INTRODUCTIONEndometriosis is a benign, chronic inflammatory gynecologicaldisorder defined by the presence of tissue implants resemblingendometrial glands and stroma outside of the uterus (Bulun, 2009).The ectopic implants are found most commonly on the ovariesand on the visceral and peritoneal surfaces within the pelvis. Asmany as 10% of women aged 30-40 can be affected, although manymore can have asymptomatic disease (Baldi et al., 2008).

The ectopic lesions often develop as invasive neoplasms andincreasing evidence suggests that endometriosis might be aprecursor of ovarian epithelial tumors (Brinton et al., 1997;Kobayashi et al., 2007; Melin et al., 2006; Nagle et al., 2008; Olsonet al., 2002; Somigliana et al., 2006). Prevention strategies forovarian cancer, the most lethal gynecologic malignancy, are aresearch priority, and early identification and treatment ofprecursor lesions is essential for long-term survival. Currenttherapy for endometriosis includes surgical and medical approachesaimed at eliminating hormonal imbalances, restoring fertility,

reducing the extent of implants and providing pain relief (Baldi etal., 2008; Bulun, 2009). Despite treatment, endometriosis oftenrecurs, leading to renewed pain and infertility. New and improvedtherapeutic approaches are needed for endometriosis, and theadjuvant potential of immune therapies can be considered.

The pathophysiology of endometriosis is poorly understood.Although the triggering molecular events are yet to be delineated,it has been proposed that impaired immune surveillance (Dunn etal., 2002) in the host may contribute actively to the pathogenesisof endometriosis (Bergqvist et al., 2001; Lebovic et al., 2001;Santanam et al., 2002). Whereas innate immunity has beenaddressed repeatedly (Maeda et al., 2002; Montagna et al., 2008;Tariverdian et al., 2007; Yamamoto et al., 2008), there have beenfew studies of the role of adaptive, antigen-specific immunity inthis disease (Antsiferova et al., 2005; Podgaec et al., 2007; Szyllo etal., 2003). Given the potential for the occurrence of ovarian cancerin a subset of endometriosis patients (Brinton et al., 1997; Kobayashiet al., 2007; Melin et al., 2007; Melin et al., 2006; Nagle et al., 2008;Ogawa et al., 2000; Olson et al., 2002; Prowse et al., 2006; Somiglianaet al., 2006; Varma et al., 2004; Yoshikawa et al., 2000), the besttarget antigens to study are those that are present in bothendometriosis and ovarian epithelial tumors. Several ovariantumor-associated antigens have been defined and are currentlybeing tested as antigen-specific immune therapies in ovariancancer. One such antigen is mucin 1 (MUC1) (reviewed by Vladet al., 2004), which is normally expressed by glandular and luminalepithelial cells of the human endometrium, and is present only inlow levels on the ovarian surface epithelium (Brayman et al., 2004).MUC1 is overexpressed in all subtypes of ovarian epithelial tumors,

RESEARCH ARTICLE

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Disease Models & Mechanisms 2, 593-603 (2009) doi:10.1242/dmm.002535Published by The Company of Biologists 2009

A conditional mouse model for human MUC1-positiveendometriosis shows the presence of anti-MUC1antibodies and Foxp3+ regulatory T cellsRaluca A. Budiu1, Iulia Diaconu2,4, Rachel Chrissluis2, Anica Dricu3,5, Robert P. Edwards1,2 and Anda M. Vlad1,2,*

SUMMARY

Endometriosis is defined by the presence of tissue implants resembling endometrial glands outside of the uterus, at ectopic sites, frequently on theovarian surface. The ectopic lesions are often invasive, resistant to therapy, and may predispose to endometrioid and clear cell ovarian tumors. Thecomplex mechanisms leading to chronic endometriosis are mediated partly by impaired immune surveillance in the host. Although innate immunityhas been addressed previously, the response of adaptive immune effectors to specific antigens has not been characterized, mostly because veryfew endometriosis antigens have been defined to date. We postulated that the mucin 1 (MUC1) glycoprotein, which is normally present on eutopichuman endometrial glands and overexpressed in endometrioid and clear cell ovarian tumors, is also present in ectopic lesions of ovarian endometriosis.Furthermore, changes in MUC1 expression in endometriosis could promote adaptive anti-MUC1 immunity that might play a role in the malignantprogression. To test our hypothesis, we crossed MUC1 transgenic mice, which express human MUC1 under the endogenous promoter, with theloxP-Stop-loxP-KrasG12D/+ (Kras) mice, in which endometriosis can be induced through Cre-loxP recombination. The double transgenic MUC1Krasmice develop benign, MUC1-positive ovarian lesions, closely resembling human endometriosis. Subsequent to disease induction, the mice generatehigh titers of IgM and IgG antibodies that are specific for MUC1. Antibodies appear early in disease and the predominance of the IgG1 subclasssuggests Th2-driven immunity. Immune phenotyping revealed an accumulation of Foxp3+ CD4 regulatory T cells (Tregs) in the draining lymphnodes at late-stage disease. Furthermore, our observations in human endometriosis showed a similar recruitment of FOXP3+ CD4 T cells. Overall,our results reveal a Th2/Treg-dominant natural immunity in endometriosis with potential implications for cancer progression.

1Department of Obstetrics, Gynecology and Reproductive Sciences, University ofPittsburgh School of Medicine and Magee Womens Research Institute, Pittsburgh,PA 15213, USA2Department of Immunology, University of Pittsburgh School of Medicine,Pittsburgh, PA 15261, USA3Karolinska Cancer Center, Department of Oncology-Pathology and KarolinskaUniversity Hospital, 171 76 Solna, Stockholm, Sweden4Present address: Cancer Gene Therapy Group, Finnish Institute for MolecularMedicine, University of Helsinki, Haartmaninkatu 8, 00290 Helsinki, Finland5Present address: Department of Biochemistry, University of Medicine andPharmacy, Bd 1 Mai, nr 62, 200322 Craiova, Romania*Author for correspondence (e-mail: vladam@upmc.edu)

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and spontaneous or MUC1 vaccine-induced immunity has beenstudied extensively in cancer patients (Cramer et al., 2005; Oei etal., 2008; Terry et al., 2007; von Mensdorff-Pouilly et al., 2000). Bycontrast, much less is known about changes in MUC1 expressionand its immunogenic properties in precursor lesions such asendometriosis.

The progression of normal tissue to premalignant and malignantlesions is often a lengthy process leading to complex molecularchanges. Whether, and how, these changes are recognized by theadaptive immune system needs to be explored.

This question, which is difficult to address in humans, requiresthe development of adequate preclinical models. Only a few animalmodels for endometriosis exist currently, such as immune-deficientanimals that have been exposed to transplanted xenogeneic humanendometriotic lesions (Awwad et al., 1999; Grummer et al., 2001;Ozawa et al., 2006), and they are not suitable for studies ofimmunoregulation. Dinulescu et al. (Dinulescu et al., 2005)engineered the first conditional murine model of endometriosisbased on the Cre-lox technology (Sauer, 1998). The loxP-Stop-loxP-KrasG12D/+ mice (LSL-Kras, referred to herein as Kras mice) developde novo, benign ovarian endometriosis-like lesions followinginjection, to the ovarian bursa, of Cre-encoding adenovirus(AdCre), which mediates DNA recombination and activation of themutated, floxed, oncogenic KrasG12D/+ allele (Dinulescu et al.,2005). The mice show lesions that are similar to humanendometriosis, although they do not express any knownendometriosis-associated epithelial antigens. The mouse Muc1

homolog (designated ‘Muc1’ to distinguish it from human MUC1)shares little homology with human MUC1 (Spicer et al., 1991) andis not expected to show the same immunogenicity as the humanmolecule. To explore MUC1 immunobiology in endometriosis, weintroduced the human molecule into the Kras model. The newlygenerated MUC1+/–loxP-Stop-loxP-KrasG12D/+ mice (for brevity,MUC1Kras mice) allowed us to make important observationsregarding MUC1 expression and MUC1-specific immunesurveillance during the development of endometriosis-like lesions.Our combined data from mice and humans identify MUC1 as amarker of glandular epithelia in endometriosis; show increasinglevels of anti-MUC1 antibodies early in the development of lesions;and show an expansion of immune suppressive T cells late inchronic disease. These findings may help explain the role of theadaptive immune system in the pathogenesis of endometriosis andits relationship to cancer.

RESULTSMUC1 expression in human endometriosisIn the human uterus (Fig. 1A), MUC1 glycoprotein is normallypresent on the apical pole of epithelial cells lining the lumen of eutopicendometrial glands (Fig. 1B). MUC1 is also expressed by the epithelialcells lining the endometrial lumen (not shown). All glandular andlumenal cells in the uterus are positive for cytokeratin 7 (Cyk7) (Fig.1C), an intermediate filament protein that is important for theproliferation of the endometrial glands during decidualization anda marker for identification of the ectopic endometrium (Norwitz et

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Fig. 1. Distribution of human MUC1 in thenormal human uterus and ovaries, and inovarian endometriosis. (A-C)Hematoxylin andeosin (H&E) staining of healthy humanendometrium shows glandular epithelia (A) thatare positive for MUC1 (B) and Cyk7 (C). Ovariesharboring ectopic lesions of endometriosis (D)show MUC1-positive glands (E) that resemblethose found in the human uterus. Glandularepithelial cells in human ovarian endometriosisare MUC1 positive (E), Cyk7 positive (F), and aresurrounded by stroma and fibrous tissue (F,arrowheads). In the normal ovary (G), MUC1 isexpressed only at low levels on the ovarian surfaceepithelia (OSE) (G, arrows). Bars, 40m. (H)MUC1transcripts quantitated by qRT-PCR: mRNA wasextracted from ovarian tissue with confirmedovarian endometriosis (E1-E9, n9), benignovarian cysts (C1-C6, n6), or from normal ovaries(N1-N3, n3). All results were normalized toGAPDH and presented as the percentage changefrom N, the average 2–Ct from the three normalovaries (see Methods), which was rendered as100%. The results shown are from one of threeexperiments with highly consistent results.

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al., 1991). Ovarian endometriosis is defined by the presence in theovaries of infiltrating glandular lesions resembling uterine glands andstroma (Fig. 1D). Similar to the uterine (eutopic) glands, the ectopic(endometriotic) glands are positive for MUC1 (Fig. 1E) and Cyk7(Fig. 1F) and are accompanied by focal, periglandular endometrioticstroma and fibrous tissue (Fig. 1F, arrowheads). This is in contrastto normal ovaries, where the only cells that express MUC1, albeit atlow levels, are the ovarian surface epithelial (OSE) cells (Fig. 1G).We confirmed the increase in MUC1 in nine cases of humanendometriosis by quantitative real-time polymerase chain reaction(qRT-PCR). All ovarian endometriosis specimens (E1-E9) showedsignificant increases in MUC1 gene transcripts (Fig. 1H) whencompared with normal ovaries from women undergoing prophylacticoophorectomies (N1-N3). By contrast, only two out of six cases ofbenign physiological ovarian cysts showed an upregulation of theMUC1 gene. We acknowledge that, in the absence of cell-specificRNA extraction, some of the variability seen may have been becauseof the tissue sampling for mRNA extraction. Nevertheless, allendometriosis specimens showed increased levels of MUC1,suggesting that MUC1 could be a marker for epithelial cells inendometriosis lesions.

Anatomical distribution of MUC1 in the gynecological tract ofhealthy MUC1Kras miceDinulescu et al. (Dinulescu et al., 2005) previously reported thatactivation of the oncogenic KrasG12D mutation in the OSE triggersendometriosis-like ovarian lesions. In order to create a model forthe human MUC1 antigen in endometriosis, we crossed the MUC1mice, which express the human protein under the endogenouspromoter, with the Kras mice, in which endometriosis can beinduced. The double transgenic MUC1Kras female mice from eachlitter were screened by PCR and selected based on the heterozygouspresence of both the MUC1 transgene and the mutant LSL-KrasG12D/+

allele (supplementary material Fig. S1). All double transgenic micedevelop and breed normally and, if unexposed to Cre recombinase,remain healthy throughout their life. The distribution of humanMUC1 protein throughout the gynecological tract of healthyMUC1Kras female mice (Fig. 2A-D) closely resembles thedistribution in humans (Fig. 1) (Chambers et al., 1994; Rowse et al.,1998). MUC1 expression in the mouse oviduct (Fig. 2A) is similar

to that seen in the human fallopian tubes (Brayman et al., 2004). Inthe eutopic endometrium of MUC1Kras mice (Fig. 2B,D), MUC1 ispresent on glandular and luminal epithelia, and is confined largelyto the apical surface, the facing lumen of the glandular ducts (Fig.2B), and the endometrial lumen (Fig. 2D). As expected, the ovaries(Fig. 2E) and endometrium (Fig. 2F) from control KrasG12D/+ micestain negatively for human MUC1. These results illustrate that thetissue distribution of the MUC1 antigen in the gynecological tractof healthy MUC1Kras mice is similar to that seen in MUC1 mice(supplementary material Fig. S2) (Rowse et al., 1998) and in humantissues (Fig. 1B,G), and endorse the suitability of these mice as a modelfor studying natural or vaccine-induced MUC1-specific immunityin endometriosis and other diseases of the gynecological tract. Wenote, however, that the normal OSE cells in both MUC1 mice(supplementary material Fig. S2B) and healthy MUC1Kras mice (Fig.2E) express more MUC1 than the human OSE cells, which showlower, yet detectable, MUC1 levels (Fig. 1G).

Injection of recombinant adenovirus in the ovarian bursa ofMUC1Kras mice leads to effective infection of the OSEIn mice, a bursa (capsule) encloses each ovary, separating it fromthe abdominal cavity. Kras activation in LSL-KrasG12D/+ (Kras) mice(Dinulescu et al., 2005) requires targeted expression of Crerecombinase in the OSE through a single injection of AdCre underthe ovarian bursa. Unlike Kras mice, the OSE cells in MUC1Krasmice express MUC1, a heavily sialylated glycoprotein that canpotentially interfere with the ability of the recombinant virus toenter the target cells (Arcasoy et al., 1997). We first confirmed thepotential of the adenovirus to infect the surface monolayer ofovarian epithelial cells in these MUC1Kras mice. Intrabursalinjection of an adenovirus encoding the lacZ reporter gene(AdLacZ) shows that the virus can successfully infect the MUC1-expressing OSE cells, resulting in blue staining of the surface of theinjected ovary upon exposure to X-gal substrate (Fig. 3A).Contralateral uninjected (control) ovaries remained stain-free (Fig.3B). Furthermore, the efficacy of infection is similar to that seenin single transgenic MUC1 (Fig. 3C) and Kras mice (Fig. 3D). Thestaining is detectable in the ovaries only, and not in adjacent tissuessuch as the uterus and/or oviducts (data not shown). Mostimportantly, the lacZ staining is confined to the OSE cell monolayer

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Fig. 2. Human MUC1 expression in thegynecological tract of healthy MUC1Kras femalemice. (A)MUC1 is detected on the ovarian (Ov)surface epithelia (OSE) and in the oviduct (Od). Aclose-up of the OSE (box in A) is shown in panel C.MUC1 is also expressed on the glandular and luminaluterine (Ut) epithelia (B and D, respectively). Healthyovaries (E) and uterus (F) from uninjected Kras micedo not express MUC1 and were used as negativecontrols for MUC1 staining. Sections of paraffin-embedded tissues (4m thick) were stained forMUC1 by immunohistochemistry using an anti-human MUC1 antibody. The positive MUC1 stainingis brown. Counterstaining with Mayer’s hematoxylinreveals the nuclei (blue). Bars, 40m.

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and no blue dye is detectable in the ovarian stroma (Fig. 3E). Overall,these results demonstrate targeted and effective incorporation ofadenovirus, despite the MUC1 presence on the OSE.

Intrabursal administration of AdCre in MUC1Kras mice leads toMUC1-positive endometriosis-like ovarian lesionsTo activate the oncogenic KrasG12D allele, we injected AdCre underthe ovarian bursa of MUC1Kras mice (n10). Kras (n5) and MUC1mice (n5) were also injected and served as controls. All injectionswere unilateral, keeping contralateral ovaries as controls. Wemonitored the occurrence of lesions at 12, 24 and 32 weeks post-AdCre injections. No lesions (either ovarian or peritoneal) weredetected in any of the mice sacrificed at the 12- or 24-week timepoints. However, all MUC1Kras mice that were kept for 32 weeksor longer (n5) developed discrete ovarian lesions. The lesions werebenign in nature and the mice showed no signs of distressthroughout the experiment. The disease developed at a similar ratein control Kras mice (Dinulescu et al., 2005).

The histological findings from two MUC1Kras mice, which wererepresentative of the experimental group, are summarized in Fig.4. The ovarian lesions consisted of endometrial glandularepithelium on the ovarian surface, dominated by the epithelialcomponent (Fig. 4A,B). These lesions are MUC1 positive (Fig. 4C,F)and stain positively for the estrogen receptor (ER) (Fig. 4D,G) andCyk7 (Fig. 4E,H), which are markers that are typically used todiagnose endometriosis (Al-Khawaja et al., 2008; Norwitz et al.,1991). Morphologically, the lesions resemble those found in Krasmice (Fig. 4I,J), which are also positive for both Cyk7 (Fig. 4K) andER (data not shown). However, as expected, the lesions are negativefor the human MUC1 protein (Fig. 4L). At late time points, theAdCre-injected MUC1 mice showed no lesions in their AdCre-

injected ovaries (supplementary material Fig. S2). A detailed MUC1expression profile in the ovaries and uteri of AdCre-injectedMUC1Kras mice is shown in Fig. 5. Normal MUC1 expression isseen on the OSE monolayer of a control non-injected (contralateral)ovary (Fig. 5A). The surface epithelium shows mostly flat cuboidalcells expressing detectable MUC1. By contrast, injected ovariesshow increased MUC1 expression on both OSE and bursalepithelial cells (Fig. 5B), with a mostly pseudostratified architecture.Subjacent to the surface lesions, MUC1-positive endometrioticlesions with glandular morphology are seen infiltrating the ovarianparenchyma (Fig. 5C,D). The de novo, ovarian, glandular epithelialimplants are similar to the eutopic uterine glands (Fig. 5E). Thetissue architecture of the glandular uterine epithelia, and of theciliated epithelia in the oviducts (Fig. 5F), remains normalthroughout the disease.

These results demonstrate that, in MUC1Kras mice, ovarianMUC1 expression changes with disease development from low tohigh expression on both OSE cells and the deep infiltratingglandular structures resembling uterine glands. Furthermore, thelesions highly resemble human endometriosis and express thehuman antigen MUC1.

Having observed changes in MUC1 protein expression indiseased mice with de novo ovarian endometriosis, we nextpostulated that MUC1 immunogenicity also changes during diseasedevelopment.

MUC1Kras mice progressing to disease show increased MUC1-specific humoral immunityTo measure naturally occurring humoral immunity to the MUC1antigen following disease induction in MUC1Kras mice, wecollected blood before, and every 3-4 weeks after, AdCre injection(Fig. 6). The increase of IgM antibodies in experimental miceoccurred early after the injection and was maintained throughoutthe disease (Fig. 6A; and data not shown). The mice also developedMUC1-specific IgG antibodies consisting mostly of the IgG1subclass (Fig. 6B). At 12 weeks post-injection, some of the MUC1(control) mice also exhibited an increase in MUC1-specific IgMantibodies, probably because of slight increases in the localexpression of MUC1 by the OSE cells in response to transitoryadenovirus infection. However, no ectopic lesions were detectedin the AdCre-injected MUC1 mice and no MUC1-expressing cells,other than the OSE cells, could be detected in their ovaries(supplementary material Fig. S2). Furthermore, the anti-MUC1 IgGresponses in experimental MUC1Kras mice were higher than incontrol injected MUC1 mice, suggesting that the isotype switchwas the result of disease induction rather than local virus-inducedearly inflammation. Furthermore, the anti-MUC1 IgM antibodytiters diminish at late time points, after adenovirus clearance. Theisotype switch from IgM to IgG1 is an indirect correlate of in vivoTh2 immunity in mice (Mizoguchi et al., 1999) and our resultssuggest that a Th2-prone environment may develop in diseasedMUC1Kras mice.

MUC1Kras mice with endometriosis show increased CD4 Foxp3 Tcells in regional lymph nodesThe balance between the various CD4 T-cell subsets can enhanceor limit disease-associated immunity. Our phenotypic analysis ofT cells from the draining (para-aortic) lymph nodes showed

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Fig. 3. Injection of recombinant adenovirus Ad5LacZ under the bursaresults in efficient infection of OSE cells. We injected 1.2�106 plaque-forming units (PFU) of AdLacZ under the bursa of MUC1Kras (A), MUC1 (C) andKras (D) mice. Representative mice from each group are shown. The mice weresacrificed 72 hours later and the ovaries were harvested for whole-mount-galactosidase detection, after exposure to X-gal substrate. The efficiency ofinfection is indicated by the indigo staining at 20 hours post-substrateexposure. Contralateral non-injected ovaries remained stain-free (B), denotinguninfected cells. (E)After exposure to X-gal, the injected ovary (shown in A)was embedded in paraffin and sectioned. Sections (4m thick) were stainedby H&E. The infected cells (blue) reside within the superficial monolayer of OSEcells (E, arrows); no blue dye can be detected in the ovarian stroma. Bar, 40m.

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increased percentages of CD4+ Foxp3+ regulatory T cells (Tregs)in MUC1Kras- and Kras-diseased mice when compared withuninjected MUC1Kras controls or injected MUC1 mice (Fig. 7A,B;supplementary material Fig. S3; and data not shown). No increasesin CD4+ Foxp3+ T cells were detected at early time points, in theabsence of lesions (not shown). In addition, the capacity of the Tcells in the spleen and regional lymph node to secrete interferon (IFN) in response to polyclonal stimulation was decreasedsignificantly in diseased mice (Fig. 7C). However, despite thesignificant increase in the percentage of Tregs in the spleens ofdiseased mice compared with controls, the increase was less severewhen compared with the increase detected in the draining lymphnodes. Overall, these results suggest that when endometrioticlesions are histologically apparent, the immune environment showsskewing towards immune suppression.

To extrapolate our findings to human lesions, we measured theexpression of FOXP3 in human endometriosis (Fig. 7D). Wedetected an increased amount of FOXP3 mRNA in six out of ninepatients (E1-E9). Only one of six patients with benign, physiologicalovarian cysts (C1-C6) showed FOXP3 transcripts. We confirmed

the presence of the FOXP3 protein in lesions by flow cytometrystaining of FOXP3+ CD4 T cells that were infiltrating the ovariesof two different endometriosis patients (Fig. 7E). In contrast to thefindings in lesions, the systemic FOXP3+ CD4 T cell percentageswere within normal limits in both patients, at 2% and 3%,respectively. These results confirm the findings in mice and show,for the first time, the recruitment of FOXP3+ T cells in lesions ofendometriosis.

DISCUSSIONVery few defined antigens for human endometriosis exist currentlyand even fewer are confirmed in preclinical animal models. Here,we describe a conditional mouse model of ovarian endometriosisexpressing the human MUC1 tumor-associated antigen. The newlygenerated MUC1Kras mice develop lesions that, in addition torecapitulating the endometriosis-like lesions seen in Kras mice(Dinulescu et al., 2005), are also positive for the human MUC1antigen, thus mimicking the human disease even more closely.Using the MUC1Kras mice, we detected important changes inovarian MUC1 expression during disease progression, identified

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Fig. 4. MUC1Kras mice develop MUC1-, ER- and Cyk7-positiveovarian endometriosis-like lesions. Six 8-week-old female micewere injected with 2.5�107 PFU of AdCre under the ovarianbursa, 36 hours after the induction of ovulation. Mice weresacrificed at 32 weeks post-AdCre injection and gynecologicaltract organs were harvested at necropsy. Panels A-H show theresults from two different MUC1Kras mice (A-E and F-H,respectively) at 32 weeks post-AdCre injection under the bursa.Paraffin-embedded ovaries from diseased mice were step-sectioned and 4m-thick sections were obtained. H&E stainingshows endometrioid glandular lesions in the injected ovary(panel B is a magnified view of the boxed lesions in A).Immunohistochemistry shows ectopic lesions that are positive forMUC1 (C,F), ER (D,G) and Cyk7 (E,H). The lesions in MUC1Kras micemorphologically resemble the ectopic glands that are seen in theovaries of Kras mice (H&E staining in I,J). The ectopic epithelialcells in Kras mice are positive for Cyk7 (K) and negative for MUC1(L). Bars, 40m.

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MUC1-specific immune responses, and revealed a Th2/Treg biasin the host.

The report by Dinulescu et al. (Dinulescu et al., 2005) was thefirst to show that activation of the oncogenic KrasG12D mutationin the OSE might be responsible for the development of ovarianendometriosis-like lesions in genetically engineered Kras (loxP-Stop-loxP-KrasG12D/+) mice. This model, however, lacks expressionof a known antigen with which antigen-specific adaptive immunitycan be studied. Although the homology between the mouse Muc1and the human MUC1 molecules is 87% in the cytosolic tail, theMuc1 extracellular domain (which is normally involved in cell-celland receptor-ligand interactions, and in immune regulation) is only34% homologous to its human counterpart (Spicer et al., 1991) andis thus not expected to undergo the same changes before and afterdisease induction. Furthermore, in vivo and ex vivo studies onmurine Muc1 are difficult to perform, mostly owing to the lack of

availability of adequate reagents. Using the newly developedMUC1Kras mice, we showed that mice with induced endometriosisdisplay important histomorphological changes in their ovaries,leading to increased ovarian MUC1 protein expression. Thisincrease has dual consequences, which are both potentiallyimportant for disease pathogenesis. On one hand, increased MUC1protein expression may provide a mechanism for cell expansionthrough increased adhesion and migration of ectopic cells; inaddition, it may confer a pro-survival advantage, owing toincreasing resistance to apoptosis (Huang et al., 2005; Raina et al.,2004; Schroeder et al., 2003). On the other hand, amplified MUC1expression renders the protein immunogenic, with increasingMUC1-specific antibody titers being detected ex vivo in MUC1Krasmice. The lower, yet detectable, levels of antibodies seen in MUC1mice were probably because of the local inflammation associatedwith AdCre infection. The virus may have triggered the secretionof several pro-inflammatory cytokines (such as IL-1 and IL-6)(Chang et al., 2002) that are responsible for the transitory increasein MUC1 expression (Li et al., 2003; Rowse et al., 1998).Nevertheless, this effect was only transient in MUC1 mice, whoseovaries remained lesion-free throughout the duration of ourexperiments. By contrast, injection of AdCre in MUC1Kras micewas followed by notable changes in the ovarian surface epitheliumand neoformation of endometriotic glands, which triggeredpersistent high levels of MUC1-specific IgM and IgG1. We postulatethat the isotype switch from IgM to IgG1 that was observed duringdisease development was because of a Th2 bias; this is in agreementwith previous reports in humans (Antsiferova et al., 2005; Podgaecet al., 2007) showing increased autoantibody levels (Gleicher et al.,1987) and signs of B-cell activation (Hever et al., 2007). However,it has been argued that chronic exposure to antibodies can promotetumors: antibodies can extravasate into the stroma and formimmune complexes that can initiate inflammatory cascadesassociated with tissue destruction (Johansson et al., 2008). Furtherstudies on the prognostic value of anti-MUC1 antibodies and theirrelationship to the risk for ovarian cancer are now being explored.

Our analysis on adaptive immunity in endometriosis alsorevealed the prevalence of Foxp3 Tregs in the regional lymph nodesof diseased mice, and revealed their increased presence in humanlesions. However, no increases above the normal limits of Tregs inthe peripheral blood of women with endometriosis were observed,suggesting that further analyses of this T-cell subset in patientsshould focus on the lesions and/or regional nodes rather than theperipheral blood. We also failed to identify Treg-induced immunesuppression of effector T cells in diseased mice, primarily owingto limited ex vivo availability of these cells for functional assays.Nevertheless, our results suggest an apparently paradoxicalassociation between immune reactivity (antibody production) andnon-reactivity (potentially owing to immune suppression), similarto findings from Willimsky et al., who demonstrated that toleranceto the antigen occurs at the premalignant stage and induces adefault  immune response (increased antibodies and T cellunresponsiveness) that is permissive for cancer progression(Willimsky et al., 2008).

In summary, although the preclinical model employed here isnot an exact genocopy or immune phenocopy of the humandisease, it reproduces closely the histomorphology seen in womenand is a valuable in vivo model for ovarian endometriosis. Current

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Fig. 5. Increased MUC1 expression in the ovaries of MUC1Kras femalemice with ovarian endometriosis-like lesions at 32 weeks post-AdCreinjection in the bursa. Paraffin-embedded tissues, namely the ovaries (Ov,A-D), uterus (Ut, E) and oviduct (Od, F), were sectioned and subjected toimmunohistochemistry for MUC1 detection. (A)MUC1 expression on the flatcuboidal OSE layer (arrowheads) and on the bursal epithelial cells (arrows) incontrol uninjected (right) ovaries. (B)The OSE shows a pseudostratifiedarchitecture and increased MUC1 expression at 32 weeks post-AdCre injection(arrowheads). The bursal epithelial cell monolayer (dotted arrow) can be seenadjacent to a stretch of cells with modified architecture (solid arrows).(C)MUC1-positive endometriotic lesions infiltrate the ovarian parenchyma.(D)Lesions are surrounded by reduced stroma (arrowheads). (E,F)MUC1expression in eutopic uterine glands (E) and in luminal cells in the oviductfrom the same mouse (F). Bars, 40m.

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therapies in endometriosis are of limited efficacy, and new andimproved venues, including immune-based approaches, are needed.The emergence of MUC1Kras mice will possibly facilitate furtherstudies on the roles of MUC1 in endometriosis, and will allow thein vivo testing of MUC1 vaccines as potential therapies inendometriosis and the prevention of ovarian cancer.

METHODSMiceLSL-KrasG12D/+ mice (B6;129-Krastm4Tyj) were obtained from theNIH mouse repository. MUC1 transgenic mice (Rowse et al., 1998)were purchased from Dr S. J. Gendler (The Mayo Clinic, Scottsdale,AZ) and subsequently bred, in house, at the animal facilities of theUniversity of Pittsburgh Cancer Institute (UPCI) and MageeWomens Research Institute (MWRI). All of the experimentalprocedures described here were approved by the InstitutionalAnimal Care and Use Committee (IACUC) of the University ofPittsburgh, Pittsburgh, PA.

To obtain MUC1Kras mice, we bred the heterozygous MUC1+/–

transgenic mice (MUC1 mice) with the heterozygous LSL-KrasG12D/+ (Kras) mice. All mice were on the agouti background.PCR genotyping of tail DNA was performed to identify the doubletransgenic mice in each litter.

PCR genotypingMouse tail DNA was isolated using a Puregene DNA purificationsystem (Gentra Systems), according to manufacturer’s instructions.To detect the presence of the wild-type Kras and mutated LSL-KrasG12D/+ gene, the REDTaq ReadyMix PCR reaction mix (Sigma)was used in a reaction volume of 30 l. PCR conditions were asfollows: 94°C for 3 minutes; 34 cycles of 94°C for 30 seconds, 60°Cfor 90 seconds and 72°C for 1 minute; followed by 5 minutes at72°C (Jackson et al., 2001; Tuveson et al., 2004). The sequences ofthe primers are: forward (KrasG12D/+) 5�-CCATGGC TTGA -GTAAGTCTGC-3� and (wild-type Kras) 5�-GTCGAC AAG -CTCATGCGGG-3�; reverse (universal) 5�-CGCAG ACTGTAG -AG CAGCG-3�.

For MUC1 detection, PCR was performed in a reaction volumeof 25 l using AmpliTaq Gold 1.5 U in 10� PCR buffer II,supplemented with 25 mM of MgCl2 and 2.5 mM of deoxynucleotidetriphosphates (dNTPs) (all from Applied Biosystems). PCRconditions were as follows: 95°C for 10 minutes; 39 cycles of 94°Cfor 1 minute, 59°C for 1 minute and 72°C for 1 minute; followed by10 minutes at 72°C (Beatty et al., 2007). The MUC1 primer sequencesare: forward 5�-CTTGCCAGCCA TAGCACCAAG-3� and reverse5�-CTCCACGTCGTGGACATTGATG-3�. The PCR products in a2% agarose gel were visualized under ultraviolet (UV) light with anUltra-Lum digital imaging system (UltraLum).

Administration of recombinant adenovirusRecombinant adenoviruses encoding for either the lacZ reportergene [Ad5CMVntLacZ (AdLacZ)] or Cre recombinase[Ad5CMVCre (AdCre)] were injected in vivo under the bursa ofsurgically exposed ovaries, according to a published protocol(Dinulescu et al., 2005). Both AdLacZ and AdCre were purchasedfrom the University of Iowa Gene Transfer Vector Core. Tosynchronize ovulation (Flesken-Nikitin et al., 2003), animals wereinjected intraperitoneally (IP) with 5 U of pregnant mare serumgonadotropin (PMSG, Sigma) and, 48 hours later, injected IP with5 U of human chorionic gonadotropin (hCG, Sigma).

For the lacZ reporter gene experiments, seven different mice(three MUC1Kras, two Kras, two MUC1) were injected under theovarian bursa with 1.2�107 PFU of AdLacZ, 36 hours after thehCG IP injection. Mice were sacrificed 72 hours post-AdLacZinjection and the organs of the gynecologic tracts (ovaries, oviducts,uteri) were harvested for X-gal staining of intact tissue (wholemounts) (Kiernan, 2007). The organs were fixed for 5 minutes atroom temperature in a fixative solution (2% formaldehyde, 0.25%glutaraldehyde in PBS) and then washed three times with PBS.The  0.2% X-gal substrate (5-bromo-4-chloro-3-indolyl-D-galactopyranoside; Invitrogen) was resuspended in N,N-dimethylformamide and then diluted to the final concentration in 2 mMMgCl2, 5 mM K4Fe(CN)6.3H2O, 5 mM K3Fe(CN)6 in Hank’sbuffered salt solution (HBSS) (all from Sigma). The indigo-stained

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Fig. 6. Natural antibody responses to MUC1 in mice with endometriosis. At 36 hours after the induction of ovulation, 6–8-week-old female mice wereinjected with 2.5�107 PFU of AdCre, under the bursa. Blood was collected by tail venipuncture, which was performed at baseline and at every 3 weeks thereafter.Serum anti-MUC1 antibodies were measured by ELISA. The results of the ELISAs for IgM (A) and IgG1 (B) are shown for three different time points: baseline (pre-injection; diamonds), 12 weeks (circles) and 32 weeks (triangles) post-injection. Serum was diluted at 1:80. Each assay was run in triplicate and results from onerepresentative assay are shown. The values shown represent the average optical density (OD) readings after subtracting the readings from control (BSA-coated)wells. Symbols: MUC1Kras (black symbols), Kras (gray symbols), MUC1 (white symbols). For each group, the readings at 12 weeks (circles) and 32 weeks (triangles)were compared with those at baseline (diamonds), and the statistical significance was calculated using the Student’s t-test (Statgraphics). *P<0.0001; **P<0.005;***P<0.01. (Other statistical calculations: at 12 weeks, IgM is higher in MUC1Kras mice than MUC1 mice, P0.002; at 32 weeks, both IgM (P0.05) and IgG1(P0.005) are higher in MUC1Kras mice than in MUC1 mice.)

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areas indicate -galactosidase activity in infected cells only and werevisualized with a Leica L2 inverted scope and a Canon digitalcamera. A lack of staining indicates uninfected cells.

For endometriosis-inducing experiments, animals underwentsurvival surgery and intrabursal injection of AdCre (2.5�107 PFU)to one ovary only (with the contralateral ovary serving as a control),at approximately 36 hours after hCG administration. Theadenovirus was delivered using a modified calcium phosphateprecipitation protocol, as described previously (Dinulescu et al.,2005).

MUC1-specific antibody detection by ELISABlood samples from experimental (MUC1Kras) and control (Krasand MUC1) mice were obtained by venipuncture of the tail vein

before AdCre injections (baseline) and then at every 4 weeks until8-10 months post-disease induction. Serum was separated bycentrifugation and tested for the presence of MUC1-specificantibodies with a MUC1-specific ELISA, as described previously(Soares et al., 2001). Briefly, 96-well Immulon 4 HBX plates (FisherScientific) were coated overnight with 10 g/ml of a 100-amino-acid-long MUC1 peptide in PBS, at room temperature. Thispeptide comprises five MUC1 tandem repeats: the amino acidsequence of one repeat is GVTSAPDTRPAPGSTAPPAH. Thepeptide was synthesized at the University of Pittsburgh CancerInstitute Peptide Synthesis Facility. Half of each plate was coatedwith control 2.5% bovine serum albumin (BSA) to serve as anegative control. The pre-coated plates were incubated with pre-diluted serum (1:80) for 1 hour at room temperature and then with

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Fig. 7. Identification of Tregs in diseased mice and inhuman endometriosis lesions. (A)Abdominal para-aortic(draining) lymph nodes from four different female micewere collected at 32 weeks post-injection and prepared forflow cytometry. Lymph node cells were stained for CD3,CD4, CD8 and Foxp3. The upper dot plots are from twodiseased MUC1Kras mice. The lower dot plots are from onediseased Kras mouse (left) and one healthy (non-injected)age-matched MUC1Kras control (right). Percentages wereobtained using FACSDiva software, after gating for CD3,subgating for CD4, and subtracting background eventsfrom a control gate using an isotype control antibody forFoxp3 (supplementary material Fig. S4). Additional resultsfrom two AdCre-injected MUC1 mice, and the gatingstategy, are detailed in supplementary material Fig. S3.(B)The percentages of Foxp3 T cells in regional (para-aortic) lymph nodes are higher in diseased mice(MUC1Kras-d, n4; and Kras-d, n4) than in healthy age-matched mice (MUC1Kras-h, n4) (*P<0.02; **P<0.01).(C)Detection of intracellular IFN by flow cytometryfollowing phorbol myristate acetate (PMA)-ionomycinstimulation of splenic T cells from healthy age-matchedMUC1Kras control mice (upper three dot plots) and fromexperimental diseased mice (lower three dot plots). Insetdot plots show the percentages of CD3+ CD4+ Foxp3T cells in spleens from the same mice. Numbers wereobtained in FACSDiva using the gating hierarchy describedin supplementary material Fig. S4. (D)Quantitation ofhuman FOXP3 gene transcripts by qRT-PCR. mRNA wasextracted from normal ovarian tissues (N1-N3, n3),ovarian endometriosis tissues (E1-E9, n9) and ovariancysts (C1-C6, n6). All results were normalized to GAPDHand presented as the percentage change from N, which isthe average of three normal ovaries, rendered as 100%.(E)Detection, by flow cytometry, of human CD3+ CD4+FOXP3 T cells that were isolated from either endometriosistissue (left dot plots) or peripheral blood (right dot plots)from the same patients.

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goat anti-mouse peroxidase-conjugated secondary antibodies(anti-IgM, anti-IgG and anti-IgG1, Sigma) for 1 hour at roomtemperature. The plates were washed three times with PBS–Tween20 (0.1%) and then incubated with the substrate O-phenylenediamine dihydrochloride tablets (Sigma) for 1 hour. Thereaction was stopped, and the absorbance was measured at 450nm with a Multiskan plate reader (Thermo Scientific). Data wasrepresented using the average of triplicate wells, after subtractingthe background readings from control wells. Data acquisition andanalysis was performed using Ascent Software for Multiskan(Thermo Scientific).

Flow cytometryImmune phenotyping of murine lymphocytes: cells were collectedfrom the para-aortic lymph node and spleen of euthanized mice,and prepared by mechanical disruption and red blood cell (RBC)lysis using RBC lysing buffer (Sigma). The cells were stained within24 hours. For intracellular cytokine detection, cells were stimulatedwith PMA (5 g/ml) and ionomycin (50 g/ml) for 6 hours in thepresence of Golgi Plug (all from BD Biosciences). After stimulation,cells were stained with fluorescent antibodies (all from BDBiosciences) for CD3 (clone 145-2C11), CD4 (clone RM4-5) andCD8 (clone 53-6.7), and then treated with Cytofix/Cytoperm (BDBiosciences). For cytokine detection, anti-mouse IFN (cloneXMG1.2) was used. Intracellular staining for Foxp3 (clone FJK-16s)was performed using a mouse Foxp3 staining kit (eBioscience). Allantibodies were diluted according to manufacturers’ instructions.

Immune phenotyping of human lymphocytes: lymphocytesinfiltrating the endometriotic lesions were isolated using enzymatictissue digestion, whereby the ovarian tissue was digested for 2-4hours at 37°C with collagenase (0.5% w/v) and DNase (2 g/l) (bothfrom Sigma), and then passed through a 70 m sieve. The single-cell suspension was washed with 10 ml of medium. Peripheral bloodlymphocytes were obtained through Ficoll (Amersham Biosciences)gradient centrifugation, according to manufacturer‘s instructions.When ready to use, the cells were plated in 96-well plates (2.5-5�105 cells/well) and stained with antibodies (BD Biosciences) tohuman CD3 (clone SK7) and CD4 (clone SK3). The cells were thenpermeabilized and stained intracellularly for FOXP3 (clonePCH101, eBioscience) using a permeabilization kit (eBioscience).Stained cells were analyzed on a LSR II flow cytometer using theFACSDiva data analysis software (BD Biosciences). The gatingstrategy and sample analysis are shown in supplementary materialFig. S4

ImmunohistochemistrySections with a thickness of 4 m were obtained from each selectedblock of formalin-fixed, paraffin-embedded ovary. Antigen retrievalwas performed in 0.1 M TRIS buffer (pH 9) for 20 minutes at 100°C.The primary antibodies used were: anti-human MUC1 (cloneHMPV, 1:250; BD Pharmingen), anti-Cyk7 (clone RCK105, 1:10;Abcam), anti-ER rabbit polyclonal IgG (MC-20, 1:100; Santa CruzBiotechnology). The following secondary antibodies were used (for30 minutes): biotinylated goat anti-mouse Igs (1:50; BDPharmingen) and labeled polymer-horseradish peroxidase (HRP),anti-rabbit (Dakocytomation), ready to use. The antigen-boundantibodies were visualized with a 3,3�-diaminobenzidine (DAB)substrate kit (BD Pharmingen) for 5-10 minutes: positive cells are

visualized in brown. Counterstaining was carried out with Mayer’shematoxylin for 30 seconds, which stains the nuclei blue. Sectionswere mounted with Permount (Fisher Scientific). Images wereacquired using a Canon PowerShot A640 digital camera attachedto a Zeiss microscope connected to a Dell workstation, using theAxioVision Rel. 4.6 imaging software.

Quantitative real-time polymerase chain reaction (qRT-PCR)Human specimens were obtained from the University of PittsburghHealth Sciences Tissue Bank, according to approved InstitutionalReview Board (IRB) protocols. RNA was extracted from 25 to 100mg of each homogenized tissue: three normal ovarian tissues (N1-N3), which were used as controls; six ovarian physiological cysts(C1-C6); and nine endometriosis tissues (E1-E9). Total RNA wasisolated with TRIzol reagent (Invitrogen) and then purified usingan RNeasy Mini kit (Qiagen), according to the manufacturer’sprotocol. A High-Capacity cDNA Archive Kit (Applied Biosystems)was used to convert up to 1 g of total RNA in a single 20 l reaction

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TRANSLATIONAL IMPACT

Clinical issueEndometriosis results from the mislocalization of cells that usually form theuterine lining, to ectopic locations. The condition can be asymptomatic, but for10% of women in their reproductive years, it is associated with pelvic pain,fatigue, and irregular or heavy periods. Whether or not it produces symptoms,endometriosis can impair fertility and increase the likelihood of miscarriageand stillbirth in women who become pregnant. Mislocalized cells often formectopic lesions, also called implants or nodules, on organs within the pelviccavity, including the ovaries, intestines and bladder. Surgery and drugs areused to reduce the extent of lesions, to promote fertility and to relieve pain,but endometriosis is often recurring and can precipitate ovarian epithelialtumors.

Endometriosis is mediated partly by the immune system but very fewendometriosis antigens have been identified. The cell surface glycoprotein,mucin 1 or MUC1, is a known ovarian tumor-associated antigen that is presentin ectopic lesions of ovarian endometriosis. However, the role of MUC1expression in endometriosis and its potential as a biomarker for ovarian cancerare unknown.

ResultsHere, the authors use double transgenic MUC1Kras mice, with inducibleovarian endometriosis, to study changes in MUC1 expression duringprogression to endometriosis. The mice develop benign, MUC1-positiveovarian lesions that closely resemble human endometriosis. MUC1 expressionin the affected ovaries increases significantly as lesions develop, stimulatingthe production of MUC1-specific antibodies. High IgM and IgG antibody titersappear early in the disease process and are maintained. During the later stagesof disease, MUC1Kras mice, like women with the disease, show an expansionof immunosuppressive Foxp3+ CD4 regulatory T cells in the draining lymphnodes. The authors suggest that developing tolerance to MUC1 duringendometriosis may inhibit immune competence and enable cancerprogression.

Implications and future directionsThe authors describe and characterize an animal model of humanendometriosis. The pattern of MUC1 expression and its impact on the immunesystem of the host should facilitate the development of novel immune-basedapproaches. MUC1Kras mice could also further clarify the role of MUC1 inendometriosis and provide an in vivo platform for testing MUC1 vaccines totreat endometriosis and prevent ovarian cancer.

doi:10.1242/dmm.004465Dise

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to single-stranded cDNA. Transcripts were quantified by real-timePCR on an ABI Prism 7700 Sequence Detector (Perkin-ElmerApplied Biosystems) with TaqMan Gene Expression Assays(Applied Biosystems). The sequence-specific primers and theTaqMan MGB probe (6-FAM dye-labeled) were from the TaqManGene Expression Assay mix (Hs00203958_m1 FOXP3 andHs00904314_g1 MUC1). For each sample, the mRNA expressionlevel was normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH, probe Hs99999905_m1;Applied Biosystems). Data analysis was performed using therelative  change Ct (threshold cycle) value method (Schmittgenand  Livak, 2008). The amount of target normalized to theendogenous reference and relative to a control (the average ofthree  normal ovaries, N) was given by 2–Ct, whereCtCtsample–Cthousekeeping gene.

Statistical analysesComparisons between ELISA readings from the sera ofexperimental mice versus control mice were performed withStatgraphics Plus software (Statistical Graphics Corp.) using theStudent’s t-test for comparisons of two means.ACKNOWLEDGEMENTSWe thank O. Finn for sharing expertise and reagents; K. Gantt, A. Lepisto and D.Dinulescu for their helpful comments and input; and J. Thaller and L. Mock forexcellent technical assistance. This work was supported by grants from theOvarian Cancer Research Fund, the Pennsylvania Department of Health and theUniversity of Pittsburgh Continuing Medical Research Fund (to A.M.V.) and fromThe Fifth Framework Programme (Grant QLGA-CT-2000-60005 to A.D.).

COMPETING INTERESTSThe authors declare no competing financial interests.

AUTHOR CONTRIBUTIONSR.A.B., A.M.V., I.D. and R.C. performed the in vivo experimental work in mice andthe ELISA measurements. R.A.B. and I.D. performed the IHC; A.M.V. performed theflow cytometry and R.A.B. performed the qRT-PCR experiments. R.P.E. provided theclinical specimens and A.D. contributed reagents. A.M.V. designed all experiments,and A.D. and R.P.E. participated in the conception, design and coordination of thestudy. The manuscript was finalized by A.M.V. with assistance from all authors.

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.002535/-/DC1

Received 30 December 2008; Accepted 15 July 2009.

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