Journal of the Neurological Sciences, 1977, 33 : 13-20
13
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
I M M U N O C Y T O C H E M I C A L S T U D I E S F O R T H E L O C A L 1 S A T I O N OF MEASLES A N T I G E N S I N M U L T I P L E SCLEROSIS P L A Q U E S A N D MEASLES V I R U S - I N F E C T E D CNS TISSUE
CEDRIC S. RAINE, JOHN W. PRINEAS, RACHEL D. SHEPPARD, BORNSTEIN and MONIQUE DUBOIS-DALCQ
MURRAY B.
Departments of Pathology ( Neuropathology), Neuroscience and Neurology ( CSR, RDS, MBB), Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, N.Y. 10461; Department of Neuroscience (JWPJ, New Jersey Medical School, Newark, N.J. 07108 and Veterans Administration Hospital, East Orange, N.J. 07010; and N1NCDS (MD-D), Bethesda, Md. 20014 (U.S.A.)
(Received 7 December, 1976)
SUMMARY Actively demyelinating central nervous system (CNS) lesions from a patient with acute multiple sclerosis (MS) were tested for measles antigens using peroxidaseconjugated antimeasles antibody. No evidence of measles antigens was found. Similarly reacted tissue from 2 patients with chronic MS also revealed no evidence of measles antigens. Identically treated and simultaneously tested measles-infected CNS cultures and human SSPE brain tissue stained strongly for measles antigens. The possible reasons underlying the failure to detect measles antigens in MS are discussed.
INTRODUCTION A role for measles virus in the aetiology of multiple sclerosis (MS) is suggested by the fact that MS patients as a group exhibit higher than normal titres of measles antibodies in the serum and cerebrospinal fluid (e.g. Adams and Imagawa 1962; Brown, Cathala, Gajdusek and Gibbs 1971; Salmi, Norrby and Panelius 1972). Utilising measles antibody coupled to horseradish peroxidase (HRP) or fluorescein, previous immunocytochemical studies have failed to demonstrate measles antigens in
This work was supported in part by grant 1001-A-1 from the National Multiple Sclerosis Society; by the Kroc Foundation; by USPHS grants NS 08952, NS 11920 and NS 03366; by a grant from the Alfred P. Sloan Foundation; by the Veterans Administration and a USPHS Research Career Development Award to Dr. Raine-NS 70265. Correspondence to: Dr. Cedric S. Raine, Department of Pathology (Neuropathology), Albert Einstein College of Medicine, 1300 Morris Park Avenue, The Bronx, N.Y. 10461, U.S.A.
14 MS lesions of undetermined age (Dubois-Dalcq, Schumacher and Sever 1973 ; Tanaka, Iwasaki and Koprowski 1975). If expression of measles antigens occurs in MS lesions, it is likely that this is restricted to the active or early stages of plaque formation. In view of this, the present study was undertaken in an attempt to detect measles antigens in very early lesions obtained from the brain of a patient with acute MS. MATERIAL AND METHODS M S tissue Autopsied brain tissue, frozen within 2-12 hr of death, was obtained from 3 MS subjects. In 2, symptoms of the disease had been present for 10 years and in 1, for 3 months before death. The latter patient, a 41-year-old female, had an illness diagnosed as an endogenous depression 3 months prior to death. Two weeks before death, she developed an unsteady gait followed by rapidly progressive neurological signs which led to quadriparesis and death. Autopsy examination in this patient revealed typical MS plaques present throughout the brain and spinal cord. All of the lesions were grossly visible and appeared pink and granular. In most lesions, histologic examination revealed demyelinated zones containing intense perivascular cuffing and large numbers of lipid-laden cells. Light and electron microscope (LM and EM) examination of Epon-embedded material confirmed the presence of acute disease with ongoing demyelination present around the hypercellular margins of many of the lesions, beyond the inflammatory zones. This forms the subject of a separate report (Prineas and Raine 1976). Antibodies Two antimeasles antibody conjugates were used. One was prepared from a pool of 2 serum samples obtained 10 weeks apart from a patient with subacute sclerosing panencephalitis (SSPE). The antimeasles antibody titres in these two samples were 1:256 (HI) and 1:512 (CF) in the first specimen, and 1:1024 (HI) and 1:512 (CF) in the second specimen. The second antimeasles conjugate was prepared from a serum sample obtained from a second patient with SSPE, the antimeasles antibody titres of which were 1 : 64 (HI) and 1 : 256 (CF). IgG was purified from an ammonium sulphate precipitate from whole serum which was dialysed against a PO4 buffer and passed through a DEAE cellulose ion-exchange column. Five mg of IgG were coupled to HRP by the two-step method of Avrameas and Ternynck (1971). The subsequent conjugate was separated into aliquots and stored at --20 °C until needed. The HRPconjugated IgG was used without absorption and after absorption with normal brain tissue. Controls Measles antibody specificity was determined using: (a) fresh-frozen uninfected brain tissue obtained at autopsy from a patient with no history of neurological illness; (b) fresh-frozen human SSPE brain tissue obtained at autopsy; and (c) measles-infected (Edmonston Strain) and normal organotypic cultures of hamster cerebeltar tissue, the
15 preparative techniques of which have been described previously (Raine, Feldman, Sheppard and Bornstein 1969). The specificity of the antimeasles antibody conjugates was confirmed by blocking experiments employing pretreatment of the tissue with uncoupled antimeasles antisera from a patient with SSPE. Procedure
Incubation with the conjugates was performed using 200 #m thick cryostat sections which were allowed to thaw in PO4 buffered paraformaldehyde containing lysine and periodate (McClean and Nakane 1973) and left for 30 min at 0-4 °C before washing in phosphate buffered saline (PBS) for 12 hr on a rocker platform at 4 °C. The cultures of hamster cerebellum were similarly fixed and washed. Following washing, the tissues were incubated for 2 hr with diluted HRP-conjugated SSPE IgG and subsequently post-fixed in 2.5 ~o PO4 buffered glutaraldehyde. The tissues were then reacted in diaminobenzidine (DAB) and prepared for LM and EM by techniques similar to those described previously by Dubois-Dalcq et al. (1973) and Dubois-Dalcq, Worthington, Gutenson and Barbosa (1975). Reacted tissues were observed by LM as unosmicated, cleared thick sections or whole mounts or as postosmicated, unstained or toluidine blue-stained 1 #m Epon sections. Electron microscopy was carried out on unosmicated and osmicated preparations which were unstained or stained with lead and uranyl salts. RESULTS In organotypic, measles-infected CNS cultures and human SSPE tissue, strongly positive staining for measles antigens was apparent after treatment with both HRPlgG conjugates and DAB substrate. This reaction could be blocked by pretreatment with unconjugated SSPE antisera. Intracytoplasmic viral inclusions stained intensely in the organotypic CNS cultures, and considerable membrane staining was evident on infected cells (Figs. 1 and 2). Intranuclear staining was rare. In the same cultures, cells displaying no positive staining could also be found. EM examination showed most measles material at the surface of cultures to be heavily coated with HRP reaction product (Fig. 3) which in unstained preparations was visualised as an electron-dense precipitate around individual virions (Fig. 4). Unstained sections of the human SSPE brain tissue showed positively reacted measles inclusions in the cytoplasm of numerous cells (Fig. 5) and occasionally in nuclei. Morphologic examination of plaques from the patient with acute MS confirmed ongoing demyelination in the periplaque area and intense perivascular cuffing within the lesions. No evidence of measles antigens could be found in these same plaques (Fig. 6). Similarly, no reaction for measles antigens was detected in the lesions from the 2 cases of chronic MS.
16
Fig. 1. A light micrograph from a whole mount of a culture of hamster cerebellum infected with Ldm o n s t o n strain measles virus for 12 days. The culture was fixed, washed and then reacted with HRPlabelled antimeasles lgG followed by diaminobenzidine. A giant cell is seen (note many nuclei in centre) containing large, positively reacting intracytoplasmic viral inclusions. The m e m b r a n e of the polykaryocyte also contains viral antigen. Note the adjacent unreacted cclls. , 600. Fig. 2. A 1 ~m, toluidine blue-stained epon section of a giant cell in a measles-infected culture shows a positive reaction for measles antigens in the cytoplasm and the membranes. Nuclei show no reaction product. Twenty-one days postinfection. 1,400.
Fig. 3. An electron micrograph taken from the surface of a CNS culture infected with measles for 12 days and then reacted with the HRP-SSPE IgG conjugate shows budding viral particles at one pole of an astrocyte (nucleus at left). Each particle is heavily stained with reaction product. The section is counterstained with lead and uranyl salts, x 22,000. Fig. 4. Higher magnification of a measles-infected CNS culture reacted with HRP-SSPE IgG shows virions coated by a thick reaction product. Note the regularly-aligned, tubular, viral nucleocapsids (arrows) beneath the viral envelope. The section is not counterstained with lead or uranyl salts, x 100,000.
18
Fig. 5. Human SSPE tissue. A 1 /tin epon section photographed with Nomarski interference optics shows positively reacted intracytoplasmic viral inclusions (arrows). The tissue spaces are ice artifacts. × 800. Fig. 6. Acute MS tissue. A preparation treated identically to Fig. 5 but taken from the periphery of an. actively demyelinating lesion from acute MS shows many macrophages containing lipid deposits and no evidence of reaction product for measles antigens, x 800.
19 DISCUSSION These experiments have failed to obtain evidence of expressed measles antigens in both chronic lesions from 2 patients with typical relapsing MS and in actively demyelinating lesions from a patient with acute MS using techniques which readily demonstrated measles antigens in measles-infected CNS cultures and autopsy tissue from a patient with SSPE. In view of the suggestion from indirect evidence from a number of disciplines (see Raine, Schaumburg, Snyder and Suzuki 1975) that measles virus may play a role in MS, it is apparent that more refined immunocytochemical procedures on MS plaques will be required to reveal the presence of measles antigens if they do, in fact, occur. In the present experiments on acute MS lesions, the possibility cannot be excluded that measles determinants were masked by endogenous IgG, although this was not the case in the human SSPE tissue. Also, these findings do not preclude an infection by a defective measles virus in MS, a phenomenon which might be demonstrated by studies similar to the above but using monospecific antisera raised against individual viral components. Finally, there exists the possibility that measles virus may be only one of a number of agents capable of including MS and it is possible that another virus was involved in the particular acute MS case under study. ACKNOWLEDGEMENTS The authors thank Dr. Robert D. Terry for his constructive criticism. The excellent technical assistance of Everett Swanson, Miriam Pakingan, Howard Finch, Fred Connell and Kathy Worthington is gratefully acknowledged. We thank Mary Palumbo for secretarial assistance. The IgG used in this study was purified and conjugated in the laboratory of Dr. Dale E. McFarlin, Chief, Neuroimmunology Branch, NINCDS, Bethesda. Md. 20014. The authors are most grateful to Dr. McFarlin for his help and discussion. The authors also thank Drs. David Fuccillo and John Sever, NINCDS, for their help in the determination of the measles antibody titres.
REFERENCES Adams, J. M. and D. T. Imagawa (1964) Measles antibodies in multiple sclerosis, Proc. Soc. exp. bioL Med., 111 : 562-566. Avrameas, S. and T. Ternynck (1971) Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration, Immunochem., 8: 1175-1179. Brown, P., F. Cathala, D. C. Gajdusek and C. J. Gibbs (1971) Measles antibodies in the cerebrospinal fluid of patients with multiple sclerosis, Proc. Soc. exp. BioL Med., 137: 956-961. Dubois-Dalcq, M., G. Schumacher and J. L. Sever (1973) Acute multiple sclerosis- - Electron microscopic evidence for and against a viral agent in the plaques, Lancet, 2: 1408-1411. Dubois-Dalcq, M., K. Worthington, B. S. Gutenson and L. H. Barbosa (1975) Immunoperoxidase labeling of subacute sclerosing panencephalitis virus in hamster acute encephalitis, Lab. lnvest., 32: 518-526. McClean, I. W. and P. K. Nakane (1973) Periodate-lysine-paraformaidehydefixative: a new fixative for immunoelectron microscopy (Abstract), J. Cell BioL, 59; 209a.
20 Prineas, J. W. and C. S. Raine (1976) Electron microscopy and immunoperoxidase studies of early multiple sclerosis lesions, Neurology (Minneap.), 26 (Suppl.): 29-32. Raine, C. S., L, A. Feldman, R. D. Sheppard and M. B. Bornstein (1969) Ultrastructure of measles virus in cultures of hamster cerebellum, J. ViroL, 4:169 181. Raine, C. S., H. H. Schaumburg, D. H. Snyder and K. Suzuki (1975) lntranuclear "paramyxoviruslike" material in multiple sclerosis, adreno-leukodystrophy and Kuf's disease, J. neuroL Sc'i., 25 : 2941. Salmi, A. A., E. Norrby and M. Panelius (1972) Identification of different measles virus-specific antibodies in the serum and cerebrospinal fluid from patients with subacute sclerosing panencephalitis and multiple sclerosis, Infect. Immun., 6: 248-258. Tanaka, R., Y. Iwasaki and H. Koprowski (1975) Ultrastructural studies of perivascular cuffing cells in multiple sclerosis brain, Amer. J. Path., 8: 467474.
13
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
I M M U N O C Y T O C H E M I C A L S T U D I E S F O R T H E L O C A L 1 S A T I O N OF MEASLES A N T I G E N S I N M U L T I P L E SCLEROSIS P L A Q U E S A N D MEASLES V I R U S - I N F E C T E D CNS TISSUE
CEDRIC S. RAINE, JOHN W. PRINEAS, RACHEL D. SHEPPARD, BORNSTEIN and MONIQUE DUBOIS-DALCQ
MURRAY B.
Departments of Pathology ( Neuropathology), Neuroscience and Neurology ( CSR, RDS, MBB), Rose F. Kennedy Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Bronx, N.Y. 10461; Department of Neuroscience (JWPJ, New Jersey Medical School, Newark, N.J. 07108 and Veterans Administration Hospital, East Orange, N.J. 07010; and N1NCDS (MD-D), Bethesda, Md. 20014 (U.S.A.)
(Received 7 December, 1976)
SUMMARY Actively demyelinating central nervous system (CNS) lesions from a patient with acute multiple sclerosis (MS) were tested for measles antigens using peroxidaseconjugated antimeasles antibody. No evidence of measles antigens was found. Similarly reacted tissue from 2 patients with chronic MS also revealed no evidence of measles antigens. Identically treated and simultaneously tested measles-infected CNS cultures and human SSPE brain tissue stained strongly for measles antigens. The possible reasons underlying the failure to detect measles antigens in MS are discussed.
INTRODUCTION A role for measles virus in the aetiology of multiple sclerosis (MS) is suggested by the fact that MS patients as a group exhibit higher than normal titres of measles antibodies in the serum and cerebrospinal fluid (e.g. Adams and Imagawa 1962; Brown, Cathala, Gajdusek and Gibbs 1971; Salmi, Norrby and Panelius 1972). Utilising measles antibody coupled to horseradish peroxidase (HRP) or fluorescein, previous immunocytochemical studies have failed to demonstrate measles antigens in
This work was supported in part by grant 1001-A-1 from the National Multiple Sclerosis Society; by the Kroc Foundation; by USPHS grants NS 08952, NS 11920 and NS 03366; by a grant from the Alfred P. Sloan Foundation; by the Veterans Administration and a USPHS Research Career Development Award to Dr. Raine-NS 70265. Correspondence to: Dr. Cedric S. Raine, Department of Pathology (Neuropathology), Albert Einstein College of Medicine, 1300 Morris Park Avenue, The Bronx, N.Y. 10461, U.S.A.
14 MS lesions of undetermined age (Dubois-Dalcq, Schumacher and Sever 1973 ; Tanaka, Iwasaki and Koprowski 1975). If expression of measles antigens occurs in MS lesions, it is likely that this is restricted to the active or early stages of plaque formation. In view of this, the present study was undertaken in an attempt to detect measles antigens in very early lesions obtained from the brain of a patient with acute MS. MATERIAL AND METHODS M S tissue Autopsied brain tissue, frozen within 2-12 hr of death, was obtained from 3 MS subjects. In 2, symptoms of the disease had been present for 10 years and in 1, for 3 months before death. The latter patient, a 41-year-old female, had an illness diagnosed as an endogenous depression 3 months prior to death. Two weeks before death, she developed an unsteady gait followed by rapidly progressive neurological signs which led to quadriparesis and death. Autopsy examination in this patient revealed typical MS plaques present throughout the brain and spinal cord. All of the lesions were grossly visible and appeared pink and granular. In most lesions, histologic examination revealed demyelinated zones containing intense perivascular cuffing and large numbers of lipid-laden cells. Light and electron microscope (LM and EM) examination of Epon-embedded material confirmed the presence of acute disease with ongoing demyelination present around the hypercellular margins of many of the lesions, beyond the inflammatory zones. This forms the subject of a separate report (Prineas and Raine 1976). Antibodies Two antimeasles antibody conjugates were used. One was prepared from a pool of 2 serum samples obtained 10 weeks apart from a patient with subacute sclerosing panencephalitis (SSPE). The antimeasles antibody titres in these two samples were 1:256 (HI) and 1:512 (CF) in the first specimen, and 1:1024 (HI) and 1:512 (CF) in the second specimen. The second antimeasles conjugate was prepared from a serum sample obtained from a second patient with SSPE, the antimeasles antibody titres of which were 1 : 64 (HI) and 1 : 256 (CF). IgG was purified from an ammonium sulphate precipitate from whole serum which was dialysed against a PO4 buffer and passed through a DEAE cellulose ion-exchange column. Five mg of IgG were coupled to HRP by the two-step method of Avrameas and Ternynck (1971). The subsequent conjugate was separated into aliquots and stored at --20 °C until needed. The HRPconjugated IgG was used without absorption and after absorption with normal brain tissue. Controls Measles antibody specificity was determined using: (a) fresh-frozen uninfected brain tissue obtained at autopsy from a patient with no history of neurological illness; (b) fresh-frozen human SSPE brain tissue obtained at autopsy; and (c) measles-infected (Edmonston Strain) and normal organotypic cultures of hamster cerebeltar tissue, the
15 preparative techniques of which have been described previously (Raine, Feldman, Sheppard and Bornstein 1969). The specificity of the antimeasles antibody conjugates was confirmed by blocking experiments employing pretreatment of the tissue with uncoupled antimeasles antisera from a patient with SSPE. Procedure
Incubation with the conjugates was performed using 200 #m thick cryostat sections which were allowed to thaw in PO4 buffered paraformaldehyde containing lysine and periodate (McClean and Nakane 1973) and left for 30 min at 0-4 °C before washing in phosphate buffered saline (PBS) for 12 hr on a rocker platform at 4 °C. The cultures of hamster cerebellum were similarly fixed and washed. Following washing, the tissues were incubated for 2 hr with diluted HRP-conjugated SSPE IgG and subsequently post-fixed in 2.5 ~o PO4 buffered glutaraldehyde. The tissues were then reacted in diaminobenzidine (DAB) and prepared for LM and EM by techniques similar to those described previously by Dubois-Dalcq et al. (1973) and Dubois-Dalcq, Worthington, Gutenson and Barbosa (1975). Reacted tissues were observed by LM as unosmicated, cleared thick sections or whole mounts or as postosmicated, unstained or toluidine blue-stained 1 #m Epon sections. Electron microscopy was carried out on unosmicated and osmicated preparations which were unstained or stained with lead and uranyl salts. RESULTS In organotypic, measles-infected CNS cultures and human SSPE tissue, strongly positive staining for measles antigens was apparent after treatment with both HRPlgG conjugates and DAB substrate. This reaction could be blocked by pretreatment with unconjugated SSPE antisera. Intracytoplasmic viral inclusions stained intensely in the organotypic CNS cultures, and considerable membrane staining was evident on infected cells (Figs. 1 and 2). Intranuclear staining was rare. In the same cultures, cells displaying no positive staining could also be found. EM examination showed most measles material at the surface of cultures to be heavily coated with HRP reaction product (Fig. 3) which in unstained preparations was visualised as an electron-dense precipitate around individual virions (Fig. 4). Unstained sections of the human SSPE brain tissue showed positively reacted measles inclusions in the cytoplasm of numerous cells (Fig. 5) and occasionally in nuclei. Morphologic examination of plaques from the patient with acute MS confirmed ongoing demyelination in the periplaque area and intense perivascular cuffing within the lesions. No evidence of measles antigens could be found in these same plaques (Fig. 6). Similarly, no reaction for measles antigens was detected in the lesions from the 2 cases of chronic MS.
16
Fig. 1. A light micrograph from a whole mount of a culture of hamster cerebellum infected with Ldm o n s t o n strain measles virus for 12 days. The culture was fixed, washed and then reacted with HRPlabelled antimeasles lgG followed by diaminobenzidine. A giant cell is seen (note many nuclei in centre) containing large, positively reacting intracytoplasmic viral inclusions. The m e m b r a n e of the polykaryocyte also contains viral antigen. Note the adjacent unreacted cclls. , 600. Fig. 2. A 1 ~m, toluidine blue-stained epon section of a giant cell in a measles-infected culture shows a positive reaction for measles antigens in the cytoplasm and the membranes. Nuclei show no reaction product. Twenty-one days postinfection. 1,400.
Fig. 3. An electron micrograph taken from the surface of a CNS culture infected with measles for 12 days and then reacted with the HRP-SSPE IgG conjugate shows budding viral particles at one pole of an astrocyte (nucleus at left). Each particle is heavily stained with reaction product. The section is counterstained with lead and uranyl salts, x 22,000. Fig. 4. Higher magnification of a measles-infected CNS culture reacted with HRP-SSPE IgG shows virions coated by a thick reaction product. Note the regularly-aligned, tubular, viral nucleocapsids (arrows) beneath the viral envelope. The section is not counterstained with lead or uranyl salts, x 100,000.
18
Fig. 5. Human SSPE tissue. A 1 /tin epon section photographed with Nomarski interference optics shows positively reacted intracytoplasmic viral inclusions (arrows). The tissue spaces are ice artifacts. × 800. Fig. 6. Acute MS tissue. A preparation treated identically to Fig. 5 but taken from the periphery of an. actively demyelinating lesion from acute MS shows many macrophages containing lipid deposits and no evidence of reaction product for measles antigens, x 800.
19 DISCUSSION These experiments have failed to obtain evidence of expressed measles antigens in both chronic lesions from 2 patients with typical relapsing MS and in actively demyelinating lesions from a patient with acute MS using techniques which readily demonstrated measles antigens in measles-infected CNS cultures and autopsy tissue from a patient with SSPE. In view of the suggestion from indirect evidence from a number of disciplines (see Raine, Schaumburg, Snyder and Suzuki 1975) that measles virus may play a role in MS, it is apparent that more refined immunocytochemical procedures on MS plaques will be required to reveal the presence of measles antigens if they do, in fact, occur. In the present experiments on acute MS lesions, the possibility cannot be excluded that measles determinants were masked by endogenous IgG, although this was not the case in the human SSPE tissue. Also, these findings do not preclude an infection by a defective measles virus in MS, a phenomenon which might be demonstrated by studies similar to the above but using monospecific antisera raised against individual viral components. Finally, there exists the possibility that measles virus may be only one of a number of agents capable of including MS and it is possible that another virus was involved in the particular acute MS case under study. ACKNOWLEDGEMENTS The authors thank Dr. Robert D. Terry for his constructive criticism. The excellent technical assistance of Everett Swanson, Miriam Pakingan, Howard Finch, Fred Connell and Kathy Worthington is gratefully acknowledged. We thank Mary Palumbo for secretarial assistance. The IgG used in this study was purified and conjugated in the laboratory of Dr. Dale E. McFarlin, Chief, Neuroimmunology Branch, NINCDS, Bethesda. Md. 20014. The authors are most grateful to Dr. McFarlin for his help and discussion. The authors also thank Drs. David Fuccillo and John Sever, NINCDS, for their help in the determination of the measles antibody titres.
REFERENCES Adams, J. M. and D. T. Imagawa (1964) Measles antibodies in multiple sclerosis, Proc. Soc. exp. bioL Med., 111 : 562-566. Avrameas, S. and T. Ternynck (1971) Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration, Immunochem., 8: 1175-1179. Brown, P., F. Cathala, D. C. Gajdusek and C. J. Gibbs (1971) Measles antibodies in the cerebrospinal fluid of patients with multiple sclerosis, Proc. Soc. exp. BioL Med., 137: 956-961. Dubois-Dalcq, M., G. Schumacher and J. L. Sever (1973) Acute multiple sclerosis- - Electron microscopic evidence for and against a viral agent in the plaques, Lancet, 2: 1408-1411. Dubois-Dalcq, M., K. Worthington, B. S. Gutenson and L. H. Barbosa (1975) Immunoperoxidase labeling of subacute sclerosing panencephalitis virus in hamster acute encephalitis, Lab. lnvest., 32: 518-526. McClean, I. W. and P. K. Nakane (1973) Periodate-lysine-paraformaidehydefixative: a new fixative for immunoelectron microscopy (Abstract), J. Cell BioL, 59; 209a.
20 Prineas, J. W. and C. S. Raine (1976) Electron microscopy and immunoperoxidase studies of early multiple sclerosis lesions, Neurology (Minneap.), 26 (Suppl.): 29-32. Raine, C. S., L, A. Feldman, R. D. Sheppard and M. B. Bornstein (1969) Ultrastructure of measles virus in cultures of hamster cerebellum, J. ViroL, 4:169 181. Raine, C. S., H. H. Schaumburg, D. H. Snyder and K. Suzuki (1975) lntranuclear "paramyxoviruslike" material in multiple sclerosis, adreno-leukodystrophy and Kuf's disease, J. neuroL Sc'i., 25 : 2941. Salmi, A. A., E. Norrby and M. Panelius (1972) Identification of different measles virus-specific antibodies in the serum and cerebrospinal fluid from patients with subacute sclerosing panencephalitis and multiple sclerosis, Infect. Immun., 6: 248-258. Tanaka, R., Y. Iwasaki and H. Koprowski (1975) Ultrastructural studies of perivascular cuffing cells in multiple sclerosis brain, Amer. J. Path., 8: 467474.
