{"paper_id":"cc3c809b-43d6-465d-bdc6-48723f5747cd","body_text":"BioMed Central\nPage 1 of 11\n(page number not for citation purposes)\nReproductive Biology and \nEndocrinology\nOpen AccessResearch\nTLR3 and TLR4 expression in healthy and diseased human \nendometrium\nSvenja Allhorn†1, Carsten Böing†2, Andrea A Koch1, Rainer Kimmig and \nIsabella Gashaw*1\nAddress: 1Institute of Anatomy II, University of Duisburg-Essen, Hufelandstr. 55, 45147 Essen, Germany and 2Department of Obstetrics and \nGynaecology, University of Duisburg-Essen, Hufelandstr. 55, 45147 Essen, Germany\nEmail: Svenja Allhorn - S_Allhorn@web.de; Carsten Böing - Carsten.Boeing@uk-essen.de; Andrea A Koch - AndreaKoch7@aol.com; \nRainer Kimmig - Rainer.Kimmig@uk-essen.de; Isabella Gashaw* - isabella.gashaw@bayerhealthcare.com\n* Corresponding author    †Equal contributors\nAbstract\nBackground: Toll-like receptors (TLRs) pl ay an essential role in the innate immune system by\ninitiating and directing immune response to  pathogens. TLRs are expressed in the human\nendometrium and their regulation might be crucial for the pathogenesis of endometrial diseases.\nMethods: TLR3 and TLR4 expression was investig ated during the menstrual cycle and in\npostmenopausal endometrium considering peritoneal endometriosis, hyperplasia, and endometrial\nadenocarcinoma specimens (grade 1 to 3). The expression studies applied quantitative RT-PCR and\nimmunolabelling of both proteins.\nResults: TLR3 and TLR4 proteins were mostly locali sed to the glandular and luminal epithelium.\nIn addition, TLR4 was present on endometrial dendritic cells, monocytes and macrophages. TLR3\nand TLR4 mRNA levels did not show significant changes during the menstrual cycle. In patients with\nperitoneal endometriosis, TLR3 and TLR4 mRNA expression decreased significantly in proliferative\ndiseased endometrium compared to controls. Interestingly, ectopic endometriotic lesions showed\na significant increase of TLR3 und TLR4 mRNA expression compared to  corresponding eutopic\ntissues, indicating a local gai n of TLR expression. Endometrial  hyperplasia and adenocarcinoma\nrevealed significantly reduced receptor levels when compared with postmenopausal controls. The\nlowest TLR expression levels were determined in poor differentiated carcinoma (grade 3).\nConclusion: Our data suggest an involvement of TLR3  and TLR4 in endo metrial diseases as\ndemonstrated by altered expression levels in endometriosis and endometrial cancer.\nBackground\nToll-like receptors (TLRs) recognize specific pathogen\nassociated molecular patterns (PAMPs) and serve an\nessential role in the innate immune system by initiating\nand directing immune response to microbial pathogens.\nHuman TLRs comprise a large family of 10 proteins with\nmember-specific activators and a complex downstream\nsignalling [1]. TLRs are expressed on various immune cells\nbut are also present on mucosal surfaces of the respiratory,\ngastrointestinal and urinary tract [1]. Applying different\nadaptor proteins such as toll-like receptor adaptor mole-\ncule 1 (TRIF, TICAM1), myeloid differentiation primary\nPublished: 7 September 2008\nReproductive Biology and Endocrinology 2008, 6:40 doi:10.1186/1477-7827-6-40\nReceived: 10 April 2008\nAccepted: 7 September 2008\nThis article is available from: http://www.rbej.com/content/6/1/40\n© 2008 Allhorn et al; licensee BioMed Central Ltd. \nThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), \nwhich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\n\nReproductive Biology and Endocrinology 2008, 6:40 http://www.rbej.com/content/6/1/40\nPage 2 of 11\n(page number not for citation purposes)\nresponse gene 88 (MyD88), myelin and lymphocyte pro-\ntein Mal, translocation associated membrane protein\n(TRAM) and sterile alpha and TIR motif containing\n(SARM), TLRs activate signalling pathways of mitogen-\nactivated protein kinases, nuclear factor kappa-B (NFêB),\nsignal transducers and activators of transcription (STATs)\nor the activator protein 1 (AP1) [1-3]. These signalling cas-\ncades result in enhanced secretion of various pro- and\nanti-inflammatory cytokines such as interferons, tumor\nnecrosis factor α (TNF α) and interleukins IL4, IL8, and\nIL12 [1,2]. Two studies have described the expression of\nhuman TLRs in epithelial cells within the female repro-\nductive tract [4,5]. Other than their importance for the\ninteraction between host and pathogen, the receptors\nmight be involved in mucosal homeostasis as described\nalready for the intestine and colon [6]. TLR3 is implicated\nin the recognition of dsRNA, mRNA and viruses [1,7],\nwhereas TLR4 is a key component of the initial injury\nresponse by reacting towards bacterial endotoxin and\nmultiple endogenous ligands [8]. Recent studies have\ndetermined the expression pattern of TLR3 [4,9-12] and\nTLR4 [4,10-14] in the human endometrium, but their\npossible involvement in the pathogenesis of endometrial\ndiseases associated with inflammation remains to be elu-\ncidated.\nEndometriosis is a common benign gynaecological condi-\ntion of reproductive aged women [reviewed in [15]]. The\ndisease is characterised by endometrial tissue fragments\noutside the uterine cavity and is associated with pelvic\npain, dysmenorrhoea, and infertility. Since aetiology and\npathogenesis remain uncertain, different theories are dis-\ncussed including altered immune function. The deregula-\ntion of immune response in endometriosis is\ncharacterised by increased number of activated macro-\nphages and their secreted products, such as growth factors,\ncytokines, and angiogenic factors [16,17]. Young et al.\nreported an increase in interleukin-8 (IL-8) production\nafter stimulating TLR3 and TLR4 in endometrial cell lines\nwith appropriate ligands [12]. IL-8 is a chemotactic acti-\nvating cytokine for leukocytes and it has been hypothe-\nsized to play a role in the growth and maintenance of\nectopic endometrial tissue [18]. Recent studies consider\nendometriosis as a process of sterile inflammation in the\npelvis, which is accompanied by elevated levels of inflam-\nmatory key regulators such as TNF α [19] or NF- κβ [20].\nBoth are known downstream targets of TLRs.\nEndometrial carcinoma is the most common gynaecolog-\nical malignancy in Europe and North America affecting\nmainly postmenopausal women [21]. In endometrial\ntumorigenesis, two different types are characterised: the\nestrogen-related adenocarcinoma (endometrioid type)\nand the non-endometrioid type such as papillary serous\nand clear cell carcinoma [21]. Adenocarcinoma accounts\nfor seventy percent of endometrial cancer and is mostly\npreceded by premalignant changes like endometrial\nhyperplasia [21]. The majority of adenocarcinoma\nexpresses steroid receptors and occur in women with risk\nfactors associated with an imbalance of estrogen and pro-\ngesterone. However, inflammation with production of\npro-inflammatory cytokines such as TNF α is known to\nplay an important role in cancer development [22]. In\nendometrial hyperplasia and adenocarcinoma the expres-\nsion of NFκB and TFNα has been demonstrated [23] indi-\ncating that the production of pro-inflammatory cytokines\nseem to play a role in endometrial tumorigenesis.\nThe present study describes the expression pattern of TLR3\nand TLR4 mRNA and proteins in healthy endometrium\nacross the menstrual cycle and in postmenopausal tissue.\nTo assess the possible involvement of these toll-like recep-\ntors in endometrial pathologies, their expression pattern\nwas also examined in endometriosis and in adenocarci-\nnoma specimens.\nMethods\nEndometrial tissues\nEndometrial tissues were obtained from 55 women with\nregular menstrual cycles (mean 28 ± 2.2 days) who were\nundergoing gynaecological procedures for benign condi-\ntions at the Department of Gynaecology, University Hos-\nTable 1: Patients' characteristics according to diagnosis at time of surgery\nn Age, mean Age, SD Indications for surgery\nPremenopausal, controls (proliferative & secretory) 27 37 8.6 fibroids (n = 7), non endometriotic ovarian cyst (n = 2), \ninfertility (n = 5), dysmenorrhoe (n = 11), pelvic pain (n = 1), \nuterine prolapse (n = 1)\nPremenopausal, non-endometriotic, menstrual 8 39 10.7 no surgery\nPremenopausal, endometriotic 20 34 6.8 endometriosis (n = 11), ovarian cyst (n = 2), infertility (n = 1), \ndysmenorrhoe (n = 6*)\nPostmenopausal, controls 8 68 10.2 fibroid s (n = 6), uterine prolapse (n = 2)\nPostmenopausal, hyperplasia 10 64 13 abnorma l endometrial thickness, supposed carcinoma\nPostmenopausal, endometrial carcinoma 16 67 12.8 abnormal endometrial thickness, endometrial carcinoma\nn = number of patients, * not including patients with known endometriosis before surgery\n\nReproductive Biology and Endocrinology 2008, 6:40 http://www.rbej.com/content/6/1/40\nPage 3 of 11\n(page number not for citation purposes)\npital Essen (table 1). In this cohort, 20 women have been\ndiagnosed with endometriosis. Menstrual effluents were\ncollected from women without proven endometriosis\nduring first three days of menstrual bleeding as described\nelsewhere [24].\nThe menstrual cycle phase was characterised by morpho-\nlogic evaluation following the criteria of Noyes et al. [25]:\nproliferative (P, controls: n = 16, endometriotic: n = 13),\nsecretory (S, controls: n = 11, endometriotic: n = 3) and\nmenstrual (M, n = 8) phase. Additionally, four prolifera-\ntive corresponding ectopic lesions were included,\nobtained from the above-characterised cohort. In the pre-\nmenopausal group, patient age ranged from 19 to 52 years\n(median: 38, detailed data in table 1).\nPostmenopausal endometrium was obtained from 34\nwomen including 10 with endometrial hyperplasia and\nanother 16 with endometrial carcinoma (table 1). The\nremaining 8 patients did not have any endometrial abnor-\nmalities and were used as the control group. Patients were\nconsidered postmenopausal if they have been in meno-\npause for at least one year. Endometrial adenocarcinoma\nspecimens were classified based on the post-operative his-\ntopathologic WHO guidelines [26] as follow: grade 1 (G1,\nwell differentiated, n = 5), G2 (moderately differentiated,\nn = 6), G3 (undifferentiated, n = 5). In the postmenopau-\nsal group, patient age ranged from 37 to 86 years (median:\n66).\nNone of the women included in the studies had received\nany hormonal treatments for at least three months preced-\ning biopsy and routinely analyzed laboratory parameters\nfrom blood samples were physiologically analogous to\nthe patient's age. Considering the leukocyte content and\nlevel of C-reactive protein, no systemic inflammation was\ndiagnosed at the time of surgery. Volunteers donating\nmenstrual effluents were healthy and without diagnosed\ninfections.\nInstitutional ethical approval was granted for all subjects,\nand all women provided written informed consent.\nAll biopsies were transferred into a buffered saline solu-\ntion directly after surgery and stored in this buffer for max-\nimal two hours until further use. A portion of the biopsy\nspecimen was fixed in 4% formalin and embedded in par-\naffin for histology and immunohistochemistry, the\nremainder was flash-frozen in liquid N\n2 for RNA extrac-\ntion.\nQuantitative real-time PCR\nIsolation of total RNA from endometrial tissue and\nreverse transcription into cDNA were carried out applying\nstandard methods as described previously [24]. Following\na DNase digest and reverse transcription, quantitative\nreal-time PCR (qPCR) reactions were performed in tripli-\ncates using an ABI Prism 7300 Sequence Detector\n(Applied Biosystems, Weiterstadt, Germany) in a total\nvolume of 20 μl containing 40 ng cDNA, 3.75 pmol gene-\nspecific primers (table 2) and SYBR Green reagent\n(Applied Biosystems) with ROX dye as passive control for\nsignal intensity. The thermal cycle profile was 10 sec at\n95°C, followed by 45 cycles of 5 sec at 95°C and 35 sec at\n60°C. Melting curve analysis allowed determination of\nthe specificity of the PCR fragments. All melting curves\nyielded one peak per PCR product.\nTo determine the copy number of PCR fragments, serially\ndiluted, gene specific standard cDNAs generated from\namplicons of TLR3, TLR4 and β-actin (ACTB) were used.\nApplying thermal block cyclers and ethidium bromide gel\nelectrophoresis, standard PCRs were conducted. Each\ngene-specific PCR resulted in one distinct band of the\nappropriate length. The amplicons were purified by using\na Qiagen kit and cDNA concentration was measured pho-\ntometrically. For each gene, five different dilutions of\nstandard cDNA were used in real time PCR. Threshold\ncycles for TLR3 signals were between 26 and 38 and for\nTLR4 between 25 and 36, respectively. Because of the\ndiversity in the RNA quality, each individual sample was\nnormalized to its ACTB mRNA content as an internal\nstandard. These relative values were used for statistics.\nImmunohistochemistry\nParaffin-embedded specimens were sectioned at 7 μm,\nrehydrated and microwaved in 0.01 M sodium citrate\nbuffer, pH 6.0, for 10 min for antigen retrieval. Immunos-\ntainings were performed on paraffin sections applying the\ndiaminobenzidine staining method with the\nVECTASTAIN Elite ABC kit (Vector Laboratories, Burlin-\ngame, CA) according to the manufacturer's protocol.\nTable 2: Oligonucleotide primers used for the quantitative real time PCR.\nGene (GenBank No.) Forward primers (position) Reverse primers (position)\nTLR3 (NM_003265) 5'-GTATTGCCTGGTTTGTTAATTG G (2059–2082) 5'-AAGAGTTC AAAGGGGGCACT (2215–2194)\nTLR4 (NM_138557) 5'-AAGCCGAAAGGTGATTGTTG (2187–2206) 5 '-CTGAGCAGGGTCTTCTCCAC (2339–2320)\nACTB (NM_001101) 5'-ACCAACTGGGACGACATGGA (302–322) 5'-CCAGAGGCGTACAGGGATAG (510–491)\nAll primers were designed using the Primer3 software.\n\nReproductive Biology and Endocrinology 2008, 6:40 http://www.rbej.com/content/6/1/40\nPage 4 of 11\n(page number not for citation purposes)\nEndogenous peroxidase activity was quenched with 0.3%\nH2O2 in methanol for 10 minutes and washed in buff-\nered saline solution (PBS). Unspecific binding of the first\nantibody was blocked by 30-minute incubation step in\nPBS containing 0.15% normal horse serum. Slides were\nincubated in a humidified chamber overnight at 4°C with\nthe monoclonal mouse-anti-human antibodies against\nTLR3 [27] and TLR4 [HTA125, [28]] at 20 μg/ml and 100\nμg/ml, respectively (Acris Antibodies, Hiddenhausen,\nGermany). Control samples were carried out by omitting\nthe primary antibody. All sections were counterstained\nwith haematoxylin and documented by using a Zeiss Axi-\nophot microscope (Zeiss, Jena, Germany) with a Nikon\nDS-U1 camera and the LUCIA Image Analysis software\n(Nikon, Tokyo, Japan).\nImmunofluorescent staining\nFrozen tissues were sectioned at 7 μm and fixed in 70%\nethanol. Unspecific binding of the first antibody was\nblocked by a 30 min incubation step in 5% BSA/PBS. The\nTLR4 antibody was incubated as described above and was\ndetected using Alexa Fluor 488-conjugated anti-mouse\nantibody (3.3 μg/ml, MoBiTec, Goettingen, Germany).\nSections were fixed in formalin (4%) for two minutes and\nthen washed in PBS. The incubations with CD14 (10 μg/\nml, mouse anti-human, BioLegend, San Diego, CA) or\nCD163 (10 μg/ml, mouse anti-human, HyCult Biotech-\nnology, Uden, The Netherlands) occurred at room tem-\nperature for 60 min. CD14 antigen is expressed on on\nmonocytes/macrophages, acting as a dendritic cells pre-\ncursor [29]. CD163 is a member of the scavenger receptor\ncystein-rich family class B and is expressed on most sub-\npopulations of mature tissue macrophages [30]. CD163 is\nhighly abundant in human placenta [31] and is present in\nshed menstrual endometrium [24]. The secondary, goat\nanti-mouse antibody was Cy3-conjugated (2.5 μg/ml,\nDianova, Munich, Germany) and was applied to the spec-\nimens for another 60 minutes. Nuclei were identified by\n4',6'-diamidino-2-phenylindole staining (DAPI, Sigma,\nMunich, Germany) using 0.1 μg/ml DAPI in methanol for\n15 min at 37°C. Negative controls were performed by\nomitting the primary antibody and were used to adjust the\nbackground fluorescence.\nAfter mounting with Mowiol (Sigma), confocal micros-\ncopy was performed using a Zeiss Axiovert 100 micro-\nscope and LSM 510 system (Zeiss, Jena, Germany). TLR4\nwas detected at 488 nm, CD14 as well as CD163 at 543\nnm, and DAPI at 366 nm, respectively.\nStatistical analysis\nExploratory data analyses, Kruskal-Wallis test for group\ncomparisons, as well as the Mann-Whitney U test for non-\nparametric independent two-group comparisons were\nperformed with the program SPSS 14 for Windows (SPSS\nInc., Chicago, IL). Differences with P < 0.05 were regarded\nas statistically significant, P < 0.01 as highly statistically\nsignificant. Values of mRNA quantification are given as\nmean ± standard deviation (SD).\nResults\nTLR3 and TLR4 expression is deregulated in peritoneal \nendometriosis\nBoth receptors were expressed in all endometrial biopsies\nwith the averaged TLR4 mRNA levels being higher (20-\nfold) than TLR3 (figure 1). This difference was the greatest\nin the shed menstrual endometrium, where TLR4 tran-\nscripts were 564-fold higher than those for TLR3. The rel-\native abundance of both transcripts did not vary\nthroughout the menstrual cycle (figure 1).\nTLR3 and TLR4 proteins were expressed mainly in the\nluminal and glandular epithelium (figure 2). Interest-\ningly, the glands presented a heterogeneous immune\nstaining for TLR3 (figure 2C, I). Indeed, the TLR3 receptor\nwas found to be locally expressed in a subset of epithelial\ncells within one gland. In addition, we report the expres-\nsion of TLR4 protein on immune cells such as monocytes\nand macrophages, in menstrual phase samples (figure 2J).\nCo-immunostainings on menstrual effluents confirmed\nthat CD14 positive dendritic cells and monocytes (figure\n2K) as well as CD163 positive resident macrophages (fig-\nure 2L) expressed TLR4 protein.\nTLR3 and TLR4 transcript are expressed in endometrium during the menstrual cycleFigure 1\nTLR3 and TLR4 transcript are expressed in \nendometrium during the menstrual cycle. Columns \nindicate mean TLR3 and TLR4 mRNA quantities from \nendometrium in proliferative (n = 16), secretory (n = 11) and \nmenstrual phase (n = 8) run in triplicates. The y-axis is scaled \nlogarithmically; error bars represent the standard deviation \nof the mean.\n\n\nReproductive Biology and Endocrinology 2008, 6:40 http://www.rbej.com/content/6/1/40\nPage 5 of 11\n(page number not for citation purposes)\nIn endometriosis, we can observe a significant decrease in\nTLR3 and TLR4 mRNA levels in eutopic tissues collected\nduring the proliferative phase, when compared to controls\n(P < 0.05; figure 3A–B). Interestingly, endometriotic\nlesions in proliferative phase showed a significant\nincrease of TLR3 mRNA expression (P < 0.05) when com-\npared with the corresponding eutopic endometrium (fig-\nure 3A). For the TLR4 transcript, a 6-fold increase was\nobserved in the endometriotic lesions in comparison with\nthe diseased eutopic endometrium ( P < 0.01; figure 3B).\nIn the endometrial tissues collected during the secretory\nphase, the TLR4 mRNA level tended to be lower in eutopic\nendometrium than in controls (P = 0.08; figure 3D).\nImmunostaining analyses confirmed these findings at the\nprotein level. In endometriosis, eutopic tissues revealed\nweaker staining for TLR3 and TLR4 proteins (figure 2B, D,\nF, H) when compared to controls (figure 2A, C, E, G). Fig-\nure 4 exemplary presents the expression of TLR3 and TLR4\nprotein in eutopic compared to ectopic endometrium\nfrom the same patient. The TLR3 (Fig. 4A) and TLR4 (Fig.\n4C) immunostaining in diseased eutopic endometrium\nwas barely detectable, whereas corresponding lesion from\nthe uterosacral ligament showed an intense staining in the\nglandular epithelium for both proteins (Fig. 4B and 4D).\nConcerning protein localization, we found TLR3 and\nTLR3 and TLR4 protein is localised to endometrial cells during the menstrual cycleFigure 2\nTLR3 and TLR4 protein is localised to endometrial cells during the menstrual cycle. TLR3 protein staining in \nhealthy late proliferative (LP) tissue was high in luminal and glandular tissue (A, brown precipitate) and lower in LP endometri-\notic tissue (B). Late secretory (LS) endometrium showed highly expressed TLR3 in the epithelium (C), but weakly in endome-\ntriosis (D). Intense staining of TLR4 proteins was shown in mid proliferative (MP) tissue (E). In late proliferative phase of \nendometriosis, TLR4 proteins were comparably lower (F). TLR4 protein was high in mid secretory (MS) normal endometrium \n(G), whereas it was decreased in endometriotic MS tissue (H). During the menstrual phase, both TLR3 (I) and TLR4 (J) were \nhighly expressed. Co-immunostaining for TLR4 (green), CD14 (K, red) and CD163 (L, red) demonstrated that TLR4 proteins \nwere expressed by CD14 positive dendritic cells and monocytes (K, yellow) and by CD163 positive macrophages (L, yellow). \nLocalisation of TLR4 to immune cells is marked by a black arrow (J) and by white arrows (K, L).\n\n\nReproductive Biology and Endocrinology 2008, 6:40 http://www.rbej.com/content/6/1/40\nPage 6 of 11\n(page number not for citation purposes)\nTLR4 proteins in glandular and epithelial cells of endome-\ntriosis patients.\nTLR3 and TLR4 are expressed in postmenopausal \nendometrium and regulated endometrial adenocarcinoma\nTLR3 and TLR4 mRNA abundance in healthy postmeno-\npausal tissues is similar to those found during the men-\nstrual cycle. In postmenopausal controls, TLR4 mRNA\nlevels were higher than those for TLR3 (P < 0.05, figure 5).\nTLR3 and TLR4 mRNA expression varied significantly\nbetween control, hyperplasia and endometrial adenocar-\ncinoma samples (Kruskal-Wallis test, P < 0.01). For both\nreceptors, we observe a significant decrease in mRNA\nabundance in endometrial hyperplasia and adenocarci-\nnoma samples, when compared to postmenopausal\nendometrium (P < 0.05; figure 5A–B). In undifferentiated\nG3 carcinoma, TLR3 and TLR4 mRNA levels were signifi-\ncantly lower than in postmenopausal controls ( P < 0.01)\nand in hyperplasic endometrial tissues ( P < 0.05, figure\n5C–D).\nTLR3 and TLR4 proteins in hyperplasia and endometrial\ncarcinoma were mostly localized to the luminal and glan-\ndular epithelium (figure 6). Additionally, we demonstrate\na discontinuous staining for TLR3 protein within epithe-\nlial glands of G1 carcinoma (figure 6C), comparable to\nthe findings in secretory and menstrual phase of premen-\nopausal women (figure 2C, I). In undifferentiated G3 car-\ncinoma, staining for TLR3 (figure 6E) and TLR4 (figure 6J)\nwas not detectable, strengthening our findings of low\nTLR3 and TLR4 mRNA abundance in G3 carcinoma (fig-\nure 5B). In accordance with staining patterns obtained\nduring the menstrual phase (figure 2J), we were able to\nfind TLR4 protein localized on immune cells (figure 6F, G,\nH, I). We performed co- immunostainings on controls\nand on malignant endometrial tissues (G2 carcinoma)\nwith antibodies for CD14 and CD163 (figure 7). TLR4\nprotein was expressed on CD14 positive dendritic cells,\nand monocytes (figure 7A, C), as well as on CD163 posi-\ntive macrophages (figure 7B, D).\nTLR3 and TLR4 mRNA expression is regulated in endometri-osisFigure 3\nTLR3 and TLR4 mRNA expression is regulated in \nendometriosis. The expression of TLR3 (A, C) and TLR4 \nmRNA (B, D) in endometrium during proliferative (n = 13, \nrun in triplicates, A, B) and secretory phase (n = 3, C, D) \nwas decreased in eutopic endometriotic endometrium when \ncompared to controls. In addition, four proliferative corre-\nsponding lesions were evaluated (A, B) showing a local \nupregulation of both receptors on ectopic sites. Columns \nrepresent the mean ratio of TLR copy number to ACTB \ncopy number. Error bars represent the standard deviation of \nthe mean. * P < 0.05; ** P < 0.01.\nTLR3 and TLR4 protein is locally induced in endometriotic lesionsFigure 4\nTLR3 and TLR4 protein is locally induced in endome-\ntriotic lesions. No specific TLR3 protein staining was seen \nin eutopic endometriotic tissue (A) whereas a high glandular \nlocalisation of the protein was detected in a gland of an \nectopic endometriotic lesion from the same patient (B). Sim-\nilarly, TLR4 was not detectable in eutopic endometrium (C) \nbut present in glandular epithelium of ectopic endometrium \nfrom the same women (D).\n\n\nReproductive Biology and Endocrinology 2008, 6:40 http://www.rbej.com/content/6/1/40\nPage 7 of 11\n(page number not for citation purposes)\nDiscussion\nIn the current study, we report that toll-like receptor 3 and\n4 expression is modulated in pathogenic alterations of the\nendometrium. We also found higher TLR4 expression lev-\nels in endometrial samples throughout the menstrual\ncycle and in postmenopausal biopsies, when compared to\nthose for TLR3. In most tissues including gut, gonads and\nplacenta, TLR3 is greater expressed than TLR4 mRNA [32].\nTLR3 recognizes RNA and viruses, whereas TLR4 mediates\nthe response to bacterial endotoxins and is activated due\nto sterile inflammation [33,34]. Thus, the predominant\nexpression of TLR4, observed in uterine tissues, might\nreflect the occurrence of sterile inflammation during the\nmenstrual cycle. Moreover, ascending bacterial pathogens\ncould contribute to the TLR4 dominance in the uterus.\nIn agreement with earlier reports [4,12], both investigated\nTLRs were mainly localized in the endometrial epithe-\nlium, the site of primary immune response in the uterus.\nIn addition, we were able to detect TLR4 protein on\nendometrial CD14 and CD163 positive immune cells. We\nfound CD14 mainly expressed within the epithelial layer,\nonly a sporadic number of CD14 positive cells was\ndetected in stromal compartment, probably representing\nthe population of monocytes. A recent study performed\non bovine endometrial cells, co-localised TLR4 transcripts\nTLR3 and TLR4 mRNA expression is decreased in endometrial adenocarcinomaFigure 5\nTLR3 and TLR4 mRNA expression is decreased in endometrial adenocarcinoma. (A-B) Columns indicate mean \nTLR3 (A) and TLR4 (B) mRNA levels from postmenopausal patients (PMP, n = 8), and those diagnosed with endometrial \nhyperplasia (HP, n = 10) and endometrial carcinoma (EnCa, n = 16). (C-D) TLR3 (C) and TLR4 (D) mRNA expression in dif-\nferent carcinoma grades compared to postmenopausal controls and hyperplastic endometrium: G1 (n = 5), G2 (n = 6) and G3 \n(n = 5). Error bars represent the standard deviation of the mean. * P < 0.05; ** P < 0.01.\n\n\nReproductive Biology and Endocrinology 2008, 6:40 http://www.rbej.com/content/6/1/40\nPage 8 of 11\n(page number not for citation purposes)\nwith CD14 mRNA and protein to stromal and epithelial\ncells [35]. CD14 is a known accessory molecule for TLR4\nand conducts a downstream signalling cascade via MyD88\n[3]. In agreement with Pioli et al., who detected TLR4,\nCD14 and MyD88 transcripts in human endometrium\n[14], we were able to co-localize TLR4 with CD14 proteins\nsuggesting the presence of both interacting receptors\nCD14 and TLR4 in the endometrial cells.\nFor the first time, we present endometrial effluents\nexpressing high levels of TLR3 and TLR4 proteins. Since\nthe period of menstruation is accompanied by an\nincreased risk of infections due to ascending microorgan-\nisms [36], we believe that the increased expression of toll-\nlike receptors may be one of the defense mechanisms used\nby the uterus. Previous studies reported that toll-like\nreceptors are also implicated in epithelial repair as\ndescribed for intestinal [6] and alveolar epithelial cells\n[37]. In damaged tissue, necrosis induced inflammation is\nthought to trigger danger signals, leading to tissue repair\nresponse through TLRs [6]. Since repair processes occur\nevery month in the uterus of premenopausal women, we\nbelieve that the interaction between hyaluronan and TLR4\nmight promote the endometrial repair. Hyaluronan is\nreleased by necrotic cells, interacts with TLR4 and acti-\nvates CD44 mediated signalling [38]. In the\nendometrium, deposition of hyaluronan has been\ndescribed in stromal compartment [39]. Moreover,\nhyaluronan has been reported to be involved in attach-\nment of endometrial cells to the mesothelium as a very\nearly step of endometriosis [40]. Further investigations of\nhyaluronan-TLR4 signalling in healthy and diseased\nendometrium would be of interest to gain insight into the\nfunctional role of TLR4 expression in the uterus.\nEndometriosis causes chronic inflammatory conditions in\nthe pelvic cavity and in the uterus. This disorder is dis-\ncussed to be accompanied by an activation of the Th2 type\nof immune response and a shift from Th1 towards Th2\ncytokine production [41]. Interestingly, Th2 cytokines\nwere shown to play an important role in balancing TLR\nsignalling in human intestinal epithelial cells by mediat-\ning downregulation of TLR3 and TLR4 expression and\nfunction [42]. This could also be the case in the diseased\neutopic endometrium, where decreased TLR levels were\nfound. It remains to be fully elucidated, if deregulation of\nTLR expression is involved in the pathogenesis of\nendometriosis or if altered TLR expression patterns are a\nconsequence resulting from the presence of endometriotic\nlesions. We could recently show that uterine gene expres-\nsion patterns are altered due to the existence of ectopic\nlesions in a non-human primate model for endometriosis\n[43,44]. Since implantation is a process accompanied by\nan inflammatory event, an impaired fertility observed in\nendometriotic women could be one consequence [45].\nContinued studies are needed to determine the role of\nTLR function in diseased endometrium, which could be a\npromising path towards a better understanding of the\npathogenesis of this disease.\nInterestingly, we found a local upregulation of both TLRs\nin peritoneal endometriotic lesions when compared to\nTLR3 and TLR4 proteins are present in postmenopausal endometriumFigure 6\nTLR3 and TLR4 proteins are present in postmenopausal endometrium. Localisation of TLR3 in normal postmeno-\npausal endometrium (A), endometrial hyperplasia (B), endometrial adenocarcinoma grade G1 (C), G2 (D) and G3 (E). Local-\nisation of TLR4 protein in normal postmenopausal endometrium (F), endometrial hyperplasia (G), endometrial \nadenocarcinoma grade G1 (H), G2 (I) and G3(J). All stained sections indicated epithelium as the preferred localisation of \nTLR3 and TLR4 proteins. TLR4 protein was additionally present in immune cells (arrows).\n\n\nReproductive Biology and Endocrinology 2008, 6:40 http://www.rbej.com/content/6/1/40\nPage 9 of 11\n(page number not for citation purposes)\neutopic endometriosis from the same patients. A recent\nstudy presented a local upregulation of CD14 and CD163\nin ovarian endometriotic lesions [46]. However, we\nobserve locally gained TLR4 expression in epithelial cells\nas demonstrated in immunohistochemical stainings. We\npropose that the sterile inflammation process, which\noccurs in the pelvic cavity upon endometriosis, is able to\nenhance the epithelial TLR4 expression and thus activate\nTLR4 is localised to immune cells of postmenopausal endometriumFigure 7\nTLR4 is localised to immune cells of postmenopausal endometrium. Co-Immunostaining of TLR4 with CD14 and \nCD163 in healthy endometrium (A, B) and in adenocarcinoma (C, D). TLR4 proteins were expressed by CD14 positive den-\ndritic cells and monocytes (A, C) and by CD163 positive macrophages (B, D). Arrows indicate the co-localisation of TLR4 \nwith the immune cells. Scale bar = 20 μm.\n\n\nReproductive Biology and Endocrinology 2008, 6:40 http://www.rbej.com/content/6/1/40\nPage 10 of 11\n(page number not for citation purposes)\nthe known downstream signalling cascade. One of the\npotentially activated TLR-downstream molecules is NF-\nκB, which was recently found as constitutively elevated in\nendometriotic lesions [20]. It is established that the acti-\nvation of NF- κB is linked to proliferation, angiogenesis\nand enhanced production of inflammatory cytokines on\nectopic sites [17]. Hence, the TLR-NF- κB cascade might\ncontribute to the chronic persistence of endometriotic\nlesions.\nIn endometrial adenocarcinoma, expression levels of the\ndownstream molecules TNF α and NF- κB were decreased\nin G2 and G3 but not in the well-differentiated grade 1\ncarcinoma [47]. Although the evidence is lacking, the\nalmost negligible levels of both toll-like receptors in G3\nendometrial adenocarcinoma may reflect lowered differ-\nentiation and possibly indicates poor prognosis.\nBoth endometriosis and endometrial adenocarcinoma are\nestrogen dependent diseases. In both conditions, TLR3\nand TLR4 were significantly decreased in diseased\nendometrium when compared to age matched controls.\nHowever, it is known that estrogen did not influence the\nexpression of either TLR3 [48] nor TLR4 [49,50] in epithe-\nlial cells of endometrium [48], retina [50] and in macro-\nphages [49]. Thus, additional factors are required to\ndecrease TLR-expression in endometriotic endometrium\nand in endometrial carcinoma.\nBesides excessive estrogen, genetic predisposition presents\none of the risk factors associated with the development of\nendometrial adenocarcinoma. In our study, we observed\nhigh inter-individual differences in TLR expression as mir-\nrored by high standard deviations. The results implicate a\npossible impact of polymorphisms on mRNA expression\nin physiologic and pathologic endometrium. Recently, a\nfunctional polymorphism of the TLR4 gene, associated\nwith impaired TLR signalling, was considered as a signifi-\ncant risk factor for gastric carcinoma [51]. Another single\nnucleotide polymorphism in 3'-untranslated region of the\nsame gene has been associated with increased risk for\nprostate carcinoma [52]. It remains to be fully elucidated,\nif genetic polymorphisms in genes encoding for toll-like\nreceptors might promote endometrial carcinogenesis.\nConclusion\nOur data suggest an involvement of TLR3 and TLR4 in\nendometrial diseases as we demonstrated altered expres-\nsion levels for both receptors in endometriosis and\nendometrial adenocarcinoma. Healthy and differentiated\nendometrium seems to require an adequate TLR3 and\nTLR4 expression. Further studies are necessary to investi-\ngate the potential function of both receptors in endome-\ntrial diseases.\nCompeting interests\nThe authors declare that they have no competing interests.\nAuthors' contributions\nSA processed tissue samples, established the TLR-assays,\ncarried out the expression analyses, analyzed data, and\ndrafted the manuscript. CB participated in the design of\nthe study, collected patients' tissues, and was involved in\nthe analyses of data. AAK was involved in tissue process-\ning and expression analyses. RK participated in the design\nand interpretation of the study. IG conceived the study,\nparticipated in its design, coordination, and analysis, and\nhelped to draft the manuscript. All authors have read and\napproved the final manuscript.\nAcknowledgements\nWe appreciate the support of Prof. Dr. Elke Winterhager, Institute of Anat-\nomy II, University of Duisburg-Essen. We thank Georgia Rauter for her \nexcellent technical assistance and Claudia Jacobs for her support by manag-\ning of patients' data.\nReferences\n1. Takeda K, Kaisho T, Akira S: Toll-like receptors.  Annu Rev Immunol\n2003, 21:335-376.\n2. Cario E, Rosenberg IM, Brandwein SL, Beck PL, Reinecker HC, Podol-\nsky DK: Lipopolysaccharide activates distinct signaling path-\nways in intestinal epithelial cell lines expressing Toll-like\nreceptors.  J Immunol 2000, 164(2):966-972.\n3. Palsson-McDermott EM, O'Neill LA: Signal transduction by the\nlipopolysaccharide receptor, Toll-like receptor-4.  Immunology\n2004, 113(2):153-162.\n4. Fazeli A, Bruce C, Anumba DO: Characterization of Toll-like\nreceptors in the female re productive tract in humans.  Hum\nReprod 2005, 20(5):1372-1378.\n5. Schaefer TM, Desouza K, Fahey JV, Beagley KW, Wira CR: Toll-like\nreceptor (TLR) expression and TLR-mediated cytokine/\nchemokine production  by human uterine epithelial cells.\nImmunology 2004, 112(3):428-436.\n6. Stenson WF: Toll-like receptors and intestinal epithelial\nrepair.  Curr Opin Gastroenterol 2008, 24(2):103-107.\n7. Kariko K, Ni H, Capodici J, Lamphier M, Weissman D: mRNA is an\nendogenous ligand for Toll-like receptor 3.   J Biol Chem  2004,\n279(13):12542-12550.\n8. Mollen KP, Anand RJ, Tsung A, Prince JM, Levy RM, Billiar TR:\nEmerging paradigm: toll-like re ceptor 4-sentinel for the\ndetection of tissue damage.  Shock 2006, 26(5):430-437.\n9. Jorgenson RL, Young SL, Lesmeister MJ, Lyddon TD, Misfeldt ML:\nHuman endometrial ep ithelial cells cyclically express Toll-\nlike receptor 3 (TLR3) and exhibit TLR3-dependent\nresponses to dsRNA.  Hum Immunol 2005, 66(5):469-482.\n10. Hirata T, Osuga Y, Hamasaki K,  H i r o t a  Y ,  N o s e  E ,  M o r i m o t o  C ,\nHarada M, Takemura Y, Koga K, Yoshino O, Tajima T, Hasegawa A,\nYano T, Taketani Y: Expression of toll-like receptors 2, 3, 4, and\n9 genes in the human endometrium during the menstrual\ncycle.  J Reprod Immunol 2007, 74(1–2):53-60.\n11. Aflatoonian R, Tuckerman E, Elliott SL, Bruce C, Aflatoonian A, Li TC,\nFazeli A: Menstrual cycle-dependent changes of Toll-like\nreceptors in endometrium.  \nHum Reprod 2007, 22(2):586-593.\n12. Young SL, Lyddon TD, Jorgenson RL, Misfeldt ML: Expression of\nToll-like receptors in human endometrial epithelial cells and\ncell lines.  Am J Reprod Immunol 2004, 52(1):67-73.\n13. Hirata T, Osuga Y, Hirota Y, Ko ga K, Yoshino O, Harada M, Morim-\noto C, Yano T, Nishii O, Tsutsumi O, Taketani Y: Evidence for the\npresence of toll-like recep tor 4 system in the human\nendometrium.  J Clin Endocrinol Metab 2005, 90(1):548-556.\n14. Pioli PA, Amiel E, Schaefer TM, Connolly JE, Wira CR, Guyre PM: Dif-\nferential expression of Toll-li ke receptors 2 and 4 in tissues\nof the human female reproductive tract.  Infect Immun 2004,\n72(10):5799-5806.\n\nPublish with BioMed Central   and  every \nscientist can read your work free of charge\n\"BioMed Central will be the most significant development for \ndisseminating the results of biomedical research in our lifetime.\"\nSir Paul Nurse, Cancer Research UK\nYour research papers will be:\navailable free of charge to the entire biomedical community\npeer reviewed and published immediately upon acceptance\ncited in PubMed and archived on PubMed Central \nyours — you keep the copyright\nSubmit your manuscript here:\nhttp://www.biomedcentral.com/info/publishing_adv.asp\nBioMedcentral\nReproductive Biology and Endocrinology 2008, 6:40 http://www.rbej.com/content/6/1/40\nPage 11 of 11\n(page number not for citation purposes)\n15. Giudice LC, Kao LC: Endometriosis.  Lancet 2004,\n364(9447):1789-1799.\n16. Kyama CM, Debrock S, Mwenda JM, D'Hooghe TM: Potential\ninvolvement of the immune system in the development of\nendometriosis.  Reprod Biol Endocrinol 2003, 1(1):123.\n17. Guo SW: Nuclear factor-kappab (NF-kappaB): an unsus-\npected major culprit in the pa thogenesis of endometriosis\nthat is still at large?  Gynecol Obstet Invest 2007, 63(2):71-97.\n18. Arici A: Local cytokines in endometrial tissue: the role of\ninterleukin-8 in the pathogenesis of endometriosis.   Ann N Y\nAcad Sci 2002, 955:101-109. discussion 118, 396–406.\n19. Iwabe T, Harada T, Terakawa N: Role of cytokines in endometri-\nosis-associated infertility.  Gynecol Obstet Invest  2002, 53(Suppl\n1):19-25.\n20. Gonzalez-Ramos R, Donnez J, Defrere S, Leclercq I, Squifflet J, Lousse\nJC, Van Langendonckt A: Nuclear factor-kappa  B is constitu-\ntively activated in peritoneal endometriosis.  Mol Hum Reprod\n2007, 13(7):503-509.\n21. Amant F, Moerman P, Neven P, Timmerman D, Van Limbergen E,\nVergote I: Endometrial cancer.  Lancet 2005, 366(9484):491-505.\n22. Coussens LM, Werb Z: Inflammation and cancer.  Nature 2002,\n420(6917):860-867.\n23. Modugno F, Ness RB, Chen C, Weiss NS: Inflammation and\nendometrial cancer: a hypothesis.   Cancer Epidemiol Biomarkers\nPrev 2005, 14(12):2840-2847.\n24. Gashaw I, Stiller S, Boing C, Kimmig R, Winterhager E: Pre-men-\nstrual regulation of the pr o-angiogenic factor CYR61 in\nhuman endometrium.  Endocrinology 2008, 149(5):2261-2269.\n25. Noyes RW, Hertig AT, Rock J: Dating the endometrial biopsy.\nFertil Steril 1950, 1(1):3-25.\n26. Silverberg SG, Kurman RJ, Nogales  F, Mutter GL, Kubik-Huch RA,\nTavassoli FA: Tumours of the uterine corpus: epithelial\ntumours and related lesions.  In Pathology and Genetics of Tumours\nof the Breast and Female Genital Organs Edited by: Tavassoli FA, Devilee\nP. Lyon: IARC Press; 2003:221-232. \n27. Furrie E, Macfarlane S, Thomson G, Macfarlane GT: Toll-like recep-\ntors-2, -3 and -4 expression patterns on human colon and\ntheir regulation by mucosal-associated bacteria.   Immunology\n2005, 115(4):565-574.\n28. Kumazaki K, Nakayama M, Yanagihara I, Suehara N, Wada Y: Immu-\nnohistochemical distribution of Toll-like receptor 4 in term\nand preterm human placentas from normal and complicated\npregnancy including chorioamnionitis.   Hum Pathol  2004,\n35(1):47-54.\n29. Koski GK, Lyakh LA, Cohen PA, Rice NR: CD14+ monocytes as\ndendritic cell precursors: diverse maturation-inducing path-\nways lead to common activation of NF-kappab/RelB.  Crit Rev\nImmunol 2001, 21:1-3.\n30. Fabriek BO, Dijkstra CD, Berg TK van den: The macrophage scav-\nenger receptor CD163.  Immunobiology 2005, 210(2–4):153-60.\n31. Blaschitz A, Weiss U, Dohr G, Desoye G: Antibody reaction pat-\nterns in first trimester placenta: implications for trophoblast\nisolation and purity screening.  Placenta 2000, 21(7):733-741.\n32. Zarember KA, Godowski PJ: Tissue expression of human Toll-\nlike receptors and differential  regulation of Toll-like recep-\ntor mRNAs in leukocytes in response to microbes, their\nproducts, and cytokines.  J Immunol 2002, 168(2):554-561.\n33. Takeda K, Akira S: Toll-like receptors in innate immunity.  Int\nImmunol 2005, 17(1):1-14.\n34. Jiang D, Liang J, Li Y, Noble PW: The role of Toll-like receptors\nin non-infectious lung injury.  Cell Res 2006, 16(8):693-701.\n35. Herath S, Fischer DP, Werling D, Williams EJ, Lilly ST, Dobson H,\nBryant CE, Sheldon IM: Expression and function of Toll-like\nreceptor 4 in the endometrial cells of the uterus.  Endocrinology\n2006, 147(1):562-570.\n36. Eschenbach DA: Acute pelvic inflammatory disease: etiology,\nrisk factors and pathogenesis.   Clin Obstet Gynecol  1976,\n19(1):147-169.\n37. Noble PW, Jiang D: Matrix regulation of lung injury, inflamma-\ntion, and repair: the role  of innate immunity.  Proc Am Thorac\nSoc 2006, 3(5):401-404.\n38. Taylor KR, Trowbridge JM, Rudisi ll JA, Termeer CC, Simon JC, Gallo\nRL: Hyaluronan fragments stimulate endothelial recognition\nof injury through TLR4.  J Biol Chem 2004, 279(17):17079-17084.\n39. Salamonsen LA, Shuster S, Stern R: Distribution of hyaluronan in\nhuman endometrium across the menstrual cycle. Implica-\ntions for implantation and menstruation.   Cell Tissue Res 2001,\n306(2):335-340.\n40. Dechaud H, Witz CA, Montoya-Rodriguez IA, Degraffenreid LA,\nSchenken RS: Mesothelial cell-associated hyaluronic acid pro-\nmotes adhesion of endome trial cells to mesothelium.   Fertil\nSteril 2001, 76(5):1012-1018.\n41. Podgaec S, Abrao MS, Dias JA Jr, Rizzo LV, de Oliveira RM, Baracat\nEC: Endometriosis: an inflammatory disease with a Th2\nimmune respon se component.   Hum Reprod  2007,\n22(5):1373-1379.\n42. Mueller T, Terada T, Rosenber g IM, Shibolet O, Podolsky DK: Th2\ncytokines down-regulate TLR expression and function in\nhuman intestinal epithelial cells.   J Immunol  2006,\n176(10):5805-5814.\n43. Gashaw I, Hastings JM, Jackson K, Winterhager E, Fazleabas AT:\nInduced endometriosis in the baboon (Papio anubis)\nincreases the expression of the proangiogenic factor CYR61\n(CCN1) in eutopic and ectopic endometria.  Biol Reprod 2006,\n74(6):1060-1066.\n44. Hastings JM, Jackson KS, Mavrogianis PA, Fazleabas AT: The estro-\ngen early response gene FOS is altered in a baboon model of\nendometriosis.  Biol Reprod 2006, 75(2):176-182.\n45. Iborra A, Palacio JR, Martinez P: Oxidative stress and autoim-\nmune response in the infertile woman.   Chem Immunol Allergy\n2005, 88:150-162.\n46. Hever A, Roth RB, Hevezi P, Mari n ME, Acosta JA, Acosta H, Rojas J,\nHerrera R, Grigoriadis D, White E, Conlon PJ, Maki RA, Zlotnik A:\nHuman endometriosis is associated with plasma cells and\noverexpression of B lymphocyte stimulator.  Proc Natl Acad Sci\nUSA\n 2007, 104(30):12451-12456.\n47. Vaskivuo TE, Stenback F, Tapanainen JS: Apoptosis and apoptosis-\nrelated factors Bcl-2, Bax, tu mor necrosis factor-alpha, and\nNF-kappaB in human endometrial hyperplasia and carci-\nnoma.  Cancer 2002, 95(7):1463-1471.\n48. Lesmeister MJ, Jorgenson RL, Young SL, Misfeldt ML: 17Beta-estra-\ndiol suppresses TLR3-induced cytokine and chemokine pro-\nduction in endometrial epithelial cells.   Reprod Biol Endocrinol\n2005, 3:74.\n49. Vegeto E, Ghisletti S, Meda C, Etteri S, Belcredito S, Maggi A: Regu-\nlation of the lipopolysaccharide signal transduction pathway\nby 17beta-estradiol in macrophage cells.   J Steroid Biochem Mol\nBiol 2004, 91(1–2):59-66.\n50. Paimela T, Ryhanen T, Mannermaa E, Ojala J, Kalesnykas G, Salminen\nA, Kaarniranta K: The effect of 17beta-estradiol on IL-6 secre-\ntion and NF-kappaB DNA-binding activity in human retinal\npigment epithelial cells.  Immunol Lett 2007, 110(2):139-144.\n51. Hold GL, Rabkin CS, Chow WH, Smith MG, Gammon MD, Risch HA,\nVaughan TL, McColl KE, Lissowska J, Zatonski W, Schoenberg JB, Blot\nWJ, Mowat NA, Fraumeni JF Jr, El-Omar EM: A functional poly-\nmorphism of toll-like receptor 4 gene increases risk of gas-\ntric carcinoma and its precursors.   Gastroenterology 2007,\n132(3):905-912.\n52. Zheng SL, Augustsson-Balter K, Ch ang B, Hedelin M, Li L, Adami HO,\nBensen J, Li G, Johnasson JE, Turner AR, Adams TS, Meyers DA, Isaacs\nWB, Xu J, Gronberg H: Sequence variants of toll-like receptor\n4 are associated with prostate cancer risk: results from the\nCAncer Prostate in Sweden Study.   Cancer Res  2004,\n64(8):2918-2922.","source_license":"CC0","license_restricted":false}