Transcutaneous ?-amyloid immunization reduces
cerebral ?-amyloid deposits without T cell
infiltration and microhemorrhage
William V. Nikolic*, Yun Bai*, Demian Obregon*, Huayan Hou*, Takashi Mori*†, Jin Zeng*, Jared Ehrhart*,
R. Douglas Shytle*‡, Brian Giunta*, Dave Morgan§, Terrence Town¶?, and Jun Tan*‡§?**
*Department of Psychiatry and Behavioral Medicine,‡Center for Excellence in Aging and Brain Repair, and§Department of Molecular Pharmacology and
Physiology, University of South Florida, Tampa, FL 33613;†Institute of Medical Science, Saitama Medical Center/School, Saitama 350-8550, Japan;
¶Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06520-8011; and **Department of Molecular Genetics, Third Medical
University, Chongqing 400038, China
Edited by Richard A. Flavell, Yale University School of Medicine, New Haven, CT, and approved November 29, 2006 (received for review October 24, 2006)
Alzheimer’s disease (AD) immunotherapy accomplished by vacci-
nation with ?-amyloid (A?) peptide has proved efficacious in AD
treatment of cerebral amyloidosis without concurrent induction of
detrimental side effects are lacking. We have developed a trans-
cutaneous (t.c.) A? vaccination approach and evaluated efficacy
and monitored for deleterious side effects, including meningoen-
cephalitis and microhemorrhage, in WT mice and a transgenic
mouse model of AD. We demonstrate that t.c. immunization of WT
mice with aggregated A?1–42plus the adjuvant cholera toxin (CT)
results in high-titer A? antibodies (mainly of the Ig G1 class) and
A?1–42-specific splenocyte immune responses. Confocal micros-
copy of the t.c. immunization site revealed Langerhans cells in
areas of the skin containing the A?1–42 immunogen, suggesting
processing. To evaluate the efficacy of t.c. immunization in reduc-
ing cerebral amyloidosis, transgenic PSAPP (APPsw, PSEN1dE9)
mice were immunized with aggregated A?1–42 peptide plus CT.
Similar to WT mice, PSAPP mice showed high A? antibody titers.
Most importantly, t.c. immunization with A?1–42plus CT resulted
in significant decreases in cerebral A?1–40,42levels coincident with
increased circulating levels of A?1–40,42, suggesting brain-to-blood
efflux of A?. Reduction in cerebral amyloidosis was not associated
with deleterious side effects, including brain T cell infiltration or
cerebral microhemorrhage. Together, these data suggest that t.c.
immunization constitutes an effective and potentially safe treat-
ment strategy for AD.
Alzheimer’s disease ? cytokine ? Langerhans cells ? vaccine
cellular neurofibrillary tangles and extracellular senile plaques
primarily composed of 40- to 42-aa ?-amyloid (A?) peptides (1). In
PDAPP transgenic mouse model of AD with A?1–42plus Freund’s
adjuvant resulted in dramatic reduction of cerebral amyloidosis.
clinical trial, patients were administered a synthetic A? peptide
(AN-1792) plus adjuvant, and ?6% of these patients developed
aseptic meningoencephalitis, most likely mediated by brain-
infiltrating activated T cells (3, 4). This serious side effect led to
suspension of the clinical trial. Furthermore, passive transfer of A?
antibodies to transgenic AD mice results in cerebral microhemor-
rhage, a potentially adverse side effect (5, 6). Uncovering of these
adverse events has redirected A? vaccination strategies toward the
goal of developing an approach that is both safe and effective.
Studies examining the brains of A?-vaccinated patients devel-
oping meningoencephalitis implicate A?-reactive T cell subsets as
major components of this deleterious response to active A? vac-
and is pathologically characterized by the presence of intra-
cination (7, 8). To subvert possible meningoencephalitis resulting
from A? vaccination, various strategies have been attempted.
Interestingly, recent works suggest that A?-derived peptides deliv-
ered intranasally (with adjuvant) to mucosal epithelial tissues
results in effective clearance of A? plaques and improvement of
cognitive function in animal models of AD. Moreover, T cell
reactivity appeared to be considerably reduced compared with
other active immunization strategies. In other studies, differential
T cell responses depended on the epitope/fragment of A? peptide
used for vaccination. Specifically, portions of the A? peptide
seemed to stimulate different T cell responses, resulting in either
proinflammatory T helper (Th) cell type 1 (Th1) responses or
antiinflammatory Th cell type 2 (Th2) responses (9, 10). Such
findings imply that A? vaccination is not only efficacious, but may
also prove to be safe and therefore a feasible strategy for AD
therapy depending on a number of factors, including route of
delivery, adjuvant choice, and A? epitope administered.
The skin is a well established effective route for vaccination,
including delivery of peptide-based vaccines (11–13). Strong hu-
moral and cellular immune responses have been elicited after
transcutaneous (t.c.) vaccination (14), largely owing to the diverse
populations of resident antigen-presenting cells (APCs) and other
immune cells in the various dermal layers. Subsets of dermal-
resident Langerhans cell (LC) precursors, known as migratory
CD14?LC precursors, are important immune regulators that
demonstrate ‘‘professional’’ APC capability, including reducing T
cell stimulatory function by producing antiinflammatory cytokines
(15). Also, skin-resident keratinocytes release the antiinflamma-
tory cytokine IL-10 in response to certain stimuli. Keratinocyte-
derived IL-10 serves to buffer harmful proinflammatory immune
activation and thereby preserves skin barrier integrity (16).
targeting A? immunotherapy to skin tissue might provide an
immunotherapeutic approach that is both efficacious and safe. In
this study we tested a t.c. A? immunization strategy using both WT
and the transgenic PSAPP (APPsw, PSEN1dE9) mouse model of
designed research; W.V.N., Y.B., D.O., H.H., T.M., J.Z., J.E., and T.T. performed research;
D.M. and T.T. contributed new reagents/analytic tools; W.V.N., Y.B., D.O., H.H., T.M., B.G.,
and J.T. analyzed data; and W.V.N., T.T., and J.T. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviations: AD, Alzheimer’s disease; A?, ?-amyloid; t.c., transcutaneous; CT, cholera
toxin; Th, T helper; LC, Langerhans cell.
?To whom correspondence may be addressed. E-mail: email@example.com or
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
February 13, 2007 ?
vol. 104 ?
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t.c. Immunization of Mice with A?1–42Peptide Plus CT Results in High
A? Antibody Titers. To determine whether t.c. administration of
A?1–42to mice could result in A? antibody production, 20 non-
transgenic C57BL/6 mice at 8 weeks of age were used for this
experiment, and 10 mice received aggregated A?1–42peptide with
CT (A?/CT), whereas the remaining 10 received CT alone. Mice
biweekly for the next 12 weeks. Blood samples were taken at weeks
0 (baseline), 4, 8, and 16 (immediately before the death of these
mice). Plasma A? antibody titers were measured by ELISA. A?
antibody isotypes were determined by an IgG isotyping assay using
an isotype-specific secondary antibody (18). A? antibodies were
first detected at week 4 in all immunized mice and dramatically
increased thereafter (Fig. 1A; **, P ? 0.001). Consistent with our
Fruend’s adjuvant (18, 19), A? antibodies of the IgG1 isotype were
produced at the highest level, whereas IgG2a antibodies directed
against A? were present in significantly lesser quantity (Fig. 1A; **,
P ? 0.001). A? IgG2b antibody was least detectable (Fig. 1 B and
titers in plasma from these mice at weeks 0, 4, 8, and 16 after
immunization. A similar pattern of results was observed, albeit CT
antibody titers were higher in plasma from mice t.c.-immunized
with either A?/CT compared with CT alone (data not shown). As
an additional control group, we injected WT mice with PBS alone
(n ? 10) in parallel but were unable to detect A? antibody titers in
these animals, confirming the specificity of our titer assay (data not
A?-Specific Immune Responses in Splenocytes from Mice t.c.-Immu-
nized with A? Plus CT. We quantified key cytokines produced by
activated T cells (IFN-?, IL-2, and IL-4) in splenocyte superna-
tants by ELISA as an indicator of immune responsiveness.
Nonspecific mitogenic stimulation of cultured splenocytes with
Con A resulted in ?2-fold increases in IFN-?, IL-2, and IL-4
production in cells from mice immunized with A?/CT or CT
versus PBS-immunized controls (Fig. 1D). No statistically sig-
nificant difference was noted between A?/CT and CT alone
groups for each cytokine (P ? 0.05). Similar results were
observed in cultured splenocytes stimulated with anti-CD3 (data
not shown). On the other hand, specific recall stimulation with
A?1–42 peptide of primary cultured splenocytes from A?/CT
t.c.-immunized mice resulted in significantly increased produc-
tion of IFN-?, IL-2, and IL-4 compared with splenocytes cul-
tured from mice immunized with CT alone (Fig. 1D; **, P ?
0.001). Importantly, regarding the antiinflammatory cytokine
IL-4, an 8-fold increase in its secretion by splenocytes from
A?/CT-immunized mice after A?1–42 recall stimulation was
observed (Fig. 1D; ##, P ? 0.001). Taken together with the
predominantly IgG1 A?-specific humoral response in A?/CT
t.c.-immunized WT mice, this IL-4 result suggests an antiinflam-
matory Th2 immune response.
t.c. Immunization with A? Plus CT Promotes Recruitment of Dermal
LCs. Skin tissues and frozen sections were prepared from WT mice
18 h after t.c. vaccination with A?/CT or CT alone, and they were
costained with antibodies against mouse CD207 (Langerin, a
pan-LC marker), mouse CD11c [a marker of an LC subset (20)],
and/or rabbit anti-human A?. A?/CT t.c. immunization resulted in
LC recruitment into dermal layers compared with CT alone or
PBS-immunized controls [Fig. 1E and supporting information (SI)
Fig. 5], where dermal LCs were much less frequently observed.
Furthermore, these LCs tended to be found in regions of the skin
that stained positive for A? peptide by 4G8 A? antibody (Fig. 1E).
These data show the migratory action of LCs in response to A?/CT
the initial immune response to A?/CT t.c. immunization.
Immune Response and Increased Circulating A?. Eighteen transgenic
PSAPP mice, which overproduce human A? and develop signifi-
cant amyloid deposits by 8 months of age (17), were immunized at
4 months of age in this study. Half of them (n ? 9) received
aggregated A?1–42peptide with CT, whereas the remaining half
received CT alone. The 16-week procedure that we used was
t.c.-immunized with aggregated A?1–42peptide plus
CT. (A) A? antibody titers were measured by ELISA.
Data are presented as mean ? SD (n ? 10) of A?
by post hoc comparison revealed significant differ-
ences in anti-A? titers when comparing week 4 to
were determined by an Ig isotyping assay and are
represented as ratios (mean ? SD; n ? 10) of IgG1 to
IgG2a (B) or IgG1 to IgG2b (C). One-way ANOVA fol-
lowed by post hoc comparison revealed significant
differences between the ratio of IgG1 and IgG2a ver-
(D) Splenocytes were individually isolated and cul-
alone, or PBS (control). These cells were stimulated
with Con A (5 ?g/ml) or A?1–42(20 ?g/ml) for 48 h.
Cultured supernatants were collected from these cells
for IFN-?, IL-2, and IL-4 cytokine analyses by ELISA.
of each cytokine over PBS control. One-way ANOVA
followed by post hoc comparison revealed significant
differences between groups for levels of each of three
cytokines [IFN-?, IL-2, and IL-4 (**, P ? 0.001)] after in
vitro A?1–42 challenge. As noted, there was also a
significant difference in cytokine levels between IL-4 and either IFN-? or IL-2 after A?1–42challenge (##, P ? 0.001). (E) To characterize dermal immune responses
to A?/CT t.c. immunization, skin tissues were prepared from nontransgenic C57BL/6 mice t.c.-immunized for 18 h with PBS (control, Top), CT alone (Middle), or
A?/CT (Bottom) as indicated and then analyzed by laser scanning confocal microscopy with the indicated antibodies (antibody 4G8 was used to reveal A?). Note
the presence of CD207?CD11c?LCs in A?-positive regions in the A?/CT t.c.-immunized group. DAPI (blue signal) was used as a nuclear counterstain in merged
images shown to the right. (Scale bar: 50 ?m.)
Generation of immune responses in WT mice
www.pnas.org?cgi?doi?10.1073?pnas.0609377104Nikolic et al.
from these mice were measured by ELISA. Significant increases in
A? antibody titers were observed in PSAPP mice t.c.-immunized
with A?/CT (P ? 0.001) (Fig. 2A). Similar to WT mice, A?
antibodies were first detected at week 4 in plasma and dramatically
increased thereafter. By contrast, these A? antibodies were not
detected in plasma from CT-vaccinated control mice (Fig. 2A).
Two weeks after the final immunization, primary splenocytes were
isolated and cultured from individual mice. Recall stimulation of
splenocytes from A?/CT t.c.- immunized PSAPP mice with A?1–42
peptide resulted in significantly increased production of IFN-? and
IL-2, and particularly IL-4 (data not shown), similar to results from
A?/CT t.c.-immunized WT mice.
In support of peripheral sink hypothesis (21, 22) we quantified
A? levels in the blood by ELISA and found significantly increased
circulating A?1–40,42in PSAPP mice t.c.-immunized with A?/CT as
early as 4 weeks after immunization (Fig. 2 B and C). Importantly,
plasma A?1–40,42levels increased rapidly to the highest values of
781 ? 118 pg/ml and 129 ? 46 pg/ml, respectively, by week 8 (2
after 16 weeks of immunization.
PSAPP Mice t.c.-Immunized with A? Plus CT Show Reduced Cerebral
Amyloidosis in the Absence of T Cell Infiltrates or Cerebral Microhe-
morrhage. Using a sandwich ELISA-based method, detergent-
soluble A?1–40,42levels were reduced by ?53% and 48%, respec-
tively (P ? 0.001) (Fig. 3A). Insoluble A?1–40,42(prepared by acid
extraction of detergent-insoluble material in 5 M guanidine) levels
were reduced by 50% and 54%, respectively, in A?/CT t.c.-
A? plaques in brains of mice that received t.c. immunization with
histochemistry (Fig. 3 D and F, respectively). At 10 months of age,
A?/CT t.c.-immunized PSAPP mice showed 42–58% (Fig. 3E) and
58–65% (Fig. 3G) reductions in 4G8 immunoreactive and Congo
red-positive A? deposits, respectively, across hippocampal and
cortical brain regions examined. Together, these results demon-
strate that t.c. immunization with A?/CT is effective in reducing
cerebral amyloidosis in PSAPP mice.
To determine whether this systemic increase in human A?1–40,42
correlation analysis and noted an inverse correlation between
plasma and brain-soluble A? (Fig. 3C). In addition, simultaneous
analysis of plasma A? levels and brain-soluble A? levels on a
mouse-by-mouse basis provided further evidence of an inverse
correlation (SI Fig. 6). These data suggest that circulating A?
antibodies play an important role in clearance of A? from brain to
not solely responsible for reduced cerebral amyloidosis, as, inter-
estingly, we detected A? antibodies in brain homogenates from
A?/CT t.c.-immunized PSAPP mice [18.87 ? 6.25 (mean ng/mg of
total protein ? SD)], whereas A? antibodies were undetectable in
PSAPP mice t.c.-immunized with CT alone (data not shown).
that additional A? clearance mechanisms (i.e., mediated by the Fc
receptor on phagocytic microglia) are operating.
induce T cell infiltration into the brain. We immunostained brain
sections from mice immunized with A?/CT, CT alone, or, as a
protein emulsified in complete Freund’s adjuvant (to induce ex-
perimental autoimmune encephalomyelitis; brains were isolated 20
days after immunization, when copious amounts of T cells have
infiltrated the brain). As shown in Fig. 4 A–C, we did not detect
CD3?T cells in brains from mice t.c.-immunized with CT or
A?/CT; however, this result was not caused by a technical issue as
T cells were detected in the positive control tissue (Fig. 4A).
with CD4 or CD8 antibodies (which stain different subsets of T
cells) and did not detect T cell infiltration in brains of mice
in our positive control tissue (data not shown).
Further, we carried out microhemorrhage analysis via Perl’s
Prussian blue stain and did not detect positive staining with this
in our positive control tissue [in this case, from mice i.p. passively
given A? antibodies (23)] (Fig. 4 D–F). As an additional indicator
of possible blood-brain-barrier breakdown, we analyzed apoli-
poprotein B (present in blood but not normally in brain) levels in
these brain tissues by Western blot and it was undetectable in the
t.c.-immunized groups (SI Fig. 7). It is noteworthy that both the
Prussian blue stain and apolipoprotein B analyses were negative in
t.c.-immunized PSAPP mice, suggesting that detection of A?
blood-brain-barrier, but rather was likely caused by physiological
entry of A? antibodies into the brain parenchyma.
To translate animal A? immunization approaches into successful
but also safe, including avoiding meningoencephalitic reactions to
A? immunization previously observed in humans (24, 25). Exper-
imental and postmortem evidence suggests that such aseptic me-
ningoencephalitis observed in AD patients after A? vaccination
resulted from CNS invasion by A?-reactive T cells (10, 25). The
requirement of A?-reactive T cells for cerebral amyloid plaque
t.c.-immunized PSAPP mice at the time points indicated. (A) Plasma A? antibody titers were measured by ELISA. Data are presented as mean ? SD (n ? 9) of A?
peptides were measured separately by A? ELISA. Data are presented as mean ? SD (n ? 9) of A?1–40or A?1–42(pg/ml plasma).*, P ? 0.05;**, P ? 0.001. Arrows
indicate each t.c. immunization with respect to the time of blood sample collection.
Nikolic et al.
February 13, 2007 ?
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unclear (8, 25, 26). A previous study using intranasal delivery of
short A?-derived peptides lacking T cell reactive epitopes with a
specific immune-modulating adjuvant (LT R192G) demonstrated
the possibility of potentiating an effective humoral anti-A? re-
sponse while minimizing A? reactive T cells (10), suggesting that
A?-reactive T cells are not necessary for an effective A? antibody
represent reasonable therapeutic potential; however, it should be
noted that anosmia/hyposmia may limit the usefulness of intranasal
A? immunization. Further evidence that A?-reactive T cells are
likely not required for A? immunotherapy efficacy comes from
passive immunization studies, which have shown that humoral
responses alone may be sufficient to effectively reduce cerebral
amyloid burden, and thereby mitigate neurodegeneration (27, 28).
Here, we investigated the potential of t.c. A? immunization for
the treatment of AD-like cerebral amyloidosis in transgenic mice.
t.c. immunization is an attractive route of delivery, as it is conve-
nient, relatively painless, and minimally invasive. This strategy is
also appealing because the epidermal and dermal immune systems
provide a unique environment for immune stimulation caused by
LC antigen presentation (29–32). Indeed, after A?/CT t.c. immu-
nization, we observed cells double-positive for CD207 and CD11c
in dermal regions that stained positive for A?, showing that these
LCs migrate to the t.c. immunization site and likely participate in
millennia, owing to constant bombardment of the skin with various
antigenic stimuli, resulting in a delicate balance between immuno-
genic and tolerogenic responses. It is noteworthy that t.c./
epicutaneous immunization has been successful in mitigating neu-
rodegenerative disease in both induced and spontaneous forms of
demyelinating disease multiple sclerosis (33, 34).
To determine the ability of A? t.c. immunization to effectively
produce A? antibodies, we began our investigation in nontrans-
antibody titers. Remarkably, the A? antibody response was ob-
of T cell infiltration. (D–F) Staining for hemosiderin was also performed to
(C and F). (A) For CD3 staining, the positive control consisted of CD3-positive
brain sections from experimental autoimmune encephalomyelitis mice. (D)
For microhemorrhage, experimental sections were compared with sections
from mouse brains suffering microhemorrhage. Each panel is representative
of staining repeated in triplicate for each brain section for either CD3 or
hemosiderin. The brain region shown for each panel is the neocortex. (Mag-
nification: CD3 staining, ?10; microhemorrhage, ?20.)
Absence of T cell infiltration or brain microhemorrhage in A?/CT
9) of A?1–40or A?1–42(pg/mg protein), and reductions for each group are indicated. (C) A significant inverse correlation (P ? 0.001) between plasma and
brain-soluble A? levels was revealed. Plasma/brain A? levels are presented as percentage mean ? SD (n ? 9) of soluble circulating/brain A? at 16 weeks after
t.c. immunization of PSAPP mice with A?/CT over CT control mice. (D) Mouse brain coronal paraffin sections were stained with monoclonal anti-human A?
(Bottom) The entorhinal cortex (EC). (E) Percentages (plaque burden, area plaque/total area) of A? antibody-immunoreactive A? plaques (mean ? SD; n ? 9)
were calculated by quantitative image analysis, and reductions for each mouse brain area analyzed are indicated. (F) Mouse brain sections from the indicated
regions were stained with Congo red. (Left) A?1–42/CT t.c.-immunized PSAPP mice. (Right) CT t.c.-immunized PSAPP mice. (G) Percentages of Congo red-stained
plaques (mean ? SD; n ? 9) were quantified by image analysis, and reductions for each brain region are indicated.
Reduction of cerebral A?/?-amyloid pathology in PSAPP mice t.c.-immunized with A?1–42/CT. (A and B) Detergent-soluble A?1–40,42peptides (A) and
www.pnas.org?cgi?doi?10.1073?pnas.0609377104 Nikolic et al.
served as early as week 4 in all immunized mice and dramatically
increased thereafter, remaining elevated through 16 weeks postini-
tial vaccination. Antibody isotype characterization demonstrated a
our previous report using an i.p. route of A? vaccination plus
and thus A? clearance without the overt proinflammatory (i.e.,
possibly contributing to autoimmune responses) Th1-type activa-
tion that typifies cellular immune responses (19, 36, 37). Accord-
ingly, to circumvent meningoencephalitic reactions, many studies
by using various strategies (38–40). The effectiveness and potential
safety of these strategies seems promising, but further investigation
is needed to confirm whether the link between Th cell responses
and meningoencephalitis in AD patients is causative.
Notwithstanding the need for these critical studies, A? immu-
nization appears to modulate immune responses based on three
major criteria: (i) tissue route of delivery, (ii) antigen epitope used
for immunization, and (iii) properties of the coadministered adju-
vant. Whether Th2 polarization in this study occurred because of
route of delivery, CT adjuvant choice, or the genetic background of
Th2 immune response (43), and our data demonstrating IgG1-
subtype antibodies produced in the greatest proportion (compared
with IgG2a or IgG2b antibodies) supports this notion. Of note, CT
antibody titers were observed (data not shown), indicating an
immunogenic response to this adjuvant. To confirm specific sys-
temic versus local immune cell activation, we analyzed primary
cultures of isolated splenocytes from t.c.-immunized WT mice and
found that A?/CT t.c. immunization conferred A?-specific T cell
response as measured by secretion of cytokines IFN-?, IL-2, and
or IL-2, further suggesting Th2 immune responses after A?/CT t.c.
where we found Th2-type cytokine responses both in vivo and ex
immune responses are likely preferred to proinflammatory Th1
responses in the A? vaccination paradigm, given that proinflam-
matory Th1 cells likely contributed to the aseptic meningoenceph-
alitis in the human clinical trial of AN-1792 (7, 44). When taken
together, these findings show that A?/CT t.c. immunization of WT
mice produces both A?-specific local LC immune response and
that are sustained throughout the immunization protocol.
To determine the potential therapeutic efficacy of A? t.c. im-
munization, 6-month-old transgenic PSAPP mice [which develop
robust amyloid pathology at 8 months of age (17)], were t.c.-
immunized against A?/CT or CT alone for 16 weeks. Results
out the 16-week immunization period only in the A?/CT-
immunized group. Interestingly, the magnitude of A? antibody
response in A?/CT t.c.-immunized mice was only about half of that
transgenic mouse models of AD are hyporesponsive to A? vacci-
nation, probably owing to overexpression of the human APP
transgene throughout their lives (45). This humoral response cor-
related with high plasma levels of A?1–40,42peptides, which peaked
?8 weeks and remained relatively constant to 16 weeks. Immuno-
histological and histochemical analyses of A?-immunoreactive
sections showed reductions by ?50% compared with CT t.c.-
immunized PSAPP mice, and a negative correlation existed be-
These results show that A?/CT t.c. immunization is effective at
mitigating cerebral amyloidosis and suggest activation of A? brain-
For AD immunotherapy approaches to be useful, they must not
only be efficacious, but such approaches must also be safe and well
tolerated. Importantly, although we did observe peripheral A?-
specific T cell responses consistent with an antiinflammatory Th2
response (characterized in vivo by IgG1 A? antibody production
and ex vivo by IL-4 secretion after A? recall stimulation of spleno-
cytes) after A?/CT t.c. immunization, no signs of aseptic menin-
goencephalitis and/or cell-mediated immunity were observed in
brains as evidenced by lack of CD3-positive T cell infiltrates.
However, we did observe evidence of humoral immunity in brain
as demonstrated by A? antibody titers in brain homogenates,
similar to data from previous reports using other modes of A?
immunotherapy (46, 47), supporting the notion that A? antibodies
cross the blood-brain-barrier. This observation was not caused by
poor perfusion at the time of death, as Perl’s stain (which normally
detects even trace amounts of iron that could be present because of
poor perfusion) results were consistently negative. Finally, other
transgenic AD mice results in cerebral microhemorrhage (5, 6).
Importantly, Perl’s stain did not show this potentially adverse side
effect in mice t.c.-immunized with A?/CT. Thus, when taken
together, t.c. immunization holds potential as an effective and safe
potential treatment strategy for AD.
Materials and Methods
Reagents. Lyophilized CT, CT antibody, Con A, and mouse CD3
was purchased from U.S. Peptides (Rancho Cucamonga, CA).
from R & D Systems (Minneapolis, MN). A?1–40 and A?1–42
ELISA kits were purchased from IBL-American (Minneapolis,
MN). Alexa-Fluor-conjugated secondary antibodies (including Al-
exa-Fluor488, Alexa-Fluor594, and Alexa-Fluor647) were purchased
from Invitrogen (Carlsbad, CA).
obtained from The Jackson Laboratory (Bar Harbor, ME). Brain
sections from mice induced with experimental autoimmune en-
for CD3 staining. Brain sections for positive microhemorrhage
by D.M. (23).
t.c. Immunization of Mice. We first t.c.-immunized WT C57BL/6
mice. These mice (n ? 10, five male/five female) were t.c.-
saline on a weekly basis for the first month. Thereafter, these mice
were continually t.c.-immunized with A?1–42(100 ?g per mouse)
and CT (5 ?g/ml) or CT alone (5 ?g per mouse) in 100 ?l of 0.9%
saline biweekly for the next 12 weeks. t.c. immunization was
performed as described (48). To ensure mice immobility for the
duration of administration for each immunization, mice were
an extra precaution not to damage the skin. The skin was then
swabbed with acetone to remove surface oils and enhance pene-
tration, allowed to air dry, and then rehydrated by swabbing with
layer to prevent unnecessary leakage of the immunization solution.
Last, 100 ?l of A?1–42in combination with CT or CT alone in 0.9%
saline was placed on the shaved region and allowed to be absorbed
for 2 h. At the end, the skin was washed with 0.9% saline and dried,
so as to remove any remaining immunization solution. Mice were
cleaned thereafter and returned to their cages. We then t.c.-
Nikolic et al.
February 13, 2007 ?
vol. 104 ?
no. 7 ?
of age by using the same procedure described above.
mice were prepared and treated as described (19). Lactate dehy-
drogenase (LDH) release assay was carried out as described (49),
and LDH was not detected in any of the wells studied.
Immunofluorescence Staining. The dorsal skin was removed by
prepared for immunofluorescence staining. The staining was cur-
ried out with the following primary antibodies: anti-mouse CD207
(Langerin; 1:250; eBioscience, San Diego, CA), anti-mouse CD11c
(1:50; Pierce Biotechnology), and/or anti-human A? antibody
(clone 4G8; 1:500) or rat anti-mouse CD3 antibodies (1:200)
overnight at 4°C, followed by appropriate secondary antibodies
conjugated with Alexa-Fluor488, Alexa-Fluor594, and/or Alexa-
Fluor647(1:500) for 45 min. Sections were then washed three times
DAPI to counterstain cell nuclei, and then viewed under a BX-51
microscope (Olympus, Center City, PA) or visualized in indepen-
dent channels with a LSM510 META confocal microscope (Zeiss,
Thornwood, NY) equipped with a two-photon laser that was used
for exciting DAPI.
A? Antibody ELISA. A? antibodies in mouse plasma and brain
homogenates were measured as described (9). A? antibodies
were represented as ng per ml of plasma (mean ? SD).
(18, 50). The optical density of both ELISAs was immediately
determined by a microplate reader at 450 nm. The ratios of IgG1
to IgG2a or IgG1 to IgG2b were calculated for each time point
from each mouse individually by using optical density values and
then the average ratio for each group (mean ? SD).
A? ELISA. Mouse brains were isolated and prepared as described
(51). This fraction represented the detergent-soluble fraction.
A?1–40,42 species were further subjected to acid extraction of
brain homogenates in 5 M guanidine buffer (52), followed by a
1:10 dilution in lysis buffer. Soluble A?1–40,42 species were
directly detected in plasma, and brain homogenates were pre-
pared with lysis buffer described above at a 1:4 or 1:10 dilution,
respectively. A?1–40,42 was quantified in these samples with
A?1–40,42 ELISA kits in accordance with the manufacturer’s
instructions. A?1–40,42was represented as pg per ml of plasma
and pg per mg of total protein (mean ? SD).
Immunohistochemistry and Image Analysis. A? immunostaining and
Congo red were performed as described (53). 4G8- and Congo
red-positive A? deposits were visualized with an Olympus BX-51
microscope. Quantitative image analysis was routinely performed.
Data are reported as percentage of immunolabeled area captured
(positive pixels) divided by the full area captured (total pixels). See
SI Text for more details.
Perl’s Prussian Blue Reaction for Ferric Ion-Hemosiderin. Sections
were deparaffinized, hydrated through descending grades of etha-
containing 20% hydrochloric acid and 10% potassium ferrocya-
nide. These sections were washed three times for 5 min with H20
and counterstained with hematoxylin solution (Sigma) for 15 s and
then mounted (23).
Statistical Analysis. Means and SDs were calculated according to
standard practice (51, 53).
This work was supported by grants from the National Institute of
Neurological Disorders and Stroke (J.T.), Alzheimer’s Association
(J.T.), and Byrd Alzheimer’s Center and Research Institute (J.T. and
R.D.S.). T.T. is supported by an Alzheimer’s Association grant.
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