1969) An ultrastructural study of experimental demyelination and remyelination, Part 1 (Acute experimental allergic encephalomyelitis in the peripheral nervous system), Lab. lnvest., 21: 105-118.
J. neurol. Sci., 1971, 14:15-20