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Marking and Quantifying IL-17A-Producing Cells In Vivo
April E. Price1,2, R. Lee Reinhardt1,2¤, Hong-Erh Liang1,2, Richard M. Locksley1,2,3*
1Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California, United States of America, 2Department of Medicine, University of
California San Francisco, San Francisco, California, United States of America, 3Department of Microbiology and Immunology, University of California San Francisco, San
Francisco, California, United States of America
Interleukin (IL)-17A plays an important role in host defense against a variety of pathogens and may also contribute to the
pathogenesis of autoimmune diseases. However, precise identification and quantification of the cells that produce this
cytokine in vivo have not been performed. We generated novel IL-17A reporter mice to investigate expression of IL-17A
during Klebsiella pneumoniae infection and during experimental autoimmune encephalomyelitis, conditions previously
demonstrated to potently induce IL-17A production. In both settings, the majority of IL-17A was produced by non-CD4+T
cells, particularly cd T cells, but also invariant NKT cells and other CD42CD3e+cells. As measured in dual-reporter mice, IFN-
c-producing Th1 cells greatly outnumbered IL-17A-producing Th17 cells throughout both challenges. Production of IL-17A
by cells from unchallenged mice or by non-T cells under any condition was not evident. Administration of IL-1b and/or IL-23
elicited rapid production of IL-17A by cd T cells, invariant NKT cells and other CD42CD3e+cells in vivo, demonstrating that
these cells are poised for rapid cytokine production and likely comprise the major sources of this cytokine during acute
immunologic challenges.
Citation: Price AE, Reinhardt RL, Liang H-E, Locksley RM (2012) Marking and Quantifying IL-17A-Producing Cells In Vivo. PLoS ONE 7(6): e39750. doi:10.1371/
Editor: Ciriaco A. Piccirillo, McGill University Health Center, Canada
Received January 26, 2012; Accepted May 29, 2012; Published June 29, 2012
Copyright: ? 2012 Price et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by AI30663 and AI078869 from the United States National Institutes of Health, the Howard Hughes Medical Institute, and the
Sandler Asthma Basic Research Center at University of California San Francisco. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: RML serves on the Scientific Review Board for Genentech/Roche and for the American Asthma Foundation. This does not alter the
author's adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: email@example.com
¤ Current address: Department of Immunology, Duke University Medical Center, Durham, North Carolina, United States of America
The cytokine interleukin (IL)-17A has a key role in immunity,
inducing the release of a variety of inflammatory cytokines as well
as chemokines that can mediate the rapid recruitment of
neutrophils. Studies with knockout mice and neutralizing anti-
bodies have revealed a role for IL-17A in immunity to various
bacterial and fungal infections and also in the induction and
propagation of several autoimmune diseases [1]. In humans,
genetic deficiencies in IL-17 receptor A or IL-17F are associated
with susceptibility to mucocutaneous infection with Candida albicans
and, to a lesser extent, Staphylococcus aureus [2]. Autoantibodies to
IL-17A and IL-17F are seen in patients with mutations in the
autoimmune regulator (AIRE) and may contribute to mucocuta-
neous candidiasis [3]. Patients with hyper-IgE syndrome associ-
ated with mutations in Stat3 have deficits in the induction of IL-
17-producing CD4+T (Th17) cells that correlate with recurrent
bacterial and fungal infections [4]. Polymorphisms in the gene
encoding the receptor to IL-23, a cytokine implicated in the
generation and maintenance of Th17 cells and in the promotion of
IL-17 secretion from innate cells [5,6,7,8], are associated with
protection against the development of inflammatory bowel disease
[9]. A more complete understanding of the biology of IL-17A,
including the temporal and cell-specific patterns of expression,
could aid in the generation of effective therapeutics targeted
towards these infectious and autoimmune conditions.
Although early work focused on IL-17A production by Th17
cells, more recent studies suggest important contributions by
innate-like lymphocytes, including cd T cells and NKT cells [10].
However, most reports use ex vivo restimulation to identify IL-17A-
producing cells, and thus potentially alter the pattern of cytokine
secretion that occurs in vivo. Several groups have generated IL-17F
reporter mice in order to bypass the need for restimulation, but
while IL-17A and IL-17F are often co-expressed, they are
differentially induced in certain models and can have distinct
functional roles [11,12,13]. More recently, IL-17A-cre reporter
mice were developed to permanently mark cells that have
activated the IL-17A locus, thus facilitating fate-tracking, and an
IL-17A-eGFP knockin mouse was used to track Th17 cells during
tolerance induced by a CD3-specific antibody and in mouse
models of sepsis [14,15]. Studies using IL-17F reporter mice and
IL-17A-cre mice, as well as studies using adoptive transfer [16,17],
have raised the possibility that Th17 cells have an unstable
phenotype, such that they lose the capacity to produce IL-17 and
begin to produce interferon (IFN)-c.
Here, we generated IL-17A reporter mice and used these mice
to examine the expression of IL-17A at rest, after bacterial
challenge, and during the development of autoimmune enceph-
alitis. To establish the relationships between IL-17A-producing
cells and IFN-c-producing cells, we additionally crossed these IL-
17A reporter mice to mice with an IFN-c reporter allele. Using
these dual-reporter mice, we quantified the numbers and types of
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cells that produce these cytokines in vivo without the need for ex vivo
Generation and validation of Smart-17A reporter mice
To assess IL-17A expression in vitro and in vivo, we generated IL-
17A reporter mice, termed Smart (Surface marker for transcrip-
tion)-17A mice (Figure 1A). In these mice, the 39 untranslated
region (UTR) of the il17a gene was modified to include an internal
ribosomal entry site (IRES) followed by a non-signaling form of the
human nerve growth factor (hNGFR) gene, resulting in IRES-
mediated translation of hNGFR when the IL-17A locus is
activated. We verified the efficacy of the Smart-17A allele by
demonstrating that hNGFR expression was specifically induced in
CD4+T cells in vitro only under Th17 polarizing conditions and
that intracellular IL-17A was found almost entirely within the
hNGFR+population (Figure 1B, Figure S1A). Thus, the hNGFR
reporter accurately marks 98% of Th17 cells identified using
standard methods of in vitro restimulation and intracellular
cytokine staining. Cells with the brightest staining for intracellular
IL-17A were also those with the highest mean fluorescence
intensity (MFI) for the hNGFR reporter. Approximately 30% of
cells were hNGFR+
but negative for intracellular IL-17A
(Figure 1B). These cells tended to have the lowest MFI for
hNGFR, consistent with their identification as cells that had
previously secreted IL-17A and continued to be marked by the
surface reporter. The half-life of the reporter on the cell surface
was approximately 24–48 hours as assessed by decay under in vitro
conditions (Figure S1B). Taken together, these results demonstrate
that the Smart-17A reporter mouse sensitively and accurately
marks cells that are induced to express IL-17A.
IL-17A expression in naı ¨ve mice
To characterize IL-17A expression in vivo, we crossed Smart-
17A mice to RORct-GFP reporter mice [18]. Expression of the
transcription factor RORct has been shown to accurately identify
both Th17 cells [19] and other lymphoid IL-17-producing cells
[10]. We anticipated that the inclusion of this second reporter
would enhance the detection of rare populations of IL-17A-
expressing cells by their concordant expression of RORct. We first
looked at CD3e+cell populations in multiple organs of naı ¨ve mice
for evidence of RORct and IL-17A expression (Figure 2). CD3e+
cells were gated as indicated in Figure S2. Invariant (i)NKT cells
were identified using a tetramer loaded with PBS-57 (an analogue
of a-galactosylceramide provided by the NIH tetramer facility),
and ''other CD3e+ cells'' were defined as cells that were CD3e+
but negative for CD4, CD8, the cd T cell receptor (TCR) and the
As previously reported, we observed constitutive expression of
the RORct GFP reporter in CD4+T cells in the lamina propria of
the small intestine and colon [19]. We also identified populations
of GFP+cd T cells in the spleen, lung, skin and small intestine,
GFP+iNKT cells in the lung, and GFP+other CD3e+cells in the
lung and skin. Despite recovery of these RORct+cells, however,
we did not observe hNGFR+cells among the CD3e+cell
populations in any of the examined organs. In all populations,
the small percentage of cells within the hNGFR+gate was similar
to background levels present in naı ¨ve wild-type mice (data not
shown). We also found no evidence of hNGFR expression by
dendritic cells, macrophages, neutrophils or a recently described,
lineage-negative, Thy1+population of innate lymphoid cells
(Figure S3) [20]. We repeated these analyses in Smart-17A mice
without the RORct-GFP allele and also in mice colonized by
segmented filamentous bacteria and obtained identical results
(data not shown). In all cases, none of the examined cell types,
including cells from intestinal tissues, constitutively expressed
hNGFR, suggesting that IL-17A is not expressed or is expressed at
levels too low to be detected using this reporter in resting mice.
IL-17A expression during Klebsiella pneumoniae infection
Klebsiella pneumoniae is a gram-negative extracellular bacterium
that is a cause of nosocomial and community-acquired pneumo-
nia. IL-17A is rapidly produced in the lungs of mice during K.
pneumoniae infection [21], and downstream signaling through the
IL-17 receptor (IL-17R) leads to the induction of a variety of
proinflammatory cytokines and chemokines that promote neutro-
phil accumulation [22]. Mice deficient in IL-17A, IL-17R or the
p19 subunit of IL-23 have increased bacterial dissemination and
increased mortality [22,23,24], supporting the use of this model to
investigate the cell types that produce IL-17A during acute
bacterial challenge.
We intranasally inoculated Smart-17A mice with a dose of K.
pneumoniae that led to mortality in a majority of infected mice by
day 5. During the first 3 days after infection, we noted a
substantial increase in the total number of CD3e+cells in the
lungs (Figure 3A). The vast majority of these cells were CD4+and
CD8+T cells, with much smaller numbers of cd T cells, iNKT
cells and other CD3e+cells. When assayed two days post-infection,
the highest percentages of hNGFR+cells were found amongcdT
cells, with lower percentages of hNGFR+iNKT cells and other
CD3e+cells (Figure 3B). A small but reproducible percentage of
CD4+T cells were also hNGFR+, whereas percentages of reporter-
positive CD8+T cells did not differ from background levels in
wild-type mice. We did not observe hNGFR expression by any
CD3e-cell populations (data not shown). Although innate-like T
cells comprised only a small portion of the total number of CD3e+
cells in the infected lung, they represented a majority of the total
hNGFR+cells (Figure 3C). cd T cells themselves comprised over
half of the hNGFR+cells (54%), followed by CD4+T cells (31%),
other CD3e+cells (9%) and iNKT cells (5%). Thus, innate-like T
cells are major sources of IL-17A during K. pneumoniae infection.
Innate-like T cells are poised to produce IL-17A in
response to proinflammatory cytokines
The discovery that innate-like T cells expressed IL-17A during
acute K. pneumoniae infection led us to investigate the potential
signals that mediate the production of this cytokine. It has been
noted that IL-1b and IL-23 can induce TCR-independent IL-17A
production from cd T cells in vitro [7] and TCR/CD1d-dependent
IL-17A production from iNKT cells in vitro and ex vivo [8]. To
determine if these findings applied to innate-like T lymphocytes in
vivo, we administered IL-1b and IL-23 intranasally to Smart-17A
mice and examined reporter expression from cells in the lungs 8 hr
later (Figure 4A). The addition of either IL-1b or IL-23
individually elicited hNGFR expression in cd T cells, iNKT cells
and other CD3e+cells, and the combination of both cytokines
resulted in a synergistic increase in hNGFR levels to levels similar
to those seen after K. pneumoniae infection. In contrast, we did not
observe induction of IL-17A production as assessed by hNGFR
expression in CD4+T cells. The brief time between cytokine
administration and IL-17A expression suggests that tissue resident
innate-like T cells are poised to produce IL-17A rapidly after
receiving signals from these cytokines. IL-1b and IL-23 subunit
p19 mRNA were induced in the lung within 1 day after K.
pneumoniae infection (Figure 4B), providing further evidence that
these cytokines may play a role in inducing acute IL-17A
production from innate-like T lymphocytes.
IL-17A-Producing Cells In Vivo
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IL-17A expression during experimental autoimmune
Experimental autoimmune encephalomyelitis (EAE) is a mouse
model of multiple sclerosis that is associated with the production of
IL-17A. IL-17A2/2mice have reduced incidence and severity of
disease [25]. IL-1R2/2mice and IL-23p192/2mice are also less
susceptible to disease [7,26]. Although Th17 cells are thought to
be the main source of IL-17A during EAE, cd T cells can also
produce this cytokine and may act to amplify IL-17A production
from Th17 cells [7,14]. Most of these studies relied on ex vivo
restimulation to assay IL-17A production, however, and thus the
cells producing IL-17A directly in vivo during the development and
propagation of EAE are not fully characterized.
To track IL-17A expression during EAE, we immunized Smart-
17A mice with myelin oligodendrocyte glycoprotein peptide
(MOG) emulsified in complete Freund's adjuvant (CFA) and
treated mice with pertussis toxin to induce disease. We then
examined CD3e+cell lineages in the draining lymph nodes (LN) at
day 6 and in the spinal cord and cerebellum (referred to as CNS)
at day 12 when mice displayed symptoms of paralysis. At both
sites, the vast majority of isolated T cells were CD4+or CD8+T
cells, with only small numbers of cd T cells, iNKT cells and other
CD3e+cells (Figure 5A). Small but reproducible percentages (1–
2%) of CD4+T cells expressed the hNGFR reporter in both the
LN and the CNS (Figure 5B). Among the remaining T cell
populations, we observed hNGFR-expressing cd T cells, iNKT
cells and other CD3e+cells (Figure 5B). The percentages of these
hNGFR+innate-like T cells were greater in the LN than the CNS.
Expression of hNGFR by CD8+T cells in both the LN and CNS
was negligible when compared to wild-type controls, and we did
not observe hNGFR expression by any CD3e2cells (data not
shown). Although the total numbers of cd T cells, iNKT cells and
other CD3e+cells were substantially lower than the total numbers
of CD4+cells, these innate-like T cells comprised the majority of
IL-17A-expressing hNGFR+cells in both the LN and CNS
(Figure 5C). In the LN, the greatest fraction of hNGFR+cells was
other CD3e+cells (36%), followed by cd T cells (29%), iNKT cells
(17%) and CD4+T cells (15%). In the CNS, the largest fractions of
hNGFR+cells were cd T cells and CD4+T cells (42% each), while
other CD3e+cells and iNKT cells together made up the remaining
16%. These data suggest that innate-like T cells are a significant
source of IL-17A during both the initiation and propagation
phases of EAE.
Figure 1. Generation of Smart-17A mice. (A) Targeting strategy for the il17a locus. For detailed description, see Materials and Methods. (B) CD4+
T cells were isolated from wild-type or Smart-17A mice and polarized under Th17 conditions for 4 days. hNGFR was detected using a surface antibody
and IL-17A was assayed using intracellular cytokine staining after restimulation. A representative flow cytometry plot is shown from .5 comparable
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Since the percentages of IL-17A-expressing CD4+T cells
observed using the Smart-17A reporter were substantially lower
than what has been reported in the literature using ex vivo
restimulation, we verified that ex vivo restimulation of cells isolated
from the LN and CNS of Smart-17A mice led to an induction of
hNGFR expression that very closely mirrored the percentages of
IL-17A+cells found using standard intracellular cytokine staining
methods (Figure 5D). This confirms that cells from Smart-17A
mice accurately report IL-17A production, and further suggests
that ex vivo restimulation can induce IL-17A production from cells
that are not actively producing the cytokine in vivo.
Differential production of IL-17A and IFN-c at effector
sites during inflammation
IFN-c is a cytokine that is expressed by multiple cell types and is
generally associated with inflammatory immune responses. Mice
deficient in IFN-c or the IFN-c receptor (IFN-cR) are more
susceptible to a variety of bacterial infections including pulmonary
infection with K. pneumoniae [27]. IFN-c has also been implicated in
the pathogenesis of autoimmune diseases, including EAE,
although the precise role of this cytokine is controversial. Thus,
mice deficient in IFN-c, IL-12 p35 subunit and IFN-cR are more
susceptible to disease [28,29,30] whereas T-bet2/2mice are
protected from disease [31]. EAE can be induced in Rag2/2mice
by the transfer of CD4+T cells that have been polarized under
either Th1 or Th17 conditions [32]. Complicating matters further,
recent studies have suggested that Th17 cells may represent an
unstable population that can convert to an IFN-c producing
phenotype under specific inflammatory conditions [33].
To explore the relationship between IL-17A and IFN-c
production during K. pneumoniae infection and EAE, we crossed
Smart-17A mice to Great reporter mice, which mark IFN-c
production by the coordinate expression of YFP downstream of an
IRES introduced into the IFN-c locus [34]. The most striking
observation in these dual reporter mice was that at the effector
sites following both challenges, either in the lungs of mice infected
with K. pneumoniae or in the CNS of mice during EAE, the
percentages of YFP+cells were substantially greater than the
percentages of hNGFR+cells among CD4+T cells, iNKT cells
and other CD3e+cells (Figure 6A). In contrast, the populations of
cells that expressed YFP and hNGFR was much more comparable
among cd T cells. As noted previously, we saw no measurable
expression of hNGFR above background levels in CD8+T cells,
although we did note a substantial population of YFP+CD8+T
cells, especially in the CNS during EAE. We observed some co-
expression of YFP and hNGFR among CD4+T cells, especially in
Figure 2. IL-17A expression in resting mice. Cells were isolated from indicated organs of Smart-17A/RORct reporter mice and levels of hNGFR
were assayed. Populations were gated as described in Figure S2. All gates were set using a wild-type mouse as a negative control. * denotes cell
populations that were too few in number to reliably assess marker expression. Representative flow cytometry plots are shown from 1 of 3
comparable experiments, each including 2–3 mice.
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the CNS of mice during EAE, where approximately half of the
hNGFR+cells also expressed YFP. However, among the innate-
like T cells, IFN-c-expressing and IL-17A-expressing cells
segregated into distinct populations. The same trends in cytokine
production were evident when we examined the total numbers of
YFP+and hNGFR+cells isolated from these effector sites
(Figure 6B). IFN-c-producing cells greatly outnumbered IL-17A-
producing cells during the first three days of K. pneumoniae infection
and during all stages of progressive EAE disease among all subsets
of CD3e+cells except for cd T cells.
Figure 3. IL-17A expression during infection with Klebsiella pneumoniae. Wild-type or Smart-17A mice were infected with 500–1000 K.
pneumoniae. (A) At the indicated time points, cells were harvested from lungs and numbers of cells were enumerated. (B) Cells were isolated from the
lungs of mice 2 days after infection and assayed for hNGFR expression. (C) The total number of hNGFR+and YFP+cells on day 2 post-infection were
calculated and the percentage attributable to each cell population is shown in a pie graph. The percentage of background staining seen in a wild-
type mouse under identical conditions was subtracted before performing all calculations to control for nonspecific staining. This experiment was
repeated 3 times with n .3 mice at each time point. For bar and pie graphs, data from independent experiments were compiled. For flow cytometry,
representative plots are shown.
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We used IL-17A reporter mice to assess IL-17A expression in
resting mice and during models of bacterial pneumonia and
autoimmune disease. We did not observe constitutive IL-17A
expression from cells in naı ¨ve mice. However, in appropriate
infectious and autoimmune models, we observed IL-17A-express-
ing CD4+T cells and greater percentages of IL-17A-expressing cd
T cells, iNKT cells and other CD3e+cells. The other CD3e+cells
were defined by their expression of CD3e+and lack of expression
of CD4, CD8, cd TCR and the CD1d-tetramer. It is possible that
these cells are type II NKT cells that do not recognize a-
galactosylceramide, or T cells that have downregulated the
expression of CD4, CD8 or the cd TCR. In all of these studies,
Figure 4. Expression of IL-17A from innate-like T lymphocytes cells can be induced by IL-1b and/or IL-23. (A) Smart-17A mice were
inoculated intranasally with PBS or with 500 ng IL-1b, IL-23 or both cytokines. Lungs were harvested 8 hr later and cells were analyzed for hNGFR
expression. Gates were set using a wild-type control. The experiment was repeated 2 times and representative data are shown. (B) Wild-type mice
were infected with K. pneumoniae and levels of IL-1b and the IL-23 subunit p19 mRNA in whole lung homogenate were measured using quantitative
PCR. Expression of GAPDH was used as a reference to define relative expression. The experiment was done twice and a representative experiment is
shown, n=3 for all groups.
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IL-17A production as assessed by the reporter was limited to
CD3e+T cells and was not expressed by myeloid cell populations.
By using novel dual cytokine reporter mice, we demonstrated that
IFN-c-expressing lymphocytes greatly outnumbered IL-17A-
expressing lymphocytes in both the acute bacterial and in the
more subacute autoimmune challenges.
We were unable to detect expression of the hNGFR reporter in
any CD3e+or CD3e2cell type in naı ¨ve mice, suggesting that IL-
Figure 5. IL-17A expression during experimental autoimmune encephalomyelitis. Wild-type or Smart-17A mice were immunized with
MOG-CFA to induce EAE. (A) At the indicated time point, cells were harvested from the draining axial, brachial and inguinal lymph nodes (LN) at day 6
or spinal cords and cerebellums (CNS) at day 12 and numbers of cells were enumerated. (B) Cells were assayed for hNGFR expression. (C) The total
numbers of hNGFR+cells were calculated and the percentage attributable to each cell population is shown in a pie graph. The percentage of
background staining seen in a wild-type mouse under identical conditions was subtracted before performing all calculations to control for
nonspecific staining. (D) Cells were isolated from the LN or CNS and immediately stained for surface markers or restimulated for 5 hr with PMA/
ionomycin and then stained for surface markers and/or intracellular expression of IL-17A. The experiments in A–C were repeated 3 times with n.2
mice at each time point. The experiment in D was repeated 2 times with n.5 mice at each time point. For bar and pie graphs, data from independent
experiments were compiled. For flow cytometry, representative plots are shown. For the CNS data, mice were excluded that did not display
symptoms of paralysis.
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Figure 6. Differential production of IL-17A and IFN-c in effector sites during inflammation. (A) Expression of hNGFR and YFP was
assessed in the lungs of mice at day 2 after infection with K. pneumoniae or in the spinal cord and cerebellum (CNS) of mice at day 12 after induction
of EAE. (B) Numbers of hNGFR+and YFP+cells were enumerated during K. pneumoniae infection or EAE disease course. EAE disease scores were
determined as described in Materials and Methods. ND = not detected. These experiment was repeated 3 times with n.2 mice at each time point or
disease score. For flow cytometry, representative plots are shown. For bar graphs, data from individual experiments were compiled.
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17A is not expressed in situ in resting mice. Segmented filamentous
bacteria (SFB), Candidatus arthromitis, have been shown to induce
IL-17A production by lamina propria T cells when introduced
into the mouse intestinal flora [35]. Cohousing Smart-17A mice
with mice obtained from Taconic Farms led to colonization by
SFB as determined by PCR, but did not induce hNGFR
expression on cells in the lamina propria of the small intestine
or colon (data not shown). Our inability to quantitate active IL-
17A production from cells in tissues of resting mice contrasts with
prior observations made with other strains of IL-17A reporter
mice. In the first report, Hirota et al. [14] crossed IL-17A-cre mice
to Rosa-flox-stop-YFP mice to generate an IL-17A fate-tracking
reporter and observed that 11% of lamina propria CD4+T cells
were constitutively YFP-positive in these mice. A study by
Esplugues et al. [15] using a second IL-17A reporter strain, which
utilized an IRES-eGFP knockin strategy more closely akin to the
construct used in the Smart-17A mice, reported that 2–4% of
lamina propria CD4+T cells constitutively expressed eGFP.
Whether the differences between our results and those using these
alternate reporter strains are technical (e.g. permissiveness of the
various IRES elements or detection limits of eGFP versus hNGFR)
is unclear, and will require direct comparisons of the strains in
Here, we crossed our Smart-17A reporter mice to RORct-GFP
reporter mice to ensure our capacity to isolate potential IL-17A-
producing cells from the relevant tissues. Although we readily
identified populations of RORct+cells in multiple organs of naı ¨ve
mice, these cells did not constitutively express the hNGFR
reporter. In line with a previous report [36], we noted substantial
populations of RORct+cd T cells in the skin and lungs of resting
mice. We also noted smaller percentages of RORct+iNKT cells in
the lung and other CD3e+cells in the lung and skin. Resident
dermal RORct+cd T cells have recently been shown to play a role
in cutaneous immunosurveillance and to contribute to skin
pathology during a mouse model of psoriasis [37,38,39]. The
localization of constitutive RORct+innate-like T lymphocytes to
the lung is intriguing as this mucosal tissue is relatively sterile and
devoid of large numbers of commensal bacteria, a feature that is
may contribute to the development of the RORct+populations in
the intestinal tract [35,40,41]. Although lung-resident innate-like
T cells did not constitutively express IL-17A as assessed by the
hNGFR reporter, cd T cells, iNKT cells and other CD3e+cells
expressed hNGFR within 24 hr after Klebsiella infection or 8 hr
after administration of IL-1b and/or IL-23. Taken together, these
data suggest that RORct+innate-like T cells accumulate at
multiple epithelial barriers, where they are poised to respond
rapidly to compromises in epithelial integrity with IL-17A
production and the subsequent recruitment of neutrophils to the
injured site.
Earlier reports used ex vivo restimulation to identify Th17 cells
induced during a variety of bacterial and fungal infections,
particularlythose initiated
[42,43,44,45,46]. However, as we demonstrate here, ex vivo
restimulation has the potential to reveal IL-17A production by
cells that may not be actively producing this cytokine in vivo
(Figure 5D). Although our studies using K. pneumoniae in Smart-17A
mice revealed activation of reporter expression in both innate-like
T cells and CD4+Th17 cells, earlier studies demonstrated
increased mortality in mice lacking cd T cells but no difference
in mortality in mice lacking abT cells [22,46], suggesting that cd T
cells may be a more important source of IL-17A during this
infection. Similar observations were made in experimental
Mycobacterium tuberculosis infection [47] and in intraperitoneal
infection with Escherichia coli [48], suggesting a more generalized
bymucosal challenges
role for IL-17A production by cd T cells in immunity to bacterial
infections. Our findings corroborate these interpretations.
Somewhat unexpected were the small percentages of IL-17A-
expressing CD4+T cells recovered from lymph nodes and spinal
cords and cerebellums during the peak of EAE disease, a canonical
experimental model for Th17 induction. Despite the relatively
small percentage of IL-17A reporter-positive CD4+T cells, the
large numbers of CD4+T cells in the inflammatory milieu was
such that CD4+T cells still comprised a sizeable portion of total
IL-17A-producing cells, making up 15% of the total in the LN and
42% in the CNS. The vast majority of effector CD4+T cells,
however, were Th1 cells as assessed by expression of the Great
IFN-c reporter allele and, based on our in vitro studies of the decay
of the reporter on Th17 cells, are unlikely to have secreted IL-17A
over the prior 1–2 days. Our findings seem to contrast with those
obtained using an IL-17A-cre/ ROSA-flox-stop-YFP fate-tracking
reporter mouse (14). In that study, the authors observed that over
half of the CD4+and cd T cells were YFP+in the spinal cords of
mice during the peak of EAE. However, cells positive for the IL-
17A-cre/Rosa-flox-stop-YFP reporter were those that expressed
the Cre recombinase from an activated IL-17A locus at any time
during their development, whereas the Smart-17A reporter
specifically marks cells that have recently expressed or are
currently expressing IL-17A. Thus, differences in construction of
these various reporter mice may account for the differences in the
experimental results.
Recent reports have raised the possibility that Th17 cells
represent an unstable transient phenotype rather than a fully
differentiated Th subset akin to that of Th1 and Th2 cells. During
the development of EAE, IL-17A+, IFN-c+and IL-17A/IFN-c
double-positive CD4+T cells have been observed in the lymph
nodes and spinal cords of immunized mice [19]. Adoptive transfer
of encephalitogenic Th17 cells purified from an IL-17F-cre BAC
transgenic mouse into RAG-22/2or wild-type recipients revealed
that a portion of these cells began to secrete IFN-c during the
progression of EAE [49]. Similarly, studies using the IL-17A-cre/
Rosa-flox-stop-YFP mice revealed that only half of the reporter-
positive fate-marked CD4+T cells were positive for intracellular
IL-17A in the spinal cords during the peak of EAE [14], providing
further evidence for a switch from an IL-17A-producing to an
IFN-c-producing phenotype. However, all of these studies used ex
vivo restimulation to assess IL-17A and IFN-c production from
isolated spinal cord cells. Our study, which relied solely on direct
ex vivo detection of cytokine production using knockin reporter
mice, provides additional evidence for the marked predominance
of IFN-c production by CD4+T cells during each of the clinical
stages of EAE. We saw only low numbers of IL-17A+cells as
compared to IFN-c+cells, and approximately half of the Th17
cells concordantly expressed IFN-c. Taken together, these findings
suggest that activation of the IL-17A locus may be important in
the early differentiation of pathogenic CD4+T cells in EAE, but
that actual production of IL-17A might not be a major mechanism
driving the neurological manifestations of the disease. Indeed,
recent reports have demonstrated critical contributions by GM-
CSF rather than IL-17 in the pathogenesis of EAE [50,51].
A consistent observation was the pronounced segregation of IL-
17A-expressing and IFN-c-expressing cells within the innate-like T
cell populations. This segregation occurred in both the infectious
and autoimmune models, raising questions about the mechanisms
of regulation that account for this exclusionary pattern of cytokine
production. IFN-c and IL-17 have been shown to limit
differentiation of Th17 and Th1 cells, respectively, and this
mutual inhibition could be operating in these innate populations as
well [52,53,54]. It has also been suggested that antigen-naive cd T
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cells predominately produce IL-17A when activated, whereas
antigen-experienced cd T cells produce IFN-c [55], which could
potentially explain the differences in cytokine-secreting popula-
tions seen in our models. Further characterization of the cells
expressing these cytokines is needed to address this question more
Cytokine reporter mice allow for the functional marking of cells
during the course of an inflammatory challenge and have provided
essential insights into the coordination of the immune response in
vivo. The Smart-17A mice and Great mice used in this study
permitted the detection of IL-17A-producing and IFN-c-produc-
ing cells in situ without restimulation. Using these mice, we
demonstrated that innate-like T cells, particularly cd T cells,
comprised major cell populations poised for acute IL-17A
production. During both K. pneumoniae infection and EAE, models
previously demonstrated to induce potent IL-17 expression, we
show that the numbers of IL-17A-producing cells were far fewer
than the numbers of IFN-c-producing cells in the same tissues,
suggesting different levels of regulation of these two inflammatory
cytokines in vivo. Although IL-17A production has been elicited
from a number of different cell types using restimulation, our
reporter system suggests that cytokine production is limited to T
cells in the models we studied. These cytokine reporter mice will
be valuable tools for future studies investigating the full
contributions of IL-17A-expressing cells to vertebrate immunity.
Materials and Methods
C57BL/6 mice and RORct-GFP reporter mice [18] were
obtained from Jackson Laboratories. Great IFN-c reporter mice
have been described [34]. Smart-17A mice were generated by first
assembling a composite selection/reporter cassette using standard
cloning procedures in the following order: 1) a floxed-neomycin-
resistance gene (floxed Neor); 2) encephalomyocarditis virus
(EMCV) internal ribosome entry site (IRES); 3) low-affinity
human nerve growth factor receptor (p75 LNGFR, also known
as CD271) cDNA (OriGene); 4) bovine growth hormone (bGH)
poly-adenylation signal (pA). This 3.1 kb selection/reporter
cassette was cloned into a basal targeting construct pKO915-DT
(Lexicon) containing diphtheria toxin (DT)a chain for negative
selection. Both 59 and 39 homologous arms used to flank the
cassette were obtained by high-fidelity PCR amplification of the
il17a locus from 129/SvJ genomic DNA. The 59 arm consists of a
1.6 kb fragment covering intron 2 and coding sequence of exon 3.
The 39 arm consists of a 2.2 kb fragment spanning the endogenous
39 UTR and downstream sequences. After sequence verification,
the NotI-linearized construct was electroporated into PrmCre ES
cells, which express Cre recombinase driven by the protamine
promoter [56]. G418-resistant ES clones were screened for
homologous recombination by Southern blot. Two independent
clones were injected into C57BL/6 blastocysts to generate
chimeras. The neomycin-resistance cassette was deleted in the
male germline by Cre-mediated recombination after breeding
male chimeras to C57BL/6 females. Mice carrying the Smart17A
allele were backcrossed 10 generations to the C57BL/6 back-
ground. In experiments utilizing Smart-17A/RORct-GFP mice,
the mice were homozygous for the Smart-17A allele and
heterozygous for RORct-GFP. Smart-17A/Great mice were
homozygous for both the IL-17A and IFN-c reporter alleles and
these dual reporter mice were used for all experiments described in
Figures 3, 4, 5, and 6.
Th17 polarization
CD4+T cells were isolated from the lymph nodes of Smart-17A
and wild-type C57BL/6 mice using MACS beads (Miltenyi
Biotech) and cultured with irradiated splenocytes from TCR-
mice. Cells were stimulated under the designated
conditions for 4 days: Th0 (50 U/ml IL-2), Th1 (50 U/ml IL-2,
5 ng/ml IL-12, 10 mg/ml anti-IL-4), Th2 (50 U/ml IL-2, 50 ng/
ml IL-4, 10 mg/ml anti-IFN-c), Th17 (3 ng/ml TGF-b, 20 ng/ml
IL-6, 10 mg/ml anti-IFN-c and anti-IL-4). Cytokines were
purchased from R&D Systems. For intracellular cytokine staining,
cells were restimulated with phorbol myristate acetate (50 ng/ml)
and ionomycin (750 ng/ml) for 5–6 hr, with monensin (3 mM)
added for the final 2 hr.
Klebsiella pneumoniae infection
K. pneumoniae (American Type Culture Collection #43816) were
cultured in Nutrient Broth (Difco) with shaking overnight at 37uC.
Cultures were diluted in Nutrient Broth and cultured for an
additional 2–3 hr until bacteria reached log phase. Bacteria were
pelleted by centrifugation, washed twice in PBS and diluted to a
final dose of 500–1,000 bacteria in 50 ml PBS. This inoculum was
administered intranasally after anaesthetizing mice with isofluor-
ane. Doses were confirmed by plating the inoculum on Nutrient
Broth agar plates and counting colonies the following day.
Induction of experimental autoimmune
Mice were immunized with 200 mg of MOG35–55 emulsified in
CFA containing 4 mg/ml Mycobacterium tuberculosis (Difco) subcu-
taneously and given 200 ng of pertussis toxin (List Biological
Laboratories) intravenously on the day of and 2 days after
immunization. Animals were graded daily according to their
clinical severity as follows: grade 0, no abnormality; grade 1, limp
tail; grade 2, limp tail and hind limb weakness (waddling gait);
grade 3, partial hind limb paralysis; grade 4, complete hind limb
paralysis; grade 5, moribund.
Cell isolation and preparation
Mice were perfused with PBS and organs were removed.
Mechanical dissociation was used to prepare single-cell suspen-
sions from lymph nodes, spleens and Peyer's patches. Lungs were
dissociated using a GentleMacs Dissociator (Miltenyi Biotech).
Small intestines and colons were cut into small pieces and
incubated in 5 mM EDTA in HBSS on stir plates 4 times for
15 min to remove the epithelial layer containing intraepithelial
lymphocytes. Intestinal pieces were incubated at 37uC in 200 U/
ml collagenase VIII (Sigma) in complete RPMI for a total of 4 30-
min incubations. Cells from all incubations were pooled and
lamina propria lymphocytes were purified over a 40%/100%
Percoll gradient. Central nervous system lymphocytes were
isolated as described (21) with modifications. Briefly, spinal cords
and cerebellums were cut into pieces and digested in 300 U/ml
Mandl units Collagenase D (Roche) and 50 U/ml DNase I
(Roche) at 37uC for 30 min. Lymphocytes were enriched by
separation on a 30%/70% Percoll gradient.
Flow cytometry
Single-cell suspensions were washed in FACS buffer (PBS, 3%
FCS, 1 mg/L NaN3), and the cell pellets were incubated for
10 min on ice with anti-CD16/CD32 monoclonal antibodies
(UCSF Antibody Core Facility). Cells were incubated for 30 min
on ice with antibodies to surface markers. As necessary, cells were
washed and stained with secondary antibodies for an additional
IL-17A-Producing Cells In Vivo
PLoS ONE | www.plosone.org10June 2012 | Volume 7 | Issue 6 | e39750
Page 11
20 min on ice. Live cells were gated using DAPI exclusion. For
intracellular cytokine staining, cells were stained for surface
markers, fixed in 2% formaldehyde in PBS for 20 min at room
temperature, washed and permeabilized in FACS buffer plus 0.5%
saponin. Cells were stained at room temperature for 30 min in
buffer containing 0.5% saponin buffer and 25% FCS. Dead cells
were excluded using a violet live/dead fixable stain (Invitrogen).
Antibodies were as designated
eBioscience), CD8 (BD, Biolegend), cd (eBioscience), CD3e
(eBioscience), CD11b (BD, Biolegend), CD19 (Biolegend), Gr1
(BD), CD11c (BD), Thy1.2 (eBioscience), Sca-1 (BD), IL-17A (BD,
eBioscience), hNGFR (LabVision), Streptavidin-PE (Invitrogen),
Streptavidin-APC (Biolegend). PBS-157-loaded CD1d tetramer
was obtained from the NIH Tetramer Core Facility. Cell counts
were performed using Count-Bright absolute counting beads
(Invitrogen). Samples were acquired on a LSRII flow cytometer
(BD Biosciences) and analyzed using FlowJo software (Tree Star).
and included: CD4(BD,
Quantitative PCR
Whole lungs were homogenized and RNA was isolated using
RNAzol (Molecular Research Center, Inc.). cDNA was prepared
using the SuperScript III First Strand Synthesis System (Invitro-
gen). Primer sequences (PrimerBank) where designated were as
follows:IL-1b: GAAATGCCACCTTTTGACAGTG,
CAGGCTA. Transcripts were quantified by incorporation of
SYBR Green (Invitrogen) on a StepOne Plus Real-Time PCR
System (Applied Biosystems) and quantified relative to the
expression of GAPDH (glyceraldehyde 3-phosphate dehydroge-
Supporting Information
Figure S1
vitro. (A) CD4+T cells were isolated from Smart-17A mice using
MACS beads and polarized under Th0, Th1, Th2 or Th17
conditions for 4 days, at which point surface hNGFR expression
was assayed by flow cytometry. This experiment was repeated 3
times and representative flow cytometry plots are shown. (B) CD4+
T cells from wild-type or Smart-17A mice were polarized under
Polarization of Smart-17A CD4 T cells in
Th17 conditions for 4 days. Cells were restimulated with PMA and
ionomycin and then washed and re-plated in wells containing no
cytokines. The percentage of hNGFR+cells were measured at
indicated time points to determine the rate of decay of the hNGFR
reporter. A representative graph is shown from two comparable
Gating of CD3e+
cytometry gating schemes for CD3e+cells used throughout this
study. (A) Gating scheme for CD4+T cells, cd T cells, iNKT cells
and other CD3e+cells. (B) Gating scheme for CD8+T cells. Plots
shown are from the mesenteric lymph node of a naı ¨ve Smart-17A
cell populations. Flow
Figure S3
Cells were isolated from the indicated organs of Smart-17A/
RORct dual reporter mice and assayed for GFP and surface
hNGFR expression. Dendritic cells were defined as CD11c+,
macrophages as CD11b+, neutrophils as CD11b+and Gr1+, and
innate lymphoid cells as lineage-negative (negative for CD3e,
CD8, CD19, CD11b, Gr1) and Thy1+. The gated innate
lymphoid cells included cells that were positive and negative for
both CD4 and Sca-1. hNGFR expression was not seen using any
gating scheme. All gates were drawn using a wild-type mouse as a
control. The experiment was repeated twice and representative
plots are shown.
IL-17A expression in CD3e2cell populations.
We thank B. Fife and S. Bailey-Bucktrout for assistance with EAE
experiments, J. Bando for help with isolation of lamina propria cells, L.
Cheng for help with isolation of skin cells, and N. Flores-Wilson for support
of the mouse colony. We thank the NIH Tetramer Core Facility for
providing the CD1d tetramer reagent.
Author Contributions
Conceived and designed the experiments: AEP RLR RML. Performed the
experiments: AEP. Analyzed the data: AEP. Contributed reagents/
materials/analysis tools: RLR HEL. Wrote the paper: AEP RML.
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subset: commoncrawl


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Timothy Geithner's new book about the financial crisis, "Stress Test," is basically an argument that the Wall Street bailout succeeded.
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Confiscating customer deposits in Cyprus banks was not a one-off, desperate idea of a few eurozone "troika" officials scrambling to salvage their balance sheets. A joint paper by the U.S. Federal Deposit Insurance Corporation and the Bank of England, dated Dec. 10, 2012, shows these plans have been long in the making.
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This profile was last updated on 10/1/12 and contains information from public web pages and contributions from the ZoomInfo community.
Chris BesseneckerWrong Chris Bessenecker?
Norma Michigan Inc.
Phone: (810) ***-****Email: c***@***.com
Local Address: St Clair, Michigan, United States
NORMA Group AG
2430 East Walton Blvd.
Auburn Hills , Michigan 48326
United States
Company Description: NORMA develops and manufactures the most innovative plastic and metal-based components and systems in the field of connecting technology. Our strong international... more
Pro2015_Iteration2_1.167[01] SEIDXWK-13


text:
49-9021 jobs near Murfreesboro, TN
HVAC ApprenticeNEW
Tradesmen International- Nashville, TN
Returning Candidate? Log back in! HVAC Apprentice All times are in Coordinated Universal Time. Req ID 2015-15166 Job Location(s) US-TN-Nashville Posted Date 3/23/2015 Category...3 days ago from Tradesmen International
HVAC ApprenticeNEW
Tradesmen International- Nashville, TN
Tradesmen International, America's elite skilled labor force, is seeking highly motivated HVAC Technician Apprentices to join our team. We are recognized internationally as a...2 days ago from Tradesmen International, Inc.
Compressed Air Service TechnicianNEW
Ingersoll Rand- Tennessee
At Ingersoll Rand we're passionate about inspiring progress around the world. We advance the quality of life by creating comfortable, sustainable and efficient environments. Our...2 days ago from Ingersoll Rand
HVAC TechnicianNEW
Manpower Group- Nashville, TN
Manpower is recruiting experienced HVAC technicians interested in a career opportunity with a Nashville-based heating, cooling, plumbing and electrical contractor. The qualified...2 days ago from Manpower Group
Fleet Technician - Refrigeration (Preferred) SyscoNEW
SYSCO- Nashville, TN
Returning Candidate? Log back in! Fleet Technician - Refrigeration (Preferred) Sysco Nashville Job ID 19856 Location US-TN-Nashville Category Accounting / Finance Type Full-Time...20 hours ago from Sysco
Laboratory Engineering Technician (Heating Products)NEW
Hayward Industries- Nashville, TN
Hayward Industries is the world's leading manufacturer and marketer of residential and commercial swimming pool equipment, with strong markets in the U.S., Canada, France, Spain,...2 days ago from Hayward Industries
HVAC Mechanic
Peabody Memphis- Tennessee
JOB OVERVIEW:Responsible for repairs, maintenance, upkeep and replacement of the property's plant systems and equipment in accordance with hotel standards. Will assist in other...22 days ago from The Peabody Memphis
General Service Technician
National Tire and Battery- Murfreesboro, TN
Our General Service Technician position provides candidates with the perfect starting point for an automotive repair career. Previous experience, while desirable, is not required.30+ days ago from National Tire and Battery
Technician, HVAC
Hiller- Murfreesboro, TN
HVAC Technician Full-Time Paid Vacations Paid Training Medical, Dental, Vision, and Life Insurance Retirement and Disability Benefits Job Responsibilites: Ability to perform all...30+ days ago from Hiller LLC
Commercial HVAC Technician
Hiller- Murfreesboro, TN
Commercial HVAC Technician Paid Vacations Paid Training Medical, Dental, Vision, and Life Insurance Retirement and Disability Benefits Job Responsibilities: Ability to perform all...30+ days ago from Hiller LLC
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Sandra Moore
member for 14 years, 9 months, 16 days
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Bio and Links
My name is Sandra Moore and I live in Mason, Michigan. I'm a wife, mother of three children and grandmother of eight grandchildren. I became interested in genealogy in 1999. I enjoy sharing my family information and photos with others and helping out anyone else in their genealogy research through cemetery records. I found out about find a grave website back in 2000 and never thought about helping to enter information or take photos at the cemetery till now. I have found others that have done so and so appreciate their help and now I want to do my part with volunteering in putting up whatever pictures I can or adding information from the cemeteries. To all that have entered names on cemeteries, my many thanks go out to you. Keep up the good work. Also if I happen to make a memorial page for a relative and you want ownership, I have no problem turning it over to you and will be more than happy to do so. Also if you happen to find any errors, please let me know and I will correct it right away. Sometimes stones are hard to read. I try to be as accurate as possible and do research on my own if I am not sure of a name or date or relationship. Please feel free to use my photos in your family tree.
Also please ask for transfers (giving me your member number) and to add any information under the Edit tab at the right hand top of the memorial page and not on my message board. It's a lot quicker and easier that way as it takes me directly to the persons' memorial page. Thanks!
Sincerely, Sandra Moore
Leave Public Message
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30,931 Memorials Added
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Added by
on Mar 26, 2015 8:43 AM
I'm Lilys grand daughter
I am Lily Miloszewski's grand daughter. I am the daughter of her daughter Josephine. I would like to add info to the memorial but don't understand how to.
Lily was named Waleria. She was born in Long Run, Jefferon, OH to Anna and Antoni Rosgaski. She was first married to Martin Miloszewski. They had three children, Andrew, Lily and Herman. She had married Frank Waskiewicz, Richard Morton then Felix Kain.
Added by
on Mar 25, 2015 10:28 PM
Thank You!
Thank you so very much! Appreciate your efforts...wonderful to get the family linked!
Added by
on Mar 16, 2015 1:17 PM
sandi lewis - malburg
RE: Jean, Edith and Kathrine Smith
That one is too young. I think it might be the Kathryn Smith 52114553. but there is no info about her.
Added by
sandi lewis - malburg
on Mar 08, 2015 10:50 AM
sandi lewis - malburg
RE: Jean, Edith and Kathrine Smith
Thank You, I will. Sandi
Added by
sandi lewis - malburg
on Mar 08, 2015 10:41 AM
RE: Ruby Maupin
Oh my goodness...I can't even find the person I was referring to! LOL I have ancestors coming out of my ears with research! LOL If I come back across it, I will let you know....:}
Added by
on Mar 06, 2015 9:26 PM
sandi lewis - malburg
Jean, Edith and Kathrine Smith
Hi Sandra,
I have spoke to you about this (my family) before, but did you get a copy of the burial plot list of those in the Oak Grove Cemetery in St. Louis? You entered my my Grandparents (great) and my great aunts and uncles. I am working on a cousin, Jean Smith 52287419, We know his first wife, Edith was buried there and she is on Find a Grave. But, we knew nothing about his second wife, Kathrine. From what her cousin tells me, she just seems to drop off the map. Might she be buried beside Jean and Edith? Sandi
Added by
sandi lewis - malburg
on Mar 06, 2015 2:11 PM
stacy thomas
RE: Margaret Susan Crosby Earegood
well her mother's maden name isnt Hayes but her mother did marry a Hayes man. Im just curious as to why it claimed Hays as her maden name when its not it was her last married name
Added by
stacy thomas
on Mar 04, 2015 6:38 PM
stacy thomas
Margaret Susan Crosby Earegood
I saw that death certificate and I noticed it said mother's maden name it wasnt the same. Margaret's mother's maden name is Barchus. So Im curious as to who the person on the death certificate listed as the mother is.
Added by
stacy thomas
on Mar 01, 2015 3:19 PM
Randall McCoach
Disbrow family
Thank you for creating the memorials, and thanks for the transfers! -- Randall McCoach, Albuquerque, N.M.
Added by
Randall McCoach
on Feb 25, 2015 4:00 PM
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Photo of Rachel Zoe and Nicole Richie
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Nicole Richie and Rachel Zoe posed with Byron Williams and Andy Lecompte at a party in LA on Thursday night celebrating Andy's salon.
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Read More Rachel ZoeHotNicole Richie
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Bearsy123 6 years
not a fan of those headbands
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not a fan of those headbands
minnie2011 6 years
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jamie143 6 years
Well.......Nicole looks pretty!
Link Love
- Find out what Taylor Swift said about her doppelgänger — Us Weekly
- Justin Guarini, is that really you?! — HuffPost Celebrity
- Here's what Outlander star Sam Heughan is like in person — Lainey Gossip
- Julia Roberts has a major problem with Nicole Kidman — Dlisted
- Lena Dunham's new written piece is causing a stir — ET
- Inside Kate Mara and Ellen Page's day at Disneyland — JustJared
- Kate Upton is showing major cleavage for Guess — Hollywood Tuna
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Like anything Madonna does, her new film W.E. has been subject to controversy since its inception. The movie, which parallels the scandalous relationship between England's Crown Prince Edward and the American Wallis Simpson with a contemporary love story, has already received mixed reviews at film festivals and screenings since its premiere at the Venice Film Festival this fall. But, again like anything Madonna does, the film looks great. The Weinstein Company's released the first official trailer, just two weeks before the film opens on December 9th. Click through to watch!
Madonna's New Love Story
Spending the Day With L.A.'s Ethical Taxidermist
Who happens to be a dead ringer for Bettie Page.
Little League Superstar Mo'ne Davis Won't Let the Haters Shake Her
No wonder The Disney Channel is making a movie about her.
A Major Broadway Star Just Joined the 'Beauty and the Beast' Cast
Could this be a hint that the movie will be a musical?
The Best Instagram Snaps of Models Working Out
They work hard to look so good.
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text:
|Name: _________________________
|Period: ___________________
This quiz consists of 5 multiple choice and 5 short answer questions through Chapter 33 | Chapter 34.
Multiple Choice Questions
1. What is Tom Crick's response to this revelation?
(a) He starts flirting intensely with Mary to outdo all the other boys.
(b) He begins to be afraid of Dick.
(c) He turns Freddie Parr in to the police constable.
(d) He reads history books when Dick is out wooing Mary.
2. What is the term Tom Crick keeps using to refer to Mary's attitude towards the world when she was an adolescent?
(a) Curious.
(b) Passive.
(c) Eager.
(d) Aloof.
3. What about the scene at the Lode does NOT remind Tom of the later scene of Freddie Parr's death?
(a) Dick standing on the wooden bridge.
(b) Freddie Parr in the water.
(c) A boat-hook lying on the riverbank.
(d) A bottle drifting in the water.
4. What does Ernest Atkinson do with the three remaining steam ships he owns?
(a) He donates them to the army to aid in the war effort.
(b) He names them after his daughter, Helen I, Helen II, and Helen III.
(c) He converts them into pleasure cruise ships.
(d) He sells them to the city of Gildsey for the use of the fire brigade.
5. Which of the following does NOT draw Mary and Tom closer together?
(a) They both have odd older brothers.
(b) They both grew up without a mother.
(c) They both escape to the ruined windmill together in the afternoons.
(d) They both take the same train to school every day.
Short Answer Questions
1. What marks the end of Ernest Atkinson's political career?
2. What sparks Tom Crick's interest in the mating habits of eels?
3. Tom confronts Price about his frequent interruptions during class. What question does Price's reluctance about history boil down to?
4. What does Tom Crick tell his students is the difference between animals and humans?
5. How does Harry Crick find out that his son Dick is in love with Mary?
This section contains 495 words
(approx. 2 pages at 300 words per page)


text:
Every year during move-out, ASU students find that they have accumulated more stuff than they can deal with. Stuff they don't want anymore. Stuff they don't have space for. Stuff that doesn't belong in the landfill.
Ditch the Dumpster does something good with all that stuff – reusing everything that can be reused, and repurposing or recycling the rest – all for the benefit of local children and families.
Donate small items. Look for Swift donation bins.
Drop off large items. Look for collection areas in your hall.
Recycle. Look for blue at ASU.
Our Mission
Our mission is to produce meaningful change: diverting waste from the landfill, supporting local non-profit organizations, and educating the ASU community about waste, recycling, and meeting social needs. Our vision is to be a university that produces zero-waste, that is socially embedded through community partnerships, and whose graduates genuinely desire to transform society.
Ditch the Dumpster
Begins Earth Day, April 22 and runs through May 10th
Ditch the Dumpster is an annual tradition at Arizona State University. The year-end donation and recycling drive collects thousands of pounds of reusable and recyclable items from students as they move out of ASU residence halls. All collected items get used in some way or another. Reusable items are donated to charity organizations that benefit children and families locally. Items that cannot be reused are repurposed or recycled. Ditch the Dumpster is a university-wide initiative, in partnership with University Housing, the Julie Ann Wrigley Global Institute of Sustainability, Grounds Services, Recycling Services, Campus Student Sustainability Initiatives, Goodwill of Arizona, and Big Brothers Big Sisters of Central Arizona.
How You Can Donate
Donate most of your unwanted goods in any Swift Charities for Children collection box. Donate your larger items, fragile items, food, and potentially messy items like detergents and cleansers in the large collection area locations. There are locations on every campus – please check the right sidebar for the one nearest you. Don't forget to use your blue bins and recycle as usual.
1. Donate Smaller Items
Drop off your smaller items in the Swift Charities for Children collection boxes. These items include:
- Clothing, shoes, and accessories
- Books, hangers, and storage bins
- Smaller household goods
- Smaller appliances (usable or not)
2. Donate Larger Items
Drop off your larger, fragile, or potentially messy items in the large collection areas. These items include:
- Furniture
- Lamps and egg-crate mattress pads
- Larger appliances and electronics (usable or not)
- Tightly-closed detergents or cleansers
- Unopened, nonperishable food
3. Recycle Items
Recycle the usual items, such as:
- Paper
- Cardboard
- Plastic
- Metal
Community Participants
Founded in 1947, Goodwill of Central Arizona is one of the oldest and largest nonprofits in the state. They fulfill their mission of putting people to work by providing no-cost job training programs, placement services, education, and post-employment support to individuals with barriers to employment. Goodwill of Central Arizona's vision is that through the work of Goodwill, all people will have the opportunity to learn, work, and achieve their greatest potential.
For nearly 60 years, Big Brothers Big Sisters of Central Arizona has operated under the belief that inherent in every child is the ability to succeed and thrive in life. As the nation's largest donor and volunteer supported mentoring network, Big Brothers Big Sisters makes meaningful, monitored matches between adult volunteers ("Bigs") and children ("Littles"), ages six through 18, in communities across Arizona. We develop positive relationships that have a direct and lasting effect on the lives of young people.
Big Brothers Big Sisters of Central Arizona's Donation Center collects more than four million pounds of clothing textiles and two million pounds of miscellaneous product each year in the Valley of the Sun. The funds generated from the sale of this product go "DIRECTLY" to fund the mentoring program at BBBS of Caz. For more information about Big Brothers Big Sisters of Central Arizona, please visit www.bbbsaz.org.
House of Refuge will receive many of the items donated at the Polytechnic collection locations. Located on the ASU Polytechnic campus, House of Refuge is a non-profit organization helping homeless families and individuals in crisis by providing transitional housing and supportive services that assist participants as they strive toward self-sufficiency and seek to attain permanent housing.
Community Participants »
Last year, ASU students contributed 32,888 lbs of stuff that was donated to charity, repurposed, or recycled. most of that was during spring move-out! We challenge you to beat last year's numbers!


text:
- OSI-Approved Open Source (264)
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- Public Domain (2)
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- Windows (207)
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Viewers Software
Free editor for PDF documents. Complete editing of PDF documents is possible with PDFedit. You can change raw pdf objects (for advanced users) or use many gui functions. Functionality can be easily extended using a scripting language (ECMAScript).6,233 weekly downloads
gPhoto is a program and library framework that lets users download pictures from their digital cameras. The libgphoto2 library gives you access to hundreds of models of digital cameras on several platforms.2,275 weekly downloads
Cross-platform CBR/CBZ (comic book) reader1,292 weekly downloads
An utility for previewing and exporting as images one or multiple set of brushes generated with Adobe Photoshop (.abr files). It makes publishing your custom brush sets on the internet really easy.297 weekly downloads
Jomic is a viewer for comic books stored in CBZ, CBR, and PDF files. Features include a two page mode, caching and support for several image formats (PNG, JFIF/JPEG, TIFF, and GIF). It is Open Source software distributed under the GNU GPL.286 weekly downloads
Sodipodi is general vector illustrating application for Linux/Unix and Windows. It uses W3C SVG as native file and in-memory image format and can do many neat things.127 weekly downloads
imgSeek - Intelligent image database
imgSeek is a photo collection manager and viewer with content-based search and many other features. The query is expressed either as a rough sketch painted by the user or as another image you supply (or an image in your collection).104 weekly downloads
Geeqie Image Viewer
Geeqie is a lightweight image viewer. It was forked from GQview. The development is focused on features for photo collection maintenance: raw format, Exif/IPTC/XMP metadata and integration with programs like UFraw, ImageMagick, Gimp, gPhoto or ExifTo The current development can be found on Gitouris (https://gitorious.org/geeqie)80 weekly downloads
ASH Viewer
ASH Viewer provides a graphical view of active session history data within the Oracle instance (versions supported: 8i/9i/10g/11g/12c).87 weekly downloads
GPU Flame Fractal Renderer
Renderer for flam3 cosmic recursive fractal flames implemented on GPU. Requires a CUDA-capable graphics card.37 weekly downloads
Treebolic is a Java suite (including library) whose purpose is to give a hyperbolic rendering of hierarchical data. A tree is rendered with nodes and edges but display space is subject to a particular curvature (hence the name).164 weekly downloads
CBViewer is a comic book viewer for comic book archives in cbz, cbr, zip, and rar formats. The interface is written in java but it requires unrar for linux or windows depending which OS you are running on. It will do zip files in java.40 weekly downloads
Java Mass JPEG Resizer Tool
The "Java Mass JPEG Resizer Tool" has following Features Resize all JPEG Images of a Directory, Create Webgalleries, Pack it to an ZIP File for E-Mail Attachments. The Tool is available in German and English.37 weekly downloads
Open SVG Viewer
The Open SVG Viewer's purpose is to allow efficient and flexible visualization of SVG documents in Java-enabled platforms, mainly in Pocket PC's.118 weekly downloads
osGraphX is a 3D file viewer based on openscenegraph.18 weekly downloads
DuMP3 - duplicate & similar file finder
DuMP3 is a duplicate and similar file finder.17 weekly downloads
Portable VNC Server
Pvnc is a portable VNC package. It can be built with extensions (e.g. open GL). It is tested with FC-6 and FC-8 and should work with other Linux distributions such as Ubuntu.38 weekly downloads
x2y - The Easy Image Converter
An open source image editor created using the Developer's Image Library (DevIL) which allows viewing images and converting images among a variety of formats.36 weekly downloads
Simple Viewer
Small and simple viewer for Linux, support multipage images, support transparency and alpha-channel in images.9 weekly downloads
CycleAtlas is a cycling diary based on a custom road atlas. It can be used to store or to plan new rides. Functions includes generation of planimetry, profiles and route time tables of rides. A map editor is included, in order to create a custom road map12 weekly downloads
WW2D allows you to explore Earth using satellite imagery, topographic maps and image from other data sources also providing large placenames and boundaries database and allowing you to install community-made add-ons for even more info about our planet.31 weekly downloads
FlightTrack is a 2D/3D GPS track viewing software for light aviation pilots using MacOS X.28 weekly downloads
Photoshop File Format Decode Library
Libpsd is the Photoshop file format decode library in C language with full source code. It mainly decodes and blends *.psd files. It supports for Adobe Photoshop CS version, and is compatible with the previous versions of Photoshop.28 weekly downloads
Zorro's Tools for Atari/GEM
Collection of tools for Atari/GEM platform. You can find more information on my web page.7 weekly downloads
Flamebird MX BennuGD IDE
Flamebird is an IDE for the open source game development language Bennugd (http://bennugd.org/) and Fenix(http://fenix.divsite.net). Our aim is to integrate in a professional environment a powerfull code editor, a project management system and editors an6 weekly downloads


text:
Stock Vector Illustration:
Set of abstract vector New Year headers, banners. Golden pattern on the black background
- Image ID: 117906982
- Copyright: kalomirael
High quality images curated from over 150 countries.
Over 40 million photos, vectors, illustrations, and more.
Simple and flat pricing by image or by month.
Over 230,000 images downloaded every day.


text:
Declining feline health: Cats get poorer healthcare than dogs
Fight that trend and attract cat owners to your practice.
Aug 01, 2008
Hot topics on dvm360
Pol on defense as Michigan veterinary board discusses negligence charges
Controversial reality TV veterinarian calls his approach 'common sense.'
Photo gallery: The top 10 veterinary schools in America, according to U.S. News
U.S. News & World Report ranks programs for the first time since 2011.
Front Desk Disasters, Episode 3: Dude looks like a lady
Everyone's favorite receptionist is at it again. Would you handle this situation differently?
Video: Flea hideouts in the house
Parasitology expert Michael Dryden, DVM, MS, PhD, reveals prime hideouts for fleas—and gives tips to clear them out of clients' homes for good.
Veterinarians: Your clients are going to Google with these cat questions
You might be surprised by what your clients are researching. Plus, get an educational client handout.


text:
Yarawaka Atama Juku
Should Female Friend Shave
Kidou Senshi Pachindaa
How Hot Is Hot Stove
Kawa no Nushi Tsuri
Is There A God Exist?
Donkey Kongu
How Sad Is Crying Widow
Chou-Gekijou-Han Keroro Gunsou Enshuu Dayo!
Mom Dad What Are You Doing
Majime ni Fumajime Kaiketsu Zorori
Where In Earth Is Carmen San Diego?
You ask how his day went and he responds, "Fine." Or, you ask what he's up to and he says, "Nothing."
Rock legend David Bowie has changed his identity with almost every album. Can you remember all these classic Bowie characters?
Let's play HIDE YOUR DRINKIES!
Video games are serious business, but Goons save room for fun stuff, like making Mega Man fan art!
A psychic terrorist travels to the 1990s to win his true love by murdering her entire wedding party!
The Something Awful front page news tackles anything both off and on the Internet. Mostly "on" though, as we're all incredible nerds.
Copyright ©2015 Rich "Lowtax" Kyanka & Something Awful LLC.


text:
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|Hotel Berghof Amaranth Oberdorfstrasse 23, 3812 Wilderswil, Switzerland (3.8 miles from St.Beatus Höhlen)
Score from 475 reviews: Very goodThe hotel enjoys an idyllic and quiet location above Wilderswil. It offers panoramic views of the Eiger, Mönch and Jungfrau mountains and of Lake Brienz and features a heated outdoor pool. More…
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|Victoria Jungfrau Grand Hotel & Spa Höheweg 41, 3800 Interlaken, Switzerland (3.6 miles from St.Beatus Höhlen)
Score from 593 reviews: Excellent There are 4 people looking at this hotel.The Victoria Jungfrau Grand Hotel & Spa features a picturesque location in Interlaken, between 2 beautiful lakes at the foot of the eternally snow-capped Jungfrau. More…
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|Parkhotel Gunten Seestrasse 90, 3654 Gunten, Switzerland (4.3 miles from St.Beatus Höhlen)
Score from 143 reviews: Excellent There is 1 person looking at this hotel.The Parkhotel Gunten is located in a private park next to Lake Thun and offers comfortable accommodations with a wonderful view. More…
$113 Average price per night
|Gästehaus Seeblick Schachenstrasse 43, 3700 Spiez, Switzerland (4.3 miles from St.Beatus Höhlen)
Score from 167 reviews: Very goodOffering panoramic views of Lake Thun, the Gästehaus Seeblick lies surrounded by a large lawn in the village of Spiez, a 5-minute walk from the lakeshore. Free Wi-Fi is available. More…
$113 Average price per night
|Hotel Adler Dorfstrasse 36, 3655 Sigriswil, Switzerland (4.0 miles from St.Beatus Höhlen)
Score from 37 reviews: Very goodIn Sigriswil, Hotel Adler is 2 miles from Lake Thun. Its restaurant features a terrace with a view of the Swiss Alps. Rooms have a balcony and free Wi-Fi. More…
$108 Average price per night
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Our special coverage of the closest race for years
Meet Malaysia's first female mixed martial artist
30 Sep 2010 22:28 GMT | Politics, Latin America, Ecuador
World leaders have pledged their support for Rafael Correa, the president of Ecuador, after a state of emergency was called amid protests by police officers angry over plans to remove some of their benefits.Correa himself sought refuge in a hospital in Quito on Thursday after being teargassed by as he attempted to speak to offficers at a barracks in the capital. Hugo Chavez, the Venezulean president, called on other leaders in the region to rally to defend his close ally, describing the unrest as "an effort to overthrow President Correa".
"Together with the people of Latin America and the Caribbean we will be vigilant and standing in solidarity [with Correa] in this historic moment," a statement from Chavez's government said.Borders closedNeighbouring Peru has closed its border with Ecuador until the situation in the country is cleared up."I am ordering all traffic halted on the northern border until the situation is clarified and authority of President Correa restored," Alan Garcia, the Peruvian president told reporters at the government palace. He said foreign ministers from across South America would try to travel to Ecuador in a bid to resolve the conflict.Garcia's actions were echoed by Colombia, which also shares a border with Ecuador. "I spoke with [Peru's president] and the two countries decided to close the borders with Ecuador as a political sign of solidarity with President Correa and with Ecuador's democracy," Juan Manuel Santos, the Colombian president, told reporters before getting on a flight for a regional meeting in Argentina on Ecuador's unrest.Speaking to Correa before the meeting of the Organisation of American States, Miguel Insulza, the head of 35-nation body, said that the president has the "full support" of the group.The US envoy to the OAS said that Washington condemned any unconstitutional attempt to unseat the democratically elected president."The US condemns any attempt to violate the democratic process and constitutional order of Ecuador," Carmen Lomellin said. "We support the democratic government in Ecuador."The Mexican foreign ministry expressed "concern over the events today in Ecuador" and called for an effort for "dialogue and conciliation". Similar expressions of support came from leaders in Chile and Paraguay.Concern for CorreaEurope has also signalled its support for the embattled Ecuadorean president."I strongly urge all parties to refrain from violence and actions which could undermine Ecuador's constitutional order and rule of law," Catherine Ashton, the European Union foreign policy chief, was quoted as saying by her spokeswoman Maja Kocijancik.
"I call for the preservation of constitutional order in Ecuador and express my full support to Ecuador's democratically elected institutions."Ban Ki-moon, the UN secretary general, said that he was deeply concerned for the "personal welfare" of Correa."The secretary general is deeply concerned about developments today in Ecuador, including reported acts of insubordination by some members of the police and military," a spokesman for Ban said.
"The secretary general is also concerned about the physical condition and personal welfare of President Rafael Correa.""He welcomes the endeavours of the Organisation of American States and other regional actors to contribute to an early, constructive resolution."
Source: Agencies
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