close

Вход

Забыли?

вход по аккаунту

?

Central and peripheral nervous system infection immunity and inflammation in the nonhuman primate model of lyme borreliosis.

код для вставкиСкачать
Central and Peripheral Nervous System
Infection, Immunity, and Inflammation in
the NHP Model of Lyme Borreliosis
Andrew R. Pachner, MD,1 Diego Cadavid, MD,1 Gale Shu, BS,1 Donna Dail, MS,1 Sarah Pachner,1
Emir Hodzic, DVM,2 and Stephen W. Barthold, DVM, PhD2
The relationship between chronic infection, antispirochetal immunity, and inflammation is unknown in Lyme neuroborreliosis. In the nonhuman primate model of Lyme neuroborreliosis, we measured spirochetal density in the nervous
system and other tissues by polymerase chain reaction and correlated these values to anti-Borrelia burgdorferi antibody
in the serum and cerebrospinal fluid, and to inflammation in tissues. Despite substantial presence of Borrelia burgdorferi, the causative agent of Lyme borreliosis, in the central nervous system, only minor inflammation was present there,
though skeletal and cardiac muscle, which contained similar levels of spirochete, were highly inflamed. Anti-Borrelia
burgdorferi antibody was present in the cerebrospinal fluid but was not selectively concentrated. All infected animals
developed anti-Borrelia burgdorferi antibody in the serum, but increased amplitude of antibody was not predictive of
higher levels of infection. These data demonstrate that Lyme neuroborreliosis is a persistent infection, that spirochetal
presence is a necessary but not sufficient condition for inflammation, and that antibody measured in serum may not
predict the severity of infection.
Ann Neurol 2001;50:330 –338
Lyme borreliosis is a protean, multisystemic disease
caused by the spirochete Borrelia burgdorferi. The organs most often affected are the skin, the joints, the
heart, and the central and peripheral nervous system.
Neurologic manifestations, known as Lyme neuroborreliosis (LNB), occur in 5 to 20% of North American
cases and in a higher percentage of infected Europeans,
and include aseptic meningitis, encephalopathy, facial
nerve palsy, radiculitis, and peripheral neuropathy.1,2
Lyme borreliosis is currently the most common
arthropod-borne disease in the United States, where
thousands of cases are reported to the Centers for Disease Control (CDC) every year.
Of the several animal models studied, the nonhuman primate (NHP) model is the most representative
of human disease3 because it features consistent neurological infection4 –7 as well as involvement of tissues
outside of the nervous system. Previous studies have
demonstrated disseminated infection but have been
limited by relatively small numbers of animals, inadequate tools to assess spirochetal presence, and incomplete assessment of the inflammation and the anti-B.
burgdorferi immune response.
From the 1Department of Neurosciences, UMDNJ-New Jersey
Medical School, Newark, NJ; and 2Center of Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of
California, Davis, CA.
Received Feb 16, 2001, and in revised form Apr 20. Accepted for
publication Apr 20, 2001.
330
© 2001 Wiley-Liss, Inc.
The goals of this study were to relate spirochetal
presence in nervous system and extraneural tissues in a
large group of NHPs to the anti-spirochetal host immune response and to histopathological evidence of inflammation. Specific questions we wished to answer
were: (1) Are there sites within the nervous system
preferentially infected by the spirochete, and how does
spirochetal density in the nervous system compare with
spirochetal density elsewhere; (2) within areas in the
nervous system, is the presence of inflammation highly
correlated with the presence of spirochetes; (3) is spirochetal load correlated with the anti-B. burgdorferi antibody response, ie do animals with more extensive infection mount higher serum antibody responses either
in the serum or cerebrospinal fluid (CSF); and (4) is
there intrathecal antibody production in the central
nervous system?
Materials and Methods
Animals
Eight male Rhesus macaques, Macaca mulatta, 3 to 4 years
of age, were anesthetized and underwent cisternal punctures
and phlebotomies as previously described.8 Housing and care
Published online Jun 18, 2001; DOI: 10.1002/ana.1093
Address correspondence to Dr A. Pachner, Department of Neurosciences, UMDNJ-New Jersey Medical School, 185 South Orange
Avenue, Newark, NJ 07103. E-mail: pachner@umdnj.edu
was in accordance with the Animal Welfare Act and the
Guide for the Care and Use of Laboratory Animals in facilities accredited by the American Association for Accreditation of Laboratory Animal Care. The study was reviewed and
approved by the New Jersey Medical School and the University of California, Davis, Animal Care and Use Committees. Complete blood count and chemistries were obtained
on all animals at each phlebotomy for monitoring purposes.
Three days prior to inoculation, dexamethasone 2mg/kg
once daily was given intramuscularly and was continued until 28 days postinoculation, at which time 1mg/kg was administered once daily for 1 week, followed by 0.5mg/kg for
2 weeks, after which no more dexamethasone was given.
This dose of dexamethasone is considered relatively mild in
NHPs, and no steroid-induced side effects, ie, fluid retention
manifested as weight gain, glucose intolerance, change in appearance, or change in behavior resulted from this protocol.
Areas of injection were documented, and tissue obtained for
analysis were in regions distant from injections.
Inoculation
Four NHPs, identified as 099, 177, 199, and 383, were injected intradermally with a total of 1ml volume, in multiple
0.1ml aliquots (containing a total of 1 million N40Br strain
spirochetes) along the dorsal thoracic midline, as in previous
studies.6,8,11,12 Four NHPs, identified as 154, 192, 211, and
242, were infected by tick feeding as below. Because there
were no differences between the routes of inoculation on any
of the measures used in this study, the eight infected animals
will be considered together as B. burgdorferi-infected NHPs.
Inoculation of Nonhuman Primates by Feeding of
Infected Ticks
Ticks were infected by placing uninfected larvae on four
C3H/HeJ mice ( Jackson Laboratory, Bar Harbor, ME) that
were inoculated intradermally with 105 N40 spirochetes 2
weeks prior to tick feeding, as previously described.25 In
brief, replete larval ticks were collected and allowed to molt
and harden into nymphs. Thirty ticks were randomly sampled from this population, and individually tested by PCR
for infection; all were positive. For analysis, ticks were individually crushed in liquid nitrogen with a plastic grinder,
and powder was used for DNA extraction with DNeasy tissue kits, according to the manufacturer’s instruction for insects (Qiagen, Valencia, CA). Infection of NHPs by tick
feeding was accomplished by glueing 1.5cm diameter polycarbonate screw-cap chambers with fabric mesh tops onto
the skin of the thoracic midline, using Nexaband glue
(MWI, Boise, ID). Eight nymphal ticks were placed into
each chamber, chambers were sealed, the NHP thorax was
wrapped with elastic bandage, then protected in a fabric vest.
Each day, NHPs were immobilized with ketamine, chambers
opened, and ticks were examined. If fewer than four ticks
were found to be attached, three additional ticks were added.
On days 3 through 5, replete ticks were collected and tested
by PCR, as described above.
Necropsy
On the morning of the necropsy, food and water were withheld. NHPs were immobilized with intramuscular ketamine
for transport, and pentobarbital was given intravenously
upon arrival to the necropsy suite. The posterior neck area
was shaved, scrubbed, and a cisternal puncture was performed to obtain CSF (3– 8ml). Blood was obtained by direct puncture from the inferior vena cava following laparotomy. A final lethal bolus of pentobarbital was then
administered and a cannula was placed in the left ventricle to
allow perfusion with saline. The animal was perfused with
1L of saline per 4kg of body weight. Fluids were drained
into the right thoracic cavity by an incision in the right
atrium and allowed to drain by gravity through an incision
in the thoracic wall. After completion of perfusion, and cessation of cardiac function, the base of the skull was entered
posteriorly, and a culture obtained of the high cervical spinal
cord using a sterile disposable biopsy needle. The head was
then removed, transported to a hood, and incisions into the
skull made using a Stryker saw, and then brain and brainstem exposed. After cultures were obtained of brain and
brainstem structures, the brain and brainstem were sectioned.
The spinal cord was then exposed in the dural sheath by
sequential removal of vertebrae by rongeuring. Cultures were
obtained after sections of spinal cord were exposed and the
dura entered using sterile scissors. The spinal cord and cauda
equina were sectioned. Samples of heart, urinary bladder,
and skeletal muscle were cultured. The heart, aorta, spleen,
testes, lymph nodes, urinary bladder, biceps, quadriceps, brachial plexus, median nerve, and sciatic nerve were then removed for processing. These were organs known to be infected in previous studies of NHP Lyme borreliosis.11-13,18
Tissues collected underwent one of three different processes:
they were frozen, embedded and frozen in OCT, or fixed for
DNA extraction, immunohistochemistry, or histology, respectively.
Polymerase Chain Reaction
DNA was extracted from tissue and polymerase chain reaction (PCR) performed using OspA and OspB primers as in
previous studies.11-13,18
On each PCR a standard curve of positive controls was
run. This curve consisted of 400, 200, 100, 50, 25, 12.5fg of
B. burgdorferi DNA extracted from cultured spirochetes, each
diluted in water; 500ng of DNA extracted from brain of uninfected Rhesus macaques was present in each tube of the
standard curve. The level of B. burgdorferi DNA of any specimen was approximated by visually comparing the band on
the gel to the readings of the standard curve. Thus, the DNA
level of any specimen was expressed as a number of femtograms of B. burgdorferi DNA/500ng of total DNA. The spirochetal load for any given tissue or NHP was determined as
the mean of the levels of DNA present within the 31 specimens tested for each animal. Spirochetal load was also analyzed as percentage of samples within a group as being positive, ie, visible as a band on PCR. Each PCR run also
included negative controls which included both water samples and DNA extracted from the brain of uninfected NHPs.
The false positivity rate was 2%. Runs in which a false positive was detected were assumed to be contaminated, and
data from those runs were not included in the data analysis.
Levels of spirochetal DNA within tissue determined by analyzing the density of the band on gel were confirmed in selected samples by analysis of the PCR product, using capture
Pachner et al: Lyme Neuroborreliosis
331
Fig 1. Spirochetal DNA in tissues.
Spirochetal density is expressed as
the mean log 10 of the femtograms
per 500ng of extracted DNA of
tissue. All samples from each tissue
were used in the calculation, including negative samples which were
assigned a score of 0.
enzyme-linked immunosorbent assay (ELISA) as previously
described.13,18
Statistical analyses were performed using DataDesk software (DataDesk, Ithaca, NY).
Interference with B. burgdorferi Polymerase Chain
Reaction by DNA Extracted from Tissue
The 500ng of DNA extracted from tissue was added to tubes
containing 400, 200, 100, 50, 25, or 12.5fg of B. burgdorferi
DNA. In each experiment, a positive control was included in
which water was added instead of tissue DNA. The degree of
interference was measured by how many logs of 2 the sensitivity curve was pushed to the right relative to the sensitivity
of the standard curve without tissue DNA. For example, an
absence of signal at 12.5, 25, and 50, but positivity at 100fg,
was read as an interference of 3, when the sensitivity of the
standard curve performed in known noninterferring DNA
was 12.5fg.
ELISA/Immunoblot
Serum and CSF antibody ELISAsa and immunoblotting
were performed as previously described8,9,18 Each plate contained a positive control, the necropsy serum of NHP 099,
at dilutions of 1 to 5000, 1 to 15000, 1 to 45000, and 1 to
135000. Sera other than the positive control were run at a
dilution of 1 to 5000. Commercial B. burgdorferi sensu
stricto nitrocellulose strips (Microbiology Reference Laboratory, Cypress, CA) were used.
Pathology
Inflammation was assessed by examination of hematoxylin
and eosin (H and E) stained tissue as described previously,11
with a score of 0 to 5, ranging from no inflammatory cells (a
score of 0) to confluent inflammation (a score of 5).
Results
Clinical Manifestations of Infection
As in previous studies in this model,8,12,13 NHPs tolerated this infection well without obvious clinical signs.
Behavior monitored by Primate Center personnel appeared normal, level of activity was similar to uninfected NHPs, and weight was maintained. Neuropsychological or activity measures were not performed.
Spirochetal Presence in Tissue
Spirochetal presence in tissue was assessed by PCR of
DNA extracted from tissues obtained at the necropsy 3
months after infection. The sensitivity of the PCR was
generally 12.5fg of B. burgdorferi DNA for the OspA
target. Results for groups of tissue are shown in Table
Table 1. PCR for B. burgdorferi in Tissues
Percent Positive Samples by PCR
Mean Density (fg B. burg. NDA/500ng Extracted DNA
Tissue Source
Needle Inoculated
Tick Inoculated
Both Groups
Needle Inoculated
Tick Inoculated
Both Groups
CNS
PNS
Heart Tissue
Other Tissues
All Tissues
22/63 (35%)
11/26 (42%)
4/17 (24%)
5/18 (28%)
42/124 (34%)
20/65 (31%)
10/27 (37%)
5/15 (33%)
6/21 (29%)
41/128 (32%)
42/128 (33%)
21/53 (39%)
9/32 (28%)
11/39 (28%)
82/252 (33%)
38
64
44
26
42
34
59
52
31
41
36
61
47
29
41
CNS ⫽ central nervous system; PNS ⫽ peripheral nervous system, including muscle; Other Tissues ⫽ bladder, skin, spleen, testis, lymph.
332
Annals of Neurology
Vol 50
No 3
September 2001
1; the mean level in the third column is the mean for
all tissues in the group, including those samples which
were negative.
Within the cerebrum, samples obtained from the
frontal and anterior parietal areas were more frequently
positive with higher levels than samples from the posterior parts of the cerebrum. Brainstem and spinal cord
had less organism than cerebrum. The highest concentration of spirochetes occurred in peripheral nervous
system tissue, particularly in the skeletal muscles. Results of the PCR analysis tissue by tissue are shown in
Figure 1. To confirm the results with OspA target,
PCRs on all specimens were performed using the OspB
target. Results were more than 90% concordant, with
lack of concordance being mainly the result of samples
in which OspA was positive and OspB negative.
Interference with Polymerase Chain Reaction by
Extracted DNA
Extraction of tissue specimens using the above techniques yielded tissue extracts of high purity as assessed
by OD 260/OD 280 ratio. When 500ng of these extracts were added to PCR of a standard curve of B.
burgdorferi DNA, interference with PCR signal was observed, but the interference varied among extracts.
Those tissue samples that were positive on the original
PCR displayed less ability to interfere. This was observed primarily with the OspA PCR in which 7 of 12
DNA samples negative on the B. burgdorferi PCR interfered with the standard curve, while only 2 of 16
initially positive DNA samples interferred. There was
no correlation between OD 260 of OD 280 ratio, a
crude assay for DNA purity, and the degree of interference (data not shown). Interference was not affected
by pretreatment of the interferring specimens with
RNase.
multiple bands visualized, the strongest being at 17,
18, 39, 41, 58, 60, and 93 kD (see Fig 2B).
Anti-B. burgdorferi Antibody Response in
Cerebrospinal Fluid
ELISA. Anti-B. burgdorferi antibody was detectable in
the cerebrospinal fluid (CSF) beginning in the second
month postinoculation (data not shown). When the serum and CSF were adjusted to the same concentration
of IgG, values were similar, consistent with absence of
significant intrathecal IgG antibody production. The
total immunoglobulin, IgG, IgA, and IgM, and total
protein concentrations in the CSF were normal for cisternal fluid.
There were no unique bands seen in
the CSF relative to those seen in the serum, and the
pattern, when corrected for immunoglobulin concentration, was similar to that in the serum (data not
shown).
IMMUNOBLOT.
H and E of Target Tissues
Inflammation was present primarily in skeletal and cardiac muscle as shown in Table 2. Brachial plexus and
peripheral nerves had a low level of inflammation but
significantly more than the central nervous system or
those tissues sampled in the group “other tissues,” including aorta, skin, spleen, bladder, testis, and lymph
node. No inflammation was present in the tissues of
uninfected NHPs, ie scores for these animals in all tissues were 0. Examples of inflammation observed in
brain and nerve are shown in Figures 3A and B, respectively.
Anti-B. burgdorferi IgG antibody began to appear at a significant level in the second month of infection, and rose through the third month (Fig 2A).
The pattern of development of IgG antibody was similar in most animals except for NHP 192, which had a
higher antibody level at all time points than the other
NHPs.
Correlations Between Spirochete Levels in Nonhuman
Primates and Antibody Response
There was no significant relationship between spirochete load and anti-spirochetal antibody response. Although NHP 192 had higher antibody levels than the
other animals, the spirochete load in NHP 192 was
similar to the other animals, being in midrange for the
group of eight NHPs. Spirochete levels were analyzed
in relationship to band densities on immunoblots, and
again there were no significant correlations between the
spirochete densities and any of the bands visualized on
the blots.
The immunoblot patterns for the
anti-B. burgdorferi IgG antibody response of all animals
were similar. In both groups, the immunoblots of the
preinoculation sera were negative, and an increasing
number of bands appeared with increasing time postinoculation. At the time of necropsy 3 months postinoculation, sera from all animals in both groups had fulfilled CDC criteria for immunoblot positivity with
Correlations Between Spirochete Levels in Tissue
and Inflammation
Correlations were assessed separately for individual animals in addition to the combined group of eight animals. Spirochetal DNA was present at similar levels in
all tissues, but inflammation was much more marked
in peripheral and skeletal muscle. A scatterplot was
generated from the spirochetal density and the inflam-
Anti-B. burgdorferi Antibody Response in Serum
ELISA.
IMMUNOBLOT.
Pachner et al: Lyme Neuroborreliosis
333
Fig 2. (A) Anti-B. burgdorferi IgG
antibody in tick- versus needleinoculated NHPs. Sera were diluted
1 to 2,500, and run on the same
day for comparison purposes on a
routine anti-B. burgdorferi IgG
ELISA. (B) Immunoblot patterns
for IgG antibody at necropsy in
tick- and needle-inoculated NHPs.
Strips 1 and 2 reacted with positive
sera from infected humans with
strong and weak IgG antibody responses, respectively, and strip 3
represents a negative control human
serum. Strips 4 to 7 are from the
needle-inoculated NHPs (99, 177,
199, and 383, respectively) and
strips 8 to 11 are from the tickinoculated NHPs (154, 192, 211,
and 242, respectively). All sera were
used at a dilution of 1 to 100.
matory index. The correlation coefficient was weakly
positive at 0.191 (Spearman Rank). It appeared from
the scatterplot that the majority of the variability was
present in those tissues without inflammation, ie an inflammatory index of 0. When these values were deleted
from the analysis, the correlation coefficient became
more significant at 0.311.
Discussion
Neurological involvement in the NHP model is
unique3 among animal models of Lyme borreliosis.
Meningitis, neuritis, and myositis are prominent features of NHP Lyme borreliosis.5,8,12,14 –16 B. burgdorferi has been cultured from the spinal cord and CSF,
PCRs are positive in skeletal muscles, peripheral nerves,
nerve root and brachial plexi, and spirochetes can be
visualized by immunohistochemistry.11 In this study,
334
Annals of Neurology
Vol 50
No 3
September 2001
the nervous system continued to be a favored site for
spirochetal invasion with PCR positivity consistently
detectable in sites throughout the neuraxis. The NHP
model, as an excellent model of human disease,3 can be
used to test hypotheses about neurological involvement
that cannot be answered in rodent models in which the
nervous system is not involved.
This manuscript presents for the first time a combined analysis of spirochetal load, immunological response to the spirochete in the CSF and serum, and
inflammation in infected tissues in a large group of animals in the NHP model of LNB. The work showed
that B. burgdorferi is widely disseminated throughout
the central and peripheral nervous system, a strong
host immune response attacks the spirochete but is unable to clear the organism, and there is widespread in-
Table 2. Inflammation in Tissues of Infected NHPs
Tissue
Brain
Brain stem
Spinal cord
Cauda equina
Brachial plexus/peripheral nerves
Skeleton muscle
Cardiac muscle
Other tissues (aorta, skin, spleen,
bladder, testis, lymph node)
Degree of
inflammationa
⬍0.1 (2/188)
⬍0.1 (2/47)
⬍0.1 (3/80)
⬍0.1 (1/13)
0.3 (14/46)
2.8 (67/24)
1.8 (74/41)
⬍0.1 (7/81)
a
Total inflammation score/number of tissues sampled.
flammation in which presence of spirochete is necessary but not sufficient to cause inflammation.
The presence of spirochetal infection was demonstrated by identifying B. burgdorferi DNA in multiple
tissues by PCR. PCR is the preferred method of detection of chronic infection in the NHP model, as other
techniques such as culture, mouse infectivity testing,
and immunohistochemical localization have inadequate
sensitivity.13 The level of spirochetal load in this group
of NHPs varied near the level of detection of B. burgdorferi DNA in tissue, ie 5 to 15fg in 500ng extracted
DNA. Some tissue samples were strongly positive by
gel electrophoresis of PCR products indicating a density 3 to 100 times higher than this cutoff value, while
other tissue samples were negative. It appears likely
that some of these “negative” tissues were false negative, since many samples of DNA extracted from PCRnegative tissues had substances that interferred with the
PCR. This problem is common in PCR studies of tissue where pathogen DNA represents an extremely
small percentage of total DNA assayed, and has been
previously reported in the NHP model of Lyme borreliosis.17
The density of spirochetes in a given tissue was determined in two ways: first, by a semiquantitative measure of samples within a tissue based on comparison to
a standard curve, and second, by sampling numerous
tissues and determining the percentage of tissue samples positive, ie; above the level of detection. Our first
hypothesis was that spirochetes would be found by
PCR diffusely through the central and peripheral nervous system, and this hypothesis was supported by the
results. Based on data from chronically immunosuppressed NHPs,18 which had demonstrated that the
densities of spirochetes were 100- to 1,000-fold higher
in skeletal and cardiac muscle than in CNS tissue, we
additionally hypothesized that the same ratio would
hold true in the immunocompetent NHPs but that the
density of spirochetes would generally be less. The density of spirochetes per milligram of tissue, or per microgram of extracted DNA, was indeed lower in our
Fig 3. Samples of inflammation in the brain (A) and sciatic
nerve (B) of infected NHPs. (A) A focal area of meningeal
thickening and inflammation in the frontal lobe. A nest of
subperineurial inflammatory cells in the sciatic nerve is seen.
immunocompetent NHPs relative to the immunosuppressed NHPs,18 but the densities were surprisingly
uniform in all tissues, with CNS spirochetal load being
essentially identical to that in other tissues, and at levels similar to those in immunosuppressed NHPs. These
data provide evidence for the theory that spirochetes in
CNS tissue are protected from immune-mediated
clearance relative to spirochetes in peripheral nerve,
skeletal muscle, and heart. This may be particularly relevant to chronic CNS Lyme disease,19 –22 in which the
organism may persist in the CNS for months to years.
The studies described in this manuscript were of animals infected for about 3 months; longer studies are
now in progress.
The primary immune defense mechanism against B.
burgdorferi infection, as with other spirochetal infections,23 is the specific humoral immune response.24,25
The anti-B. burgdorferi antibody response in our stud-
Pachner et al: Lyme Neuroborreliosis
335
ies displayed characteristics seen previously.8,9 There
was an initial IgM response followed by a switch to
IgG. The amplitude of the IgG response by ELISA and
the number of B. burgdorferi proteins identified on immunoblot increased with increasing duration of infection. The height and complexity of the IgG response in
NHP Lyme borreliosis is likely important for spirochetal clearance because NHPs treated with steroids
who mount a high titer IgM response with a negligible
IgG response have very high levels of spirochetes18 in
tissues other than central nervous system, relative to
animals who mount a typical strong IgG response to
disseminated infection.
One of the questions posed at the outset of these
studies was whether spirochetal load was consistently
correlated with the anti-B. burgdorferi antibody response. A reasonable hypothesis would be that because
the presence of the spirochete theoretically drives the
specific antibody response, the higher the level of antibody, the greater the spirochetal load. The only animal with an antibody response significantly different
than the other NHPs was NHP 192, which had a
higher antibody response detected by ELISA of the serum. However, the spirochetal load in this NHP was
essentially identical to the others. Conversely, NHP 177,
the only NHP with a substantially lower spirochetal load
relative to the others, had a similar antibody level to the
other NHPs. Thus, it appeared that there was no correlation between spirochetal load and level of antibody as
measured by ELISA. Interestingly, the immunoblots of
the serum antibody response in all NHPs looked very
similar. Thus, the elevated antibody seen on the ELISA
of the serum of NHP 192 may have resulted from antibodies directed against nonprotein antigens not detected on immunoblots, an antibody reactivity previously described in Lyme borreliosis.26
Another question we wished to answer was: Within
areas in the nervous system, is the presence of inflammation highly correlated with the presence of spirochetes? These data confirm an association, but it is not
a close one. Thus, although the presence of spirochetes
is a necessary condition, it is not sufficient. For instance, the cerebrum had a large load of spirochetes
relative to other organs, but had no inflammation.
Other organs, such as skeletal and cardiac muscle, had
very high levels of inflammation with spirochetal levels
similar to that of brain. However, it is still possible that
a correlation does exist between spirochetal load and
inflammation, as skeletal and cardiac muscle may have
had much higher levels of spirochetes prior to
immune-mediated clearance. Thus, the low levels in
muscle relative to the levels seen in immunosuppressed
NHPs18 may indicate that the necropsy tissue assayed
in this experiment was the result of substantial, but incomplete, clearance of the spirochete from these tissues. For example, spirochete numbers and inflamma-
336
Annals of Neurology
Vol 50
No 3
September 2001
tion may have been more highly correlated at earlier
time points postinfection.
The inflammatory response was assessed in tissues
obtained at necropsy by examining muliple organs
stained with hematoxylin and eosin. The inflammation
was similar to that described in previous studies in the
NHP model11,27 for most tissues. Inflammatory infiltrates in cardiac muscle have been composed of T cells
and plasma cells and to a lesser degree B cells. There
have been few studies of inflammation in tissues of humans with Lyme borreliosis, but those published have
been consistent with the NHP model in showing multifocal collections of lymphocytes and plasma cells.27
Cardiac muscle inflammation has been consistent in
the NHP model of Lyme borreliosis, similar to the
mouse model,28,29 while skeletal muscle inflammation,
though not as prominent previously in NHPs injected
with N40Br, has been seen in other studies of LNB in
NHPs infected with other B. burgdorferi strains.15 Why
inflammation tends to occur more in cardiac and skeletal muscle relative to other organs is unclear but may
be an initial preferential tropism of the spirochete to
collagen-rich muscle followed by immune-mediated incomplete clearance of the spirochete. B. burgdorferi is
known to have an affinity for collagen.28,30
In the immune response to other pathogens, intrathecal antibody production is associated with improved
clearance of organisms from the CNS.31,32 Another hypothesis that we tested in this study was that selective
intrathecal IgG antibody production would not be
prominent, despite the presence of large amounts of B.
burgdorferi within the central nervous system. This hypothesis was based on the fact that N40Br is a B.
burgdoferi sensu stricto strain of the pathogen, and in
human studies of Lyme neurological involvement this
genogroup of the pathogen, intrathecal IgG antibody
production33 has not been prominent, in contrast to
the pathogen of Lyme meningitis in Europe, B. garinii,
in which selective concentration of intrathecal IgG antibody is a consistent feature.34,35 Previous to this
study, we had found anti-B. burgdorferi IgG antibody
in the CSF of our infected NHPs,8,13 but had not
tested specifically whether this was more than would be
predicted by passive diffusion from the serum. In the
current work we documented that intrathecal IgG antibody production was not present and that the antibody present in the CSF was likely because of diffusion
from the serum; these data are consistent with the human disease. The factors that determine which types of
infections in the nervous system induce intrathecal antibody production are unknown. Studies are currently
underway in our laboratory to assess the presence of
intrathecal antibody production in NHPs infected with
a B. garinii strain of B. burgdorferi, a European strain.
Our studies also compared two different routes of
inoculation, needle and tick, and found them to be
comparable. The optimum route for inoculation in animal models of Lyme borreliosis has been controversial,
with evidence that both needle inoculation and tick inoculation result in systemic infection,36,37 although immune responses to the organism may be different38 – 40
and vaccine studies may show different results for protection for the challenge by the two routes.41 Tick inoculation has significant methodological disadvantages
relative to needle inoculation. Investigators who perform tick inoculations must have experience with the
insects and facilities that will support ticks. The time
required for tick inoculations is greater than for needle
injection, because initially mice need to be infected by
needle; the ticks then need to be fed on the infected
mice prior to putting the ticks on NHPs. Finally, the
inoculum entering the NHP cannot be quantitated,
and may vary from one NHP to the next. Because of
these problems, most investigators working with animal models of Lyme borreliosis use needle inoculation,
which is simple and fast and requires no special facilities. However, tick inoculation has some theoretical
benefits. For instance, some investigators feel that infection with laboratory-reared ticks is more “natural”
than needle inoculation. In addition, others feel that
expression of B. burgdorferi proteins in the infected animal is likely to be more similar to expression in ticks
rather than culture medium. However, in the only
study relevant to this issue, B. burgdorferi from
laboratory-reared ticks behaved more similarly to cultured spirochetes than to field-sampled ticks,42 indicating significant complexities in delivery of infection by
ticks.
In this study, the NHPs underwent transient immunosuppression with the corticosteroid dexamethasone
during the initial phase of the infection, at a dose considered mild for NHPs. This technique has been utilized to maximize the yield of infection because in previous studies in this model, some immunocompetent
NHPs spontaneously cleared infection within the first
few months, presumably from the rapid development
of high-titered anti-B. burgdorferi IgG.18 This mild immunosuppression protocol, previously used in this
model,8,13 resulted in all eight NHPs in these 2 groups
becoming infected, and allowed the use of a minimum
of experimental animals for these studies.
These results in the NHP model have direct relevance to the diagnosis and therapy of Lyme neuroborreliosis in humans.2 The height of the antibody response to B. burgdorferi within an outbred population
such as humans or NHPs, infected with the same
strain and amount of spirochete, will be variable. Thus,
the relative amplitude of the antibody response is likely
not to be useful clinically to determine severity of inflammation. The magnitude of inflammation bears
only a slight association with spirochete density, and
CNS tissues had minimal inflammation despite sub-
stantial spirochetal densities; thus, measures of inflammation such as CSF pleocytosis are not highly correlated with spirochete load. Our studies were performed
with an American isolate of N40Br. Because the clinical characteristics of Lyme neuroborreliosis are different
in Europe,35 and the strains of spirochete are substantially different there, the above conclusions may not be
able to be applied to European LNB. Studies are now
in progress in NHPs infected with a European strain.
In summary, the studies outlined identify the central
and peripheral nervous system as major reservoirs of
spirochetal infection and demonstrate that a strong,
well-developed anti-B. burgdorferi humoral immune response does not clear spirochetes from the animal during months of infection, especially in the nervous system. Spirochetal presence is a necessary but not
sufficient condition, for inflammation. Despite the
presence of the spirochetes in CNS tissue, there is little
inflammation and no intrathecal antibody production
in the CNS, in contrast to strong systemic antibody
production and marked inflammation in muscle.
Supported by the National Institutes of Health (NO1-AI 95358
and RO1-NS34715 to A.R.P.).
References
1. Pachner AR, Steere AC. The triad of neurological manifestations of Lyme disease. Neurology 1985;35:47–53.
2. Pachner AR. Borrelia burgdorferi in the nervous system: the
new “Great Imitator.” Ann NY Acad Sci 1988;539:56 – 64.
3. Coyle PK. Neurological Lyme disease: is there a true animal
model? Ann Neurol 1995;38:560 –562.
4. Philipp MT, Aydintug MK, Bohm RP, et al. The early and
early disseminated phases of Lyme disease in the Rhesus
monkey: a model for the infection in humans. Infect Immun
1993;61:3047–3059.
5. Roberts ED, Bohm RP, Cogswell FB, et al. Chronic Lyme disease in the Rhesus monkey. Lab Invest 1995;72:146 –160.
6. Pachner AR, Amemiya K, Delaney E, et al. Interleukin 6 is
expressed at high levels in the CNS in Lyme neuroborreliosis.
Neurology 1997;49:147–152.
7. Roberts ED, Bohm RP, Lowrie RC, et al. Pathogenesis of Lyme
neuroborreliosis in the rhesus monkey: the early disseminated
and chronic phases of disease in peripheral nervous system.
J Infect Dis 1998;178:722–732.
8. Pachner AR, Delaney E, O’Neill T, Major E. Inoculation of
non-human primates with the N40 strain of Borrelia burgdorferi leads to a model of Lyme neuroborreliosis faithful to the
human disease. Neurology 1995;45:165–172.
9. Pachner AR, Ricalton N, Delaney E. Comparison of polymerase chain reaction with culture and serology in murine experimental Lyme borreliosis. J Clin Microbiol 1994;31:208 –214.
10. Dressler F, Whalen JA, Reinhardt BN, Steere AC. Western
blotting in the serodiagnosis of Lyme disease. J Infect Dis 1993;
167:392– 400.
11. Cadavid D, O’Neill T, Schaefer H, Pachner AR. Borrelia burgdorferi localizes to the leptomeninges, perineurium, and perimysium in the non-human primate model of Lyme neuroborreliosis. Lab Invest 2000;80:1043–1054.
12. Pachner AR, Delaney E, O’Neill T. Neuroborreliosis in the
Pachner et al: Lyme Neuroborreliosis
337
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
non-human primate: Borrelia burgdorferi persists in the central
nervous system. Ann Neurol 1995;38:667– 679.
Pachner AR, Zhang W, Schaefer S, O’Neill T. Detection of
active infection in nonhuman primates with Lyme neuroborreliosis: comparison of PCR, culture and a bioassay. J Clin
Microbiol 1998;36:3243–3247.
Philipp MT, Johnson BJB. Animal models of Lyme disease:
pathogenesis and immunoprophylaxis. Trends Microbiol 1994;
2:431– 437.
England JD, Bohm RP, Roberts ED, Philipp MT. Lyme neuroborreliosis in the rhesus monkey. Semin Neurol 1997;17:53–
56.
England JD, Bohm RP, Roberts ED, Philipp MT. Mononeuropathy multiplex in rhesus monkeys with chronic Lyme disease. Ann Neurol 1997;41:375–384.
Cogswell FB, Bantar CE, Hughes TG, et al. Host DNA can
interfere with detection of Borrelia burgdorferi in skin biopsy
specimens by PCR. J Clin Microbiol 1996;34:980 –982.
Pachner AR, Amemiya K, Bartlett M, et al. Lyme borreliosis in
rhesus macaques—effect of corticosteroids on spirochetal load
and isotype switching of anti-Borrelia burgdorferi antibodies.
Clin Diagn Lab Immunol 2001;8:225–232.
Ackermann RE, Gollmer E, Rehse-Kupper B. Progressive
Borrelien-Enzephalomyelitis. Chronische manifestation der
Erythema-chronicum-migrans Krankeit am Nervensystem.
Dtsch Med Wochenschr 1985;110:1039 –1042.
Logigian EL, Kaplan RF, Steere AC. Chronic neurological
manifestations of Lyme disease. N Engl J Med 1990;323:1438 –
1444.
Pachner AR, Duray P, Steere AC. Central nervous system manifestations of Lyme disease. Arch Neurol 1989;46:790 –795.
Logigian EL, Kaplan RF, Steere AC. Successful treatment of
Lyme encephalopathy with intravenous ceftriaxone. J Infect Dis
1999;180:377–383.
Cadavid D, Pachner AR. Spirochetal infections of the CNS. In:
Griggs RC, Joynt RJ, eds. Clinical neurology. Philadelphia:
Lippincott-Raven, 1998.
Johnson RC, Kodner C, Russel M. Passive immunization of
hamsters against experimental infection with the Lyme disease
spirochete. Infect Immun 1986;53:713–714.
Barthold SW, Feng S, Bockenstedt LK, et al. Protective and
arthritis-resolving activity in serum from mice infected with
Borrelia burgdorferi. Clin Infect Dis 1997;25:S9 –S17.
Wheeler CM, Garcia Monco JC, Benach JL, et al. Nonprotein
antigens of Borrelia burgdorferi. J Infect Dis 1993;167:665– 674.
England, JD, Bohm RP, Roberts ED, Philipp MT. Mononeuropathy multiplex in Rhesus monkeys with chronic Lyme disease. Ann Neurol 1997;41:375–384.
Pachner AR, Basta J, Hulinska D. Localization of Borrelia
burgdorferi in murine Lyme borreliosis by electron microscopy.
Am J Trop Med Hyg 1995;52:128 –133.
338
Annals of Neurology
Vol 50
No 3
September 2001
29. Barthold SW, deSouza MS, Janotka JL, et al. Chronic Lyme
borreliosis in the laboratory mouse. Am J Pathol 1993;143:
959 –972.
30. Sigal LH. Lyme disease: a review of aspects of its immunology
and immunopathogenesis. Annu Rev Immunol 1997;15:63–92.
31. Griffin D, Levine B, Tyor W, et al. The role of antibody in
recovery from alphavirus encephalitis. Immunol Rev 1997;159:
155–161.
32. Tyor WR, Wesselingh S, Levine B, Griffin DE. Long term intraparenchymal Ig secretion after acute viral encephalitis in
mice. J Immunol 1992;149:4016 – 4020.
33. Steere AC, Berardi VP, Weeks KE, et al. Evaluation of the intrathecal antibody response to Borrelia burgdorferi as a diagnostic test for Lyme neuroborreliosis. J Infect Dis 1990;161:1203–
1209.
34. Wilske B, Scierz G, Preac-Mursic V, et al. Intrathecal production of specific antibodies against Borrelia burgdorferia in patients with lymphocytic meningoradiculitis. J Infect Dis 1986;
153:304 –314.
35. Hansen K, Lebech AM. The clinical and epidemiological profile of Lyme neuroborreliosis in Denmark, 1985–1990: a prospective study of 187 patients with Borrelia burgdorferi-specific
intrathecal antibody production. Brain 1992;115:399 – 423.
36. Levin M, Levine JF, Yang S, et al. Reservoir competence of the
southeastern five-lined skink (Eumeces inexpectatus) and the
green anole (Anolis carolinensis) for Borrelia burgdorferi. Am J
Trop Med Hyg 1996;54:92–97.
37. Shih CM, Liu LP. Accelerated infectivity of tick-transmitted
Lyme disease spirochetes to vector ticks. J Clin Microbiol 1996;
34:2297–2299.
38. Roehrig JT, Piesman J, Hunt AR, et al. The hamster immune
response to tick-transmitted Borrelia burgdorferi differs from
the response to needle-inoculated, cultured organisms. J Immunol 1992;149:3648 –3653.
39. Golde WT, Kappel KJ, Dequesne G, et al. Tick transmission of
Borrelia burgdorferi to inbred strains of mice induces an antibody response to P39 but not to outer surface protein A. Infect
Immun 1994;62:2625–2627.
40. Aydintug MK, Gu Y, Philipp MT. Borrelia burgdorferi antigen
that are targeted by antibody-dependent, complement-mediated
killing in the rhesus monkey. Infect Immun 1994;62:4929 –
4937.
41. Hagman KE, Yang X, Wikel SK, et al. Decorin-binding protein
A (DbpA) of Borrelia burgdorferi is not protective when immunized are challenged via tick infestation and correlates with
the lack of DbpA expression by B. burgdorferi in ticks. Infect
Immun 2000;68:4759 – 4764.
42. Brunet LR, Spielman A, Fikrig E, Telford SR III. Heterogeneity of Lyme disease spirochetes within individual vector ticks.
Res Microbiol 1997;148:437– 445.
Документ
Категория
Без категории
Просмотров
1
Размер файла
1 077 Кб
Теги
central, periphery, mode, nervous, inflammation, primate, borreliosis, nonhuman, lyme, system, infectious, immunity
1/--страниц
Пожаловаться на содержимое документа