In situ hybridization for detection of nocardial 16S rRNA: Reactivity within intracellular inclusions in experimentally infected cynomolgus monkeys--and in Lewy body-containing human

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Experimental Neurology 184 (2003) 715–725

In situ hybridization for detection of nocardial 16S rRNA: reactivity

within intracellular inclusions in experimentally infected cynomolgus

monkeys—and in Lewy body-containing human brain specimens

Gail Chapman,a,1 Blaine L. Beaman,a David A. Loeffler,b Dianne M. Camp,b

Edward F. Domino,c Dennis W. Dickson,d William G. Ellis,e Ibsen Chen,e

Susan E. Bachus,f and Peter A. LeWittb,g,h,*

aDepartment of Medical Microbiology and Immunology, University of California School of Medicine, Davis, CA 95616, USAbWilliam Beaumont Hospital Research Institute, Royal Oak, MI 48073, USA

cDepartment of Pharmacology, The University of Michigan Medical School, Ann Arbor, MI 48104, USAdMayo Clinic, Jacksonville, FL 32224, USA

eDepartment of Pathology, University of California School of Medicine, Davis, CA 95616, USAfPsychology Department and Krasnow Institute, George Mason University, Fairfax, VA 22030, USA

gDepartment of Neurology, Wayne State University School of Medicine, Detroit, MI 48201, USAhDepartment of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, Detroit, MI 48201, USA

Received 23 January 2003; revised 23 June 2003; accepted 1 July 2003

Abstract

Our previous studies found that experimental infection of BALB/c mice with the Gram-positive bacterium Nocardia asteroides induced a

parkinsonian-type syndrome with levodopa-responsive movement abnormalities, loss of nigrostriatal dopaminergic neurons, depletion of

striatal dopamine, and intraneuronal inclusions in the substantia nigra (SN) with an appearance similar to Lewy bodies. In the present study,

an in situ hybridization technique was developed to detect nocardial 16S ribosomal RNA (rRNA), using a Nocardia-specific probe (B77).

Cerebral cortical specimens from cynomolgus monkeys were examined for the presence of nocardial RNA 48 h, 3.5 months, and 1 year after

experimental infection with N. asteroides. Hybridization reactions were detected within Nocardia-like structures 48 h after infection and

within intracellular inclusion bodies (immunoreactive for a-synuclein and ubiquitin) in one of two 3.5-month-infected monkeys. The in situ

hybridization procedure was then applied in a blinded fashion to 24 human SN specimens with Lewy bodies and 11 human SN specimens

without Lewy bodies (including five normal controls). Hybridization reactions were detected in nine Lewy body-containing specimens and

none of the others. Reactivity was limited to inclusions with the appearance of Lewy bodies, with the exception of one specimen in which

intracellular reactivity was also observed in Nocardia-like structures. These results suggest a possible association between Nocardia and

neurodegenerative disorders in which Lewy bodies are present.

D 2003 Elsevier Inc. All rights reserved.

Keywords: Dementia with Lewy bodies; In situ hybridization; Lewy bodies; Neurodegeneration; Nocardia; Parkinson’s disease

Introduction (PD) occurs in association with spherical eosinophilic

The loss of dopaminergic neuronal cell bodies in the

substantia nigra (SN) pars compacta in Parkinson’s disease

0014-4886/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/S0014-4886(03)00337-6

* Corresponding author. Division of Neurology, William Beaumont

Hospital Research Institute, 3811 West Thirteen Mile Road, Royal Oak, MI

48073. Fax: +1-248-355-3857.

E-mail address: [email protected] (P.A. LeWitt).1 Present address: NAVMEDRSCHU#3, PSC 452, Box 157, FPO

09835-0007, Egypt.

inclusions termed Lewy bodies. The pathophysiology lead-

ing to these ubiquitin- and a-synuclein-immunoreactive

structures is not known, but is likely to be related to

abnormal aggregation of these proteins (Trojanowski and

Lee, 1998). Though the combination of SN Lewy bodies

and localized neuronal dropout is pathognomonic for PD,

Lewy bodies are also present elsewhere in the PD brain

(Forno, 1986). The density of Lewy bodies in neocortical

and limbic regions is greatly increased in PD patients with

concomitant dementia, in comparison to PD patients with

G. Chapman et al. / Experimental Neurology 184 (2003) 715–725716

normal cognition (Apaydin et al., 2002). Morphological

differences are typically present between nigral and cortical

Lewy bodies in the PD brain; the former contain a distinct

central core and peripheral halo, while the latter inclusions

often lack these characteristics and may appear simply as

homogeneous pink rounded bodies when stained with

hematoxylin–eosin (Forno et al., 1996; Kosaka et al.,

1984). Lewy bodies are also found in neurological disorders

other than typical PD, including dementia with Lewy bodies

(DLB) (Kosaka et al., 1984), subacute sclerosing panence-

phalitis (Gibb et al., 1990), and multiple system atrophy

(Tison et al., 1995). These inclusions may also be present in

relatively low numbers in clinically normal elderly subjects

(Forno, 1986).

Intraneuronal inclusions resembling Lewy bodies have

been described in various experimental animal models of

PD, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

(MPTP)-treated squirrel monkeys (Forno et al., 1986, 1995),

rotenone-treated rats (Betarbet et al., 2000), and transgenic

mice (Tu et al., 1996) and Drosophila (Feany and Bender,

2000). We have investigated another experimental model for

generating Lewy body-like inclusions, namely systemic

infection of mice with the Gram-positive, acid-fast bacteri-

um Nocardia asteroides. In an earlier publication, we

reported that intraneuronal inclusions resembling Lewy

bodies were present in the SN of BALB/c mice 1 month

after experimental infection with N. asteroides strain GUH-

2 (Kohbata and Beaman, 1991). These mice also developed

levodopa-responsive motor impairments. Subsequent stud-

ies showed additional findings characteristic of PD in these

animals, including depletion of striatal dopamine (Hyland et

al., 2000) and apoptotic cell death of tyrosine hydroxylase-

immunoreactive SN neurons (Tam et al., 2002). These

neurodegenerative changes were unlikely to be the result

of chronic nocardial infection, because no cellular inflam-

matory response was observed and the organism could not

be cultured from the brain by day 14 postinfection (Kohbata

and Beaman, 1991). A robust infection occurred in the SN

in the first 48 h after systemic administration, however.

Electron microscopic studies indicated that Nocardia ad-

hered to endothelial cells in the SN, then invaded into and

grew within neurons (Beaman and Ogata, 1993). Recent in

vitro studies in our laboratory (Camp et al., 2003) have

suggested that nocardial secretory products may be involved

in the loss of dopaminergic neurons. Neurodegenerative

changes in the SN have also been reported in nonhuman

primates (cynomolgus monkeys) acutely infected with

Nocardia (Beaman et al., 2000). Because of the neurochem-

ical, pathophysiological, and behavioral similarities between

subjects with PD and animals experimentally infected with

N. asteroides, and because of the possibility that environ-

mental factors (including infectious agents) may be involved

in the etiology of PD, as suggested by numerous investi-

gators (Di Monte et al., 2002; Takahashi and Yamada, 2001;

Tanner, 1989; Tsui et al., 1999; Uitti and Calne, 1993), we

have initiated studies to determine whether PD might be

associated with nocardial CNS infection. In the present

study, we initially characterized the inclusions resulting

from experimental infection with N. asteroides in cynomol-

gus monkeys, then investigated for the presence of nocardial

RNA in SN specimens from subjects with disorders in

which Lewy bodies are present.

Materials and methods

Animal housing and maintenance

Cynomolgus macaque monkeys were maintained at the

animal care facilities of the University of Michigan (Ann

Arbor, MI) and the California Regional Primate Center of the

University of California at Davis (Davis, CA). The monkeys

housed at the University of Michigan were for long-term

studies related to the development of intraneuronal inclusion

bodies within the brain. The monkeys at the University of

California at Davis were utilized for short- and long-term

studies involving development and testing of the in situ

hybridization procedure for detection of Nocardia in brain

specimens. All monkeys were housed under similar condi-

tions, fed similar diets, and maintained and monitored in a

similar manner according to the procedures described in the

Guide for the Care and Use of Laboratory Animals (National

Research Council, National Academy Press, 1996).

Experimental infection of monkeys with N. asteroides

A log-phase cell suspension of N. asteroides strain GUH-

2 in Hank’s Balanced Salt Solution (6 � 106 colony-

forming units [CFU] in 1 ml) was injected into the right

saphenous vein of monkeys following induction of anesthe-

sia with ketamine. Three minutes later, 1–2 ml of blood was

removed from the left saphenous vein, diluted 1:10 in sterile

distilled water, and plated on trypticase soy agar for CFU

quantification. The monkeys at the University of Michigan

were infected initially with N. asteroides strain I-38-SYN, a

mutant of strain GUH-2; because no neurological abnor-

malities developed, they were infected again 1.5 months

later with strain GUH-2. These monkeys were coded 93-R-

204 and 93-R-205, and those at the University of California

were coded M2, M5, and M6. Monkey M2 was euthanized

48 h after infection and the brain was perfused with 3%

glutaraldehyde–1.5% paraformaldehyde. This animal

served as the positive control for development of the in situ

hybridization procedure. Monkey M5 was euthanized 1 year

postinfection. Monkey M6, the negative control for the in

situ experiments, received culture medium alone and was

euthanized after 48 h. Monkeys 93-R-204 and 93-R-205

became listless on postinfection day 5, necessitating treat-

ment with trimethoprim–sulfamethoxazole. The animals

recovered, although 93-R-204 developed a persistent tremor

in one arm. These animals were euthanized 3.5 months after

infection with strain GUH-2.

G. Chapman et al. / Experimental Neurology 184 (2003) 715–725 717

Light and electron microscopy studies on monkey brain

specimens

Monkeys were euthanized and the brains were removed.

Coronal brain slices were prepared from monkeys 93-R-

204 and 93-R-205. Small portions of these specimens were

frozen (� 70jC) for subsequent culturing, then the coronal

slices were placed into fixative (3% glutaraldehyde–1.5%

Table 1

In situ hybridization reactivity of human substantia nigra specimens with a Nocardi

Brain bank Age Gender Clinical findings

Parkinsonism De

Normal controls

BTRC 78 M no no

BTRC 51 M no no

BTRC 59 M no no

UCD 63 M no no

UCD 61 M no no

Neuropathology without Lewy bodies

BTRC 88 F no ye

UCD 76 M yes ye

UCD 68 F no ye

UCD 61 M no ye

UCD 69 F no ye

UCD 19 M no ye

Neuropathology with Lewy bodies in substantia nigra

BTRC 84 M yes un

UCD 85 M yes ye

UCD 82 M yes ye

UCD 79 M yes ye

EIN 69 F no ye

EIN 84 M yes ye

EIN 94 M no ye

UCD 71 F yes ye

EIN 77 M no ye

BTRC 81 F unknown un

UCD 82 M no ye

UCD 84 F unknown ye

UCD 76 M no ye

UCD 85 M yes ye

UCD 70 M yes ye

UCD 71 M no ye

UCD 82 F no ye

UCD 80 F no ye

EIN 66 M no ye

EIN 86 F yes no

EIN 85 F no ye

EIN 78 F yes no

EIN 70 M no ye

EIN 82 F no ye

Brain Bank: BTRC = Harvard Brain Tissue Resource Center (McLean Hospita

University of California at Davis.

Abbreviations: AD, Alzheimer’s disease; DLB, dementia with Lewy bodies; PD,a Marked neuronal loss in substantia nigra, with neurofibrillary tangles; no Lew

features.b Lewy bodies, neurofibrillary tangles, and extensive loss of pigmented neuro

parkinsonism.c Increased numbers of non-neuritic senile plaques in cortex and hippocampus; fe

paraformaldehyde). After several days in the fixative,

specimens from cerebral cortex, thalamus, caudate, and

putamen were cut into 0.5 � 2.0 mm segments. (SN

specimens were not available for this study.) Histochemical

and immunocytochemical studies were performed on speci-

mens from all of these regions; in situ hybridization studies

were performed only on the cerebral cortex. These tissues

were postfixed in 1% osmium tetroxide in phosphate buffer

a-specific probe (B77) complementary to 16S rRNA of Nocardia asteroides

Neuropathological diagnosis B77 reactivity

mentia

normal negative

normal negative

normal negative

normal negative

normal negative

s AD negative

s Pick’s disease, AD negative

s AD, cerebral infarcts, possible

post-encephalitic parkinsonisma

negative

s Pick’s disease negative

s cerebral infarcts negative

s Lafora disease negative

known PD, AD 3+

s DLB 3+

s post-encephalitic parkinsonism,

possible PDb

2 +

s DLB, AD, cerebral infarcts 2 +

s DLB, AD 2+

s DLB, pathological agingc 2 +

s DLB, AD 2+

s DLB 1+

s DLB, pathological agingc 1 +

known AD with Lewy bodies negative

s AD with Lewy bodies negative

s PD, AD negative

s DLB negative

s DLB negative

s DLB negative

s DLB, AD negative

s DLB negative

s DLB negative

s DLB, pathological agingc negative

DLB, pathological agingc negative

s DLB, pathological agingc negative

DLB, pathological agingc negative

s DLB, AD negative

s DLB, pathological agingc negative

l); EIN = Albert Einstein College of Medicine (New York, NY); UCD =

Parkinson’s disease.

y bodies; marked rigidity in upper extremities, but no other parkinsonian

ns in substantia nigra; clinical history compatible with post-encephalitic

w or no neurofibrillary tangles.

G. Chapman et al. / Experimental Neurology 184 (2003) 715–725718

(pH 7.4) for 1 h, rinsed, incubated in 0.5% uranyl acetate

for 30 min, rinsed again, dehydrated through graded

ethanol baths, and embedded in Med-cast epoxy resin

(Ted Pella, Inc., Redding, CA). After polymerization, thick

sections (approximately 1-Am-thick) were cut and stained

with methylene blue for detection of inclusions with the

light microscope. The tissue blocks in which inclusions

were observed were trimmed and sectioned for electron

microscopy. Thin (gold to silver) sections were placed on

copper grids (300 mesh), stained for 15 min with 0.5% (w/

v) uranyl acetate in 50% methanol–water (v/v), rinsed, and

stained for 5 min with 0.1% lead citrate. After washing in

deionized water, the sections were visualized and photo-

graphed using a Philips model 400 electron microscope

operated at 80 kV. Additional specimens from the fixed

coronal sections were paraffin-embedded. Sections (6–10

Am) were placed on glass slides, deparaffinized, and

stained by the following methods (Luna, 1968): hematox-

ylin–eosin, the Brown and Brenn modification of the

Gram stain, Ziehl–Neelsen stain for acid fastness, periodic

acid-Schiff (PAS) for carbohydrates, Grocott’s methena-

mine silver stain for glycolipids, von Kassa stain for

calcium, and Perl’s Prussian blue iron stain. Immunocyto-

chemical staining was also performed for localization of

ubiquitin and a-synuclein, as described by Gai et al.

(2000).

Human brain specimens

Paraffin-embedded histological sections were prepared

from 35 postmortem formalin-fixed SN specimens, includ-

ing Lewy body-containing disorders (PD, dementia with

Lewy bodies [DLB], and Alzheimer’s disease [AD] with

Lewy bodies), other neurodegenerative disorders, and

normal controls. Reviewing clinical and neuropathological

reports allowed us to make a determination as to the

presence of Parkinsonism and/or dementia, and a probable

neuropathological diagnosis (Table 1). In some cases,

determination of the definitive neuropathological diagnosis

was difficult (i.e., some specimens had severe neuronal

loss in the SN, sufficient for a diagnosis of PD, but no

Lewy bodies were detected in the SN, whereas others had

findings of PD and AD, or AD-type changes together

with Lewy bodies). Therefore, specimens were simply

grouped as normal controls (n = 5), neuropathology

without Lewy bodies (n = 6), or neuropathology with

Lewy bodies in the SN, as shown by hematoxylin–eosin

staining and/or immunocytochemical staining for ubiquitin

or a-synuclein (n = 24). All slides lacked diagnostic

identification and were number-coded for analysis in a

blinded fashion. No cases were known to have had prior

nocardial infection. These specimens were obtained from

the Department of Neuropathology, Albert Einstein Col-

lege of Medicine, Yeshiva University (New York, NY),

the Department of Pathology, University of California at

Davis, and the Harvard Brain Tissue Resource Center

(McLean Hospital, Belmont, MA). Specimens from Ye-

shiva University and the University of California at Davis

were paraffin-embedded, sectioned, and mounted on slides

at these institutions, whereas the paraffin-embedded speci-

mens from the Harvard brain bank were sectioned and

mounted on slides at Sinai Hospital (now Sinai-Grace

Hospital, Detroit, MI).

In situ hybridization

Tissue preparation

Procedures described by Chapman and Beaman (1999)

for in situ labeling were followed. Briefly, tissues were

deparaffinized in xylene, rehydrated through decreasing

ethanol concentrations, washed in 0.1 M Tris buffer, pH

7.4, and dried at room temperature. Rinsing with Tris

buffer was also performed following all subsequent steps.

The specimens were delipidated in 0.5% Triton X-100 in

Tris buffer (15 min), then deproteinated with proteinase K

(10 Ag/ml in Tris buffer with 50 mM EDTA, 30 min, 37jC)and incubated in 0.1 M glycine in 0.2 M Tris buffer, pH 7.4

(15 min, room temperature). The samples were then incu-

bated with levamisole (10 mM) to inactivate endogenous

alkaline phosphatase. After extensive rinsing in diethylene

pyrocarbonate (DEPC)-treated double-deionized water

(ddH2O), specimens were rinsed in Tris buffer then incu-

bated with acetic anhydride (0.25% in 0.1 M triethanol-

amine–0.9% NaCl, pH 8) on a rocking platform for 10 min

at room temperature to reduce nonspecific binding of the

oligonucleotide probe to charged molecules in the tissue.

Tissues were washed in 2� standard saline citrate (SSC;

1� SSC = 0.15 M sodium citrate, 0.015 M sodium

chloride, pH 7.0) for 5 min at room temperature, then

dehydrated and further delipidated through increasing con-

centrations of ethanol and chloroform, respectively. Sec-

tions were then air-dried and stored in a desiccator box at

4jC.

Synthesis of oligonucleotide probes

A deoxynucleotide sequence complementary to the high-

ly conserved 16S ribosomal RNA (rRNA) was synthesized

on a Model 394 DNA–RNA Synthesizer (Applied Biosys-

tems, Foster City, CA). The sequence of the probe, termed

B77, was 5V-GCTCGTGTACCCCGAAGGGCCTTAC-3Vat positions 104–69 using the Escherichia coli numbering

system of Brosius et al. (1978). The reverse sequence,

termed B68, was synthesized as a negative control. These

oligonucleotide sequences were desalted using Nap-5 col-

umns (Pharmacia Biotech, Piscataway, NJ). Effluents from

the columns were collected, diluted 1:100, and the OD260

was determined. The concentration of the oligonucleotide

sequence was determined via the following formula: (6.25

nmol/OD unit/ml) � (dilution factor) � (OD260).

G. Chapman et al. / Experimental Neurology 184 (2003) 715–725 719

Specificity of oligonucleotide probe sequences

A BLAST database search revealed that a variety of

nocardioform actinomycetes had 100% nucleic acid homol-

ogy with probe B77. There was also indication of homology

with a closely related microorganism, Rhodococcus. Because

the taxonomy of nocardioform actinomycetes is in flux, the

true taxonomic status of actinomycetes in the BLAST data

bank cannot be accurately determined at present. Most

species of Rhodococcus were named Nocardia before 1990,

and differentiation of members of these two genera is often

difficult (Beaman and Beaman, 1994). Nevertheless, the only

pathogenic organisms with 100% homology to probe B77

were Nocardia spp. No homologies were found to mamma-

lian nucleic acid sequences. The reverse sequence, probe

B68, included a 16-base sequence with homology to human

chromosome 20, but it did not hybridize to human DNA or to

histological sections from human or monkey brain specimens

(Table 2; Figs. 2B and F).

Digoxigenin labeling of probes

Both the primary (B77) and reverse (B68) oligonucleotide

sequences were 3’ end-labeled with the DIG/Genius 6 Ol-

igonucleotide Tailing Kit (Boehringer Mannheim Corp.,

Indianapolis, IN) and precipitated as described by the man-

ufacturer. Briefly, 100 pg of oligonucleotide was added to 20

Al of reaction mixture containing dATP, Dig-dUTP, terminal

Table 2

Probe B77 reactivity to bacterial and mammalian DNA

Source designation or strain Dot blot

reactivity

Nocardia asteroides GUH-2 (human strain) positive

Nocardia asteroides ATCC 10905 positive

Nocardia asteroides ATCC 14759 positive

Nocardia asteroides ATCC 19247 (type strain) positive

Nocardia farcinica ATCC 3318 (type strain) positive

Nocardia brasiliensis 17E (human strain) positive

Nocardia brasiliensis ATCC 29296 (type strain) positive

Tsukamurella paurometabola ATCC 8368 (type strain) negative

Gordona bronchialis ATCC 25592 (type strain) negative

Streptomyces somaliensis human-UCD (lab strain) negative

Actinomyces israelii human serotype 1 negative

Rhodococcus species 4277 (soil) negative

Mycobacterium tuberculosis ATCC 25177 (strainH37Ra) negative

Actinomadura madurae human-UCD (lab strain) negative

Mycobacterium smegmatis ATCC 19420 negative

Mycobacterium gordonae ATCC14470 (type strain) negative

Mycobacterium avium intracellular-human-UCD

(lab strain)

negative

Escherichia coli XL1 Blue negative

Human midbrain DNA negative

Mouse BALB/c tail DNA negative

The reverse complement sequence probe B68 was negative in all reactions.

Abbreviations: ATCC, American Type Culture Collection; UCD, Univer-

sity of California School of Medicine, Davis, Department of Medical

Microbiology and Immunology culture collection.

transferase, CoCl2, and reaction buffer. Unlabeled control

oligonucleotide was included in a separate tailing reaction.

The mixture was incubated at 37jC � 15 min, then the

reaction was terminated by placing it on ice and adding

EDTA. The labeled probes were precipitated by adding

glycogen, lithium chloride, and ethanol. After incubation at

� 70jC for 30 min, the probes were pelleted by centrifuga-

tion (14,000 � g), washed with 70% ethanol, dried under

vacuum, and resuspended in 50-Al DEPC-treated ddH2O.

The yield of digoxigenin-labeled probes was determined

using the DIG/Genius 3 Nucleic Acid Detection Kit (Boeh-

ringerMannheim). The tailed primers were stored at � 20jC.

Prehybridization and in situ hybridization

Prehybridization and in situ hybridization were per-

formed as described by Lewis et al. (1993) with slight

modifications. Prehybridization solution was prepared by

combining 474 Al of formamide, 200 Al of 20� SSC, 20 Al of50� Denhardt’s solution, 200 Al of 20% dextran sulfate, 50

Al of freshly denatured herring sperm (stock solution con-

centration = 500 Ag/ml), and 25 Al of yeast tRNA (stock

solution concentration = 250 Ag/ml), bringing to a final

volume of 1 ml with DEPC H2O. Thirty-five microliters of

the prehybridization solution was applied to each section

after slowly warming (>10 min) the tissue to 37jC. A cover

slip was placed over the sample to allow even spreading of

the solution and to reduce evaporation during incubation in a

humidified chamber (2–5 h, 37jC). Excess hybridization

buffer was then removed by gentle rinsing with 2� SSC.

After brief draining, 35 Al of the digoxigenin-labeled probe

(probe concentration = 20 fmol/Al in prehybridization solu-

tion) was applied. The hybridization mixture was incubated

overnight in a humidified chamber at 37jC. Slides were thenimmersed in 2� SSC to remove cover slips, followed by

washes in 2� SSC (1 h, room temperature), 1� SSC (1 h,

room temperature), and 0.5� SSC (50jC for 10 min, then

room temperature for 30 min).

Detection of the digoxigenin-labeled probe by in situ

hybridization

Detection of the hybridized probe in tissue sections was

also performed as described by Lewis et al. (1993) with minor

modifications. Following post-hybridization washes, slides

were rinsed in Tris buffer, then nonspecific binding of the

anti-digoxigenin antibody (see below) was blocked by incu-

bation with Tris buffer (100 mMTris HCl, 150 mMNaCl, pH

7.5) with 2% normal sheep serum (Sigma Co., St. Louis, MO)

and 0.3% Triton X-100. Sections were incubated with cover

slips for 5 h in a humidified chamber at room temperature

with alkaline phosphatase-conjugated anti-digoxigenin-Fab

fragments (Boehringer Mannheim) in Tris buffer with 1%

sheep serum and Triton X-100 (‘‘buffer 1’’). Slides were

rinsed briefly in buffer 1 to remove cover slips, then washed

with the same buffer for 30 min at 25jC on an orbital shaker.

G. Chapman et al. / Experimental N720

Slides were then washed in pH 9.5 Tris buffer (100 mM Tris

HCl, 100 mM NaCl, 50 mMMgCl2) (‘‘buffer 2’’) for 10 min

at 25jC. Chromagen solution containing nitroblue tetrazoli-

um, 5-bromo-4-chloro-3-indolyl-phosphate, and levamisole

in buffer 2 was added to slides, followed by incubation at

room temperature in the dark for 22 h. (Fresh chromagen was

added after 12 h.) Immunoreactivity was stopped by rinsing

in Tris–EDTA buffer, pH 8.0. Slides were dehydrated in 70%

and 95% ethanol, counterstained with ethanolic eosin,

brought to 100% ethanol, cleared with xylene, and cover

slips were adhered with Permount.

Fig. 1. Characterization of inclusion bodies in the monkey brain at 3.5 months aft

204, hematoxylin–eosin stain. Arrow points to an eosinophilic inclusion body (

indicating an immunoreactive Lewy body (scale bar = 10 Am). (C) Cortex of mo

inclusion (scale bar = 10 Am). (D) Cortex of monkey 93-R-204, ubiquitin staining

monkey 93-R-204, electron micrograph. Microfilaments similar to those present

Assessment of in situ hybridization

Two investigators (GC and BLB) independently

reviewed the B77- and B68-probed slides in a blinded

fashion using light microscopy. Each section was initially

scanned with the low power (10�) objective, and areas of

possible hybridization were then examined under oil

immersion (100� objective). Reactivity was scored in a

semi-quantitative manner as follows: negative = no reac-

tivity observed; 1+ = one to two reactions observed in

each section; 2+ = three to four reactions observed in each

eurology 184 (2003) 715–725

er infection with N. asteroides strain GUH-2. (A) Cortex of monkey 93-R-

scale bar = 10 Am). (B) Cortex of a DLB specimen, a-synuclein staining,

nkey 93-R-204, a-synuclein staining. Arrow points to an immunoreactive

. An immunoreactive inclusion is seen (scale bar = 10 Am). (E) Caudate of

in Lewy bodies are indicated by the arrow (scale bar = 1 Am).

G. Chapman et al. / Experimental N

section; 3+ = five or more reactions observed in each

section. To be accepted as a positive specimen, reactivity

with probe B77 had to be present in association with no

reactivity to probe B68 in the same region of an adjacent

section. If a B77-positive inclusion body could not be

located in the serial section treated with B68, then it was

not counted.

Fig. 2. In situ hybridization of monkey and human brain specimens to Nocardia-sp

Cortex of monkey M2, 48 h postinfection, hybridized with probe B77. Reactivity

from same specimen as (A) above, using the reverse complement sequence, probe B

10 Am). (C) Cortex of monkey M2, 48 h postinfection. An intracellular hybridizati

Hybridization reactivity is seen to intracellular coccobacillary, Nocardia-like struc

from the SN of the same PD specimen. Hybridization to an inclusion body, possibl

shown in (E) above but incubated with the reverse sequence, primer B68; no hybrid

Hybridization reaction to probe B77 within an inclusion body (scale bar = 10 Am)

with B77, demonstrating the variation in appearance of these inclusions (scale bar

inclusion with hybridization reactivity to probe B77 is seen (scale bar = 10 Am).

Determination of probe hybridization specificity

Probe specificities were evaluated by dot blot hybridiza-

tion analysis of PCR products using seven non-nocardial

reference strains, eight different isolates of Nocardia, and

two mammalian DNA specimens. PCR was performed using

the universal prokaryotic primers pA (Arnoldi et al., 1992)

eurology 184 (2003) 715–725 721

ecific probe B77 and the reverse oligonucleotide sequence, probe B68. (A)

is present within nocardial filaments (scale bar = 10 Am). (B) Serial section

68, as the primer. No staining of the nocardial filaments is seen (scale bar =

on reaction to N. asteroides (scale bar = 10 Am). (D) SN of a PD specimen.

tures; compare with panel (C) above (scale bar = 10 Am). (E) Another field

y a Lewy body (scale bar = 10 Am). (F) Serial section of the same inclusion

ization reactivity is seen (scale bar = 10 Am). (G) SN from a DLB specimen.

. (H) A different inclusion body in SN from the same specimen, hybridized

= 10 Am). (I) Cortex of monkey 93-R-204, 3.5 months after infection. An

Nine hybridization-positive inclusions were observed in this tissue section.

G. Chapman et al. / Experimental Neurology 184 (2003) 715–725722

and pE (Lane et al., 1985), coding for procaryotic 16S rRNA

at positions 8–27 and 926–908, respectively (E. coli num-

bering system of Brosius et al., 1978). These primers flanked

the region of 16S rRNA to be screened by probe B77,

resulting in an 893-bp fragment for nocardiae. PCR was

performed using 30 pmol each of primers pA and pE, with

either 12 Al of boiled sample preparation or 0.5–1 Ag of

genomic DNA. The reaction was carried out on a Perkin

Elmer Thermocycler (Perkin Elmer Life Sciences, Inc.,

Boston, MA) for 30 cycles (95jC � 1 min, 55jC � 1

min, 72jC � 1 min) with final extension at 72jC � 5 min.

Twenty microliters of each PCR reaction product was

electrophoresed on an 0.8% agarose gel, stained with ethi-

dium bromide, and observed by UV illumination. Dot blot

analysis was also performed on each PCR reaction product.

Three microliters of PCR product was placed on a Zetaprobe

nylon membrane (BioRad Laboratories, Hercules, CA). The

membrane was air-dried, denatured (0.5 M NaOH, 1.5 M

NaCl for 20 min), neutralized (0.5 M Tris HCl, pH 8.0/1.5 M

NaCl, 20 min), washed in this same buffer for 5 min, then

UV cross-linked (Genelinker, BioRad) and air-dried. Two

hundred nanograms of each in situ probe (B77 and B68)

were 5V end-labeled with 75 ACi-32P-ATP using T4 kinase

(Promega Corp., Madison, WI). The 20-Al kinase reaction

mixture was added to 40-ml prehybridization solution (5�SSC, 20 mM Na2HPO4, pH 7.2, with 7% SDS and 14%

formamide). After 30–60 min in prehybridization solution at

60jC, hybridization was performed for 2–4 h at the same

temperature. Blots were then rinsed in 2� SSC with 0.1%

SDS, washed in the same buffer for 10 min at 60jC followed

by 1� SSC with 0.1% SDS (10 min, 60jC) and 0.5� SSC

with 0.1% SDS (10 min, 60jC). Autoradiography was

performed on photographic film (Kodak XOMATAR, East-

man Kodak Co., Rochester, NY) at � 70j C for 25 h.

Results

Histopathological findings in experimentally infected

monkeys

No Gram-positive organisms were observed in brain

specimens from monkeys 93-R-204 and 93-R-205, who

were euthanized 3.5 months post-infection. However,

Nocardia was cultured from the brains of both animals.

Granulomatous cellular infiltrates were occasionally ob-

served, as well as small, Gram-negative, weakly acid-fast

spherical bodies similar to nocardial L-forms. Eosinophilic,

primarily intracellular inclusion bodies were also present

(Fig. 1A). Many of these inclusions were immunoreactive

for ubiquitin and a-synuclein (Figs. 1C and D) and resem-

bled the cortical Lewy bodies commonly observed in DLB

(Galasko, 2001); for comparison, Fig. 1B shows a cortical

Lewy body from a patient with the diagnosis of DLB.

Others were target-like, resembling the classical Lewy

bodies typically found in the PD SN. The monkey inclu-

sions were not reactive to PAS, Van Kassa calcium, Prussian

blue iron, or methenamine silver staining (data not shown).

Hematoxylin–eosin staining revealed marked variation in

their appearance, ranging from strongly eosinophilic (sim-

ilar to Lewy bodies) to basophilic, and varying in diameter

from 1 Am to more than 15 Am. No inclusions were

observed in brain specimens from monkeys M6 (not

infected), M2 (infected for 48 h), or M5 (infected for 1

year).

Electron microscopy revealed that the inclusions in the

monkey were composed of a compact array of filaments

(Fig. 1E, arrow) similar to those reported in Lewy bodies

(Gai et al., 2000). However, the Nocardia-induced inclusion

bodies in monkeys appeared to be more circumscribed (Fig.

1E) than reported for cortical Lewy bodies in humans

(McKeith et al., 1996).

Specificity of probe B77

The specificity of probe B77 was evaluated by dot blot

hybridization analysis of PCR products using nocardial

isolates, non-nocardial reference strains, and mouse and

human DNA. The eight nocardial strains were positive to

the probe whereas the non-nocardial strains and two mam-

malian DNA specimens were negative (Table 2). No reac-

tivity was observed with the reverse oligonucleotide

sequence, probe B68.

Hybridization of probe B77 in monkey cerebral cortex

specimens

Reactivity to digoxigenin-labeled probe B77 in the 48-

h infected monkey M2 was observed in filamentous struc-

tures resembling Nocardia (Figs. 2A and C). These struc-

tures were Gram-positive. The presence of Nocardia in

these specimens was confirmed by culture and electron

microscopy as reported previously (Beaman et al., 2000).

No B77 reactivity was observed in tissues from the nonin-

fected monkey, M6, nor was reactivity detected to either the

reverse probe (B68) (Fig. 2B) or nonlabeled probe B77 in

any specimens. Brain specimens from the 3.5-month

infected monkey, 93-R-204, displayed B77 reactivity within

some, but not all, of the inclusions described above (Fig.

2I); occasional extracellular reactivity was also noted. Hy-

bridization reactivity was not detected in the other 3.5-

month infected monkey, 93-R-205, although Nocardia was

also cultured from the cerebral cortex of this animal and

inclusion bodies similar to those observed in monkey 93-R-

204 were present. Reactivity was also not observed in the 1-

year infected monkey, M5.

Hybridization of probe B77 in human brain specimens

Reactivity to digoxigenin-labeled probe B77 was detected

in 9 of the 35 human SN specimens (Table 1). Two were

scored as 3+ (zfive reactions observed in the tissue section),

G. Chapman et al. / Experimental Neurology 184 (2003) 715–725 723

five were scored as 2+ (three or four reactions), and two were

scored as 1+ (one or two reactions). Decoding of the results

revealed that reactivity was limited to specimens containing

Lewy bodies in the SN. No reactivity with the reverse

oligonucleotide sequence probe 68 was observed (Fig. 2F).

Hybridization reactions, some resembling Nocardia-like

coccobacillary structures, were observed within inclusions

with an appearance similar to cortical Lewy bodies (Figs.

2D, E, G, and H), although they were in the SN. Similar to

our findings for monkey 93-R-204, the hybridization reac-

tions appeared to be primarily intracellular.

Discussion

The similarities between Lewy bodies and the inclusions

that were observed in our earlier study of Nocardia-infected

mice (Kohbata and Beaman, 1991) led to the present

investigation. Similar inclusions were present in the two

cynomolgus monkeys infected 3.5 months previously with

N. asteroides. Some of these inclusions resembled Lewy

bodies in the PD SN, with a dense core and peripheral halo,

whereas others lacked a distinct central core and more

closely resembled cortical Lewy bodies. Light and electron

microscopic observations indicated that most of the inclu-

sions were intracellular. Though the morphology of some of

the inclusion-bearing cells appeared similar to neurons,

staining for cell surface antigens was not performed. There-

fore, the cell type in which these inclusions were present is

uncertain. Some inclusions had an eosinophilic appearance

after staining with hematoxylin–eosin, similar to Lewy

bodies, while others were more basophilic and resembled

Michaelis–Gutmann bodies. These latter inclusions are

found in malakoplakia, a rare inflammatory condition of

unknown etiology occurring predominantly in the urinary

bladder and less commonly in other organs including the

brain (Gal et al., 1987; Ho, 1989; Rickert et al., 2000).

Relative to the present study, it is of interest to note that

infections with Nocardia and Rhodococcus have been

reported in cases of malakoplakia (Blackshear, 1970; Shin

et al., 1999).

Observation of the inclusions in the two 3.5-month

infected monkeys revealed some similarities and some

differences with Lewy bodies. The eosinophilic inclusions

were immunoreactive for a-synuclein and ubiquitin, and did

not stain for carbohydrates, similar to Lewy bodies (Forno,

1986). However, they also did not stain for calcium, iron, or

glycolipids, whereas some studies of Lewy bodies have

found staining for one or more of these substances (Cas-

tellani et al., 2000; Forno, 1996; Jellinger et al., 1990;

Kimula et al., 1983).

Hybridization of probe B77 to structures similar in

appearance to intact Nocardia in the cerebral cortex of

one of the 48-h infected monkeys M2 indicated that the in

situ hybridization technique could successfully be used to

detect nocardial RNA in brain specimens. Reactivity to

probe B77 within intracellular inclusions in one of the

3.5-month infected monkeys 93-R-204 confirmed the cul-

ture findings that the organism had persisted in the brain.

The failure of probe B77 to hybridize to the cortex from the

other 3.5-month infected monkey, despite a positive bacte-

rial culture and the presence of the inclusions described

above, was not explained. Nevertheless, the successful

application of the in situ hybridization technique in brain

specimens from monkeys M2 and 93-R-204 permitted the

subsequent investigation of the presence of nocardial RNA

in the human brain specimens.

Hybridization with probe B77 was detected in 9 of the 24

SN specimenswith Lewy bodies. These results are unlikely to

be ‘‘false positives’’ because reactivity was not observed in

adjacent sections hybridized with unlabeled probe B77 or its

reverse nucleotide. Further, detection of hybridization reac-

tions within specimens from all three brain banks in this study

(Table 1) argues strongly against the possibility that cross-

contamination of specimens during tissue processing could

have accounted for these results. (As stated in Materials and

methods, the dissection, sectioning, and mounting of brain

tissues were performed at three different institutions.)

BLAST analysis showed no significant homology between

these probe sequence and mammalian nucleic acid sequen-

ces. Because this analysis detected some evidence for ho-

mology to other actinomycetes, the possibility that probe B77

may have hybridized to an organism closely related to

Nocardia cannot be ruled out, although Nocardia species

were the only pathogens with 100% homology to it. The

hybridization reactions were observed primarily within in-

tracellular inclusions, though occasional extracellular reac-

tivity was also noted. Both intracellular and extracellular

Lewy bodies are present in PD and DLB (Iseki et al., 2000;

Yamada et al., 1991); further studies are required to determine

if the inclusions in which in situ hybridization reactivity to the

nocardial cDNA probe was present are, in fact, Lewy bodies.

Nocardia is widely distributed in soil, water, and vege-

tation (Beaman, 1992; Beaman and Beaman, 1994, 2000).

As a result, human exposure to this organism occurs

frequently (Hubble et al., 1995; Kohbata and Shimokawa,

1993). Serum samples were not available from the human

subjects whose brain specimens were used in these studies,

although anti-Nocardia antibodies were detected in the

experimentally infected (3.5-month-infected) monkeys (data

not shown). Because of the high percentage of seropositive

individuals in the normal population, it is unlikely that

serology would have been of value in detection of persistent

nocardial infection in the subjects in this study. In addition,

15% of individuals with culture-confirmed nocardial infec-

tions are seronegative (Hubble et al., 1995). Although most

clinical infections with Nocardia occur in immunocompro-

mised individuals, subjects with normal immune function

can also contract nocardiosis, usually from puncture

wounds or by inhalation. The organism has a propensity

for infecting the brain, and approximately one-quarter of

human infections involve the CNS (Beaman and Beaman,

G. Chapman et al. / Experimental Neurology 184 (2003) 715–725724

1994), typically resulting in the formation of abscesses or

granulomas (Beaman and Beaman, 1994, 2000). These

pathological findings are not generally present in disorders

in which Lewy bodies are present, and to our knowledge,

there have been no published reports of bacteria being

cultured from PD brain specimens. However, nocardiae

can convert to cell wall-deficient L-forms, which are not

detectable with standard culture procedures (Beaman and

Beaman, 1994). These L-forms have been isolated from

cerebrospinal fluid of patients with chronic nocardial CNS

infections (Beaman, 1982) and have also been detected in

mice that developed persistent head shaking following

experimental nocardial infection (Beaman, 1992; Beaman

and Scates, 1981). A recent study described acid-fast

spherical structures with a similar appearance to filterable

nocardiae in SN specimens from three subjects with PD

(Kohbata et al., 1998). These reports suggest that conver-

sion to an L-form may allow nocardial persistence, includ-

ing as a subclinical infection, in the human brain. Whether

the in situ hybridization reactivity in some of the PD and

DLB specimens may be due to the presence of L-forms is

not known; future studies employing both standard and L-

form-specific culture conditions, as well as PCR (which is

able to detect nocardial L-forms (Qasem et al., 2001), are

indicated to address this issue. Immunocytochemical stain-

ing is another technique that should be of value in evalu-

ating the presence of Nocardia in human brain specimens.

However, our efforts to immunostain Nocardia within the

brain in experimentally infected animals have thus far been

unsuccessful (Beaman, unpublished results). We suspect

that following nocardial invasion of neurons, the multiple

layers of membranes (as many as 30) that are seen by

electron microscopy to surround the organism (Beaman,

1993) may prevent adequate penetration of antisera.

Lewy body formation is likely to have multiple etiolo-

gies, and the process by which these structures are formed is

poorly understood. Factors that are thought to contribute to

this process include abnormal phosphorylation and incom-

plete proteolysis of neurofilament proteins (Trojanowski and

Lee, 1994), a-synuclein aggregation with neurofilament

subunits (Trojanowski and Lee, 1998), and co-localization

of parkin, a ubiquitin ligase, with aggregated a-synuclein

(Schlossmacher et al., 2002). The presence of Nocardia and/

or its neurotoxic secretory products (Camp et al., 2003)

within neurons may influence a-synuclein aggregation.

Though the results in the present study suggest the possi-

bility of an association between Nocardia and Lewy body-

containing disorders, we caution that independent confir-

mation of these findings, using a variety of methodologies,

is required before arriving at definitive conclusions.

Acknowledgments

Thanks are expressed to the Department of Neuro-

pathology, Albert Einstein College of Medicine (New York,

NY), the University of California at Davis Tissue Reposi-

tory (Davis, CA, supported by NIH grants AG10129 and

AG12435), and to the Harvard Brain Tissue Resource

Center (Belmont, MA, supported by USPHS grant MH/NS

31862) for providing brain specimens and reports. Thanks

are also expressed to Dr. Anders A.F. Sima (Wayne State

University) for helpful discussions regarding the manu-

script. This investigation was supported by grants to B.L.B.

(RO1-AI42144, from the National Institute of Allergy and

Infectious Diseases, NIH) and to P.A.L. (R01-ES010793

from the National Institute of Environmental Health

Sciences, NIH, and a Center of Excellence grant from the

National Parkinson Foundation, Miami, FL). Portions of this

manuscript involving the methodology for the in situ

detection of nocardial 16S rRNA were from the doctoral

dissertation of Gail Chapman.

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