Acta Neuropathologica
Acta neuropath. (Berl.) 37, 263-265 (1977)
9 by Springer-Verlag1977
Filamentous and Multilamellated Cytoplasmic Inclusions in Progressive Multifocal Leukoencephalopathy J. J. H a u w and R. Escourolle Laboratoire de Neuropathologie Charles Foix, H6pital de la Salp~tri+re, 47 Bd de l'H6pital, F-75634Paris, Cedex 13, France
Summary. Inclusions identical to those described in multiple sclerosis (MS) brain by Prineas (1975) have been seen by ultrastructural study of cerebral tissue in two elsewhere reported cases of Progressive Multifocal Leukoencephalopathy (PML). The meaning of these formations, which have been suggested to be related to a special process of demyelination in MS, is discussed in the light of the hypotheses concerning the demyelination mechanism in PML. Whatever might be their significance, these cytoplasmic non viral inclusions are not specific for MS. Key words: Progressive multifocal leukoencephalopathy - Multiple sclerosis - Demyelination - Electron microscopy.
Recent ultrastructural studies demonstrated the presence of cytoplasmic inclusions in multiple sclerosis (MS) brain. These m e m b r a n e - b o u n d structures made of filamentous and multilamellated profiles might represent the morphological ground of a special demyelinating process (Prineas, 1975). We have observed quite similar structures in two cases of another demyelinating disease, Progressive Multifocal Leukoencephalopathy (PML).
In the second case (Castaigne et al., 1973), a cerebral biopsy was directly fixed in glutaraldehyde. The procedure for electron microscopy was similar to the one described fot the 1st case.
Results The m o r p h o l o g y of both cases was similar and will be described together. However, poorer preservation of the formalin-fixed tissue of case I was noted. The inclusions consisted of aggregates of linear and curved serpentine-like structures (Figs. 3 - 9 ) . They were approximatively 16 n m wide and could be followed o v e r distances varying from 2 0 0 - 5 0 0 nm, and, in a few occasions, up to 1 0 0 0 - 1500 nm. N o central core was seen on transverse sections of these structures. Although, on longitudinal sections, they usually consisted of indistinct linear filamentous-like profiles, occasional pentalaminar appearance was observed in case 2 (Figs. 8 and 9). These linear profiles, often grouped into 1 0 - 30 units bundles, were generally entrapped in m e m b r a n e - b o u n d inclusions. They were seldom associated with a m o r p h o u s lipid inclusions. The cells and the processes bearing these cytoplasmic inclusions were frequently difficult to classify. Although some of them were easily identified as phagocytes, others were obviously not and could belong to any type of glial cells.
Discussion Materials and Methods The clinical, biological and pathological characteristics of the two cases have been reported elsewhere. In the first case (Escourolle et al., 1973), the electron microscopic study was performed on previously formalin-fixed autopsy brain tissue. After 70 days of fixation, blocks of brain tissue were rinced in Sorensen's buffer, post-fixed in 3.5 ~ glutaraldehyde for 1 h and in 2~ OsO~ for 1 h, and embedded in epon 812. Thin sections were contrasted by uranyl acetate and lead citrate and examined with a Hitachi HU I1A electron microscope.
There is no doubt that the diagnosis of the two cases reported is PML. The clinical and pathological features of the first case are typical. These include the ultrastructural observation of elongated and round structures, sometimes grouped into paracristalline formations, the morphology of which is similar to that of Papova virions (Escourolle et al., 1973) (Fig. 1). In the second case, the initial diagnosis of PML, made on the typical findings from the biopsy (including the presence
264
Acta neuropath. (Berl.) 37 (1977)
Fig. 1 Case 1. Intranuclear round papova virions, some of them slightly out of focus. Formalin-fixed brain tissue. The scale (0.2 g) is the same for every figures
Fig.2 Case 2. Intranuclear round and elongated particles. Cerebral biopsy Fig. 3
Case 1. Intracytoplasmic non viral filamentous serpentine-like structures. The disrupted membrane binding the inclusion is seen (arrows) Fig. 4
Case 1. Another inclusion at higher magnification Fig. 5
Case 2. Low magnification of a cytoplasmic membrane-bound cytoplasmic inclusion Fig. 6
Case 2. Another inclusion made of clearly distinct linear profiles Fig. 7
Case 2. Enlargement of a part of the Figure 6. No distinct laminated appearance of the linear profiles is seen at this higher magnification Fig.8
Case 2. A cytoplasmic membrane-bound inclusion contains various serpentine-like profiles, some of them exhibit a pentalaminar structures (arrow), best seen in Figure 9 Fig.9
Case 2. Enlargement of a part of Figure 9. Arrows: pentalaminar appearance of the serpentine-like profiles
o f r o u n d and elongated virus-like particles by ultrastructural study; Fig.2) was confirmed at autopsy. F u r t h e r m o r e , a p a p o v a virus was isolated f r o m cultivated embryonic h u m a n brain cells inoculated by the biopsy specimen (Castaigne et al., 1973, C a t h a l a et al., 1973). It was typed as a JC virus by N a r a y a n et al. (1974). Inclusions such as those described in this report have never been described in P M L . T h e y are identical to the "filamentous serpentine-like inclusions" observed by Prineas (1975) in glutaraldehyde fixed tissue o f early a u t o p s y M S brain. A l t h o u g h Prineas (1975) does n o t describe the pentalaminar appearance that
we have occasionally seen in the biopsy specimen, Suzuki et al. (1969) have reported intracytoplasmic aggregates o f lamellated structures with a periodicity averaging 6 0 - 7 5 A in M S brain biopsies. The significance o f these n o n viral m e m b r a n e - b o u n d filamentous inclusions seen b o t h in P M L and in MS is poorly understood. Prineas (1975) suggest they are likely to be m e t a c h r o m a t i c lipids. H e thinks that they m a y be related to a peculiar process o f demyelination. H e states that "the m e c h a n i s m o f demyelination in multiple sclerosis m a y be an unusual one that involves a progressive reduction in the n u m b e r o f myelin
J. J. Hauw and R. Escourolle: Cytoplasmic Inclusions in Progressive Multifocal Leukoencephalopathy
lamellae around nerve fibers in the vicinity of cells of uncertain origin that contain filamentous and multilamellated cytoplasmic inclusions unlike the usual pleiomorphic inclusions seen in myelin phagocytes". Until recently, the demyelination in PML was assumed to be directly related to the obvious oligodendroglial viral lesions characteristics of this disease (zu Rhein, 1969). Recently, Chou et al. (1975) have proposed a more complicated process involving the production of basic protein induced by the papova infection and the immune response of lymphocytes already sensitized to the same basic protein in patients with malignant and chronic disease. It should be noted that, in case 2, immunological investigations showed an immunodepression for both the humoral and cell-mediated immunities and, in contrast, an hyperactivity versus central nervous system antigen (Castaigne et al., 1973). In conclusion, whatever might be their significance, these filamentous and multilamellated cytoplasmic inclusions are not specific for multiple sclerosis since they are present in PML. Acknowledgements. We thank Dr. B. Berger for helpful discussions and Dr. C. de Baecque for correction of the English manuscript. Misses M. Asselineau and M. Tong skilful technical assistance and typewriting by Mrs. Etchebehere are acknowledged. This research was supported by grants from the Fondation pour la Recherche M6dicale Fran9aise and the Association pour la Recherche sur la Scl6rose en Plaques.
265
References Castaigne, P., Escourolle, R., Derouesne, C., Cathala, F., Hauw, J. J., Duclos, H., Goust, J. M. : Un cas de leucoenc6phalopathie mnltifoeale progressive. Etude anatomo-clinique et immunologique. Isolement/t partir d'une biopsie c6r6brale d'une nouvelle souche de virus Papova. Rev. neurol. 128, 85-94 (1973) Cathala, F., Hauw, J.J., Escourolle, R.: Isolement et 6tude ultrastructurale d'un virus de type Papova non neutralis6 par le s6rum de r&f6rence anti-SV 40 au cours de la leucoenc~phalopathie multifocale progressive. C. R. Acad. Sci. (Paris) (D) 276, 1081 -- 1084 (1973). Chou, S. M., Oguchi, K., Holden, E. M., Tarsy, D.: Unusual ultrastructural findings in progressive multifocal leukoencephalopathy. J. Neuropath. exp. Neurol. 34, 8 1 - 82 (1975) Escourolle, R., Hauw, J. J., Signoret, J. L., Lhermitte, F.: Leucoenc+phalopathie multifocale progressive au cours d'une tuberculose ganglionnaire. Nouv. Presse M6d. 2, 1277-1281 (1973) Narayan, O., Penney, J. B., Johnson, R. T., Herndon, R. M., Weiher, L. P.: Etiology of progressive multifocal leukoencephalopathy. Identification of Papova-virus. New Engl. J. Med. 289, 1278-1282 (1973) Prineas, J. : Pathology of the early lesion in multiple sclerosis. Human Path. 6, 531-554 (1975) Suzuki, K., Andrews, J. M., Waltz, J. M., Terry, R . D . : Ultrastructural studies of multiple sclerosis. Lab. Invest. 20, 444-454 (1969) Zu Rhein, G. M.: Association of papova-virions with a human demyelinating disease (progressive multifocal leukoencephalopathy). Progr. reed. Virol. II, 185-247 (1969)
Received October 18, 1976/Accepted October 22, 1976
Acta neuropath. (Berl.) 37, 263-265 (1977)
9 by Springer-Verlag1977
Filamentous and Multilamellated Cytoplasmic Inclusions in Progressive Multifocal Leukoencephalopathy J. J. H a u w and R. Escourolle Laboratoire de Neuropathologie Charles Foix, H6pital de la Salp~tri+re, 47 Bd de l'H6pital, F-75634Paris, Cedex 13, France
Summary. Inclusions identical to those described in multiple sclerosis (MS) brain by Prineas (1975) have been seen by ultrastructural study of cerebral tissue in two elsewhere reported cases of Progressive Multifocal Leukoencephalopathy (PML). The meaning of these formations, which have been suggested to be related to a special process of demyelination in MS, is discussed in the light of the hypotheses concerning the demyelination mechanism in PML. Whatever might be their significance, these cytoplasmic non viral inclusions are not specific for MS. Key words: Progressive multifocal leukoencephalopathy - Multiple sclerosis - Demyelination - Electron microscopy.
Recent ultrastructural studies demonstrated the presence of cytoplasmic inclusions in multiple sclerosis (MS) brain. These m e m b r a n e - b o u n d structures made of filamentous and multilamellated profiles might represent the morphological ground of a special demyelinating process (Prineas, 1975). We have observed quite similar structures in two cases of another demyelinating disease, Progressive Multifocal Leukoencephalopathy (PML).
In the second case (Castaigne et al., 1973), a cerebral biopsy was directly fixed in glutaraldehyde. The procedure for electron microscopy was similar to the one described fot the 1st case.
Results The m o r p h o l o g y of both cases was similar and will be described together. However, poorer preservation of the formalin-fixed tissue of case I was noted. The inclusions consisted of aggregates of linear and curved serpentine-like structures (Figs. 3 - 9 ) . They were approximatively 16 n m wide and could be followed o v e r distances varying from 2 0 0 - 5 0 0 nm, and, in a few occasions, up to 1 0 0 0 - 1500 nm. N o central core was seen on transverse sections of these structures. Although, on longitudinal sections, they usually consisted of indistinct linear filamentous-like profiles, occasional pentalaminar appearance was observed in case 2 (Figs. 8 and 9). These linear profiles, often grouped into 1 0 - 30 units bundles, were generally entrapped in m e m b r a n e - b o u n d inclusions. They were seldom associated with a m o r p h o u s lipid inclusions. The cells and the processes bearing these cytoplasmic inclusions were frequently difficult to classify. Although some of them were easily identified as phagocytes, others were obviously not and could belong to any type of glial cells.
Discussion Materials and Methods The clinical, biological and pathological characteristics of the two cases have been reported elsewhere. In the first case (Escourolle et al., 1973), the electron microscopic study was performed on previously formalin-fixed autopsy brain tissue. After 70 days of fixation, blocks of brain tissue were rinced in Sorensen's buffer, post-fixed in 3.5 ~ glutaraldehyde for 1 h and in 2~ OsO~ for 1 h, and embedded in epon 812. Thin sections were contrasted by uranyl acetate and lead citrate and examined with a Hitachi HU I1A electron microscope.
There is no doubt that the diagnosis of the two cases reported is PML. The clinical and pathological features of the first case are typical. These include the ultrastructural observation of elongated and round structures, sometimes grouped into paracristalline formations, the morphology of which is similar to that of Papova virions (Escourolle et al., 1973) (Fig. 1). In the second case, the initial diagnosis of PML, made on the typical findings from the biopsy (including the presence
264
Acta neuropath. (Berl.) 37 (1977)
Fig. 1 Case 1. Intranuclear round papova virions, some of them slightly out of focus. Formalin-fixed brain tissue. The scale (0.2 g) is the same for every figures
Fig.2 Case 2. Intranuclear round and elongated particles. Cerebral biopsy Fig. 3
Case 1. Intracytoplasmic non viral filamentous serpentine-like structures. The disrupted membrane binding the inclusion is seen (arrows) Fig. 4
Case 1. Another inclusion at higher magnification Fig. 5
Case 2. Low magnification of a cytoplasmic membrane-bound cytoplasmic inclusion Fig. 6
Case 2. Another inclusion made of clearly distinct linear profiles Fig. 7
Case 2. Enlargement of a part of the Figure 6. No distinct laminated appearance of the linear profiles is seen at this higher magnification Fig.8
Case 2. A cytoplasmic membrane-bound inclusion contains various serpentine-like profiles, some of them exhibit a pentalaminar structures (arrow), best seen in Figure 9 Fig.9
Case 2. Enlargement of a part of Figure 9. Arrows: pentalaminar appearance of the serpentine-like profiles
o f r o u n d and elongated virus-like particles by ultrastructural study; Fig.2) was confirmed at autopsy. F u r t h e r m o r e , a p a p o v a virus was isolated f r o m cultivated embryonic h u m a n brain cells inoculated by the biopsy specimen (Castaigne et al., 1973, C a t h a l a et al., 1973). It was typed as a JC virus by N a r a y a n et al. (1974). Inclusions such as those described in this report have never been described in P M L . T h e y are identical to the "filamentous serpentine-like inclusions" observed by Prineas (1975) in glutaraldehyde fixed tissue o f early a u t o p s y M S brain. A l t h o u g h Prineas (1975) does n o t describe the pentalaminar appearance that
we have occasionally seen in the biopsy specimen, Suzuki et al. (1969) have reported intracytoplasmic aggregates o f lamellated structures with a periodicity averaging 6 0 - 7 5 A in M S brain biopsies. The significance o f these n o n viral m e m b r a n e - b o u n d filamentous inclusions seen b o t h in P M L and in MS is poorly understood. Prineas (1975) suggest they are likely to be m e t a c h r o m a t i c lipids. H e thinks that they m a y be related to a peculiar process o f demyelination. H e states that "the m e c h a n i s m o f demyelination in multiple sclerosis m a y be an unusual one that involves a progressive reduction in the n u m b e r o f myelin
J. J. Hauw and R. Escourolle: Cytoplasmic Inclusions in Progressive Multifocal Leukoencephalopathy
lamellae around nerve fibers in the vicinity of cells of uncertain origin that contain filamentous and multilamellated cytoplasmic inclusions unlike the usual pleiomorphic inclusions seen in myelin phagocytes". Until recently, the demyelination in PML was assumed to be directly related to the obvious oligodendroglial viral lesions characteristics of this disease (zu Rhein, 1969). Recently, Chou et al. (1975) have proposed a more complicated process involving the production of basic protein induced by the papova infection and the immune response of lymphocytes already sensitized to the same basic protein in patients with malignant and chronic disease. It should be noted that, in case 2, immunological investigations showed an immunodepression for both the humoral and cell-mediated immunities and, in contrast, an hyperactivity versus central nervous system antigen (Castaigne et al., 1973). In conclusion, whatever might be their significance, these filamentous and multilamellated cytoplasmic inclusions are not specific for multiple sclerosis since they are present in PML. Acknowledgements. We thank Dr. B. Berger for helpful discussions and Dr. C. de Baecque for correction of the English manuscript. Misses M. Asselineau and M. Tong skilful technical assistance and typewriting by Mrs. Etchebehere are acknowledged. This research was supported by grants from the Fondation pour la Recherche M6dicale Fran9aise and the Association pour la Recherche sur la Scl6rose en Plaques.
265
References Castaigne, P., Escourolle, R., Derouesne, C., Cathala, F., Hauw, J. J., Duclos, H., Goust, J. M. : Un cas de leucoenc6phalopathie mnltifoeale progressive. Etude anatomo-clinique et immunologique. Isolement/t partir d'une biopsie c6r6brale d'une nouvelle souche de virus Papova. Rev. neurol. 128, 85-94 (1973) Cathala, F., Hauw, J.J., Escourolle, R.: Isolement et 6tude ultrastructurale d'un virus de type Papova non neutralis6 par le s6rum de r&f6rence anti-SV 40 au cours de la leucoenc~phalopathie multifocale progressive. C. R. Acad. Sci. (Paris) (D) 276, 1081 -- 1084 (1973). Chou, S. M., Oguchi, K., Holden, E. M., Tarsy, D.: Unusual ultrastructural findings in progressive multifocal leukoencephalopathy. J. Neuropath. exp. Neurol. 34, 8 1 - 82 (1975) Escourolle, R., Hauw, J. J., Signoret, J. L., Lhermitte, F.: Leucoenc+phalopathie multifocale progressive au cours d'une tuberculose ganglionnaire. Nouv. Presse M6d. 2, 1277-1281 (1973) Narayan, O., Penney, J. B., Johnson, R. T., Herndon, R. M., Weiher, L. P.: Etiology of progressive multifocal leukoencephalopathy. Identification of Papova-virus. New Engl. J. Med. 289, 1278-1282 (1973) Prineas, J. : Pathology of the early lesion in multiple sclerosis. Human Path. 6, 531-554 (1975) Suzuki, K., Andrews, J. M., Waltz, J. M., Terry, R . D . : Ultrastructural studies of multiple sclerosis. Lab. Invest. 20, 444-454 (1969) Zu Rhein, G. M.: Association of papova-virions with a human demyelinating disease (progressive multifocal leukoencephalopathy). Progr. reed. Virol. II, 185-247 (1969)
Received October 18, 1976/Accepted October 22, 1976
