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We assessed whether the intracellular bacterium Chlamydia pneumoniae was present in post-mortem brain samples from patients with and without late-onset Alzheimer's disease (AD), since some indirect evidence seems to suggest that infection with the organism might be associated with the disease. Nucleic acids prepared from those samples were screened by polymerase chain reaction (PCR) assay for DNA sequences from the bacterium, and such analyses showed that brain areas with typical AD-related neuropathology were positive for the organism in 17/19 AD patients. Similar analyses of identical brain areas of 18/19 control patients were PCR-negative. Electron- and immunoelectron-microscopic studies of tissues from affected AD brain regions identified chlamydial elementary and reticulate bodies, but similar examinations of non-AD brains were negative for the bacterium. Culture studies of a subset of affected AD brain tissues for C. pneumoniae were strongly positive, while identically performed analyses of non-AD brain tissues were negative. Reverse transcription (RT)-PCR assays using RNA from affected areas of AD brains confirmed that transcripts from two important C. pneumoniae genes were present in those samples but not in controls. Immunohistochemical examination of AD brains, but not those of controls, identified C. pneumoniae within pericytes, microglia, and astroglia. Further immunolabelling studies confirmed the organisms' intracellular presence primarily in areas of neuropathology in the AD brain. Thus, C. pneumoniae is present, viable, and transcriptionally active in areas of neuropathology in the AD brain, possibly suggesting that infection with the organism is a risk factor for late-onset AD.
A-K Ultrastructural analyses of brain tissues from AD patients to identify EB and RB of C. pneumoniae. Brain tissues from AD patients PCR-positive for the organism were prepared for EM and IEM analysis as described in Materials and methods; the antichlamydial Ab used in the latter analysis was a mAb targeting a C. pneumoniae outer membrane protein (see text). A Typical inclusion body with presumptive EB and RB as seen in EM analysis of temporal cortex of patient AD8; sizes of chlamydia-like bodies typically range from 0.2 to 1.0 µm, which is characteristic for this organism (bar = 0.5 µm). B Pear-shaped presumptive EB of C. pneumoniae as identified in EM analysis of the temporal cortex of patient AD14 (bar = 0.2 µm). C Another typical inclusion body with presumptive EB and RB as seen in EM analysis of temporal cortex of patient AD8 (bar = 1.0 µm). D IEM analysis of the temporal cortex of patient AD8, showing C. pneumoniae EB labelled with the antisurface protein mAb (arrow, 15-nm gold particles) (bar = 0.25 µm). E IEM analysis of the hippocampus of patient AD7, showing RB labelled with the anti-surface protein mAb (arrows, 15-nm gold particles) (bar = 1.0 µm). F, G Higher magnification of IEM analyses from additional sections of the hippocampus of patient AD7, showing RB labelled with the anti-surface protein mAb (F, 15-nm gold particles; G, 5-nm gold particles, arrowheads) (bars = 0.25 µm). H, I IEM analysis of the temporal cortex of two different control brains (H, C18MS; I, C8) using the anti-surface protein mAb (bars = 1.0 µm). Note that no structures were immunolabelled in these sections. J Low magnification EM of a typical uninfected astrocytoma (SW1088) cell in culture, obtained from the ATCC (bar = 1.0 µm). K High magnification EM of an astrocytoma (SW1088) cell infected with the laboratory strain of C. pneumoniae (TW-183), also obtained from the ATCC. A pear-shaped organism characteristic of C. pneumoniae is apparent in the center of the cell (arrow) (bar = 0.5 µm)
… 
A-J Immunohistochemical analysis of brain tissues from control (non-AD) and AD patients using two primary mAb, one targeting a C. pneumoniae surface protein and the second targeting the chlamydial LPS. Preparation of tissues and details of the primary and secondary Abs employed are described in Materials and methods. A Immunolabelling of perivascular cells (arrows) using the anti-LPS mAb; tissue is from the temporal cortex of patient AD4 (bar = 50 µm). B Immunolabelling of apparent glial cells (arrowheads) in the dentate gyrus of patient AD4, using the anti-LPS mAb (bar = 50 µm). C Immunolabelling of tissue from the temporal cortex from a nonAD control patient (C17MS, Table 1) using the anti-LPS mAb (bar = 50 µm). D Immunolabelling of perivascular cells (arrows) using the anti-surface protein mAb; tissue is from the temporal cortex of patient AD2 (bar = 25 µm). E Immunolabelling of apparent glial cells (arrowheads) in the temporal cortex of patient AD7 using the anti-surface protein mAb (bar = 50 µm). F Immunolabelling of tissue from the dentate gyrus from a non-AD control patient (C18MS, Table 1) using the anti-surface protein mAb (bar = 50 µm). G Control immunolabelling of a section from the dentate gyrus of patient AD5 using normal mouse serum as primary Ab (bar = 100 µm). H Control immunolabelling of a section from the temporal cortex of patient AD7 using normal rabbit serum as primary Ab (bar = 100 µm). I Immunolabelling of a section from the temporal cortex of patient C17MS using anti-CD54 mAb (bar = 100 µm). J Immunolabelling of a section from the temporal cortex of patient AD7 using anti-CD54 mAb (bar = 100 µm)
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Abstract We assessed whether the intracellular bacte-
rium Chlamydia pneumoniae was present in post-mortem
brain samples from patients with and without late-onset
Alzheimers disease (AD), since some indirect evidence
seems to suggest that infection with the organism might be
associated with the disease. Nucleic acids prepared from
those samples were screened by polymerase chain reaction
(PCR) assay for DNA sequences from the bacterium, and
such analyses showed that brain areas with typical AD-re-
lated neuropathology were positive for the organism in
17/19 AD patients. Similar analyses of identical brain ar-
eas of 18/19 control patients were PCR-negative. Electron-
and immunoelectron-microscopic studies of tissues from
affected AD brain regions identified chlamydial elemen-
tary and reticulate bodies, but similar examinations of non-
AD brains were negative for the bacterium. Culture stud-
ies of a subset of affected AD brain tissues for C. pneu-
moniae were strongly positive, while identically per-
formed analyses of non-AD brain tissues were negative.
Reverse transcription (RT)-PCR assays using RNA from
affected areas of AD brains confirmed that transcripts from
two important C. pneumoniae genes were present in those
samples but not in controls. Immunohistochemical exam-
ination of AD brains, but not those of controls, identified
C. pneumoniae within pericytes, microglia, and astroglia.
Further immunolabelling studies confirmed the organisms’
intracellular presence primarily in areas of neuropathology
in the AD brain. Thus, C. pneumoniae is present, viable,
and transcriptionally active in areas of neuropathology in
the AD brain, possibly suggesting that infection with the
organism is a risk factor for late-onset AD.
Key words Chlamydia · Alzheimers disease ·
Inflammation · Dementia · Infection
Introduction
Alzheimers disease (AD) is a severe mental health prob-
lem affecting upwards of 4 million people in the United
States [44]. Studies have established that AD appears in
two distinct manners: an early-onset form and a late-on-
set, sporadic form. Incidence of the latter increases with
age, and AD is now thought to be the most important sin-
gle cause of senile dementia. Estimates of the prevalence
of late-onset AD based on epidemiologic evidence vary
Med Microbiol Immunol (1998) 187: 23–42 © Springer-Verlag 1998
Received: 4 May 1998
Brian J. Balin · Hervé C. Gérard · E. James Arking
Denah M. Appelt · Patrick J. Branigan
J. Todd Abrams · Judith A. Whittum-Hudson
Alan P. Hudson
Identification and localization of
Chlamydia pneumoniae
in the Alzheimer’s brain
ORIGINAL INVESTIGATION
B. J. Balin · E. J. Arking · D. M. Appelt
Department of Pathology and Laboratory Medicine,
MCP-Hahnemann School of Medicine,
Allegheny University of the Health Sciences,
New College Building, Broad and Vine Streets,
Philadelphia, PA 19102, USA
H. C. Gérard · P. J. Branigan · A. P. Hudson (
½)
1
Department of Microbiology and Immunology,
MCP-Hahnemann School of Medicine,
Allegheny University of the Health Sciences,
2900 Queen Lane, Philadelphia, PA 19129, USA;
and Medical Research, Department
Veterans Affairs Medical Center,
University and Woodland Avenues,
Philadelphia, PA 19104, USA
J. T. Abrams
Department of Dermatology,
MCP-Hahnemann School of Medicine,
Allegheny University of the Health Sciences,
New College Building, Broad and Vine Streets,
Philadelphia, PA 19102, USA
J. A. Whittum-Hudson
2
Ocular Immunology Laboratories,
Wilmer Ophthalmological Institute,
Johns Hopkins University School of Medicine,
600 North Wolfe Street,
Baltimore, MD 21287, USA
H. C. Gérard and E. J. Arking contributed equally to the work pre-
sented here. B. J. Balin and A. P. Hudson are equivalent senior au-
thors for this publication.
Present addresses:
1
Department of Immunology and Microbiology, Wayne State Uni-
versity School of Medicine, Gordon H. Scott Hall, 540 East Canfield
Avenue, Detroit, MI 48201, USA
e-mail: ahudson@med.wayne.edu; Tel.: 313-993-6641; Fax: 313-
577-1155
2
Department of Medicine/Division of Rheumatology, Lande Medi-
cal Research Building, 550 East Canfield Avenue, Detroit, MI 48201,
USA
[25], and incidence of the disease appears to differ with the
population examined; however, at least half of the total
cases of dementia in the elderly may be attributable to AD
[11, 25]. Thus, AD is a significant mental health concern
and will increase in importance with the continued aging
of the population.
The causes underlying the signature neuropathology
seen in essentially all AD patients, i.e., the neurofibrillary
tangles (NFTs) and neuropil threads (NTs) comprised of
modified tauprotein and the neuritic senile plaques (NSPs)
comprised of deposits of
β
-amyloid peptide (A
β
), are
poorly understood. tau is a normal component of the neu-
ronal cytoskeleton, and evidence suggests that its abnor-
mal deposition in NFTs, NTs, and dystrophic neurites re-
sults from various types of aberrant post-translational mod-
ification of the protein [1, 2, 49, 91]. It is not clear whether
NFTs are primary lesions in AD, or whether their forma-
tion is a response to other neuronal injuries [29]; some data
suggest that the level of NFT accumulation correlates more
closely with the degree of cognitive impairment in AD pa-
tients than do other factors [60]. Regardless, A
β
deposi-
tion appears to be critical in the neuronal degeneration pro-
cess observed in AD [84]. In the early-onset, familial form
of the disease (FAD), mutations in the amyloid precursor
protein gene (
β
APP) are associated with increased A
β
de-
position and early onset of symptoms [98]. Mutations in
genes encoding the proteins presenilin-1 (PS-1) and pre-
senilin-2 (PS-2) also lead to increased A
β
deposition in
FAD patients and in an animal model [22, 48, 51, 85]. Mu-
tations in PS-1, PS-2, and
β
APP account for most early-
onset, FAD cases [19, 48]. Late-onset AD is, however, far
more prevalent than is FAD, and while the latter is almost
certainly genetically based, the former is not. One risk fac-
tor identified for late-onset disease is the heterozygous or
homozygous presence of the APOE
ε
4 allele [83, 84]. Not
all patients expressing
ε
4 develop AD, but it has been re-
ported that the allele increases risk for the disease several-
fold, and that its presence is associated with earlier onset
and more rapid progression in FAD [20, 83]. One recent
study suggested, however, that
ε
4 may be a risk factor pri-
marily in individuals younger than 70 years of age and that
it may be involved in only a minority of late-onset AD
cases [6].
A well-recognized aspect of late-onset AD neuropathol-
ogy is the presence of inflammation at sites of NSP and
NFT deposition. The association between elevated levels
of proinflammatory cytokines and the inflammatory/im-
mune processes characteristic of AD-related pathology has
been demonstrated by several laboratories [3, 35]. Inter-
leukin (IL)-1
β
[35], IL-1
α
, IL-6, and tumor necrosis fac-
tor alpha (TNF-
α
), all of which are produced by astrocy-
tes and microglia, are elevated in AD [3] and may account
for reported damage to microvessels in the disease [37, 68].
One recent study correlated microglial over-expression of
IL-1 and activated astroglia with formation and evolution
of NFTs in AD [86]. Cytokine receptor expression on mi-
croglia has been shown to differ in AD and normal brain,
and such differences in expression probably reflect the in-
creased presence of reactive glia in the AD brain [82, 90].
Moreover, activated microglia produce reactive oxygen
species, such as NO, which can kill cells by inhibition of
glycolysis, the tricarboxylic acid cycle, and DNA synthe-
sis [97]. Production of NO may be a factor in the degen-
erative process in AD, since lipoperoxidation, glycoxida-
tive and other oxidative protein modifications have all
been implicated in the pathogenesis process of this dis-
ease [88].
The neuropathology characteristic of both early- and
late-onset AD is similar, but the detailed initiating events
leading to that pathology remain to be elucidated for late-
onset disease [18, 53]. Neurologic disease can be caused
by microorganisms, and infection with agents that do not
target the nervous system directly can elicit neuropatho-
logic side effects [8, 42]. Because of this, several groups
have attempted to establish a causal relationship between
viral infection and sporadic AD, but no such link has been
demonstrated; e.g., measles virus, various lentiviruses,
adenovirus, and others have been dismissed as agents as-
sociated with late-onset AD (e.g., [26, 58, 76]). Various
bacteria, including Chlamydia psittaci and Coxiella bur-
netii (e.g., [79]), have been dismissed also, although this
was based on negative serologic evidence. One study iden-
tified herpes simplex virus type 1 (HSV-1) infection as a
risk factor for development of AD in people expressing
APOE
ε
4 [41]; however, it is not clear how and under what
circumstances this virus might interact with the allele or
its gene product to produce or promote disease. In addi-
tion to viruses and bacteria, unconventional agents (i.e.,
prions) have been considered in the pathogenesis of AD
and discarded [59, 96]. The possible roles of environmen-
tal factors, including diet and acute or extended exposure
to aluminum, have also been investigated, but no defini-
tive role has been demonstrated [27, 44]. Interestingly, a
recent study has suggested that head trauma may be asso-
ciated with A
β
deposition in individuals carrying the
APOE
ε
4 allele [72].
Chlamydia pneumoniae is an intracellular bacterium
that is a respiratory pathogen, initially infecting the oral
and nasal mucosa [32]. This organism is a significant agent
in acute respiratory infections, including pneumonia, si-
nusitis, and bronchitis [31, 32]. Recent studies have im-
plicated C. pneumoniae also in more severe and chronic
pulmonary pathologies, including sarcoidosis and chronic
obstructive pulmonary disease [32, 33]. Epidemiologic
analyses have suggested that prevalence of infection with
C. pneumoniae is high in all adult populations studied, and
that it may increase with increasing age [36, 40, 50]. In
the Western world where population densities are rela-
tively low, children under the age of 510 years rarely
show significant levels of anti-C. pneumoniae antibodies
(Abs), although lack of such Abs does not always reflect
actual infection rate (e.g., [7]). Incidence of these Abs
rises with increasing age [31, 50], and it has been reported
that Ab titers against C. pneumoniae peak in the 6th7th
decades in most populations studied; e.g., men 60 years
and older in Seattle showed a prevalence rate of 70% in
one study [31, 50]. Some research suggests that virtually
everyone is infected with C. pneumoniae at some time dur-
24
ing his/her lifetime, and that reinfection may be common
(e.g., [50]).
Immunopathology is a general hallmark of chlamydia-
induced disease. In vivo sites of chlamydial infection
show chronic inflammation and generally contain T cells,
monocytes/macrophages, and, at some sites, B lympho-
cytes [9294]. It is not clear whether chlamydial infec-
tion elicits the inflammatory response directly by upreg-
ulating cytokine production in infected or neighboring
cells, or whether the immune response to infected cells
drives inflammation via influx of cytokine-producing
lymphocytes/macrophages. Late after development of
chronic inflammation, Th1/Th2 CD4
+
cells, as well as
CD8
+
cells and macrophages, are likely to be present at
the site of inflammation; these have all been detected in
synovia of patients with C. trachomatis-induced reactive
arthritis [14, 87] and in the conjunctivae of trachoma pa-
tients [78]. Interferon (IFN)
γ
upregulates and/or induces
production of many cytokines, and IFN
α
/
β
and IL-12 can
upregulate IFN
γ
and other cytokine expression [4]. Evi-
dence suggests that when persistent chlamydial infection
is initiated, that state is maintained in part by host pro-
ducts such as IFN
γ
, which are synthesized to clear the or-
ganism (see [57] for review).
Infection with C. pneumoniae has been implicated re-
cently in several non-pulmonary clinical entities, includ-
ing meningoencephalitis and atherosclerosis [15, 30, 46,
69]. A causal relationship between chlamydial infection
and the latter remains controversial, but a number of stud-
ies have reported a correlation between serum anti-C. pneu-
moniae Ab titers and coronary artery disease; moreover,
the bacterium has been identified by several methods in
atheromatous plaques (e.g., [16, 69]), although not all such
studies have succeeded in doing so. Importantly, while not
yet directly demonstrated, one report has suggested that
the organism may be associated with infections of the cen-
tral nervous system (CNS) [46] and with cerebrovascular
disease [95], again on the basis of serologic data. Because
a recent report [39] postulated a relationship among ath-
erosclerosis, APOE
ε
4 expression, and late-onset AD, be-
cause inflammation is characteristic of the AD brain and
is also a hallmark of chlamydial infection, and because of
its reported ubiquity in older adults, we investigated a pos-
sible relationship between infection with C. pneumoniae
and sporadic AD. We demonstrate here that this bacterium
is present, viable, and metabolically active in brain areas
showing neuropathology in most AD patients studied, but
that it is not found in similar brain regions of non-AD pa-
tients.
Materials and methods
Patient samples
Post-mortem tissue samples from various brain regions of patients
with and without AD were obtained from the Harvard Brain Tissue
Resource Center (Boston, Mass., USA), through Dr. Gail Johnson of
the University of Alabama Brain Resource Center (Birmingham,
Ala., USA), through Dr. William Hill of the Medical College of Geor-
gia (Augusta, Ga., USA), and from the MCP-Hahnemann School of
Medicine Department of Pathology (Philadelphia, Pa., USA). All
samples from patients diagnosed as late-onset AD were confirmed
at autopsy by histopathologic examination by a certified neuropa-
thologist, using standard criteria (NINDS/CERAD; [64]). Samples
from non-AD patients were age-matched as well as possible to those
of AD patients, and each was examined histologically for NSPs and
NFTs and confirmed as non-AD. Average age of control patients was
72.6 years; that of AD patients was 77.7 years. All AD and non-AD
samples were screened by polymerase chain reaction (PCR) assay
without knowledge of AD status. Virtually all AD and many control
samples were examined by electron and immunoelectron microsco-
py (EM, IEM, respectively) and/or immunohistochemistry; howev-
er, each sample could not be subjected to every analysis owing to
limited tissue availability. Table 1 summarizes information for con-
trol patients from whom samples were obtained; Table 2 provides
congruent data for AD patients.
Preparation and analysis of nucleic acids
Nucleic acids were prepared from tissue samples as described [9]; 1 µg
nucleic acids was used in each PCR screening assay. Elementary
bodies (EB) of C. pneumoniae strain TW-183 were obtained from
the American Type Culture Collection (ATCC), and DNA was pre-
pared from them for control amplifications. PCR assays to screen for
C. pneumoniae chromosomal DNA targeted two genes, each in an
independent system. In one, primers targeting the 16S ribosomal
RNA (rRNA) gene were used as described [28]. The second assay
targeted the chlamydial major outer membrane protein (MOMP) gene
(ompA) and used primers designed using GeneRunner software (Has-
tings Software, Hastings, N.Y., USA); those primer sequences were
derived from bases 2643 and 548567 (outer) and bases 115135
and 444462 (inner) of the C. pneumoniae MOMP coding sequence
[61]; they were analyzed for sequence specificity via “Blast” com-
parison with all DNA sequences in GenBank and tested extensively
under various assay conditions. Cycling for PCR assays was done in
a Barnstead/Thermolyne instrument, and products were analyzed on
2% agarose gels. Positive signal in the 16S rRNA-directed assay is
a band of 463 bp; that in the MOMP-directed assay is a band of
347 bp. All assays were independently done in duplicate by two dif-
ferent investigators on different days and in a fully blinded fashion.
Extreme care was taken in all assays to avoid cross-contamination
of both nucleic acid samples to be analyzed and reaction mixtures;
such measures included preparation of nucleic acids in a laboratory
separate from those in which PCR or reverse transcription (RT)-PCR
assays were set up and use of eight different biologic hoods, each in
a different laboratory, for setting up reactions. Results of screening
for each sample were always consistent between the 16S- and
MOMP-directed PCR assays. Primers for PCR assays targeting se-
quences from C. trachomatis, Borrelia burgdorferi, Mycoplasma
pneumoniae, and M. hominis were as described [9, 52]. APOE gen-
otypes were determined as described [38].
For RT-PCR assays, RNA was prepared from total nucleic acids
by digestion with RNase-free DNase1 (RQ1; Promega Biotech, Mad-
ison, Wis., USA); purity was assessed by PCR using primers below,
without RT. RT reactions used 1 µg total RNA, were done as described
[67], and employed nested primer systems for the gene specifying a
Mr=76000 protein containing an epitope specific for C. pneumoni-
ae (outer: bases 160–184, 1066–1086; inner: bases 373–392,
799–822; [74]), and the 3-deoxy-alpha-
D
-manno-octulosonic acid
transferase (KDO transferase) gene from the organism (outer: bases
132–152, 946–965; inner: bases 527–549, 797–815; [54]). As an inter-
nal control for the quality of RNA analyzed, we employed an RT-PCR
assay targeting transcripts from the human nuclear gene specifying
subunit 4 of cytochrome oxidase (COIV), the terminal complex in the
mitochondrial electron transport chain (bases 184–213, 319–348; [55,
71]). Primers for the C. pneumoniae transcripts were designed and
their specificity confirmed as given above. As with the PCR screen-
ing assays, precautions taken in preparation of RNA to be analyzed
and in the analyses themselves included independent preparation of
25
RNA from separate portions of the same samples by different inves-
tigators in different laboratories and biologic hoods, and use of sev-
eral hoods and reserved sets of pipettes for preparation of reaction
mixtures. Positive signals for the C. pneumoniae Mr=76000 protein
and KDO transferase RT-PCR assays are bands of 449 bp and 288 bp,
respectively. Product size for the COIV-directed assay is 164 bp. Iden-
tity of PCR and RT-PCR products was confirmed by hybridization.
Electron-microscopic and immunoelectron-microscopic analyses
Brain tissues from AD/non-AD patients were immersion-fixed in 4%
paraformaldehyde in phosphate-buffered saline (PBS) and cut into
1-mm
3
blocks. Blocks were osmicated in 1% OsO
4
prior to propy-
lene oxide/resin infiltration and embedment in Embed-812 (Electron
Microscopy Sciences, Fort Washington Pa., USA). Sections were cut
26
Patient Sex Age at Cause of death
b
Time PCR
a
APOE
c
death PM
(years) (hours) CB HP Tcx Other
C1 F 74 Respiratory failure 14 Pcx+
ε
3/
ε
3
C2 F 58 Multi-organ failure 4
ε
3/
ε
3
C3 F 90 Myocardial infarction 9 na
ε
3/
ε
3
C4 M 57 Cardiac arrest 11
ε
3/
ε
4
C5 M 66 Sepsis 8
ε
3/
ε
4
C6 F 78 Pulmonary disease 5 na
ε
3/
ε
3
C7 F 83 Renal failure 3 na
ε
3/
ε
3
C8 M 71 Aortic aneurysm 6 na
ε
3/
ε
3
C9 F 81 Respiratory failure 8 na
ε
3/
ε
3
C10 M 50 Adenocarcinoma 10 na
ε
3/
ε
3
C11 M 68 Adenocarcinoma 4 na
ε
3/
ε
4
C12 F 90 Lacunar infarcts 4 na
ε
3/
ε
4
C13 F 84 Pneumonia 7 na
ε
3/
ε
3
C14 M 64 Respiratory failure 10 na na Pfcx–
ε
2/
ε
3
C15 M 74 Respiratory failure 5 na na Pfcx–
ε
3/
ε
3
C16 M 77 Congestive heart failure 5 na na Pfcx–
ε
3/
ε
3
C17MS
d
M71 na 17
ε
2/
ε
3
C18MS F 74 Sepsis 7
ε
3/
ε
3
C19MS M 69 Cancer 3
ε
3/
ε
3
a
Independent PCR assays targeting the 16S rRNA and ompA genes of C. pneumoniae from each pa-
tient. All samples were negative in both assays except for the Pcx sample for patient C1, which was
weakly PCR-positive. We confirmed this positivity using the nested amplification systems for the 76 kDa
protein and the KDO transferase genes given in Materials and methods. A fully consistent set of sam-
ples was not available for the PCR and other analyses described for most patients studied here
b
Proximal cause of death, given as listed on autopsy report
c
APOE genotype was determined by the method of Hixson and Vernier [38]
d
Patients indicated as MS had multiple sclerosis
Table 1 Summary of control
patient characteristics and
Chlamydia-directed PCR
results (PM post-mortem,
CB cerebellum, HP hippocam-
pus, TCx temporal cortex, Pcx
parietal cortex, Pfcx prefrontal
cortex, na not available)
Patient Sex Age at Cause of death
b
Time PCR
a
APOE
c
death PM
(years) (hours) CB HP Tcx Other
AD1 F 82 Cardiac arrest 11 + na
ε
3/
ε
3
AD2 F 85 Cardiac arrest 7 + na
ε
3/
ε
4
AD3 F 81 Cancer 10 na
ε
3/
ε
3
AD4 M 87 Cardiac arrest 6 + na
ε
3/
ε
4
AD5 F 77 Cancer 15 na
ε
3/
ε
3
AD6 M 68 Sepsis 7 + + Pcx+
ε
3/
ε
3
AD7 F 82 Cardiac arrest 12 + na
ε
3/
ε
4
AD8 F 61 Lung carcinoma 23 na + Fcx+
ε
4/
ε
4
AD9 F 78 Pneumonia 22 + na +
ε
2/
ε
4
AD10 F 86 Heart failure 11 + na +
ε
3/
ε
4
AD11 F 70 Gangrenous bowel 9 na + Pcx+
ε
3/
ε
4
AD12 F 79 Sepsis 16 na +
ε
3/
ε
3
AD13 F 70 Aspiration pneumonia 24 na +
ε
4/
ε
4
AD14 F 87 Pneumonia 8 + +
ε
3/
ε
3
AD15 F 90 Atherosclerosis 9 na +
ε
3/
ε
4
AD16 M 67 Systemic infection 8 + na na Pfcx+
ε
3/
ε
4
AD17 F 78 Respiratory failure 6 na na Pfcx+
ε
3/
ε
3
AD18 F 74 Pneumonia 3 na na Pfcx+
ε
3/
ε
4
AD19 F 78 Renal failure 4 + na na Pfcx+
ε
3/
ε
3
a
Independent PCR assays targeting the 16S rRNA and ompA genes of C. pneumoniae from each pa-
tient. All samples indicated as positive were unequivocally positive in both assays (see Materials and
methods)
b
Proximal cause of death, given as listed on autopsy report
c
APOE genotype was determined by the method of Hixson and Vernier [38]
Table 2 Summary of AD
patient characteristics and
Chlamydia-directed PCR
results (Fcx frontal cortex)
on a Sorvall Porter-Bloom MT2B ultramicrotome, post-stained with
2% uranyl acetate, and viewed/photographed on a Zeiss 10 electron
microscope. For pre-embed IEM, 1-mm
3
blocks of tissue were fixed
overnight in 0.05% glutaraldehyde, 0.1% saponin in 0.2 M phosphate
buffer pH 7.0. After fixing, tissues were blocked for 2 h in 100 mM
ammonium chloride in 0.2 M phosphate buffer pH 7.0, followed by
incubation with a monoclonal Ab (mAb) (clone RR402; [77]; Wash-
ington Research Foundation, Seattle, Wash., USA) specific for an as
yet unidentified outer surface protein of C. pneumoniae diluted 1:10
in 0.2 M phosphate buffer pH 7.0, 5% cold water fish gelatin (CWFG;
Sigma Chemical Co., St. Louis, Mont., USA), at room temperature
for 15 min. Following rinses in 0.2 M phosphate buffer containing
5% CWFG, 20 mM glycine, tissues were incubated overnight at 4°C
with 5 nm or 15 nm gold-conjugated anti-mouse secondary Ab
(Amersham Life Sciences, Arlington Heights, Ill., USA) diluted 1:50
in 0.2 M phosphate buffer. Subsequently, they were rinsed in 0.2 M
phosphate buffer and refixed in 2% glutaraldehyde solution, followed
by immersion in 1% OsO
4
for 30 min at room temperature. Tissues
were dehydrated in ethanols, contrast enhanced with 2% uranyl ac-
etate, and processed for embedment in epon resin. Thin sections were
prepared, viewed, and photographed as above. For post-embed IEM,
thin sections of tissues embedded in Epon 812 were etched with 7%
H
2
O
2
for 60–75 s and rinsed with ddH
2
O. Sections were treated with
citric acid buffer (pH 6.0) for 2 min, rinsed with ddH
2
O, and incu-
bated for 1 h with a primary anti-outer surface protein mAb (undi-
luted; also clone RR402 obtained from Dako Corp., Carpenteria,
Calif., USA). Sections were rinsed in ddH
2
O, blocked for 2 min with
1% CWFG in PBS, 0.1% acetylated bovine serum albumin (BSA).
Sections were incubated with 15 nm gold-conjugated mouse secon-
dary Ab (Amersham) diluted 1:5. Then sections were rinsed, post-
stained with 2% uranyl acetate, and viewed.
Immunohistochemical analyses
Formalin-fixed paraffin-embedded blocks of tissue were cut into
7–10µm sections and prepared for immunohistochemical analysis
as described [2]. Briefly, after standard rehydration and antigen re-
trieval, tissues were blocked for endogenous peroxidase before
blocking for 1 h with 5% dried milk solids in Tris buffer, and again
with 1% CWFG. Sections from formalin-fixed tissues were incubat-
ed with the primary mAb targeting the surface-exposed protein of
C. pneumoniae (Washington Research Foundation; 1:501:250 di-
lution in 5% dried milk solids) or with the commercial, identical mAb
from the Dako Corp. (1:5 dilution in 5% dried milk solids). In relat-
ed studies, a mAb targeting the chlamydial lipopolysaccharide (LPS)
was used (1:50–1:250 dilution in 5% dried milk solids; gift of
Dr. S. Tirrell, Chiron Diagnostics). In yet other studies, consecutive
tissue sections were immunolabelled with anti-surface protein mAb
and the PHF-1 mAb targeting the PHF-tau protein [34]. In all cases,
slides were incubated with primary Ab for 14 h at 4°C, washed
with Tris buffer at room temperature, incubated with goat anti-mouse
or goat anti-rabbit IgG as secondary Ab (Amersham; 1:200 dilution
in 5% dried milk solids) for a minimum of 1 h at room temperature
or overnight at 4°C. After rinsing, tissues were incubated with
ClonoPAP (mouse or rabbit; Sternberger Monoclonals Inc., Balti-
more, Md., USA; 1:200 dilution in 5% dried milk solids) for 30 min.
Slides were washed in Tris buffer before/after development with
0.05% DAB (Sigma) in 0.01% H
2
O
2
for 8 min at room temperature.
Tissues were dehydrated with ethanols/xylenes and mounted in Per-
mount (Fisher Biotech, Pittsburgh, Pa., USA). For double immuno-
labelling, a primary (anti-mouse) mAb targeting glial fibrillary acid-
ic protein (GFAP; SMI21, Sternberger Monoclonals) was used in
conjunction with a rabbit primary polyclonal Ab (pAb) targeting the
chlamydial LPS. In complementary studies, a rabbit primary pAb
targeting inducible nitric oxide synthase (iNOS; Transduction La-
boratories, Lexington, Ky., USA) and the anti-C. pneumoniae sur-
face protein mAb were used. Secondary Abs were either anti-mouse
IgG conjugated to horse radish peroxidase (HRP; Amersham; 1:200
dilution) specific for the mAb, or anti-rabbit IgG conjugated with
fluorescein isothiocyanate (FITC; Sigma; 1:20–1:50 dilution). In
all double-immunolabelling experiments, primary Abs were added
to sections simultaneously for 14 h. Following rinsing and reblock-
ing, sections were incubated with secondary Abs overnight at 4°C,
rinsed and treated with mouse ClonoPAP, and processed for immu-
noperoxidase as above. Sections were examined using an Olympus
BX60 microscope with epifluorescence (courtesy Dr. D. Simon, Ms.
K. Wallace). In all immunohistochemical analyses, appropriate con-
trols were done in parallel in each staining experiment; these includ-
ed staining with normal mouse and/or rabbit serum, staining with an
irrelevant primary Ab (anti-CD54; PharMingen, San Diego, Calif.,
USA), or omission of primary Ab.
Culture analyses for C. pneumoniae
Culture analyses for C. pneumoniae were done by infection of a hu-
man monocyte/macrophage cell line (THP-1; obtained from Dr. Da-
vid Bernard, Allegheny University of the Health Sciences). Approx-
imately 0.5 g of frozen brain tissue from temporal cortex of two pa-
tients previously determined to be C. pneumoniae-positive by PCR
and immunohistochemical analyses, as well as congruent tissue sam-
ples from two control brains, were subjected to three freeze-thaw cy-
cles; tissues were homogenized and sonicated between cycles. From
these samples, 100 µl of homogenate were mixed with 3×10
6
THP-1
cells in 2 ml 10% fetal calf serum in RPMI-1640, 0.5% HEPES buf-
fer (pH 7.0). The homogenate and cells were centrifuged at 500×g
for 30 min, diluted to 10 ml volume in medium, then cultured for 72 h.
Following this initial culture incubation, 1 ml of culture supernatant
was used for a second passage on 3×10
6
fresh THP-1 cells; remain-
ing cells from the initial 72-h incubation were separated from debris
by centrifugation on Ficoll-Paque (Pharmacia-Biotech, Piscataway,
N.J., USA) gradients. Cells from both first and second culture pas-
sages were subjected to immunocytochemical analyses to confirm
infection of the cultured THP-1 cells. Cells for immunocytochemis-
try were diluted to 2.5×10
5
/ml in Hanks balanced salt solution, and
0.2 ml were cytospun onto ProbeOn+ (Fisher Biotech) at 500×g for
5 min, using a Shandon Cytocentrifuge III. Cells were fixed for at
least 1 h at room temperature with Streck Tissue Fixative (S.T.F.
tm
Streck Laboratories Inc., Omaha, Neb., USA), followed by a rinse
with 1× Automation buffer (Biømeda, Foster City, CA, USA). After
endogenous peroxidase deactivation, immunocytochemistry was
performed using the Biostain Super ABC Mouse/Rat kit (Biømeda)
as described by the manufacturer. Anti-surface protein mAb specif-
ic for C. pneumoniae (DAKO) and the genus-specific anti-LPS mAb
(DAKO, IMAGEN-Chlamydia Kit) were each diluted 1:10 in Ab
Diluting buffer (Biømeda) and incubated for 30 min at 37°C, fol-
lowed by 9 rinses with Automation buffer (Biømeda). Similar incu-
bations and washes were performed for the secondary anti-mouse bi-
otinylated Ab and ABC reagents. After a 1-min incubation with per-
oxidase enhancer, Ab binding was detected with the HRP chromag-
en Diaminobenzidine Cobalt (Biømeda) by 510 min incubation at
room temperature. Slides were prepared with Crystal-Mount
(Biømeda) for light microscopy. EM analyses were also performed
on THP-1 cells infected with C. pneumoniae isolated from brain tis-
sues. For such studies, cells were centrifuged at 500×g for 5 min in
1.5 ml micro-centrifuge tubes and fixed in 4% paraformaldehyde,
0.1% glutaraldehyde overnight at 4°C, then embedded and processed
for EM, as described above. Cytospins for EM were fixed with 4%
paraformaldehyde, 0.1% glutaraldehyde and subjected to the “pop-
off” technique [12], then processed for EM.
In vitro infection of cultured astrocytoma cells with a standard
strain of C. pneumoniae
As an external control for in vivo immunohistochemical and other
analyses, we infected an astrocytoma cell line in vitro with C. pneu-
moniae strain TW-183. SW1088 astrocytoma cells were obtained
from the ATCC; 4×10
3
cells were cultured in L-15 Liebovitz’s
Medium in 2- and 4-well chamber slides (Lab-Tec, NUNC, Naper-
ville, Ill. USA) and allowed to adhere overnight. Non-adherent cells
were removed by aspiration, and 400 IFU of strain TW-183 (ATCC)
C. pneumoniae were added to each well in 400-µl aliquots, followed
27
28
Fig. 1AF Representative control- and patient-directed PCR
screening analyses of total DNA prepared from AD and control pa-
tients, using primer sets independently targeting the C. pneumoniae
16S rRNA gene and the ompA gene. DNA was prepared, and reac-
tions performed and visualized, as given in Materials and methods.
A description of all primer sets employed is also given in the meth-
ods section. A Control PCR analyses demonstrating the specificity
of primers targeting the C. pneumoniae 16S rRNA gene. Lane 1:
100-bp size standards; lanes 25, 7, 9: PCR analyses using the C.
pneumoniae 16S rRNA-directed primers using purified DNA from
2, C. pneumoniae EB; 3, astrocytoma (SW1088) cells in culture; 4,
C. pneumoniae (TW-183)-infected SW1088 cells in culture; 5, C.
trachomatis (serovar C)-infected HeLa cells; 7, C. psittaci (GPIC)-
infected tissue from a guinea pig genital tract; 9, culture-grown Es-
cherichia coli; lane 6: PCR analysis targeting the C. trachomatis 16S
rRNA gene using primers specific for that gene and purified DNA
from HeLa cells experimentally infected with that organism (sero-
var C); lane 8: PCR analysis targeting the C. psittaci 16S rRNA gene
using primers specific for that gene and purified DNA from a guin-
ea pig genital tract experimentally infected with that organism; lane
10: PCR analysis targeting the E. coli 16S rRNA gene using a set of
pan-bacteria primers and purified DNA from cultures of that organ-
ism. B Control PCR analyses demonstrating the specificity of prim-
ers targeting the C. pneumoniae ompA gene. Lane 1: 100-bp size
standards; lanes 25, 7, 9: PCR analyses using the C. pneumoniae
ompA-directed primers using purified DNA from 2, C. pneumoniae
EB; 3, astrocytoma (SW1088) cells in culture; 4, C. pneumoniae
(TW-183)-infected SW1088 cells in culture; 5, C. trachomatis (ser-
ovar C)-infected HeLa cells; 7, C. psittaci (GPIC)-infected tissue
from a guinea pig genital tract; 9, culture-grown E. coli; lane 6: PCR
analysis targeting the C. trachomatis omp1 gene using primers spe-
cific for that gene and purified DNA from HeLa cells experimental-
ly infected with that organism (serovar C); lane 8: PCR analysis tar-
geting the C. psittaci omp1 gene using primers specific for that gene
and purified DNA from a guinea pig genital tract experimentally in-
fected with that organism; lane 10: PCR analysis targeting the E. co-
li 16S rRNA gene using a set of pan-bacteria primers and purified
DNA from cultures of that organism. C Control PCR analysis dem-
onstrating that chromosomal DNA from several relevant bacterial
species is not present in total DNA from representative control and
AD brain tissue samples. Primers targeting sequences in C. trachom-
atis, B. burgdorferi, M. pneumoniae, and M. hominis are described
by centrifugation of slides at 500×g for 30 min. Cultures were
brought up to 1 ml with medium and placed in a 5% CO
2
incubator
for 3 days. Cultures were then fixed for either light microscopy with
S.T.F. (Streck) and immunolabelled as given above, or they were
fixed for EM analysis with 4% paraformaldehyde, 0.1% glutaralde-
hyde for 24 h at 4°C. Samples for EM were prepared for “pop-off”
and examined on a Zeiss 10 electron microscope.
Results
C. pneumoniae DNA in the Alzheimers brain
To examine whether C. pneumoniae is present in the brains
of late-onset AD patients, we used two independent PCR
assays to assess DNA from the organism in post-mortem
brain tissues from 19 late-onset AD and 19 non-AD con-
trol patients. From each AD and control patient, we
screened tissue from one or more brain areas that typically
show AD-related neuropathology (e.g., hippocampus, tem-
poral cortex) and from cerebellum, a region usually less
affected or unaffected. The representative control PCR re-
sults given in Fig. 1 demonstrate that neither the C. pneu-
moniae 16S rRNA-directed assay (Fig. 1A) nor the ompA-
directed assay (Fig. 1B) employed for screening of brain
tissue nucleic acids amplify DNA sequences from other
chlamydial species, from other common bacteria, or from
human host cells. As external controls, nucleic acids from
all patient samples were also screened by PCR for DNA
from C. trachomatis, Borrelia burgdorferi, Mycoplasma
pneumoniae, and M. hominis; all samples were negative
for each organism (Fig. 1C). Relevant clinical and other
information for all patients is summarized in Table 1 (con-
trol patients) and Table 2 (AD patients). APOE genotype
determination showed that 11/19 AD patients (58%) had
at least one
ε
4 allele, and 2 patients were homozygotes.
This allele was present in 4/19 non-AD controls (21%);
each of these four patients was heterozygous for
ε
4. PCR
screening results for C. pneumoniae chromosomal DNA
sequences for all AD and control patient samples are also
summarized in Tables 12, and representative assays are
given in Fig. 1DF. All samples except one (parietal cor-
tex, patient C1) from non-AD control individuals were neg-
ative for C. pneumoniae DNA sequences by PCR (Table 1;
Fig. 1DF), including those from patients with multiple
sclerosis. Of the 19 late-onset AD patients, samples from
17 (90%) were PCR-positive in assays examining hippo-
campus, temporal cortex, and/or other areas (Table 2;
Fig. 1DF); DNA sequence determination and analysis of
selected PCR products from ompA-directed assays of AD
patient materials confirmed that these products were de-
rived from the authentic C. pneumoniae gene (data not
shown). Nucleic acids from cerebellum of 4 AD patients
(AD9, AD10, AD16, AD19; see Fig. 1F) were also PCR-
positive for C. pneumoniae DNA; each of these tissue sam-
ples showed severe neuropathology on histologic exam-
ination. All samples from affected brain areas of patients
AD3 and AD5 were PCR-negative for C. pneumoniae
(Fig. 1F), and microscopic examination showed that these
two brains exhibited less severe neuropathology than did
all other AD brains studied (see Discussion). Thus, PCR
screening indicates that DNA from C. pneumoniae is com-
mon in areas of the AD brain exhibiting disease-related
neuropathology, but it is uncommon in unaffected or less
affected areas, in a large majority of patients studied. Such
sequences are extremely rare in congruent brain regions
from non-AD patients of similar age.
Ultrastructural localization of C. pneumoniae
in the Alzheimers brain
To confirm the PCR screening results and to visualize the
organism in affected CNS regions, we analyzed brain sec-
tions from AD and non-AD patients using EM and IEM.
In all species of Chlamydiae, the extracellular, infectious
form of the organism, the EB, alternates with the intracel-
lular, vegetative growth form (reticulate body, RB; [89]).
Survey of tissue sections by EM revealed that areas of the
hippocampus, temporal cortex, and/or other regions of AD
brains with neuropathology contained structures whose
morphology was consistent with that of both EB and RB
29
in Materials and methods, as are the preparation and reaction condi-
tions for the analyses. Lane 1: 100-bp size standards; Lanes 2, 3:
PCR analyses targeting a C. trachomatis chromosomal DNA se-
quence using primers specific for that gene and purified DNA from
2, HeLa cells infected with C. trachomatis (serovar C); 3, uninfect-
ed HeLa cells; lanes 47: PCR analysis of purified total DNA from
hippocampus of patient C2 using primers targeting chromosomal
DNA sequences from 4, C. trachomatis; 5, B. burgdorferi; 6, M.
pneumoniae; 7, M. hominis; lanes 811: PCR analysis of purified
total DNA from hippocampus of patient AD14 using primers target-
ing chromosomal DNA sequences from 8, C. trachomatis; 9, B. burg-
dorferi; 10, M. pneumoniae; 11, M. hominis; lane 12: control PCR
analysis using the B. burgdorferi primer set on purified DNA from
that organism. D PCR analyses targeting the C. pneumoniae 16S
rRNA gene in control and AD patient samples. Lane 1: 100-bp size
standards; lanes 212: 16S rRNA-directed PCR screening of puri-
fied DNA from 2, astrocytoma (SW1088) cells in culture; 3, C. pneu-
moniae (TW-183)-infected SW1088 cells; 4, parietal cortex of pa-
tient C1; 5, cerebellum of patient C1; 6, temporal cortex of patient
C1; 7, cerebellum of patient C6; 8, temporal cortex of patient C6; 9,
cerebellum of patient AD1; 10, hippocampus of patient AD1; 11,
cerebellum of patient AD7; 12, hippocampus of patient AD7. E PCR
analyses targeting the C. pneumoniae ompA gene in control and AD
patient samples. Lane 1: 100-bp size standards; lanes 212: ompA-
directed PCR screening of purified DNA from 2, astrocytoma
(SW1088) cells in culture; 3, C. pneumoniae (TW-183)-infected
SW1088 cells; 4, parietal cortex of patient C1; 5, cerebellum of pa-
tient C1; 6, temporal cortex of patient C1; 7, cerebellum of patient
C6; 8, temporal cortex of patient C6; 9, cerebellum of patient AD1;
10, hippocampus of patient AD1; 11, cerebellum of patient AD7; 12,
hippocampus of patient AD7. F PCR screening analyses of various
brain areas from several representative patient samples using the C.
pneumoniae 16S rRNA- and ompA-directed primer systems. Lane 1:
100-bp size standards; lanes 2, 4, 6, 8, 10, 12: 16S rRNA-directed
PCR screening of purified DNA from 2, C. pneumoniae (TW-183)-
infected astrocytoma (SW1088) cells in culture; 4, cerebellum of pa-
tient C10; 6, temporal cortex of patient C10; 8, cerebellum of patient
AD9; 10, temporal cortex of patient AD9; 12, cerebellum of patient
AD3; lanes 3, 5, 7, 9, 11, 13: ompA-directed PCR analyses of puri-
fied DNA from 3, C. pneumoniae (TW-183)-infected astrocytoma
(SW1088) cells in culture; 5, cerebellum of patient C10; 7, tempo-
ral cortex of patient C10; 9, cerebellum of patient AD9; 11, tempo-
ral cortex of patient AD9; 13, cerebellum of patient AD3
30
(Fig. 2; see [65]). Usually, these structures were localized
within intracellular inclusions (e.g., Fig. 2A, C). A mem-
brane with internal electron-dense material often could be
distinguished (Fig. 2B; [65]), and we frequently identified
structures resembling RB in the process of cell division
(Fig. 2E). Sizes of the chlamydia-like bodies observed
ranged from 0.2 to 1.0 µm in diameter, characteristic for
typical C. pneumoniae EB and RB [65]. Chlamydia-like
bodies observed by EM analysis in brain tissues from AD
patients were virtually identical to objects identified in
control EM studies of human astrocytoma cells infected in
vitro with a standard C. pneumoniae strain obtained from
the ATCC (see e.g., Fig. 2J, K). The data in Fig. 2 are rep-
resentative of the ten AD brains analyzed by EM; we found
no similar objects in EM analyses of tissues from six con-
trol patients, except in sections from parietal cortex of pa-
tient C1. Thus, ultrastructural studies of areas of AD brains
showing neuropathology, but not congruent areas from
control brains, reveal forms consistent in size and morphol-
ogy with those typical of C. pneumoniae.
To confirm that the chlamydia-like bodies were, in fact,
C. pneumoniae, we employed IEM to examine PCR-posi-
tive brains previously analyzed by EM. For these analy-
ses, we used a highly specific mAb targeting an outer sur-
face protein of C. pneumoniae; organisms binding that
mAb were visualized via gold-conjugated secondary Ab.
IEM results representative of the 10 PCR-positive AD pa-
tients analyzed are included in Fig. 2. The anti-surface pro-
Fig. 2A–G
tein mAb labelled EB (Fig. 2D) and RB (Fig. 2E, G), con-
firming the presence of both developmental forms of the
organism in the AD brain. C. pneumoniae cells showed
variable distribution of immunoreactivity including par-
tial/complete circumferential labelling, and immunola-
belled bacteria were observed often in sections displaying
neurodegenerative changes typical of AD (see below).
Background labelling was extremely low, and examination
of sections from seven PCR-negative non-AD patients
showed no significant or specific labelling (Fig. 2H, I).
The only normal components seen in tissue sections that
resembled the objects in Fig. 2 were lysosomal dense bod-
ies within dystrophic neurites, but these were never la-
belled with the anti-surface protein mAb. Thus, IEM anal-
ysis of affected brain regions of AD patients confirms that
C. pneumoniae is present in those tissues; such bacteria are
31
Fig. 2A–K Ultrastructural analyses of brain tissues from AD pa-
tients to identify EB and RB of C. pneumoniae. Brain tissues from
AD patients PCR-positive for the organism were prepared for EM
and IEM analysis as described in Materials and methods; the anti-
chlamydial Ab used in the latter analysis was a mAb targeting a
C. pneumoniae outer membrane protein (see text). A Typical inclu-
sion body with presumptive EB and RB as seen in EM analysis of
temporal cortex of patient AD8; sizes of chlamydia-like bodies typ-
ically range from 0.2 to 1.0 µm, which is characteristic for this or-
ganism (bar=0.5 µm). B Pear-shaped presumptive EB of C. pneu-
moniae as identified in EM analysis of the temporal cortex of patient
AD14 (bar=0.2 µm). C Another typical inclusion body with pre-
sumptive EB and RB as seen in EM analysis of temporal cortex of
patient AD8 (bar=1.0 µm). D IEM analysis of the temporal cortex
of patient AD8, showing C. pneumoniae EB labelled with the anti-
surface protein mAb (arrow, 15-nm gold particles) (bar=0.25 µm).
E IEM analysis of the hippocampus of patient AD7, showing RB la-
belled with the anti-surface protein mAb (arrows, 15-nm gold par-
ticles) (bar=1.0 µm). F, G Higher magnification of IEM analyses
from additional sections of the hippocampus of patient AD7, show-
ing RB labelled with the anti-surface protein mAb (F, 15-nm gold
particles; G, 5-nm gold particles, arrowheads) (bars=0.25 µm). H,
I IEM analysis of the temporal cortex of two different control brains
(H, C18MS; I, C8) using the anti-surface protein mAb (bars=1.0
µm). Note that no structures were immunolabelled in these sections.
J Low magnification EM of a typical uninfected astrocytoma
(SW1088) cell in culture, obtained from the ATCC (bar=1.0 µm).
K High magnification EM of an astrocytoma (SW1088) cell infect-
ed with the laboratory strain of C. pneumoniae (TW-183), also ob-
tained from the ATCC. A pear-shaped organism characteristic of C.
pneumoniae is apparent in the center of the cell (arrow) (bar=0.5
µm)
32
absent in materials from congruent brain regions of non-
AD patients.
Immunohistochemical localization of C. pneumoniae
in the Alzheimers brain
To identify specific area(s) and host cell types within which
the bacterium resides in the brain, we performed immuno-
histochemical analysis of tissues from affected regions of
AD brains and congruent regions from non-AD brains. In
these analyses, we used two primary mAbs; the first was
a genus-specific mAb targeting the LPS of Chlamydiae,
and the second was the outer surface protein-specific mAb
employed in IEM studies. In sections of hippocampus and
temporal cortex from AD brains, a consistent pattern of
immunolabelling was observed in perivascular regions of
small- to medium-sized blood vessels in the neuropil
(Fig. 3A, D). In most sections, specific labelling also ap-
peared in microglia- and astroglia-like cells (Fig. 3B, E).
In congruent sections from brains of the six non-AD pa-
tients examined, no immunolabelling was observed using
either the anti-LPS or anti-surface protein mAb (Fig. 3C,
F). Control stainings of similar regions of AD brains with
normal mouse (Fig. 3G) and normal rabbit sera (Fig. 3H)
produced no labelling at all; other control stainings using
irrelevant primary mAbs (e.g., anti-CD54) also produced
essentially no labelling (Fig. 3I, J). We examined similar
brain sections from the two AD patients PCR-negative for
C. pneumoniae, and both were immunonegative for the or-
ganism, even though they showed mild AD-related neuro-
pathology. Staining of AD brain sections sometimes re-
vealed cellular processes, presumably from pericytes (see
below), enclosing/abutting the abluminal surface of blood
vessels. The pattern of immunoreactivity was consistent in
the ten AD brains examined, although some differences in
level of immunopositivity were evident; this latter prob-
ably reflected a difference in bacterial load in the tissues
examined but also may indicate sample variability. Thus,
immunohistochemical analysis confirms the presence of
C. pneumoniae in affected AD brain regions and localizes
the bacterium to apparent non-neuronal cells.
Culture of C. pneumoniae from Alzheimers brain tissues
Laboratory culture of an organism from a putative site of
infection is considered to be the gold standard for demon-
stration that the organism is indeed present at that site. We
therefore attempted to culture C. pneumoniae from homog-
enates of two AD (AD7, AD14) and two control non-AD
(C3, C7) brains, using the human monocyte cell line de-
signated THP-1 as host; previous studies have demon-
strated that monocyte cell lines, such as THP-1, are sus-
ceptible to infection by the organism [73]. Immunocyto-
chemistry using the anti-surface protein and anti-LPS mAb
was performed on cytospun THP-1 cells after culture with
brain homogenates. THP-1 cells incubated with homogen-
ates from temporal cortex of each control brain were com-
33
Fig. 3A–J Immunohistochemical analysis of brain tissues from
control (non-AD) and AD patients using two primary mAb, one tar-
geting a C. pneumoniae surface protein and the second targeting the
chlamydial LPS. Preparation of tissues and details of the primary and
secondary Abs employed are described in Materials and methods. A
Immunolabelling of perivascular cells (arrows) using the anti-LPS
mAb; tissue is from the temporal cortex of patient AD4 (bar=50 µm).
B Immunolabelling of apparent glial cells (arrowheads) in the den-
tate gyrus of patient AD4, using the anti-LPS mAb (bar=50 µm).
C Immunolabelling of tissue from the temporal cortex from a non-
AD control patient (C17MS, Table 1) using the anti-LPS mAb
(bar=50 µm). D Immunolabelling of perivascular cells (arrows) us-
ing the anti-surface protein mAb; tissue is from the temporal cortex
of patient AD2 (bar=25 µm). E Immunolabelling of apparent glial
cells (arrowheads) in the temporal cortex of patient AD7 using the
anti-surface protein mAb (bar=50 µm). F Immunolabelling of tis-
sue from the dentate gyrus from a non-AD control patient (C18MS,
Table 1) using the anti-surface protein mAb (bar=50 µm). G Con-
trol immunolabelling of a section from the dentate gyrus of patient
AD5 using normal mouse serum as primary Ab (bar=100 µm). H Con-
trol immunolabelling of a section from the temporal cortex of patient
AD7 using normal rabbit serum as primary Ab (bar=100 µm). I Im-
munolabelling of a section from the temporal cortex of patient
C17MS using anti-CD54 mAb (bar=100 µm). J Immunolabelling of
a section from the temporal cortex of patient AD7 using anti-CD54
mAb (bar=100 µm)
pletely negative for C. pneumoniae using either mAb for
immunolabelling (e.g., Fig. 4A). Host monocytes from
cultures incubated with homogenates from temporal cor-
tex of each AD patient, in contrast, displayed strong im-
munopositivity using both mAbs after one passage (72 h;
Fig. 4B) and after two passages (7 days; Fig. 4C). Approx-
imately 10% of cultured THP-1 cells were immunoposi-
tive for C. pneumoniae after 72-h culture, and ~70% were
positive after the second passage. We further visualized C.
pneumoniae inclusions within these infected host cells by
EM, using duplicate cytospin preparations of homogenate-
infected THP-1 cells as starting material; these studies
demonstrated classic morphology of the organism after
both first (Fig. 4D) and second passages (Fig. 4E; see
[65]). Thus, infectious C. pneumoniae were present in au-
topsy brain tissues from the two AD patients subjected to
culture analysis, but the organism was not present in con-
gruent tissues from the two-non-AD control patients stud-
ied.
Transcriptional activity of C. pneumoniae
in the Alzheimers brain
The culture analyses above clearly demonstrate that C. pneu-
moniae is viable during CNS infection, at least for the two
AD samples so examined. Interestingly, recent work has
indicated that in some cases, RNA can remain intact in
brain tissues for several hours post-mortem [41, 45]. To
confirm and extend the culture observation of viability and
infectivity, and to initiate analysis of transcriptional activ-
ity in C. pneumoniae during CNS infection, we performed
RT-PCR assays on additional control and AD patient samples.
In these analyses we targeted mRNAs from the C. pneu-
moniaegene encoding the KDO transferase enzyme, which
34
Fig. 4A –E Immunocytochemical and electron-microscopic analy-
ses of C. pneumoniae infection in cultured THP-1 monocytic cells
incubated with tissue homogenates of non-AD control and AD tis-
sues. Preparation of tissues as well as details of the culture condi-
tions are given in Materials and methods. A THP-1 cells incubated
with a tissue homogenate of temporal cortex from control brain C7
followed by immunolabelling with the anti-surface protein mAb for
C. pneumoniae (bar=100 µm). B THP-1 cells incubated for 3 days
with tissue homogenate of temporal cortex from patient AD14. These
cells were immunolabelled with the anti-surface protein mAb, which
revealed cytoplasmic inclusions of C. pneumoniae (arrows) (bar=50
µm). C THP-1 cells incubated for 7 days with tissue homogenate of
hippocampus from patient AD7. Numerous inclusions of C. pneu-
moniaewithin these cells were immunolabelled with the anti-surface
protein mAb (arrowheads) (bar=50 µm). D EM analysis of a THP-1
cell infected with C. pneumoniae (arrowheads) following 3-day cul-
ture with a tissue homogenate from temporal cortex of patient AD14
(bar=1.0 µm). E EM analysis of a THP-1 cell infected with C. pneu-
moniae following 7-day culture with a tissue homogenate from the
hippocampus of patient AD7 (bar=1.0 µm)
is required for bacterial LPS synthesis, and the gene speci-
fying a Mr=76000 protein, which includes a species-spe-
cific epitope of the organism. As an internal control for the
quality of RNA analyzed, we employed an RT-PCR assay
targeting the human nuclear gene (COIV) specifying sub-
unit 4 of cytochrome oxidase, the terminal complex in the
mitochondrial electron transport chain. We prepared RNA
from hippocampus or temporal cortex of patients AD2,
AD14, C5, and C18MS, and we analyzed those prepara-
tions for the two C. pneumoniae, as well as the host COIV,
messengers. The representative control assays given in Fig.
5AC demonstrate that neither of the C. pneumoniae-di-
rected RT-PCR primer systems amplifies any sequences
from human cells, and that the quality of RNA preparations
analyzed was adequate for these studies. Experimental re-
sults presented in Fig. 5A, B demonstrate that RNA from
brain regions showing neuropathology in the two AD pa-
tients contained both of the targeted bacterial transcripts.
In contrast, the assays shown in Fig. 5C demonstrate that
RNA prepared from congruent regions of two non-AD
brains was negative for these bacterial mRNAs. We also
showed that RNA prepared from cerebellar tissue of pa-
tients AD2 and AD14, regions PCR-negative for C. pneu-
moniae DNA in these patients (Fig. 5C; Table 2), was neg-
ative for each of the bacterial messengers. Thus, C. pneu-
moniae expresses at least some genes during CNS infection
of AD patients, confirming that such infections involve me-
tabolically active, vegetatively growing bacteria.
35
Fig. 5A–C Control- and patient-directed RT-PCR analyses of to-
tal RNA prepared from two AD and two control patients, using prim-
ers targeting mRNAs from the C. pneumoniae KDO transferase gene
and the gene specifying a Mr=76000 protein. Quality of RNA prep-
arations was examined in RT-PCR reactions targeting the human host
cell mRNA specifying subunit 4 of cytochrome oxidase (COIV).
RNA was prepared and reactions performed and visualized as de-
scribed in Materials and methods. A Analyses targeting the C. pneu-
moniae KDO transferase mRNA. Lane 1: 100-bp size standards;
lanes 2, 3: control PCR analysis for the KDO gene itself using 2,
purified C. pneumoniae DNA and 3, purified DNA from astrocyto-
ma (SW1088) cells in culture; lane 4: control PCR analysis target-
ing the COIV sequence in purified DNA from cultured SW1088 cells;
lane 5: control PCR analysis targeting the KDO transferase sequence
in purified DNA from C. pneumoniae (TW-183)-infected SW1088
cells; lanes 6, 8: RT-PCR analysis targeting the chlamydial KDO
transferase mRNA in total RNA from hippocampus of patient AD2
6,without reverse transcription and 8, with reverse transcription prior
to amplification; lane 7: RT-PCR targeting host COIV mRNA in RNA
from hippocampus of patient AD2; lanes 9, 11: RT-PCR analysis tar-
geting the chlamydial KDO transferase mRNA in total RNA from
hippocampus of patient AD14 9, without reverse transcription and
11,with reverse transcription prior to amplification; lane10: RT-PCR
targeting host COIV mRNA in RNA from hippocampus of patient
AD2. B Analyses targeting the C. pneumoniae Mr=76000 protein
mRNA. Lane 1: 100-bp size standards; lanes 2, 3: control PCR anal-
ysis for the Mr=76000 gene using 2, purified C. pneumoniae DNA
and 3, purified DNA from astrocytoma (SW1088) cells in culture;
lane 4: control PCR analysis targeting the COIV sequence in puri-
fied DNA from cultured SW1088 cells; lane 5: control PCR analy-
sis targeting the Mr=76000 protein gene in purified DNA from C.
pneumoniae (TW-183)-infected SW1088 cells; lanes 6, 8: RT-PCR
analysis targeting the chlamydial Mr=76000 mRNA in total RNA
from hippocampus of patient AD2 6, without reverse transcription
and 8, with reverse transcription prior to amplification; lane 7: RT-
PCR targeting host COIV mRNA in RNA from hippocampus of pa-
tient AD2; lanes 9, 11: RT-PCR analysis targeting the chlamydial
Mr=76 000 mRNA in total RNA from hippocampus of patient AD14
9, without reverse transcription and 11, with reverse transcription
prior to amplification; lane 10: RT-PCR targeting host COIV mRNA
in RNA from hippocampus of patient AD14. C Control- and patient-
directed RT-PCR analyses of total RNA prepared from two control
patients and from PCR-negative regions of the two AD patients. Lane
1: 100-bp size standards; lanes 2, 3: PCR analyses targeting sequenc-
es in 2, the KDO transferase gene and 3, the Mr=76 000 protein gene,
both using purified DNA from C. pneumoniae (TW-183)-infected
SW1088 cells in culture; lanes 4, 5: RT-PCR analysis targeting the
chlamydial KDO transferase mRNA using total RNA from hippo-
campus of control patient C4 4, without reverse transcription and 5,
with reverse transcription prior to amplification; lane 6: RT-PCR tar-
geting host COIV mRNA in RNA from hippocampus of patient C4;
lanes 7, 8: RT-PCR analysis targeting the Mr=76000 mRNA using
total RNA from hippocampus of control patient C2 7, without re-
verse transcription and 8, with reverse transcription prior to amplifi-
cation; lane 9: RT-PCR targeting host COIV mRNA in RNA from
hippocampus of patient C2; lane 10: KDO transferase mRNA-di-
rected RT-PCR using total RNA from cerebellum of patient AD2;
lane 11: RT-PCR for host COIV mRNA using RNA from cerebel-
lum of patient AD2; lane 12: Mr=76000 mRNA-directed RT-PCR
using total RNA from cerebellum of patient AD14; lane 13: RT-PCR
for host COIV mRNA using RNA from cerebellum of patient AD14
Host cells for C. pneumoniae in the Alzheimers brain:
relationship to neuropathology
We used double immunolabelling to identify the chla-
mydia-bearing cell types observed above and to define the
relationship between infected cells and NFTs/NSPs. Dou-
ble labelling of tissue sections from several AD brains us-
ing an anti-chlamydial LPS pAb and an anti-GFAP mAb
[24] showed that astroglial cells are a common host for
C. pneumoniae in the AD brain. Figure 6 presents repre-
sentative double immunolabelling results for the 12 AD
patients analyzed. Cells identified by the anti-LPS pAb
(Fig. 6A) co-localize with cells expressing GFAP (Fig. 6B);
astroglial cells so identified were sometimes slightly hy-
pertrophied, as has been reported for such cells in the area
of NSPs [23]. Similarly, iNOS is an enzyme produced by
36
Fig. 6AH Double immunol-
abelling of AD brain tissues for
identification of specific host
cell types harboring C. pneu-
moniae, plus controls from AD
patient tissues and the cultured
astrocytoma cell line SW1088.
Preparation of tissues and cells
is given in Materials and meth-
ods. A, B Tissue section from
the temporal cortex of patient
AD4 double immunolabelled
for the chlamydial LPS (A,
white arrowheads) and GFAP
(B, black arrowheads); labelled
cells in A were detected using
an FITC-conjugated secondary
Ab, while those in B were iden-
tified with the DAB chromo-
gen. C, D Tissue section from
the temporal cortex of patient
AD4 double immunolabelled
for iNOS (C, white arrow-
heads) and the chlamydial sur-
face protein (D, black arrow-
heads); labelled cells in C were
detected with an FITC-conju-
gated secondary Ab, while
those in D were identified with
the DAB development (A–D,
bars=50 µm). E Immunofluo-
rescence of iNOS labelling in
pericytes in the hippocampus of
patient AD7 (bar=100 µm).
F Representative section from
the temporal cortex of patient
AD7 demonstrating minimal
autofluorescence (bar= 100
µm). G Cultured astrocytoma
cells (SW1088) infected with
the laboratory strain of C.
pneumoniae (TW-183) after 3-
day culture and immunola-
belled with the anti-surface
protein mAb (bar=25 µm).
H Uninfected control astrocyto-
ma cells immunolabelled with
the anti-surface protein mAb,
showing no immunoreactivity
(bar=25 µm)
activated microglia [62, 63]. Figure 6 also presents a rep-
resentative section from an AD brain in which an anti-
iNOS pAb (Fig. 6C) was used to identify C. pneumoniae-
containing cells labelled with the anti-surface protein mAb
(Fig. 6D). Thus, infected host cells include astroglia and
activated microglia. The chlamydia-infected cells sur-
rounding blood vessels in the AD brain are probably per-
icytes, as suggested by data in Fig. 3A, D. Although no
single surface protein uniquely characterizes pericytes, we
used the anti-iNOS pAb in combination with the anti-outer
surface protein mAb to identify this cell type, since expres-
sion of this enzyme is induced in LPS-activated pericytes
(Fig. 6E; [17]). In control stainings, comparable tissue sec-
tions of AD and control brains were examined for autoflu-
orescence using an irrelevant primary mAb (anti-CD54) or
no primary Ab at all; in both cases, the level of autofluo-
rescence was minimal, particularly in lipofuscin-contain-
ing neurons (e.g., Fig. 6F). Control experiments in which
a human astrocytoma cell line (SW1088) was infected with
the index C. pneumoniae strain TW-183 confirm that as-
trocytes are, in fact, infectible with this organism (see Fig.
6G, H). These infected cells provide an important exter-
nal control for use of the anti-C. pneumoniae surface pro-
tein mAb used in the immunohistochemical analyses pre-
sented above. Thus, these results indicate that at least three
cell types, astroglia, microglia, and pericytes, harbor C.
pneumoniae in the AD brain.
We also investigated whether host cells infected with
C. pneumoniae in the CNS were located primarily in re-
gions showing typical AD-related neuropathology (NFTs/
NSPs), or whether such cells were distributed randomly
throughout the brain. The PHF-1 mAb was used to iden-
tify neuritic pathology in the AD brain [34]. Figure 7
presents typical consecutive tissue sections from temporal
cortex of an AD patient, labelled with the anti-tau mAb
PHF-1 (Fig. 7A) and anti-outer surface protein (Fig. 7B)
mAb. Staining with the latter demonstrated the presence
of chlamydia-infected glial cells near PHF-tau protein de-
position in neurites within NSPs in the eight AD patients
so analyzed. For the same patients, similar labelling of
brain areas with few or no NSP showed virtually no cells
harboring C. pneumoniae (e.g., Fig. 7C, D); in sections
from seven non-AD brains examined, no infected cells or
AD-related pathology were observed (see e.g., Fig. 3).
Thus, chlamydia-infected astroglia and microglia in the
AD brain are concentrated primarily in regions of PHF-1-
positive neuritic pathology.
Discussion
C. pneumoniae is an intracellular respiratory pathogen that
has been associated with several unexpected pathologies,
possibly including some related to nervous system func-
37
Fig. 7A–D Areas of neuropathology in the AD brain show immu-
noreactivity for C. pneumoniae. Preparation of consecutive tissue
sections from the temporal cortex of patient AD7 was as given in
Materials and methods. A Neuritic pathology characteristic of AD
was demonstrated with the PHF-1 mAb (arrows). B Consecutive tis-
sue section from the same sample demonstrating immunolabelled
presumptive glial cells (arrows) infected with C. pneumoniae immu-
nolabelled with the anti-C. pneumoniae surface protein mAb. C Weak
PHF-1 immunolabelling in the temporal cortex of patient AD7 in
which few neuritic plaques and tangles were present. D Weak immu-
nolabelling of a congruent tissue section from the temporal cortex of
patient AD7 with the anti-C. pneumoniae surface protein mAb. (Bars
for all panels=50 µm)
tion [46, 95]. Infection with the organism has also been re-
ported to be associated with atherosclerosis [69], a disease
postulated in one recent study to be linked with late-onset
AD [39]. These observations, in combination with others
suggesting increased prevalence of C. pneumoniae infec-
tion with age [50], and the inflammation associated with
both AD [3, 35] and infection by this bacterium [9294],
suggested to us that this organism might be associated with
sporadic, late-onset AD. In the work presented here, we
demonstrate that DNA from the bacterium is present in
brain areas showing neuropathology in a large majority of
AD patients, but that it is essentially absent from congru-
ent regions in non-AD control brains. Ultrastructural anal-
yses of AD brains identified chlamydia-like objects, and
these were confirmed to be C. pneumoniae by IEM. Im-
munohistochemical analyses of AD brains showed that
cells both adjacent to and distant from the vasculature are
infected, and double immunolabelling identified these host
cells as microglia, astroglia, and pericytes. Significantly,
homogenates of AD brain tissues, but not those from con-
trol individuals, productively infected a monocytic cell line
in culture with C. pneumoniae, and this indication of vi-
ability of the organism in brain tissue was confirmed and
extended by RT-PCR assays targeting C. pneumoniae
mRNA. Further immunolabelling studies showed that
chlamydia-infected cells are concentrated in areas of neu-
ritic pathology in the AD brain. Together, these results in-
dicate that metabolically active, infectious C. pneumoniae
are found in the AD brain primarily in regions and areas
showing AD-related neuropathology.
Pneumonia is a common cause of death among AD pa-
tients [13], although one study indicated that it may be
most prevalent among those with advanced dementia [47].
In the AD patients available to us for study, proximal
causes of death included many normally seen in advanced
age, with pneumonia a relatively rare occurrence; indeed,
causes of death do not appear to us to be much different
between our control and AD patient groups. Available
clinical data for the patients studied did not identify any
with pneumonia for which C. pneumoniae was the con-
firmed etiologic agent. Regardless, our PCR screening
showed C. pneumoniae DNA to be present in brain in a
high proportion of AD patients examined. In the studies
presented here, all control and AD brain samples were
screened by PCR; most AD and selected non-AD control
specimens were also examined by EM/IEM and/or immu-
nohistochemical methods when enough tissue was avail-
able to do so. Results of these latter analyses were always
consistent with PCR data and internally consistent among
samples. Given our data, it seems unlikely that dissemi-
nation of C. pneumoniae to the brain is a general occur-
rence with increasing age, since we found evidence of the
bacterium in only one brain region from a single non-AD
patient. Moreover, distribution of the organism in AD
brains was not general, but rather was concentrated in re-
gions of neuropathology. Because the number of AD pa-
tients studied was limited, however, the question of
whether CNS infection with C. pneumoniae is directly re-
sponsible, either fully or in part, for the neuropathology
characteristic of AD, or whether the organism is merely
an opportunistic invader of an organ damaged by other
means, remains completely open. More study involving
larger AD and control patient populations will be required
to address this issue (see also below).
The observations presented do not establish a causal re-
lationship between acute or chronic infection with C. pneu-
moniae and development of late-onset AD. Rather, our data
demonstrate that many cells in areas of the AD brain show-
ing typical neuropathology harbor the organism in a high
proportion of patients. Aside from the issue of whether
chlamydial infection of the brain is directly reponsible for
initiating AD-related pathology, however, such acute or
chronic infections may explain, at least in part, some char-
acteristics of the disease. For example, inflammation is
common in the AD brain in areas of neuropathology, and
proinflammatory cytokines, including IL-1
β
, IL-1
α
, IL-6,
and TNF-
α
, have been shown to be elevated in the AD
brain [3, 35]. Indeed, some studies have indicated that ad-
ministration of non-steroidal anti-inflammatory drugs is
beneficial in the treatment of AD (e.g., [10]). Such inflam-
mation, currently thought to result from A
β
deposition, has
been advanced prominently as a pathogenic mechanism in
the disease [56]. Inflammation has been implicated, of
course, as an important factor in many diseases [80], and
A
β
deposition may well be responsible in part for that ob-
taining in the AD brain. However, chlamydial infection has
long been known to engender powerful immunopathogenic
responses [31, 50], and infection by C. pneumoniae may
also contribute to the inflammation observed in this CNS
context. Our results show the presence of chlamydial LPS,
a well-known proinflammatory molecule, in regions of AD
pathology, as well as production of the bacterial mRNA
specifying a critical enzyme required for its synthesis.
Moreover, microglia and astroglia appear to function fre-
quently as host cells for C. pneumoniae in the AD brain.
Microglia are the resident tissue macrophages of the brain,
and once activated they, like astroglia, are a source of pro-
inflammatory cytokines, including IL-1
β
, TNF
α
, IL-6,
and others (e.g., [62, 63]). Our observations further indi-
cate that chlamydia-infected glial cells are concentrated
primarily in areas of AD pathology, areas which usually
show inflammation. Thus, even if brain infection with C.
pneumoniae is not the initiating event in the neuropatho-
genesis ending in AD, but rather solely an opportunistic
spread of the organism to an already damaged organ, such
infections may well exacerbate or accelerate disease path-
ogenesis.
The observations presented here indicate that C. pneu-
moniae-infected cells are often and selectively present in
areas of neuropathology in the AD brain, but these data
provide no insight into whether the infecting organisms
identified represent an acute or a chronic, persistent infec-
tion. Such persistent infection could contribute signifi-
cantly to exacerbation of the neuropathogenic process via
induction of inflammation; recent studies from several la-
boratories have indicated that C. trachomatis, a sister-spe-
cies to C. pneumoniae, can persistently infect at least some
anatomic sites. For example, we and others have shown
38
that following initial genital infection with C. trachoma-
tis, dissemination occurs to the synovium (e.g., [9]); in that
latter site the organism enters a biologic state in which pro-
duction of the MOMP is severely attenuated, completion
of the life cycle to generate new EB occurs at a low rate,
and in which the organism resides intracellularly for ex-
tended periods within monocytes/macrophages [5, 70] (see
also [57]). Given the ability of C. trachomatis to enter such
a culture-negative persistent state, and given the difficult
and often erratic culture-positivity for C. pneumoniae in
several other disease contexts, we were somewhat sur-
prised at the strong culture-positivity shown by this organ-
ism in the two brain samples analyzed here. However, the
soft consistency of brain tissue and the consequent ease of
extraction may have permitted the isolation of infectious
organisms more readily. Both the laboratory culture stud-
ies and the bacterial transcript analyses indicate that CNS
infection by C. pneumoniae is overt, i.e., that the organ-
ism in this context can and does complete its life cycle to
generate new infectious EB. It will be of significant inter-
est to screen brain tissues from other AD and control pa-
tients by culture and transcript analysis to distinguish
whether overt infection within the CNS by C. pneumoniae
is the general rule, or whether this organism can generate
long-term persistent infections in the same manner as does
its more extensively studied sister species, C. trachoma-
tis. In this regard, we are currently analyzing RNA prep-
arations from brain tissues of several AD patients to deter-
mine the relative levels of ompA-derived and other mRNAs
from C. pneumoniae to define their abundance during CNS
infection.
Previous studies have identified no relationship be-
tween late-onset AD and infection with other pathogens,
with the possible exception of HSV-1. Infection with this
virus in the presence of APOE
ε
4 was suggested to be a
risk factor for sporadic AD in the large patient population
examined, but the percentage of patients showing HSV-1
DNA in brain was far lower than that reported here for C.
pneumoniae [41]. Our assessment of APOE genotype for
our patient populations is consistent with it being a risk
factor for AD, especially in combination with chlamydial
infection of the CNS. Presence of at least one
ε
4 allele in
most AD patients PCR-positive for C. pneumoniae may
suggest that this gene product allows or promotes CNS in-
fection in some manner, a contention that seems consistent
with results from the HSV-1 report [41]. More study will
be required to assess this possibility, but we note that the
two AD patients studied here who were PCR-negative for
C. pneumoniae were
ε
3 homozygotes, and they displayed
the least severe neuropathology of the AD patients exam-
ined. The patients with the most severe neuropathology had
chlamydial DNA in the temporal lobe, prefrontal cortex,
and cerebellum; three of these latter four patients possessed
an
ε
4 allele.
Immunohistochemical staining demonstrated that chla-
mydia-infected cells are associated with blood vessels in
the brain, suggesting that dissemination of the organism
from the site of primary infection involves the vasculature.
In studies unrelated to those here, we showed that the prin-
cipal hosts for C. trachomatis are monocytes/macrophages
during persistent synovial infection [5]; this suggested
that dissemination from the genital system to the joint
might be via blood, and we demonstrated this chlamydial
species in blood monocytes from several patients with
early arthritis (H. R. Schumacher, A. P. H. unpublished
observations). No data bearing on the means of dissemi-
nation of C. pneumoniae to the brain are available yet, of
course; however, the organism has been identified in cir-
culating mononuclear cells in a rabbit model of infection
with the organism [66]. Blood-borne transport of C. pneu-
moniae EB must also remain in consideration as a means
by which dissemination to the brain occurs. The mecha-
nism(s) by which EB, or infected cells, might pass the
blood-brain barrier was not investigated here and thus re-
mains to be determined; however, evidence suggests that
this barrier may be compromised in AD patients (e.g.,
[43]). C. pneumoniae is a respiratory pathogen, infecting
the oral and nasal mucosa, and still another route of entry
into the brain that should be considered is transport to the
brain via components of the olfactory system (e.g., [21,
75, 81]. More work will be required to distinguish among
these possibilities.
While the observations presented here do not establish
a causal relationship between CNS infection with C. pneu-
moniae and development of late-onset AD, the association
between chlamydia-infected glial cells and areas of neuro-
pathology in this neurodegenerative condition suggests
that further investigation of such a relationship would be
of some interest. These cell types are known to be directly
involved in the inflammatory response within the AD
brain. In addition to a possible role in the inflammatory
process, however, infection of glial cells may result in al-
teration of mechanisms that regulate production and de-
position of A
β
, APOE
ε
4 gene product, and heparan sul-
fate proteoglycans; moreover, such infection might well
initiate membrane damage through oxidative and excito-
toxic mechanisms that could contribute significantly to the
pathology characteristic of AD. Studies are currently under
way to investigate whether chlamydia-infected microglia,
astroglia, and/or pericytes are involved in aberrant produc-
tion of A
β
via alteration of
β
APP, PS-1, or PS-2 expres-
sion, or via modification of processing of the
β
APP gene
product in infected cells, using appropriate C. pneumon-
iae-infected cell lines in culture. With regard to any pos-
sible causal relationship between chlamydial infection of
the brain and development of late-onset AD, it will also be
of importance to examine specimens from individuals with
other neurodegenerative diseases, e.g. Parkinson’s disease,
to determine whether dissemination of C. pneumoniae to
the brain occurs in these conditions, or whether it is con-
fined to those with late-onset AD. In this disease, involve-
ment of the APOE
ε
4 gene product in chlamydial infec-
tion of the brain must also be addressed. Regardless of the
results of such studies, however, our results do appear to
suggest that CNS infection by C. pneumoniae may be a
risk factor for development of sporadic late-onset AD, or
at least for some aspects of the pathology associated with
this neurodegenerative disease.
39
Acknowledgements This work was supported by grants AR-42541
(A. P. H.), EY-03324 (J. A. W.-H.), and AG-10160 (B. J. B.) from
the National Institutes of Health. We thank Dr. James England and
Mr. Dennis Tritinger (Department of Pathology, MCP-Hahnemann
School of Medicine) for departmental funds allocated to support this
project in its initial stages. We are grateful to Dr. William Hill and
Dr. Gail Johnson, The Harvard Brain Tissue Resource Center (sup-
ported in part by PHS grant MH/NS31862), and the autopsy service
of the Allegheny University of the Health Sciences for tissues pro-
vided for this work. Drs. A. Arking, J. LiPuma, T. R. Kleyman, and
R. Adler provided useful discussion of the manuscript, for which we
are grateful. Finally, we thank the families who donated tissues to
the several sources listed above for research into Alzheimers dis-
ease, without which the work presented here would not have been
possible.
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... Most of the population were infected during childhood period, and the vast majority of the adult people have serologic evidences of past infection [24]. Biological evidences of CP in atheromatous plaques, and even in influenced areas of Alzheimer's disease patients brain, cogitate about that agent could not be a just respiratory tract infection agent [25,26]. ...
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... These are formed when A reacts with ordinary tau protein and, by a documented process, converts it to hyperphosphorylated tau (p-tau). 15 P-tau leads to destruction of the dendrites that leads to apoptosis. 16 The A, in this instance, was produced simultaneously with the formation of the biofilms inside the neurons. ...
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... These are formed when A reacts with ordinary tau protein and, by a documented process, converts it to hyperphosphorylated tau (p-tau). 15 P-tau leads to destruction of the dendrites that leads to apoptosis. 16 The A, in this instance, was produced simultaneously with the formation of the biofilms inside the neurons. ...
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Chlamydiae are important human pathogens responsible for a wide range of infections and diseases. Chronic infections can be particularly devastating, as shown by diseases caused by Chlamydia trachomatis, such as trachoma and post-infectious tubal infertility, or possibly associated with Chlamydia pneumoniae, such as atherosclerosis. Cytokines are likely to be involved for the early resolution of chlamydial infections. However, recent data suggest a role played by cytokines in establishing chlamydial persistence, thus possibly contributing to chronic disease through immunopathological responses. Recent information from in-vitro cell culture systems and experimental animal models is discussed. Moreover, some new evidence about the effector functions of other immune mediators is presented. (C) Lippincott-Raven Publishers.