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Distribution of papovavirus myelin-associated glycoprotein and myelin basic protein in progressive multifocal leukoencephalopathy lesions.

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Distribution of Papovavirus,
Myelin-Associated Glycoprotein,
and Myelin Basic Protein in Progressive
Mdtkocal Ieukoencephalopathy Lesions
Yasuto Itoyama, MD," Henry deF. Webster, M D , ' Nancy H. Sternberger, PhD," Edward P. Richardson, Jr, MD,:
Duard L. Walker, MD,$ Richard H. Quarles, PhD,? and Billie L. Padgett, PhD$
To study how viruses interact with oligodendroglia and produce demyelination, we immunostained paraffin and
epon sections of lesions from patients with progressive multifocal leukoencephalopathy (PML) with antisera to
papovaviruses, oligodendroglial myelin-associated glycoprotein (MAG), and myelin basic protein (MBP) according
to the peroxidase-antiperoxidasemethod. In paraffin sections from a rapidly progressive case of PML, hyperimmune
JC virus antiserum stained single oligodendroglia which were located in white matter that appeared normal histologically and stained normally with MAG and MBP antisera. In zones surrounding areas of demyelination, viruscontaining oligodendroglia were most numerous and MAG staining of periaxonal regions was decreased, but there
was little change in MBP staining. In demyelinated regions, both MAG and MBP staining were severely altered;
also there was much less JC virus staining. In tissue from three other chronic cases, viral antiserum stained fewer
oligodendrocytes and the differences in MAG and MBP staining were much less striking. In epon sections from two
biopsies of central nervous system tissue, we studied the electron microscopic appearance of oligodendroglia that
also had been stained by JC virus antiserum. Virions were present in all nuclei and in some cytoplasmic regions. The
results suggest that changes in MAG distribution are useful indicators of early oligodendroglial abnormalities
which can cause myelin breakdown.
Itoyama Y, Webster HdeF, Sternberger NH, Richardson EP Jr, Walker DL, Quarles RH, Padgett BL:
Distribution of papovavirus, rnyelin-associated glycoprotein, and myelin basic protein in progressive
rnultifocal leukoencephalopathy lesions. Ann Neuroi 11:396-407, 1082
Progressive multifocal leukoencephalopathy (PML),
a central nervous system (CNS) papovavirus infection [18, 25, 30, 34, 37, 381, is characterized by multiple foci of demyelination in patients with defective
immune responses [ l , 21-23, 391. A t the margins of
lesions where there is active breakdown of myelin
sheaths, oligodendroglia contain virus particies [ 15,
25,37, 381 and are morphologically abnormal [I, 231.
Since PML was first described [ 11, numerous investigations have shown that this progressive CNS demyelinative process is caused by virally induced
changes in oligodendroglia [371.
In this immunochemical study of PML lesions, we
compared the distribution of papovavirus, myelin
basic protein (MBP), and myelin-associated glycoprotein (MAG). Papovavirus was found mainly in
oligodendrocytes, and, as expected, the distribution
of M B P and myelin corresponded closely [ 8 ] . MAG,
which was described by Quarles, Everly, and Brady
[19, 201, is a constituent of oligodendroglia [lo, 27,
331. It is localized in periaxonal regions of mature
myelin sheaths [lo, 271, and its distribution is altered
in histologically normal white matter that surrounds
multiple sclerosis lesions [lo]. H e r e we show that
normal-appearing white matter around PML lesions
contains many oligodendroglia infected with papovavirus and that it stains abnormally with M A G
antiserum. T h e findings suggest that changes in M A G
distribution are useful indicators of early oligodendroglial abnormalities that can cause myelin breakdown. A summary of our results has been presented
and published [ 1I].
From the "Laboratory of Neuropathology and Neuroanatomical
Sciences and the +Developmental and Metabolic Neurology
Branch' NrNCDS' Natlonal 'nstitutes
Bethesda, MD
20205, the $Charles S. Kubik Laboratory of Neuropathology,
Massachusetts General Hospital, Boston, MA 021 14, and the
$Department of Medical Microbiology, University of Wisconsin
Medical School, Madison, WI 53706.
Received June 9, 1981, and in revised form July 23. Accepted for
publication July 26, 198 1.
Address reprint requests to Dr Webster, National Institutes of
Health, Bldg 36, Bcthesda, MD 20205,
396 0364-5134/82/040396-12XO1.25 @ 1981 by the American Neurological Association
Patient Data
Patient No., Age (yr), and Sex
PML Duration
Associated Disease
1. 58, M
2. 56, F
3. 13, F
4. 61, M
Chronic lymphocytic leukemia (2 1 yr)
Histiocytic lymphoma ( 5 yr)
Chronic lymphatic leukemia ( 5 yr)
Chronic lymphatic leukemia (14 mo)
Material a n d Methods
Human Tissue
We studied C N S tissue from four patients with PML who
were 56 to 7 3 years of age at the time of autopsy (Table).
The clinical and pathological findings in three of them have
been reported (Patient 1 in [24];Patient 3, as Case 2 , in [l];
and Patient 4 in [35]).The interval from death to autopsy
was less than 24 hours, and the brains were processed for
paraffin sectioning according to routine procedures that
have been described [8].Sections 6 p m thick were cut and
mounted serially on numbered slides so that the distribution of papovavirus, MAG, and MBP could be compared in
adjacent serial sections. To relate their distribution to the
histopathological findings, we also studied sections stained
with hematoxylin and eosin, cresyl violet, the Heidenhain
and Luxol fast blue methods for myelin, and the Bodian
method for axons. C N S tissue from patients who had no
clinical or pathological evidence of neurological disease was
processed in the same way; these serially mounted sections
served as controls.
In Patients 1 and 2 , cerebral biopsies were obtained two
weeks before death. Parts of these tissue samples were
fixed in glutaraldehyde, postfixed in osmium tetroxide, dehydrated in ethanol, and embedded in epon. Alternating
semithin (1 p m ) and thin sections were cut so that we could
compare the distribution of immunostained virus, MAG,
and MBP in oligodendrocytes and nearby myelinated fibers
with the fine structure of the same cells and fibers in adjacent thin sections.
Immunostaining Procedure
W e used our modification [8] of the peroxidase-antiperoxidase method [26] with rabbit antisera to papovaviruses, to human glial cells, and to M A G [27] and goat
antiserum to MBP [ 8 ] for our immunostaining experiments. Briefly, after the paraffin sections had been immersed in xylene, a xylene-alcohol mixture, absolute alcohol, and 95% alcohol, they were stored in 0.5 M Tris
buffer at 4°C. Then the sections were covered by the following Tris-buffered solutions: (1) normal sheep (rabbit)
serum, ( 2 ) rabbit (goat) antiserum to the antigen being
studied, (3) sheep antirabbit (rabbit antigoat) IgG, ( 4 ) rabbit (goat) peroxidase-antiperoxidase, and ( 5 ) 3,3'-diaminobenzidine hydrochloride and hydrogen peroxide. Some
sections were counterstained with Luxol fast blue or hematoxylin. The final steps included immersion in osmium tetroxide, dehydration, and addition of coverslips.
Semithin (1 p m ) epon sections of the cerebral biopsies
were treated with sodium ethoxide and hydrogen peroxide
[ 2 , 291 to remove the plastic and etch the tissue. Then they
were immunostained with the described antisera according
to the peroxidase-antiperoxidase method.
Two types of antisera to papovavirus were used in these
experiments. Hyperimmune antiserum to JC virus was
raised in rabbits by injecting JC virus in Freunds complete
adjuvant subcutaneously and into hind foot pads, and
boosting three weeks later with an intraperitoneal injection
of virus suspended in saline 171. Serum collected one and
two weeks after the intraperitoneal injection had a
hemagglutination-inhibition titer of 1 : 1,024,000 against 8
hemagglutination units of JC virus, and it reacted strongly
against JC virus, BK virus, and simian virus 40 (SV40) in
immunofluorescence tests. Because JC virus used for antiserum production was grown in human fetal brain cells,
control serum was produced by immunizing rabbits with an
extract of cultured human fetal brain cells using the same
immunization schedule as that employed for hyperimmune
JC virus antiserum.
Monospecific antisera against SV40, JC, and BK viruses
were raised in rabbits by giving one intravenous injection of virus and collecting serum 10 days later [7]. Sera
were selected that lacked cross-reacting antibody by
hemagglutination-inhibition and indirect immunofluorescence tests. All rabbit sera were absorbed with human fetal
brain cells to minimize nonspecific reactions.
Antiserum against JC virus T antigen consisted of a pool
of sera from hamsters bearing tumors induced by JC virus.
Production and specificities of the rabbit antiserum to
M A G [27] and the goat antiserum to MBP [ 8 ] have been
described. As controls for staining specificity, sections were
incubated in step 2 with either serum collected from the
rabbit (or goat) prior to immunization o r absorbed antiserum in which the specific antibodies had been removed
by precipitation with purified M A G (or MBP) in amounts
sufficient to eliminate all anti-MAG (or anti-MBP) reactivity detectable by radioimmunoassay.
Papovavirus Localization
In the paraffin-embedded material, hyperimmune JC
virus antiserum stained many more cells in sections
from Patient 1 than in sections from the other three
patients. In all cases, most of the immunostained cells
were oligodendroglia. In histologically normal areas
of white or gray matter that were far removed from
demyelinated regions, staining was limited to nuclei
of single, randomly scattered oligodendrocytes (Fig
1). Although most of them were located in white
matter, there also were a few in deeper cortical
ltoyama et al: Papovavirus, MAG, and B P in PML Lesions
398 Annals of Neurology Vol 11 No 4 April 1982
layers, either among other glia or next to neurons
(Fig 1). We did not observe a relationship between
blood vessels and these single stained cells; the latter
appeared to be distributed randomly.
In transition zones between histologically normal
white matter and outer margins of demyelinated
areas, the density of stained oligodendroglia increased (described more fully later). Stained
oligodendroglia were found in clusters, and staining
often extended from the nucleus into cytoplasm and
processes (see Fig 1). Oligodendroglial nuclei that
were immunostained were larger, and when staining
also was present in the cytoplasm, oligodendroglial
swelling was much more severe (see Fig 1).
In outer margins of demyelinated regions, there
were fewer stained oligodendrocytes and many of the
irregular fragments of immunostained material probably were remnants of necrotic infected cells. These
areas also contained astrocytes and macrophages with
very densely stained nuclei and punctate cytoplasmic
In central regions of demyelinated zones, relatively
few immunostained cells were seen, and they were
either macrophages or astrocytes. No oligodendroglia or myelin sheaths were found in these areas.
In tests of staining specificity, adjacent paraffin
sections were treated with preimmune serum instead
of hyperimmune antiserum. In these sections, no
staining was observed (see Fig 1).
We used epon-embedded tissue to study the electron microscopic appearance of immunostained cells
that had been identified in an adjacent semithin section. Virtually all cells with immunostaining re-
F i g I. The sections shown in A through D are micrographs of
paraffin sections from Patient 1 , immunostained with 1 :SO0
hyperimmuneJ C virus antiserum and counterstained with
hematoxylin. An area between cortex and white matter is
shown in A . An enlarged oligodendrocyte nucleus (arrow) is
densely stained; there is no staining of neurons (n), astrocytes
(a), or endothelial cells (e). I n B, a densely stained swollen
nucleus (arrow) belongs to a neuronal satellite oligodendrocyte.
In the white matter shown in C there are three interfascicular
oligodendroglia (arrows) with densely stained nuclei and
cytoplasm. Immunostained remnants af a necrotic oligodendrocyte are present in the right lower corner. (A-C, X1,OOO.) The
same oligodendrocytes (arrows) are shown stained by hyperimmune JC virus antiserum (D) and unstained ( E ) when the
adjacent section was treated with preimmune serum. The section in E, like those in A through D, also was counterstained
with hematoxylin. (D, E, X500.) In the semithin epon section
of the biopsy specimen taken from Patient 2 ( F ) , a swollen
oligodendrocyte nucleus is densely stained by 1 :500 J C virus
antiserum. (X2,OOO.) An electron micrograph of the same
nucleus is shown in G. Numerous virions (") with the characteristic appearance of papovavirus are present. ( X 19,000.)
stricted to their nuclei were oligodendrocytes (see
Fig 1). These nuclei, as well as other immunostained
cells and extracellular material, all contained numerous virions identical in appearance to those previously described in PML (see Fig 1) [15, 371.
When paraffin and epon sections of PML lesions
were treated with monospecific antisera to JC, SV40,
and BK viruses at concentrations from 1 : 1 to 1:50,
no staining was observed. We also studied cultured
glial cells that had been infected with JC, SV40, or
BK virus. They were fixed in formalin, processed for
paraffin sectioning, and treated with the same concentrations of the described monospecific antisera.
Whether ethanol was used for dehydration or not, no
staining was observed. Finally, monospecific antiserum to the T antigen did not stain cells in sections
of PML lesions or in formalin-fixed glial cultures that
had been infected with JC, SV40, or BK virus.
BP and M A G lmmunostaining
When serial sections stained with MBP antiserum,
Lux01 fast blue, or according to the Heidenhain
method were compared, the lesions corresponded
closely in contour and size. In central areas of lesions,
MBP staining was absent or was found only as scattered dots in macrophages or astrocytes; no myelin
sheaths were present (Fig 2). More peripherally, the
intensity and distribution of MBP and myelin sheath
staining were similar except at the margins of lesions.
These areas contained many degenerating sheaths
that stained more intensely with B P antiserum than
those in normal-appearing white matter (see Fig 2)
In contrast to the distribution of MBP, areas of decreased MAG staining were much larger (Figs 2, 3).
As in multiple sclerosis [lo], the decrease in MAG
immunostaining extended beyond the margins of
demyelinated areas into regions that stained normally
with MBP antiserum and contained myelin sheaths
that appeared normal (see Figs 2, 3). Also, in lesions
from Patient 1, which were judged to be more acute
from their microscopic appearance, the difference in
the areas of decreased MAG and MBP staining was
much greater (see Figs 2, 3).
Correlation of M A G , BP, and Papovavirus Localization
In order to compare the distributions of papovavirus, MAG, and MBP, we studied acute lesions in Patient l and more chronic ones in Patient 4. First,
using our serial sections, we traced the PML lesions
detected histologically, the perimeters of B P and
MA G immunostaining, and the location of cells immunostained by hyperimmune JC virus antiserum
(Fig 4 ) . In and around each histological lesion it was
easy to identify four zones (Fig 4). In zone 1, the
white matter appeared normal histologically and
Itoyama et al: Papovavirus, MAG, and B P in PML Lesions
Fig 2. Serial parafjn sections from Patient 4, immunostained
with 2 3 0 0 MBP antiserum (A and C), 1 :250 MAG antiserum (B and D), and 1 :500 J C virus antiserum (E). At
low magnification (A), MBP staining is decreased or absent in
demyelinated areas; more intense staining is present at the
margins. The areas of decreased M A G staining are much
400 AnnaIs of NeuroIogy Vol 11 No 4 April 1982
larger (B). (A, B , ~ 2 6 . Sections
from the same area at the
edge of a demyelinated zone are shown at higher magnification
in C , D , and E. The area containing sheaths normally stained
by MBP antiserum (C) stains abnormally when treated with
MAG antiserum (D);it also contains oligodendroglia immunostained by JC virus antiseram ( E l . (C-E, X220.)
Fig 3. Serial paraffin sections from Patient I , immunostained
b y I :500BP antiserum (A)and I :250 MAG antiserum
(B). In this more acute case, decreased MBP staining is present
in areas of demyelination (A).Surrounding these demyrlinated
zones, MAG staining is decreased in large areas of white matter that stain normally with MBP antiserum (B).The distribution of virally infected cells in the outlined area is illustrated in Figure 5 . ( ~ 2 1 . )
stained normally with MAG and MBP antisera. In
zone 2, MAG immunostaining was decreased but
MBP staining was normal. Zone 3, the area of active
myelin breakdown, was at the edge of histologically
detected demyelination. It also stained less intensely
with MAG antiserum (lightly stippled zone in Fig 4).
Because there was histological evidence of active
myelin breakdown, the MBP staining often was increased [8, 101. If demyelination was much more
chronic, this zone was narrower and more sharply
demarcated. Thus, the transition from normal to decreased BP staining was in zone 3. In zone 4, histological stains showed severe or complete demyelination. Both MAG and MBP staining were greatly
decreased or absent.
To supplement the tracings and assess the distribution of JC virus-infected cells more quantita-
tively, we selected three representative areas around
the circumference of an acute PML lesion from Patient l . These areas were similar to the one illustrated
in Figure 5 and included the four zones just described. In each area, we used a 50x objective and an
ocular grid to count the total number of glial cells,
the number of immunostained oligodendroglia, and
the number of macrophages; we also calculated the
percentage of glial cells that were infected oligodendrocytes (Fig 6). As might be expected, zone 1 (normal white matter) had the highest glial cell count, the
lowest number and percentage of infected oligodendroglia, and no macrophages. But in zone 2 (decreased MAG), the number of infected oligodendroglia was highest (15% of glial cells) and the total
number of glial cells present was only slightly decreased. In zone 3 (transition from normal to abnormal MBP distribution), JC virus-positive oligodendrocytes still constituted 15% of all glial cells
even though counts of both were lower. This zone of
early active demyelination also contained a large
number of macrophages. Finally, in zone 4 (demyelinated, decreased or absent MAG and MBP), there
were many more macrophages, even fewer glial cells,
and, with the exception of zone 1, the lowest number
and percentage of infected oligodendrocytes.
Itoyama et al: Papovavirus, MAG, and BP in PML Lesions
F i g 4. Tracings of an acute lesion (Patient 1) and a more
chronic one (Patient 4). In the outer zone, shown in black Cfor
counts see F i g 6, zone l ) , white matter stains normally with
M A G and MBP antisera and appears normal in histological
sections. This zone contains fewer virally infected oligodendrocytes in the more chronic lesion. In the next zone, shown with
stippling (for counts see F i g 6, zone 2), myelin sheaths stain
normally with MBP antiserum; M A G staining i s decreased.
This zone contains the highest density of infected oligodendrocytes and is larger in the more acute lesion. The next zone,
shown between dashed lines f o r counts see F i g 6, zone 31, zncludes the area of active myelin breakdown. MAG staining is
decreased, MEP staining is abnormal, and there are fpwer virally infected cells. The central zone, shown in white (for
counts see Fig 6, zone 41, is demyelinated. MAG and MBP
staining are decreased or absent, and there are the fewest
virus-containing cells.
F i g 5 . Micrographs ofparafjn section stained with 1 :500 JC
virus antiserum, This section is serial t o the two shown in
Figure 3. A includes the area outlined in Figure 3 and shows
the distribution of infected cells from normaf white matter near
the cortical margin (top) to the demyelinated area at the bottom. The most infected cells are in the center, where, as seen in
Figure 3, M A G staining is decreased and the pattern of MBP
from the
staining is almost entirely nomzal. ( ~ 7 4 .Proceeding
top, representativeareas from the strip illustrated in A are
shown at higher magnification in B through E . In B (zone l ) ,
the white matter is normally stained by M A G and MBP
antisera; only a few oligodendroglial nuclei are immunostained. In C (zone 21, M A G staining is decreased; nuclei,
cytoplasm, and processes of many oligodendrocytes are swollen
and densely stained. In D (zone 3), there is active demyelination. This band is located at the edge of decreased MBP staining and contains a swollen immunostained oligoakndroglial
nucleus (arrow) and immunostained deposits i n degenerating
cytoplasm and extracellularly. A t the upper left, immunostained cell remnants are next to an astrocyte nucleus. In the
demyelinated region (zone 4) shown in E, immunostaining is
occasionally present in the nucleus and cytoplasm of very large
astrocytes. (B-E, ~ 1 , 0 0 0 . )
Annals of Neurology
Vol 11 No 4
April 1982
Itoyama et al: Papovavirus, MAG, and BP in PML Lesions
MA G occurs before demyelination begins, since
myelin sheaths in the same area are normal when
stained by MBP antiserum. Later, as oligodendroglia
degenerate, myelin sheaths break down and defective
immune responses promote rapid spread of virus to
adjacent oligodendroglia.
The origin and CNS entry site remain important
unsolved problems in PML. Dorries et a1 [ 5 ] studied
this question by using in situ hybridization with JC
virus carrier R N A and autoradiography. They found
silver grains over oligodendroglia, astrocytes, and
possible endothelial cells, and suggested that JC virus
replication in endothelial cells might be important in
the pathogenesis of PML [ 51. Instead of entering the
CNS from vessels at the onset of PML, papovavirus
infection could occur early in life and persist for
many years in randomly scattered oligodendroglia
until activated by loss of normal immune responses.
Padgett and Walker [17] showed that 65% of their
sample population had acquired antibodies to JC
virus before age 15. Our results appear more consistent with this latter possibility. As noted, we did not
observe any staining in endothelial cells, and neither
the infected oligodendroglia occurring singly in normal white matter nor the oligodendroglial clusters
found in the zone of decreased MAG staining were
located near vessels.
JC, BK, and SV40 viruses can be distinguished in
frozen sections of unfixed PML tissue [ 16, 3 11 and in
methanol-fixed, infected glial cell cultures [ 3 11 by
using monospecific antisera and immunofluorescence
techniques. Since the peroxidase-antiperoxidase
method is as sensitive as the immunofluorescence
techniques used in these studies, we think fixation
with formalin or glutaraldehyde was responsible for
our failure to obtain staining with monospecific antisera to each of these viruses and the T antigen.
In multiple sclerosis, zones of morphologically
normal white matter around demyelinated areas also
stain less intensely with MAG antiserum [lo]. Since
MA G is synthesized in oligodendroglia and is
localized in their periaxonal membranes [ 10,271, the
decreased staining probably is due to loss or degradation of MAG molecules that are needed to maintain
the integrity of the myelin sheath. Another possibility is that the immunoreactivity of MAG is altered
or its accessibility for reaction with antiserum is
changed. Limitations in the sensitivity of the
peroxidase-antiperoxidase method appear unlikely
since MAG was detected readily with lower concentrations of antiserum and, in these experiments, the
area of decreased staining did not change. Therefore, we suggested that an intracellular transport defect, a “dying-back’’ type of change, or some other
pathological alteration in a few oligodendroglia could
produce decreased MAG staining in a relatively large
Lesion Zones in PML
F i g 6. The numbers of glial cells (A), numbers ofJC virusinhcted oligodendroglia (B), percentages of total glial cells that
are infected oligodendroglia (C),and numbers of infiltrating
macrophages i n zones 1 through 4 (see Fig 4) of a P M L lesion
from Patient I . Cells included in all four zones were counted in
three (A-Cj or two (0)different areas around the lesion’s
perimeter by using a 50 x objective and an ocular grid.
Our results in PML clearly show that a large majority
of the cells stained by JC virus antiserum are
oligodendroglia. The distribution of virally infected
oligodendrocytes in demyelinated areas and surrounding white matter suggests the sequence of
changes diagrammed in Figure 7. Active papovavirus
infection probably begins in the oligodendroglial
nuclei which are randomly scattered in white matter
that stains normally with MAG and MBP antisera
and also appears normal histologically. Then virus
spreads to oligodendroglial cytoplasm and processes.
MAG decreases rapidly because it is synthesized in
the perikaryon and must be transported to the inner
margins of all myelin sheaths that are maintained by
the cell. This change in periaxonal oligodendroglial
404 Annals of Neurology Vol 11 No 4 April 1982
F i g 7. Sequence of changes in oligodendroglial virus distribution, oligodendroglial MAG (located periaxonally), and myelin
sheath MBP in PML. As papovuviruj spreads from the
oligodendroglial nucleus (top left) to cytoplasm (top right),
there is a change in periaxonal M A G (lower left) that occws
before myelin breakdown begins (lower right) (see text).
volume of white matter [ 101. Our study of PML was
designed to test this hypothesis, and the results demonstrated a clear relationship between decreased
MAG staining and the number of virally infected
oligodendroglia in areas where myelin sheaths still
stained normally with MBP antiserum. Thus, our
PML evidence provides additional support for the
concept that M AG is a marker for myelin-forming
oligodendroglia and can be a sensitive, useful indicator of oligodendroglial changes that precede
myelin breakdown.
Our studies of these and other CNS myelin lesions
also suggest that glial and myelin targets in demyelinating diseases can be identified by immunostaining lesions with MAG and myelin protein antisera.
Hexachlorophene intoxication is known to produce fluid-filled vacuoles in myelin sheaths [ 121. We
observed normal periaxonal MAG staining and normal MBP staining of compact myelin lamellae that
surrounded vacuoles [36]. These results are consistent with the fact that the process is reversible and
little demyelination is seen [ 321. When experimental
allergic encephalomyelitis was induced in Lewis rats
b y CNS tissue, the areas of decreased MBP and
MAG staining observed in adjacent sections were
similar [9]. This result suggests that in the type of
experimental allergic encephalomyelitis we studied,
myelin is the primary target. As noted, the pattern of
MAG and MBP staining observed in lesions associated with multiple sclerosis [ 101 and PML was quite
different. Since decreased MA G staining extended
beyond the margins of demyelinated areas into
normal-appearing white matter, we think oligodendroglia are the primary targets in both of these diseases and that demyelination probably is a secondary
Finally, this is one of the first studies to use the
peroxidase-antiperoxidase method to demonstrate
the distribution of a virus in paraffin- and eponembedded sections of nervous tissue. Our observations supplement those in earlier reports [ 3 , 4 , 61
and demonstrate that this method has several important advantages. First, its sensitivity and specificity
are very high. Oligodendroglial staining was detected
easily at antiserum dilutions below 1: 1,000, and
cells that were immunostained in a semithin section
contained virions when an adjacent thin section was
examined by electron microscopy. Another important advantage is that sections can be counterstained
after immunostaining. For example, by using hematoxylin, immunostained cells can be identified
by their nuclear and cytoplasmic morphology. Or, if
Luxol fast blue is used, the distribution of virus can
be related to areas of histological change in myelin
sheaths. Another useful feature is that paraffin-
Itoyama et al: Papovavirus, MAG, and BP in PML Lesions
embedded blocks are the largest available source of
stored human pathological tissue. Specific staining
can be detected in routinely processed autopsy tissue
after more than 20 years and does not fade during
counterstaining, photography, or subsequent storage.
The advantages of the peroxidase-antiperoxidase
method are especially important when neurological
diseases are being investigated. For diagnostic studies
of biopsy or autopsy specimens, other direct o r indirect immunostaining methods using fluorescent [ 3 , 4 ,
281 or peroxidase [13, 141 labeled antisera are simpler and often provide the required information. But
more specific and sensitive methods, such as the one
used here, are needed for investigations designed to
show whether there is selective involvement of a
particular cell type or whether t h e distribution of infected cells is related to certain tracts, nuclei, or
mesenchymal elements. Finally, we hope the sensitivity and specificity of the peroxidase-antiperoxidase method will prove especially useful in
studies of CNS diseases of unknown cause. Since
there are fewer problems with nonspecific staining
and much less antiserum is needed than with other
methods, investigators who use the peroxidaseantiperoxidase method may be successful in detecting the relatively small amounts of virus that may be
present in diseases such as multiple sclerosis.
Supported in part by Grant AI-11217 from the US Public Health
Drs Karl-Erik Astrom and Dennis Landis generously provided
epon-embedded biopsy tissue from Patients 1 and 2 for us to immunostain and study by electron microscopy. Dr Steven Cohen
kindly supplied us with antiserum to MBP. We appreciate the excellent technical assistance provided by Mrs Sophia Grabinski.
1. h r o m K-E, Mancall EL, Richardson EP Jr: Progressive multifocal leuko-encephalopathy. Brain 8 ~ 9 3 - ll l , 1958
2. Baskin DG, Erlandsen SL, Parsons JA: Immunocytochemistry
with osmium-fixed tissue. 1. Light microscopic localization of
growth hormone and prolactin with the unlabeled antibodyenzyme method. J Histochem Cytochem 27:867-872, 1979
3. Budka H , Lassmann H , Popow-Kraupp T: Measle: virus antigen in panencephalitis. An immunomorphological study stressing dendritic involvement in SSPE. Acta Neuropathol (Berl)
(in press)
4. Budka H , Popow-Kraupp T: Rabies and herpes simplex viral
encephalitis. An immunohistological study on site and distribution of viral antigens. Virchows Arch [Pathol Anat]
390:353-364, 1981
5. Dorries K, Johnson RT, ter Meulen V: Detection of polyoma
virus DNA in PML-brain tissue by (in situ) hybridization. J
Gen Virol 42:49-57, 1979
6. Gerber MA, Shah KV, Thung SN, et al: Immunohistochemical demonstration of common antigen of polyomaviruses in
routine histologic tissue sections of animals and man. Am J
Clin Pathol 73:794-797, 1980
7. Hogan TF, Padgett BL, Walker DL, et al: Rapid detection and
identification of JC virus and BK virus in human urine using
406 Annals of Neurology Vol 11 No 4 April 1982
immunofluorescence microscopy. J Clin Microbiol 11:178183, 1980
8. Itoyama Y, Sternberger N H , G e s MW, et al: Immunocytochemical method to identify myelin basic protein in
oligodendroglia and myelin sheaths of the human nervous
system. Ann Neurol 7:157-166, 1980
9. Itoyama Y, Sternberger N H , Quarles RH, et al: Immunocytochemical observations on demyelinating lesions in
experimental allergic encephalomyelitis (EAE). Soc Neurosci
Abstr 5:512, 1979
10. Itoyama Y, Sternberger N H , Webster HdeF, et al: Immunocytochemical observations on the distribution of
myelin-associated glycoprotein and myelin basic protein in
multiple sclerosis lesions. Ann Neurol 7:167-177, 1980
11. Itoyama Y, Walker DL, Richardson EP Jr, et al: Papovavirus,
myelin-associated glycoprotein and myelin basic protein in
progressive multifocal leukoencephalopathy (abstract). J
Neuropathol Exp Neurol 39:363, 1980
12. Kimbrough RD, Gaines TB: Hexachlorophene effects on the
rat brain: study of high doses by light and electron microscopy. Arch Environ Health 23:114-118, 1971
13. Kumanishi T, Hirano A: An immunoperoxidase study on
herpes simplex virus encephalitis. J Neuropathol Exp Neurol
37~790-795, 1978
14. Kumanishi T, In S: SSPE: immunohistochemical demonstration of measles virus antigen(s) in paraffin sections. Acta
Neuropathol (Berl) 48:161-163, 1979
5 . Mazlo M, Tariska I: Morphological demonstration of the
first phase of polyomavirus replication in oligodendroglial
celis of human brain in progressive multifocal leukoencephalopathy (PML). Acta Neuropathol (Berl) 49:133-143,
6. Narayan 0, Penney JB, Johnson RT: Etiology of progressive
multifocal leukoencephalopathy. Identification of papovavirus. N Engl J Med 289:1278-1282, 1973
7. Padgett BL, Walker D L Prevalence of antibodies in human
sera against J C virus, an isolate from a case of progressive
multifocal leukoencephalopathy. J Infect Dis 127:467-470,
18. Padgett BL, Walker DL, ZuRhein GM: Cultivation of
papova-like virus from human brain with progressive multifocal leucoencephalopathy. Lancet 1:1257-1260, 197 l
19. Quarles RH: Glycoproteins in myelin and myelin-related
membranes. In Margolis RU, Margolis RK (eds): Complex
Carbohydrates of Nervous Tissue. New York, Plenum, 1979,
PP 209-233
20. Quarles R H , Everly JL, Brady RO: Evidence for the close association of a glycoprotein with myelin in rat brain. J
Neurochem 2 1: 117 7-1 191, 1973
2 1. Richardson EP Jr: Progressive multifocal leukoencephalopathy. N Engl J Med 265:815-823, 1961
22. Richardson EP Jr: Progressive multifocal leukoencephalopathy. In Brain WR, Norris FH Jr (eds): The Remote
Effects of Cancer on t h e Nervous System; The Proceedings
of a Symposium. New York, Grune & Stratton, 1965, pp
23. Richardson EP Jr: Progressive multifocal leukoencephalopathy. In Vinken PJ, Bruyn GW (eds): Handbook of
Clinical Neurology. Vol 9, Multiple Sclerosis and Other Demyelinating Diseases. Amsterdam, North-Holland, 1970, pp
24. Richardson EP Jr, DeGirolami U: Neurologic disorder and
pleural effusion after 21 years of chronic lymphocytic
leukemia. N Engl J Med 286:1047-1054, 1972
25. Silverman L, Rubinstein LJ: Electron microscopic observations on a case of progressive multifocal leukoencephalopathy. Acta Neuropathol (Berl) 5:215-224, 1965
26. Sternberger LA, Hardy P H Jr, Cuculis JJ, et al: The unlabeled
antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex
(horseradish peroxidase-antihorseradish peroxidase) and its
use in identification of spirochetes. J Histochem Cytochem
18~315-333, 1970
27. Sternberger NH, Quarles RH, Itoyama Y, et al: Myelinassociated glycoprotein demonstrated immunocytochemically
in myelin and myelin-forming cells of developing rat. Proc
Natl Acad Sci USA 76:1510-1514, 1979
28. Tomlinson AH, Chinn IJ, MacCallum FO: Immunofluorescence staining for the diagnosis of herpes encephalitis. J Clin
Pathol 27:495-499, 1974
29. Trapp BD, Itoyama Y, Sternberger N H , et al: Immunocytochemical localization of Po protein in Golgi membranes
and myelin of developing rat Schwann cells. J Cell Biol
9011-6, 1981
30. Walker DL: Progressive multifocal leukoencephalopathy: an
opportunistic viral infection of the central nervous system. In
Vinken PJ, Bruyn G W (eds): Handbook of Clinical Neurology. Vol 34, Infections of the Nervous System. Amsterdam,
North-Holland, 1978, pp 307-329
31. Walker DL, Padgett BL, ZuRhein GM, et al: Current study of
an opportunistic papovavirus. In Zeman W, Lennette EH
(eds): Slow Virus Diseases. Baltimore, Williams & Wilkins,
1974, pp 49-58
32. Webster HdeF, Tabira T, Reier PJ: Examination of the developing nervous system of Xenopus tadpoles with differen-
tial interference microscopy: A new assay procedure for
neurotoxicologists. In Roizin L, Shiraki H , Grcevic N (eds):
Neurotoxicology. New York, Raven, 1977, pp 403-41 1
Webster HdeF, Trapp BD, Sternberger N H , et al: Myelin
forming glial cells: morphological and immunocytochemical
observations. In Garrod DR, Feldman JD (eds): Symposium
on Development in the Nervous System. Cambridge, England, Cambridge University Press, 1981, pp 265-288
Weiner LP, Herndon RM, Narayan 0,et al: Isolation of virus
related to SV40 from patients with progressive multifocal
leukoencephalopathy. N Engl J Med 286:385-390, 1972
Weitzman S, Kaufman S, Wolpow E, et al: Simultaneous fungal and viral infection of the central nervous system. Am J
Med Sci 276:127-132, 1978
Winchell KH, Sternberger N H , Quarles RH, et al: Myelinassociated glycoprotein and basic protein in hexachlorophene
lesions (abstract). In Proceedings of the 1lth Annual Meeting
of the American Society for Neurochemistry, Houston, TX.
Lawrence, KS, University of Kansas Press, 1980, p 184
ZuRhein GM: Association of papova-virions with a human
demyelinating disease (progressive multifocal leukoencephalopathy). Prog Med Virol 11:185-247, 1969
ZuRhein GM, Chou SM: Particles resembling papovaviruses
in human cerebral demyelinating disease. Science 148:
1477-1479, 1965
ZuRhein GM, Padgett BL, Walker DL: Progressive multifocal
leukoencephalopathy in a child with severe combined immunodeficiency. N Engl J Med 299.256-257, 1978
Itoyama et al: Papovavirus, MAG, and BP in PML Lesions
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multifocal, progressive, associates, distributions, papovavirus, lesions, basic, protein, glycoprotein, myelin, leukoencephalopathy
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