Cross-reactive memory CD4+ T cells alter the CD8+ T-cell response to heterologous secondary dengue virus infections in mice in a sequence-specific manner.

Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA.
Viral immunology (Impact Factor: 1.45). 07/2009; 22(3):215-9. DOI: 10.1089/vim.2008.0089
Source: PubMed


Dengue virus is the causative agent of dengue fever and the more-severe dengue hemorrhagic fever (DHF). Human studies suggest
that the increased risk of DHF during secondary infection is due to immunopathology partially mediated by cross-reactive memory
T cells from the primary infection. To model T cell responses to sequential infections, we immunized mice with different sequences
of dengue virus serotypes and measured the frequency of peptide-specific T cells after infection. The acute response after
heterologous secondary infections was enhanced compared with the acute or memory response after primary infection. Also, the
hierarchy of epitope-specific responses was influenced by the specific sequence of infection. Adoptive-transfer experiments
showed that memory T cells responded preferentially to the secondary infection. These findings demonstrate that cross-reactive
T cells from a primary infection alter the immune response during a heterologous secondary infection.


Available from: Coreen M Beaumier
Cross-Reactive Memory CD4
T Cells Alter the CD8
T-Cell Response to Heterologous Secondary Dengue
Virus Infections in Mice in a Sequence-Specific Manner
Coreen M. Beaumier
and Alan L. Rothman
Secondary dengue virus (DENV) infection is a major factor contributing to the risk for severe disease, an effect
that depends upon the sequence of infection with different DENV serotypes. We previously reported sequence-
dependent effects of secondary DENV infection on CD8
T-cell responses in mice. To further evaluate the effect
of infection sequence, we analyzed DENV-specific CD4
T-cell responses and their relationship to the CD8
T-cell response. Serotype cross-reactivity of CD4
T-cell responses also depended upon the sequence of sero-
types in this model. Furthermore, adoptive transfer of memory CD4
T cells altered the response of memory
T cells to secondary infection. These data demonstrate the interaction of different components of the T-cell
response in determining the immunological outcome of secondary DENV infection.
engue virus (DENV) is a mosquito-borne flavivirus
and the causative agent of dengue fever (DF) and den-
gue hemorrhagic fever (DHF). There are four immunologi-
cally and antigenically distinct serotypes of DENV: DENV-1,
DENV-2, DENV-3, and DENV-4. There is a significantly
higher risk of DHF during heterologous secondary DENV
infections (5,12). Dengue disease severity also depends on the
sequence of infection by the serotypes (1,4,12). Immuno-
pathological mechanisms have been proposed to explain the
enhanced risk for disease during secondary infection, in-
cluding a role for cross-reactive memory T lymphocytes.
To investigate the immune responses to secondary DENV
infections in a model amenable to experimental manipula-
tion, we infected Balb=c mice sequentially with heterologous
DENV serotypes and measured T-cell cytokine responses (3).
In this model, secondary infections enhanced peptide-specific
T-cell responses due to activation of serotype cross-
reactive memory T-cells. We showed differences in epitope-
specific immune responses depending on infection sequence.
We utilized this mouse model to further study the mecha-
nisms that affect the response to different sequences of
DENV serotypes.
Among the sequences tested, we observed that DENV-2-
immune mice subsequently challenged with DENV-1 showed
a marked increase in the immune response to peptide D1=3
NS3 (GYISTRVGM), the variant in DENV-1 and DENV-3 of
the immunodominant H-2K
-restricted epitope in the NS3
protein (11). In contrast, there was no increase in response to
this peptide in DENV-2-immune mice rechallenged with
DENV-3. This phenomenon was of interest since the amino
acid sequence for this peptide was the same for both DENV-1
and DENV-3. Both DENV-1 and DENV-3 induced compara-
bly low CD8
T-cell responses to this epitope in primary
infection. We therefore hypothesized that the differential re-
sponse in secondary DENV-1 versus DENV-3 infections in
DENV-2-immune mice reflected differences in the CD4
T-cell response. CD4
T-cell help has been shown to be es-
sential for the generation of effective CD8
T-cell memory
To assess serotype-dependent differences in the CD4
T-cell response, we examined the magnitude of this response
to primary and secondary DENV infections. Balb=c mice 4–6
wk of age ( Jackson Laboratories, Bar Harbor, ME) were
immunized with 210
pfu IP of DENV-1 (strain Hawaii),
DENV-2 (strain New Guinea C), or DENV-3 (strain CH53489).
For secondary infection, DENV-2-immune mice were chal-
lenged 28 d after the primary infection with 210
pfu IP of
DENV-1 or DENV-3. Nine days post primary or secondary
infection, the mice were sacrificed and splenectomized and
single cell splenocyte suspensions were made. Kinetics of the
Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, Worcester, Massachusetts.
Author’s current address: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes
of Health, Bethesda, Maryland.
Volume 22, Number 3, 2009
ª Mary Ann Liebert, Inc.
Pp. 215–219
DOI: 10.1089=vim.2008.0089
Page 1
FIG. 1. Dengue-specific CD4
T-cell responses in primary and secondary dengue virus (DENV) infections. (A and B)
Intracellular IFN-g staining was performed on splenocytes from mice that were infected with primary DENV-1, primary
DENV-2, primary DENV-3, or primary DENV-2 followed by secondary DENV-1 or DENV-3. CD4
T-cell responses to whole
virus antigens were measured. The frequencies from cells stimulated with control Vero cell antigen were subtracted from those
stimulated with viral antigen. (A ) Representative IFN-g staining in response to inactivated DENV antigens. The bottom
number in each panel represents the frequency of the IFN-g response with the media background subtracted, and the top
number represents the frequency of the IFN-g response with the Vero antigen control subtracted. (B) Comparison of CD4
T-cell responses in primary versus secondary dengue virus infections. Bars show medians and 95% confidence intervals. P
values were calculated by the Mann-Whitney test. Asterisks denote responses with significant differences between immuni-
zation groups by ANOVA ( p < 0.05); post-hoc testing showed that mice with secondary DENV-1 infection had significantly
higher responses than the primary DENV-1, DENV-2, or DENV-3 infection groups for all antigens, and significantly higher
responses than the secondary DENV-3 infection group for DENV-2 antigen. These data were generated in three individual
experiments (n ¼ 8 mice per group). (CF) Bulk culture cell lines were generated from splenocytes from mice that were
administered DENV-2 28 d earlier. Cells were stimulated with inactivated DENV-1 (circles) or DENV-3 (diamonds) antigens.
Day 7 (C and D) and day 14 (E and F) post-stimulation, cells were tested for IFN-g production to whole virus antigen for all
four serotypes by intracellular cytokine staining. (C) Representative intracellular cytokine staining of bulk culture CD4 T cells
on day 7. The bottom number in each panel represents the frequency of the IFN-g response with the media background
subtracted, and the top number represents the frequency of the IFN-g response with the Vero antigen control subtracted. (D)
Cross-reactivity of memory CD4 T-cell responses from DENV-2–immune mice on day 7. The median responses are re-
presented by the black bars. (E) Representative intracellular cytokine staining of bulk culture CD4 T cells on day 14. The
bottom number in each panel represents the frequency of the IFN-g response with the media background subtracted, and the
top number represents the frequency of the IFN-g response with the Vero antigen control subtracted. (F) Cross-reactivity of
memory CD4 T-cell responses from DENV-2–immune mice on day 14. The median responses are represented by the black
bars. P values were calculated by the Mann-Whitney test (N ¼ 4 individual cultures from individual animals per group for D;
n ¼ 3 individual cultures from individual animals per group for F).
Page 2
T-cell responses were previously optimized (3). In addition,
homologous rechallenge was also previously found to result
in no change in the T-cell response, presumably due to neu-
tralizing antibodies (3).
We measured the frequency of antigen-specific IFN-g
T cells using intracellular cytokine staining (8). Al-
though Roehrig et al. reported several candidate helper T-cell
epitopes on the DENV E protein (10), we did not find any
significant and reproducible responses to overlapping peptides
corresponding to the E protein. Therefore, we tested the CD4
T-cell response to whole virus antigens using glutaraldehyde-
fixed DENV-infected Vero cell lysates corresponding to each
serotype, as previously described (8). Splenocytes (510
were incubated overnight with a 1:20 dilution of DENV or
control antigen, PMA=ionomycin, or media alone with
5 mL=mL brefeldin A (GolgiPlug; BD Bioscience) at 378C. Cells
were permeabilized and stained with anti-mouse CD3e anti-
mouse CD4a-FITC, and anti-mouse IFN-g-APC. Data were
acquired on a FACSCalibur in the UMMS Flow Cytometry
Core. A small lymphocyte gate was drawn on forward and
side-scatter low populations and further gated on CD3
DENV-specific CD4
T-cell IFN-g responses were compa-
rably low during primary infection regardless of the infecting
serotype (Fig. 1A–B). Responses in mice that received DENV-
3 after DENV-2 were slightly higher (0–6.9% of CD4
T cells),
whereas the highest responses (1.9–10.0%) were observed in
mice that received DENV-1 after DENV-2 (Fig. 1A). In all
groups, stimulation with DENV-4 and DENV-3 antigens in-
duced the highest percentages of IFN-g-producing CD4
FIG. 2. Transfer of memory CD4
T cells affects the CD8
T-cell response to secondary dengue virus (DENV) infections.
and CD8
T cells were isolated from DENV-2-immune mice. The CD8
T cells alone or in combination with the CD4
T cells were transferred IV to naı
ve mice. The following day, the mice were administered either DENV-1 or DENV-3.
Splenocytes were harvested on day 9 post-infection. IFN-g responses to CD8
T-cell epitopes D1=3 NS3 and D2=4 NS3 were
measured by intracellular cytokine staining. (A ) Representative IFN-g staining in response to peptides D1 =3 NS3 and D2=4
NS3. The number in each panel represents the frequency of the IFN-g response with the media background subtracted. (B)
Comparison of CD8
T-cell responses after adoptive transfer of memory CD8
cells alone or both memory CD4
and CD8
cells. Symbols show individual mice; medians are represented by the horizontal bars. P values <0.1 calculated by the Mann-
Whitney test are shown. P values <0.05 are considered significant. These data were generated from three individual ex-
periments (n ¼ 6 per group).
Page 3
cells. IFN-g responses to DENV-1 or DENV-2 antigens were
not detectable above background after primary DENV infec-
tion. A preferential IFN-g response to heterologous serotypes
was observed for human DENV epitope-specific CD8
T cells
(2); however, while the comparison of murine CD4
responses to each antigen across immunization groups is
valid, we interpret any comparison of responses to antigens of
different serotypes with caution, as these experiments used
crude cell lysates and we were unable to standardize the
content of the relevant DENV antigens. With secondary
DENV-1 infection, however, there was a marked increase in
response to all four DENV serotypes. The difference in the
frequency of IFN-g
T cells in response to DENV-2 antigen
stimulation was statistically significant; this finding is notable
since the primary infection was DENV-2. These results dem-
onstrate a differential CD4
T-cell response between sec-
ondary DENV-1 and secondary DENV-3, especially to the
DENV-2 antigen.
A potential explanation for the enhanced DENV-2–reactive
T-cell response in secondary DENV-1 infection com-
pared to secondary DENV-3 infection was that greater cross-
reactive proliferation of DENV-2–reactive CD4
T cells would
occur in response to DENV-1 than to DENV-3. To test this
hypothesis, bulk culture lines were generated from splenocytes
of DENV-2–immune mice in the memory phase (day 28). To
mimic secondary DENV infection, splenocytes (310
) were
stimulated with DENV-1 or DENV-3 antigen at 1:100 dilution
in RPMI medium supplemented with 10% FBS and 50 U=mL
rmIL-2 (BD Bioscience). The cells were assayed for DENV-
specific IFN-g production on days 7 (Fig. 1C and D) and 14 (Fig.
1E and F) post-stimulation by intracellular cytokine staining.
Cell lines showed the highest responses to DENV-2 antigen
(Fig. 1 D and F). In addition, the frequency of DENV-specific
T cells was higher in DENV-1 antigen-stimulated
cultures than in DENV-3 antigen-stimulated cultures; this
difference was statistically significant on day 14 of culture.
These results suggest that DENV-2-specific memory CD4
cells are more cross-reactive to DENV-1 than DENV-3. Sur-
prisingly, by day 14, there was minimal IFN-g response to
DENV-1 and DENV-3 antigens, which were used for stimu-
lation of the lines. This suggests that the responding cells had
higher avidity for DENV-2 than other serotypes, even those
used for in vitro expansion. Fig. 1 shows a preferential expan-
sion of DENV-2-specific memory T cells by a heterologous
DENV-1 stimulus in vivo and in vitro, suggesting original an-
tigenic sin.
Lastly, we hypothesized that memory CD4
T cells gen-
erated from primary DENV-2 infection that are cross-reactive
with DENV-1 and=or DENV-3 provide help for CD8
T cells
upon secondary infection. Therefore, we next examined
whether adoptive transfer of memory CD4
T cells affected
the CD8
T-cell response to secondary infection. CD4
T cells were isolated (*90% purity) from DENV-2-im-
mune splenocytes by negative selection using magnetic beads
(Miltenyi Biotec, Gladbach, Germany). We injected combi-
nations of CD8
) and CD4
) T cells from
DENV-2-immune mice IV in 100 mL into naı
ve mice. The mice
were infected the following day with 210
pfu IP of DENV-1
or DENV-3. Nine days later, the mice were sacrificed and
T-cell IFN-g responses to the D1=3 NS3 and D2=4 NS3
(GYISTRVEM) peptides were measured by intracellular cy-
tokine staining. Cells were stimulated for 5 h at 378C with
10 mg=mL peptide. Mice that received both memory CD4
and CD8
T cells and were then challenged with either
DENV-1 or DENV-3 infection showed a higher CD8
response to the D2=4 NS3 peptide than mice that received
only CD8
T cells prior to challenge (Fig. 2). The transfer of
both memory CD4
and CD8
T cells also yielded a modest,
although not statistically significant, increase in the CD8
T-cell response to the D1=3 NS3 peptide in mice challenged
with DENV-1, but not in mice challenged with DENV-3.
These data indicate that the transfer of memory CD4
T cells
augmented the DENV-specific memory CD8
T-cell response
to a subsequent heterologous DENV challenge.
Our data parallel the epidemiologic observation that the
DENV serotype infection sequence (serotype and=or strain)
influences disease outcome (1,4,13), and identify one poten-
tial mechanism for this effect. Upon secondary DENV in-
fection, the cross-reactive DENV-specific CD4
memory cells
are stimulated by antigen from the secondary infection.
These CD4
T cells then augment the response of memory
T cells. In patients, this could result in an increase in
the production of inflammatory cytokines and an increased
risk for severe disease. The effect of CD4
T cells on the
T-cell response in DENV infections has not been ad-
dressed in clinical studies; therefore the relationship between
this mouse model and DENV infection of humans remains
speculative. There may also be other mechanisms that influ-
ence the memory CD8
T-cell response. However, it is known
that CD4
T cells have an important role in the priming of
memory CD8
T cells (6,7,9,14). This mechanism could also
be applicable to other diseases such as HIV and influenza.
This work was supported by the National Institutes of
Health grants U19 AI57319 and P30 DK032520. Its content
are solely the responsibility of the authors and do not neces-
sarily represent the official views of the NIH.
We thank Jurand Janus for the propagation of viruses
and preparation of antigens, and Dr. Sharone Green and
Dr. Anuja Mathew for their advice and guidance.
Author Disclosure Statement
The authors state that no competing financial interests exist.
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Address reprint requests to:
Dr. Alan L. Rothman
University of Massachusetts Medical School
55 Lake Avenue North
Room S6-862
Worcester, Massachusetts 01655
Received November 6, 2008; accepted February 2, 2009.
Page 5
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  • Source
    • "Some of the above findings have been replicated in mouse models of DENV infection. Sequential infection of immunocompetent mice with different DENV serotypes has recapitulated skewing of the DENV-specific T cell response [48, 49]. Additional studies have shown differences in the ability of epitope-specific T cells to clear antigen-presenting cells in vivo [50]. "
    [Show abstract] [Hide abstract] ABSTRACT: Dengue viruses (DENV) are mosquito-borne viruses that cause significant morbidity. The existence of four serotypes of DENV with partial immunologic cross-reactivity creates the opportunity for individuals to experience multiple acute DENV infections over the course of their lifetimes. Research over the past several years has revealed complex interactions between DENV and the human innate and adaptive immune systems that can have either beneficial or detrimental influences on the outcome of infection. Further studies that seek to distinguish protective from pathological immune responses in the context of natural DENV infection as well as clinical trials of candidate DENV vaccines have an important place in efforts to control the global impact of this re-emerging viral disease.
    Full-text · Dataset · Dec 2013
  • Source
    • "Severe dengue virus infections occur when a host immune to one serotype contracts an infection with another serotype. The resultant pathology, in the form of dengue hemorrhagic fever and shock syndrome, has been proposed to involve cross-reactive memory T cells that may be of high affinity to the first virus but cross-reactive at low affinity to the second virus infection [59,60]. A highly focused response to a CD8 cross-reactive response between IAV and hepatitis C virus (HCV) [61] in acutely infected HCV patients was associated with severe fulminant hepa- titis [62] . "
    [Show abstract] [Hide abstract] ABSTRACT: Recent epidemiological studies have shown that, in addition to disease-specific effects, vaccines against infectious diseases have nonspecific effects on the ability of the immune system to handle other pathogens. For instance, in randomized trials tuberculosis and measles vaccines are associated with a substantial reduction in overall child mortality, which cannot be explained by prevention of the target disease. New research suggests that the nonspecific effects of vaccines are related to cross-reactivity of the adaptive immune system with unrelated pathogens, and to training of the innate immune system through epigenetic reprogramming. Hence, epidemiological findings are backed by immunological data. This generates a new understanding of the immune system and about how it can be modulated by vaccines to impact the general resistance to disease.
    Full-text · Article · May 2013 · Trends in Immunology
  • Source
    • "DENV is divided up into four distinct serotypes (DENV 1-4) (Halstead, 1989; Morens, 1994) which serologically cross-react but do not provide neutralizing antibody. DENV has also been shown to encode variable CD8 T-cell epitopes that cross-react between the sero-types (Spaulding et al., 1999; Zivny et al., 1999; Mongkolsapaya et al., 2003; Bashyam et al., 2006; Beaumier et al., 2008). DENV infection can cause a wide array of disease presentations ranging from asymptomatic upward to dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. "
    [Show abstract] [Hide abstract] ABSTRACT: Heterologous immunity is a mechanism by which immunological memory within an individual, developed in response to a previous infection, plays a role in the immune response to a subsequent unrelated infection. In murine studies, heterologous immunity facilitated by cross-reactive CD8 T-cell responses can mediate either beneficial (protective immunity) or detrimental effects (e.g. enhanced lung and adipose immunopathology and enhanced viral titers) (Selin et al., 1998; Chen et al., 2001; Welsh and Selin, 2002; Nie et al., 2010; Welsh et al., 2010). Protective heterologous immunity results in enhanced clearance of virus during a subsequent infection with an unrelated pathogen. Such is the case when mice are immunized with lymphocytic choriomeningitis virus (LCMV) and subsequently challenged with Pichinde virus (PV) or vaccinia virus (VACV) (Selin et al., 1998). However, heterologous immunity may also mediate enhanced immunopathology as mice immunized with influenza A virus (IAV) and challenged with LCMV show increased viral titers and enhanced lung immunopathology (Chen et al., 2003). The role heterologous immunity plays during infection is not limited to the murine system. In fact, there have now been several reports of enhanced immunopathology due to heterologous immunity during human infections, involving viruses such as IAV, Epstein-Barr Virus (EBV), hepatitis C virus (HCV), and dengue virus (DENV) (Mathew et al., 1998; Wedemeyer et al., 2001; Acierno et al., 2003; Nilges et al., 2003; Clute et al., 2005; Urbani et al., 2005). Interestingly, in all reported cases in humans, heterologous immunity mediated enhanced immunopathology. Upon infection with EBV the clinical presentation can range from asymptomatic to severe, occasionally fatal, acute infectious mononucleosis (AIM) (Crawford et al., 2006b; Luzuriaga and Sullivan, 2010) which is marked by a massive CD8 lymphocytosis. This lympho-proliferative effect in AIM was shown to be partially mediated by reactivation of cross-reactive IAV-M1 58-66 (IAV-GIL) specific CD8 memory T-cells in HLA-A2 patients reacting to the EBV-BMLF1 280 (EBV-GLC) epitope (Clute et al., 2005). Interestingly, EBV infects ~90% of individuals globally by the third decade of life, establishing a life-long infection (Henle et al., 1969). However, it is unknown why 5-10% of adults remain EBV-sero-negative (EBV-SN), despite the fact that the virus infects the vast majority of the population and is actively shed at high titers even during chronic infection (Hadinoto et al., 2009). Here, we show that EBV-SN HLA-A2+ adults possess cross-reactive IAV-GIL/EBV-GLC memory CD8 T-cells that show highly unique properties. These IAV-GIL cross-reactive memory CD8 T-cells preferentially expand and produce cytokines to EBV antigens at high functional avidity. Additionally, they are capable of lysing EBV-infected targets and show the potential to enter the mucosal epithelial tissue, where infection is thought to initiate, by CD103 expression. This protective capacity of these cross-reactive memory CD8 T-cells may be explained by a unique T-cell receptor (TCR) repertoire that differs by both organization and CDR3 usage from that in EBV-seropositive (EBV-SP) donors. The composition of the CD8 T-cell repertoire is a dynamic process that begins during the stochastic positive selection of the T-cell pool during development in the thymus. Thus, upon egress to the periphery a naïve T-cell pool, or repertoire, is formed that is variable even between genetically identical individuals. This T-cell repertoire is not static, as each new infection leaves its mark on the repertoire once again by stochastically selecting and expanding best-fit effectors and memory populations to battle each new infection while at the same time deleting older memory CD8 T-cells to make room for the new memory cells (Selin et al., 1999). These events induce an altered repertoire that is unique to each individual at each infection. It is this dynamic and variable organization of the T-cell repertoire that leads to private specificity even between genetically identical individuals upon infection with the same pathogens and thus a different fate (Kim et al., 2005; Cornberg et al., 2006a; Nie et al., 2010). It is this private specificity of the TCR repertoire that helps explain why individuals with the same epitope specific cross-reactive response, but composed of different cross-reactive T-cell clones, can either develop AIM or never become infected with EBV. Our results suggest that heterologous immunity may protect EBV-SN adults against the establishment of productive EBV infection, and potentially be the first demonstration of protective T-cell heterologous immunity between unrelated pathogens in humans. Our results also suggest that CD8 T-cell immunity can be sterilizing and that an individual’s TCR repertoire ultimately determines their fate during infection. To conclusively show that heterologous immunity is actively protecting EBV-SN adults from the establishment of a productive EBV infection, one would have to deliberately expose an individual to the virus. Clearly, this is not an acceptable risk, and it could endanger the health of an individual. A humanized mouse model could allow one to address this question. However, before we can even attempt to address the question of heterologous immunity mediating protection from EBV infection in humanized mice, we must first determine whether these mice can be infected with, and build an immune response to the two viruses we are studying, EBV and IAV. We show here that these mice can indeed be infected with and also mount an immune response to EBV. Additionally, these mice can also be infected with IAV. However, at this time the immune responses that are made to these viruses in our established humanized mouse model are not substantial enough to fully mimic a human immune response capable of testing our hypothesis of heterologous immunity mediating protection from EBV infection. Although the immune response in these mice to EBV and IAV infection is not suitable for the testing of our model the data are promising, as the humanized mouse model is constantly improving. Hopefully, with constant improvements being made there will be a model that will duplicate a human immune system in its entirety. This thesis will be divided into 5 major chapters. The first chapter will provide an introduction to both general T-cell biology and also to the role of heterologous immunity in viral infection. The second chapter will provide the details of the experimental procedures that were performed to test our hypothesis. The third chapter will describe the main scientific investigation of the role of heterologous immunity in providing natural resistance to infection in human subjects. This chapter will also consist of the data that will be compiled into a manuscript for publication in a peer-reviewed journal. The fourth chapter will consist of work performed pertaining to the establishment of a humanized mouse model of EBV and IAV infection. The establishment of this model is important for us to be able to show causation for protection from EBV infection mediated by heterologous immunity.
    Preview · Article · Mar 2012
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