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J. Exp. Med. Vol. 206 No. 6 1219-1225
BRIEF DEFINITIVE REPORT
The smallpox vaccine consists of live vaccinia vi-
rus (VACV) and is considered the gold standard
of vaccines, as it has led to the complete eradica-
tion of a lethal infectious disease from the human
population. Recent fears that smallpox might be
deliberately released in an act of bioterrorism have
led to renewed efforts to better understand the
disease mechanism and to develop a safer vaccine.
Approximately 50% of US residents were born
after the regular smallpox vaccination was discon-
tinued in 1972. Thus, these unimmunized people
are vulnerable to smallpox. The population land-
scape is very different between now and 36 yr
ago, with two-to-three times more frequent inci-
dence of atopic dermatitis in the current popula-
tion (1). Individuals with atopic dermatitis are
excluded from smallpox vaccination because of
their propensity to develop eczema vaccinatum, a
disseminated vaccinia infection (2).
Atopic dermatitis is a chronic inflammatory
skin disease (3). The etiology of this disease is
multifactorial, and involves complex interactions
between genetic and environmental factors.
The skin in a preatopic dermatitis state has been
postulated to have hypersensitivity to environ-
mental triggers, resulting from a defective skin
barrier that allows the penetration of allergens
and microbial pathogens (4). The acute phase is
characterized by eczematous skin lesions with
an infiltration of Th2 cells. The chronic phase
is characterized by lichenification of skin and
an infiltration of Th1 cells. As recent studies
have established IL-17– and IL-22–producing
CD4+ T cells as a distinct class of helper T cells
(Th17), Th17 cells are also implicated in the
acute but not the chronic phase (5, 6). Despite
the progress in our understanding of atopic
dermatitis pathogenesis (7) and immune responses
to VACV (8), it is not understood why atopic
dermatitis patients are susceptible to develop-
ing eczema vaccinatum (9).
Y. Tomimori, K. Yumoto, and S. Hasegawa contributed
equally to this paper.
K. Yumoto’s present address is NASA Ames Research Center,
Moffett Field, CA 94035.
Inhibition of NK cell activity by IL-17 allows
vaccinia virus to induce severe skin lesions
in a mouse model of eczema vaccinatum
Yuko Kawakami,1 Yoshiaki Tomimori,1 Kenji Yumoto,1 Shunji Hasegawa,1
Tomoaki Ando,1 Yutaka Tagaya,4 Shane Crotty,2,3 and Toshiaki Kawakami1,3
1Division of Cell Biology, 2Division of Vaccine Discovery, and 3Center for Infectious Disease, La Jolla Institute for Allergy
and Immunology, La Jolla, CA 92037
4Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
Threats of bioterrorism have renewed efforts to better understand poxvirus pathogenesis
and to develop a safer vaccine against smallpox. Individuals with atopic dermatitis are
excluded from smallpox vaccination because of their propensity to develop eczema vaccina-
tum, a disseminated vaccinia virus (VACV) infection. To study the underlying mechanism of
the vulnerability of atopic dermatitis patients to VACV infection, we developed a mouse
model of eczema vaccinatum. Virus infection of eczematous skin induced severe primary
erosive skin lesions, but not in the skin of healthy mice. Eczematous mice exhibited lower
natural killer (NK) cell activity but similar cytotoxic T lymphocyte activity and humoral
immune responses. The role of NK cells in controlling VACV-induced skin lesions was dem-
onstrated by experiments depleting or transferring NK cells. The proinflammatory cytokine
interleukin (IL)-17 reduced NK cell activity in mice with preexisting dermatitis. Given low
NK cell activities and increased IL-17 expression in atopic dermatitis patients, these results
can explain the increased susceptibility of atopic dermatitis patients to eczema vaccinatum.
© 2009 Kawakami et al. This article is distributed under the terms of an Attribu-
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The Journal of Experimental Medicine
MOUSE MODEL OF ECZEMA VACCINATUM | Kawakami et al.
primary lesion peaked at days 7–8 (Fig. 1, B and C), and the
lesion began to subside by day 11. Unlike eczematous mice,
most normal mice failed to develop skin lesions after VACV
infection, and even when developed, their skin lesions were
much milder (Fig. 1, B and C). Virus titers in the lesional skin
of eczematous mice were 300–10,000 times higher than those
of normal mice over an observation period of 14 d (Fig. 1 D).
In erosive skin lesions of eczematous mice, epithelial layers
were separated from the rest of the skin and more leukocytes
infiltrated the diseased dermis (Fig. 2, A and B). Pock-like
satellite lesions distant from inoculation sites were rarely seen
(only 3 cases out of 230 eczematous mice and 0 out of 187
normal mice). Although weight loss was observed in a small
number of both eczematous and normal mice, there was no
correlation with skin conditions (unpublished data). Unlike the
intradermal infection at eczematous skin lesions, intranasal in-
fection or intradermal infection at distant normal skin sites
failed to induce clinical conditions (e.g., weight loss, survival,
and size of skin lesions) distinctly different between eczematous
In this study, we have established a mouse model of ec-
zema vaccinatum using a strain of mice that are prone to de-
velop eczematous skin lesions, characterized their immune
responses to VACV infection, and showed the importance of
NK cells in early suppression of VACV-induced severe ec-
zema vaccinatum–like skin lesions.
RESULTS AND DISCUSSION
We initially focused on establishing experimental conditions
in which infection with VACV induces differential clinical
outcomes between mice with and without eczematous skin
lesions. Skin lesions were induced on the backs of dermatitis-
prone NC/Nga mice (10) by epicutaneous treatment of shaved
skin with a mite extract and staphylococcal enterotoxin B
(SEB), as described previously (11). This treatment induced
elevated serum IgE levels and eczematous skin lesions (Fig. 1 A)
(11). Skin lesions with maculopapular rash started to appear
on the infected site on day 2–3 after infection in eczematous
mice and developed into severe skin erosion. The size of the
Figure 1. Induction of erosive primary skin lesions in VACV-infected eczematous mice. (A) Eczematous skin lesions were induced by repeated
Der f/SEB (D/B) treatments, and mice with a clinical score of ≥8 were infected intradermally with VACV (eczematous group). A cohort (normal group)
of mice with healthy skin was also infected at the same anatomical site. (B) Typical eczematous (right) and normal (left) mice are shown on day 6
after infection. (C) The size of erosive skin lesions. Shown is a representative of at least 15 experiments using 4–10 mice in each group. (D) Virus titers
in the infected skin (n = 7 mice per group). Shown are results representative of four independent experiments. Data represent means and SEM values.
*, P < 0.05; **, P < 0.01; and ***, P < 0.001 versus normal mice. ND, not detected.
JEM VOL. 206, June 8, 2009
BRIEF DEFINITIVE REPORT
Because of the importance of NK cells in rapid antiviral
defense (12, 13), we quantified their numbers and activities.
NK cells were more abundant in primary skin lesions in ec-
zematous than normal mice (Fig. 2, C and D). Importantly,
NK cell cytotoxic activity in the spleen was lower in eczema-
tous mice on days 2 and 3 after infection (Fig. 3 A). We mea-
sured expression of molecules involved in NK killing activity
by flow cytometry and found that the proportions of splenic
NK cells expressing granzyme B, perforin, and IFN- were
significantly lower in eczematous mice (Fig. 3 B).
IgM and IgG responses against VACV were similar be-
tween the eczematous and normal cohorts (Fig. S1 A). Consis-
tent with this, IL-4 mRNA levels in lymph nodes were not
reduced in eczematous mice for the initial 7 d after infection
(Fig. S1 B). Killing activity of CD8+ T cells and their expres-
sion of granzyme B, perforin, and IFN- in day 7 spleens did
not show differences between the two cohorts (unpublished
data). These results suggest that adaptive immunity does not
play a major role in causing differential skin outcomes of VACV
infection between the eczematous and normal mice, although
these arms of immunity are critical in the control of virus infec-
tion in vaccinia-infected mice (8).
The role of NK cells in this eczema vaccinatum model
was assessed by depletion studies. First, dermatitis was in-
duced in NC/Nga mice. 1 d before infection and on d 3 after
infection, mice were intravenously injected with anti–asialo
GM1 (AGM1) or control rabbit serum (NRS). Treatment with
AGM1 serum drastically reduced the numbers of NK1.1+
cells in the spleens (73–89% reduced as evaluated by flow
cytometry) and suppressed NK cell activity in spleens in day
3–infected normal mice (normal/AGM1 group) compared
with NRS-treated normal mice (normal/NRS group; Fig. S2).
In contrast, AGM1 treatment did not significantly reduce
the already low NK cell activity in day 3–infected eczema-
tous mice. Substantially higher virus titers were observed in
lesional skins (Fig. 3 C) and lungs (not depicted) of AGM1-
treated normal mice than those of NRS-treated normal mice.
Importantly, 14 out of 16 mice in the normal/AGM1 group
exhibited erythematous papules at the inoculation site by day
6, whereas only 1 out of 13 mice in the normal/NRS group
developed such a lesion. Some normal/AGM1 and eczematous/
AGM1 mice developed satellite lesions as well (Fig. 3 E). Ec-
zematous mice developed larger erosive skin lesions at the
site of virus inoculation than noneczematous mice (Fig. 3 D).
These primary lesions in NK-depleted eczematous mice were
significantly larger than lesions in control eczematous mice
(Fig. 3 D). As AGM1 treatment might affect other cell types
besides NK cells (14), we performed a second experiment in
which we depleted NK cells by administering anti-NK1.1
mAb. Results were similar to those with AGM1 (Fig. S3).
To complement the NK depletion experiments, we per-
formed adoptive transfer of NK cells to determine whether
activated NK cells could rescue NC/Nga mice from eczema
vaccinatum. NK cells were obtained by culturing splenocytes
in IL-15 for 4 d. The cultured cells, composed of a >95%
CD3 NK1.1+ population (Fig. 3 F, inset), were intravenously
and normal mice (unpublished data). Unlike the Western Re-
serve strain used throughout this study, intradermal infection
with the same dose of ACAM2000, the licensed vaccine cloned
from Dryvax, caused much milder skin lesions compared with
Western Reserve–induced skin lesions (unpublished data).
Figure 2. Histology of skin lesions before and after VACV infec-
tion. (A) Hematoxylin and eosin–stained skin tissues are shown for normal
and eczematous mice before and 7 d after virus infection. Bar, 1 mm.
(B) CD4+, CD8+, and Mac-1+ cells were stained by immunohistochemistry
and mast cells were stained with toluidine blue. Data represent means and
SEM values of cell numbers per high-power field (HPF; n = 8 each group).
(C) Immunohistochemical staining of NK cells with anti-Ly49G2 (clone
4D11) mAb. Bar, 0.1 mm. (D) Ly49G2 (4D11)+ NK cells were enumerated
with six mice per cohort. Shown are results representative of six indepen-
dent experiments. Data represent means and SEM. *, P < 0.05; **, P < 0.01;
and ***, P < 0.001. NS, not significant.
MOUSE MODEL OF ECZEMA VACCINATUM | Kawakami et al.
of splenocytes showed increased mRNA expression of
IL-17A and the cytokines involved in Th17 development (IL-6,
TGF-, IL-21, and IL-23) and effector functions (IL-21 and
IL-22) (Fig. 4 A). IL-17A and IL-6 mRNAs were also in-
creased in lesional skins of uninfected eczematous mice,
whereas IL-17A, IL-6, and IL-23 mRNAs were increased in
draining lymph nodes of eczematous mice (Fig. 4 A). Consis-
tent with these mRNA results, lymph nodes contained an in-
creased number of Th17 cells in eczematous mice (Fig. 4 B).
In contrast with Th17-related cytokines, surface expression of
NK cell receptors such as NKG2D, NKG2A/C/E, Ly49A/D,
and Ly49I/G was comparable in eczematous and normal mice
transferred to eczematous or normal mice. Transfer of NK cells
either totally suppressed the development of erosive skin lesions
or greatly reduced skin lesion sizes (Fig. 3 F). The activated
NK cells also delayed the kinetics of lesion development in the
subset of mice that eventually developed erosive skin lesions.
Therefore, the NK depletion and transfer experiments demon-
strate a critical role for NK cells in protecting mice from devel-
oping VACV-induced erosive skin lesions and satellite skin
lesions in this NC/Nga mouse model of eczema vaccinatum.
NK cell function is under the control of various cyto-
kines, including IL-6 and IL-10, which each inhibit NK cell
activity. The proinflammatory cytokine IL-17 is produced by
Th17 cells (15). In eczematous mice, real-time PCR analysis
Figure 3. Reduced NK cell activity was critical for the development of VACV-induced erosive skin lesions in eczematous mice. (A) NK cell
cytotoxic activity of splenocytes on day 2 after infection was measured using YAC-1 cells as target cells at the indicated effector-to-target ratios. Shown
are results representative of 5 independent experiments. Data represent means and SEM values. *, P < 0.05; and ***, P < 0.001 versus uninfected mice.
###, P < 0.001 versus eczematous mice. (B) Flow cytometric analysis of CD3 NK1.1+ NK cells expressing granzyme B, perforin, or IFN- in spleens.
Shown are results representative of 8 independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (C) Virus titers in spleens from AGM1- or
NRS-treated mice. (D) Sizes of primary skin lesions in AGM1- or NRS-treated mice. *, comparison between AGM1- and NRS-treated mice; #, compari-
son between AGM1-treated normal and eczematous mice; and §, comparison between NRS-treated normal and eczematous mice (one, two, and three
symbols indicate P < 0.05, 0.01, and 0.001, respectively. (E) Virus infection in AGM1-treated normal mice induced erythematous papules at the inocula-
tion site (indicated by the arrow; top) and satellite lesions. Satellite lesions were also induced in AGM1-treated eczematous mice (indicated by arrow-
heads). Satellite lesions in both normal/AGM1 and eczematous/AGM1 mice were confirmed to contain live virus. Shown in C–E are representative
results of three independent experiments (n = 4–8 mice per group). (F) Adoptive transfer of cultured NK cells. Sizes of primary skin lesions are shown. The
inset shows the purity of transferred NK cells as analyzed by flow cytometry. Shown are results representative of three independent experiments. Data in
A and C–F represent means and SEM values; data in B represent means and SD values.
JEM VOL. 206, June 8, 2009
BRIEF DEFINITIVE REPORT
Consistent with these changes, viral loads in the spleen and
lesional skin were lower in IL-17–neutralized mice (Fig. 4 F).
Furthermore, when the NK cells were depleted by AGM1
antibody, the effect of anti–IL-17 antibody treatment on the
incidence and lesion size (Fig. 4 G) and viral titers (Fig. S4)
was almost abrogated, indicating that effects of IL-17 neutral-
ization are exerted through the regulation of NK cells. Con-
sistent with these in vivo findings, the expression of killing
Neutralization of IL-17A in eczematous mice with anti–
IL-17 antibody caused a delay in the onset of skin lesions after
virus infection, and the lesion size was significantly smaller on
days 2 and 3 after infection (Fig. 4 C). Although the number and
the percentage of NK cells in the spleen and at the lesion site
were not changed by anti–IL-17 antibody treatment (Fig. 4 D),
the proportions of NK cells expressing granzyme B, perforin,
and IFN- were increased in IL-17–neutralized mice (Fig. 4 E).
Figure 4. Role of IL-17A in reduced NK cell cytotoxicity in eczematous mice. (A) mRNA expression of IL-17A and Th17-related cytokines was
analyzed by real-time PCR (spleen) or semiquantitative RT-PCR analysis (skin and draining lymph node). Values were normalized against those of
normal mice. Shown are results representative of two independent experiments (n = 4–6 mice). *, P < 0.05; and **, P < 0.01 by the Student’s t test.
(B) CD3+CD4+IL-17+ Th17 cells were enumerated in draining lymph nodes. (C) The onset of skin lesion development was delayed (left) and the size of
primary skin lesions was smaller (right) in mice treated with anti–IL-17 mAb. **, P < 0.01 versus control. (D) NK cells in spleens and lesional skin were
enumerated by flow cytometry and immunohistochemistry, respectively. (E) Splenic NK cells expressing granzyme B (GzmB), perforin (Pfn), or IFN-
were analyzed by flow cytometry in mice treated with anti–IL-17 or control mAb day 2 after infection. (F) Virus titers were measured on day 7.
Shown are representative results from three independent experiments. *, P < 0.05; and **, P < 0.01 versus control. (G) Mice were NK-depleted by
AGM1 injection 1 d before VACV infection. Anti–IL-17 antibody was also intraperitoneally injected 2 h after AGM1 injection. After VACV infection,
anti–IL-17 was injected on days 1 and 3, and AGM1 was injected on days 2 and 5. Skin lesion development was observed and lesion size was mea-
sured for 6 d. The result is a representative of two independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 versus rat IgG2a–injected
mice. #, P < 0.05 versus anti–IL-17 treated, NK-nondepleted mice. Data represent means and SEM values.
MOUSE MODEL OF ECZEMA VACCINATUM | Kawakami et al.
strong indications that NK cell defects are involved (17, 18,
27). Our data now show that critical failures in NK cell–me-
diated immunity allow for disastrous early spread of vaccinia
after cutaneous infection, and these NK cell defects are re-
lated to the immunosuppressive effects of IL-17A.
MATRIALS AND METHODS
Mouse infection. NC/Nga mice (10) were used in all animal experiments.
Eczematous skin lesions were induced as described previously (11). In brief,
mice were shaved on the back and dermatitis was induced by two rounds of
a week-long treatment with Dermatophagoides farinae extract (Der f; Greer
Laboratories) and SEB (Sigma-Aldrich). During this treatment, their back was
occluded with a bandage, which was removed the following week. Mice with
a clinical score of ≥8 (eczematous group) were intradermally injected with 106
PFU per 3 µl of VACV (Western Reserve strain) at the center of skin lesions.
A cohort (normal group) of age and sex-matched mice with healthy skin was
also infected at the same anatomical site. Clinical scores of eczematous skin le-
sions are based on severity (0, no signs; 1, mild; 2, intermediate; 3, severe) of
four signs (redness, bleeding, eruption, and scaling). Scoring was performed in
a blind manner. The virus was prepared by repeated (three times) freeze–thaw
cycles of infected HeLa cells in DMEM/1% FCS followed by centrifugation.
Uninfected HeLa cell extract was used as control. Virus titers were measured
by plaque-forming assays on Vero cells. All of the mouse experiments were
approved by an Institutional Review Board of the La Jolla Institute for Allergy
Histology. CD4+ and CD8+ T cells, Mac-1+ monocytes/macrophages, and
Ly49G2 (4D11)+ NK cells were detected by immunochemical staining. Mast
cells were stained by toluidine blue, and eosinophils and neutrophils were
detected by hematoxylin and eosin or Congo red staining.
NK cell assay. NK cell activity was measured using YAC-1 cells as target
cells, with effector-to-target ratios (12.5:1, 25:1, and 50:1) in spleen cells iso-
lated on day 2 or 3 after infection.
NK cell cultures. Splenocytes from NC/Nga mice were negatively se-
lected by MACS beads (Miltenyi Biotec) or the EasySep mouse NK cell en-
richment kit (StemCell Technologies Inc.). These NK-enriched cells were
cultured in RPMI 1640 with 10% FBS containing 500 ng/ml IL-15 for 4 d.
The purity of NK cells was checked by flow cytometry after staining with
anti-NK1.1 and anti-CD3 antibodies before the use for adoptive transfer.
For in vitro NK cell assays, purified splenic NK cells or whole splenocytes
were cultured in RPMI 1640 with 10% FBS containing 20 ng/ml IL-4 and/or
50 ng/ml IL-17A or IL-17F with or without 2 µg/ml anti–IL-17RA for 2 d,
followed by flow cytometry.
Flow cytometry. Single-cell suspensions of splenocytes or lymph node
cells were surface stained with anti-NK1.1 and CD3. The cells were then
effector molecules in cultured splenic NK cells was reduced
by IL-17A in a dose-dependent manner (Fig. S5), but not by
IL-17F (Fig. 5). IL-17A reduced the expression of killing ef-
fectors induced by IL-4 (Fig. 5), IL-2, IL-12, IL-15, or IL-18
(Fig. S6). The survival of these NK cells was not affected by
IL-17A or IL-17F (unpublished data). These results collec-
tively suggest that IL-17A plays a critical role in lowering NK
cell activity in eczematous mice.
IL-15 is required for the proliferation and activation of
NK cells (16). Antibody-mediated neutralization of IL-15
caused more severe skin lesions in VACV-infected normal
mice compared with the control cohort (Fig. S7). However,
IL-15 neutralization in eczematous mice did not induce sig-
nificant differences in skin lesion development. Although the
mRNA level of IL-15 is not significantly different between
normal and eczematous mice (unpublished data), the results
of IL-15 neutralization further confirm that NK cell activity
is critical for early protection from skin lesion development.
Our NC/Nga infection model does not exhibit all of the
expected features of human eczema vaccinatum. For instance,
NC/Nga mice with eczematous skin lesions exhibited func-
tional but not numerical defects in NK cells, unlike atopic
dermatitis patients, who have defects in both number and
function (17, 18). Nevertheless, this model exhibits key fea-
tures of atopic dermatitis observed in humans, including de-
fective NK cell killing activity (17, 18) and high IL-17A
expression (5, 6). IL-6 and TGF- are required for induction
of Th17 cells, and IL-23 is required for the establishment of
Th17 cells (19, 20). IL-21 is produced by Th17 cells and ex-
erts critical functions in Th17 cell differentiation (21–23).
Th17 cells were more abundant and the Th17-related cyto-
kines were increased in eczematous mice, suggesting that
Th17 cells may be involved in reducing NK cell killing activ-
ity. The NK cell–suppressive function of IL-17A observed in
our in vitro and in vivo studies was consistent with an earlier
IL-17 study (24), although it is possible that the increased
IL-17A and Th17-related cytokines might also contribute to
VACV-induced inflammation via the enhanced immuno-
pathology. Our results also support the conclusion that NK
cells are important in controlling early local and systemic
spreading of VACV in mice (25, 26). Although atopic der-
matitis is still only partially understood in humans, there are
Figure 5. IL-17A but not IL-17F reduces NK cell cytotoxicity in vitro. Splenic NK cells were incubated with the indicated cytokines for 48 h before
flow cytometric analysis of NK cells expressing granzyme B, perforin, or IFN-. Shown are results representative of two independent experiments. Data
represent means and SEM values. **, P < 0.01; and ***, P < 0.001 versus IL-4 by one-way analysis of variance.
JEM VOL. 206, June 8, 2009
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fixed, permeabilized, and stained with anti–granzyme A, –granzyme B,
-perforin, or –IFN-. Data were acquired with a FACSCalibur (BD) and
analyzed using FlowJo software (Tree Star, Inc.).
RT-PCR. Skin tissues were taken from infection sites or erosive areas by
punch biopsy, and axillary lymph nodes and spleens were also isolated. Total
RNAs were isolated with TRIzol reagent (Invitrogen) and used as a template
to prepare cDNAs. PCR products were analyzed by agarose gel elctrophoresis.
Quantitative PCR was performed using a LightCycler 480 (Roche).
Statistical analysis. Statistical analysis in each independent experiment was
performed with an unpaired, two-way analysis of variance using Prism soft-
ware (GraphPad Software, Inc.), otherwise noted. P < 0.05 was considered
Online supplemental material. Fig. S1 shows antibody responses. Fig. S2
depicts AGM1 effects on NK cells. Fig. S3 shows anti-NK1.1 effects. Fig. S4
depicts viral titers in anti–IL-17– and AGM1-treated mice. Fig. S5 shows in
vitro effects of IL-17A on NK cell mediators. Fig. S6 depicts the effects of
various cytokines on NK cell mediators. Fig. S7 shows skin lesions in anti–
IL-15–treated mice. Online supplemental material is available at http://www
The authors thank M. McCausland for excellent technical assistance, and Drs. W.M.
Yokoyama and L.L. Lanier for helpful discussion.
This study was supported in part by Atopic Dermatitis and Vaccinia Network
National Institutes of Health (NIH)/National Institute of Allergy and Infectious
Diseases contract N01 AI40030 to T. Kawakami, and NIH grants AI63107 and
AI77953 to S. Crotty. This study is publication no. 1071 from the La Jolla Institute
for Allergy and Immunology.
The authors declare no competing financial interests.
Submitted: 18 December 2008
Accepted: 4 May 2009
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