A distinct cohort of the TGFβ superfamily members expressed in human endometrium regulate decidualization

BACKGROUND Successful blastocyst implantation requires the differentiation of human endometrial stromal cells (HESC), a process known as decidualization. Activin A, a transforming growth factor β (TGFβ) superfamily member, enhances HESC decidualization and localizes to decidual cells in human endometrium. Other TGFβ superfamily members, including BMP2, BMP4, BMP7, GDF5, GDF8, GDF11, TGFβs and Nodal, may also play a role during decidualization. This study aimed to identify these TGFβ family members in human endometrium, and to determine whether they are involved in human decidualization.
IntroductionThroughout each menstrual cycle the human endometrium undergoes morphological and physiological changes, in preparation for pregnancy should the cycle include conception (Loke et al., 1995). An absolute requirement for successful implantation is the differentiation and proliferation of endometrial stromal cells into enlarged, phenotypically different decidual cells, a process termed endometrial decidualization (Tang et al., 1994; Salamonsen et al., 2003). This process is initiated near the spiral arterioles (Bell, 1991), and occurs under the influence of progesterone (Psychoyos, 1973), although cAMP appears to be essential for priming the endometrial stromal cells to the actions of progesterone; hence both pathways are absolutely required for decidualization (Gellersen and Brosens, 2003). It is now clear that the decidualization response is mediated by a complex array of bioactive molecules, including IL-11, prostaglandin E2, relaxin and activins (Frank et al., 1994; Robb et al., 1998; Jones et al., 2002; Dimitriadis et al., 2005).The transforming growth factor β (TGFβ) superfamily is a large family of proteins that encompasses the sub-families TGFβs, activins, bone morphogenetic proteins (BMPs) and growth differentiation factors (GDFs). Overall, this superfamily of proteins exhibits functional diversity, with biological roles in cell differentiation, proliferation, apoptosis and tissue remodelling, all consistent with various reproductive processes (Jones et al., 2006).Activins consist of two β subunits, βA and βB, that homo/heterodimerize to form activin A, activin B and activin AB, respectively. The activin β subunits are produced in the glandular epithelium during the proliferative and secretory phases, but in stromal cells only following decidualization during the mid-late secretory phase (Otani et al., 1998; Jones et al., 2000; Mylonas et al., 2004). High levels of dimeric activin A are secreted by cAMP-treated endometrial stromal cells. Addition of activin A to decidualizing cells in vitro significantly increased the secretion of prolactin (PRL) and IGFBP-1 (decidual cell markers), suggesting activin A drives decidualization (Jones et al., 2002; Tierney and Giudice, 2004). This decidualization response can be neutralized by follistatin, the naturally occurring activin antagonist (Jones et al., 2002; Tierney and Giudice, 2004). In addition to its tight regulation of activin (Shimonaka et al., 1991; Schneyer et al., 1994), follistatin can also bind and regulate other TGFβ members including the BMPs and GDFs (Table I).Table I.Inhibitors of TGFβ superfamily: ligand–receptor blocking.LigandFollistatinActivin-M108ASB431542Binds ligands and blocks association with type II receptorsActivin type II receptor (ActRIIA/B) antagonistInhibitor of TGFβ and activin type I receptorsActivin A++++ (Schneyer et al., 2003)++ (Harrison et al., 2004)+++ (Inman et al., 2002)Activin B++ (Schneyer et al., 2003)+++a+++ (Inman et al., 2002)GDF 8++ (Amthor et al., 2004)+++a+++ (Inman et al., 2002)GDF11++ (Gamer et al., 1999)+++a+++ (Inman et al., 2002)BMP2+ (Iemura et al., 1998)−−BMP4+ (Iemura et al., 1998; Glister et al., 2004)−−BMP7+ (Iemura et al., 1998; Glister et al., 2004)(−/+)−GDF5(+)−−Nodal−(+++)+++ (Inman et al., 2002)TGFβ1−−+++ (Inman et al., 2002)TGFβ2−−+++ (Inman et al., 2002)TGFβ3−−+++ (Inman et al., 2002)aC.A. Harrison (unpublished observations); (), anticipated activity.BMPs have been identified in the rodent uterus (Cunningham et al., 1995; Ozkaynak et al., 1997; Fitzpatrick et al., 1998; Zhao et al., 1999; Ying and Zhao, 2000; Erickson et al., 2004; Lee et al., 2007; Li et al., 2007). In pregnant mice the temporospatial localization of BMPs 2, 4, 6 and 7 suggested roles during pregnancy (Ying and Zhao, 2000). BMP2 is present within the decidual area at implantation sites (Ying and Zhao, 2000; Li et al., 2007) and plays a role in decidualization in vitro (Li et al., 2007). Importantly, conditional ablation of uterine BMP2 in mice resulted in ineffective decidualization and disrupted pregnancy outcome (Lee et al., 2007). In a human model of endometrial stromal cell (HESC) decidualization in vitro, BMP2 mRNA increased during decidualization and addition of BMP2 to cultures accelerated PRL mRNA expression (a measure of the extent of decidualization). BMP2 protein has not been examined in vivo in human endometrial tissue. The only GDFs identified in the uterus are GDF9 and GDF10, detected by northern analysis in both human and mouse (Cunningham et al., 1995; Fitzpatrick et al., 1998; Zhao et al., 1999): no studies have shown GDF proteins.TGFβ isoforms (1–3), which cannot bind follistatin, have also been localized in human endometrial stroma (Jones et al., 2006), although it is not clear whether any isoform is specific to decidualized cells or increases as the cells decidualize. The TGFβs transmit signal through the TGFβ type I, II and III receptors, all of which are present on endometrial stromal cells (Chegini et al., 1994; Dumont et al., 1995). Microarray studies have demonstrated increases in both TGFβ1 and TGFβ2 mRNA with HESC decidualization in vitro (Popovici et al., 2000; Tierney et al., 2003), while in women treated with medroxyprogesterone acetate (MPA) for 10 days, stromal mRNA and protein increased for TGFβ3 but not TGFβ1 (Reis et al., 2002). Whether the TGFβs play a functional role during human decidualization remains unknown.Given that the TGFβ superfamily of proteins are such common and crucial differentiative and proliferative factors, it is surprising that very few members of the family, including the GDFs and BMPs, have been examined in the human endometrium, particularly in the mid-secretory phase when differentiation of stromal cells is initiated. The high level of redundancy between TGFβ family members and the promiscuity of receptor–ligand interactions would suggest that it is likely that a number of other family members are involved in decidualization and in the other processes occurring in this highly dynamic tissue.In this study, the presence of BMPs (BMP2, BMP4, BMP7), GDFs (GDF5, GDF8/Myostatin, GDF11), Nodal and TGFβs were examined in vivo by immunohistochemistry in secretory phase endometrial tissue, and in vitro for mRNA expression in both non-decidualized and decidualized HESC. Inhibitors with different specificities were administered to HESC to elucidate whether activin is the major family member driving decidualization or whether other family groupings (BMPs, GDFs, TGFβs) might equally contribute to the process. As these are secreted factors, individual ligands were measured in conditioned medium from non-decidualized and decidualized HESC to assess whether they increased with decidualization. The effect of the secreted ligands on decidualization was also examined. It is important to identify which TGFβ members are important during decidualization since this process is critical for implantation and the establishment of pregnancy.
Materials and MethodsTissue collectionsEndometrial biopsies (n = 18) were collected by dilatation and curettage from fertile women who were scheduled for tubal ligation or were undergoing testing for tubal patency. Tissues were assessed by a pathologist and had no obvious endometrial pathology. The women had no steroid treatment or other medication for at least 2–3 months before the collection of tissue. Written and informed consent was obtained from all women participating in the study, and the protocols were approved by Monash Medical Centre Human Ethics Committee.ImmunohistochemistryImmunohistochemical analysis was performed using a total of nine endometrial tissue biopsies from fertile women, confirmed by Noyes criteria (Noyes et al., 1975) as mid-late secretory phase tissue (POD 6–12). For each of the antibodies, activin βA and βB, BMP2, BMP4, BMP7, GDF5, GDF8, GDF11, Nodal and TGFβ1, n = 4–5 different tissue biopsies were used. For immunolocalization in first trimester placental tissue (kindly provided by Professor Euan Wallace, Obstetrics and Gynaecology, Monash University, Melbourne, Australia), n = 2 different biopsies were used per antibody. Briefly, 5 µm sections of formalin-fixed, paraffin-embedded tissues were dewaxed and rehydrated. For each antibody an antigen retrieval step was required, which involved microwave exposure or trypsin digestion (0.01% in CaCl2for 10 min at 37°C). Endogenous hydrogen peroxidase activity was quenched using 3% H2O2 in dH2O for 10 min at room temperature. Non-specific binding was prevented by pre-incubation of tissue sections with a non-immune block [5% fetal calf serum (FCS), 2% normal human serum in 0.1% Tween/Tris-buffered saline (TBS) with addition of 10% normal horse serum for BMP2, BMP4, BMP7, GDF5, GDF8; 10% normal swine serum for Nodal; normal goat serum for activin βA and βB, GDF11, TGFβ1]. Primary antibodies were against activin βA and activin βB (400 and 600 µg/ml, respectively; both gifts provided by W. Vale, Salk Institute, La Jolla, CA, USA); BMP2, BMP4, BMP7, GDF5, GDF8, Nodal (200 µg/ml; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); GDF11 (1 mg/ml; Orbigen, San Diego, CA, USA); TGFβ1 (μg/ml; Abcam, Cambridge, UK). Specificity for these antibodies has been previously published (Nadiri et al., 2004; Kiyono and Shibuya, 2003; Mendler et al., 2000; McPherron et al., 1999; Garba and Relinger, 2001). For GDF5 and Nodal, the specificity has been shown by western blot analysis (unpublished observation). Antibodies were diluted to 2–4 µg/ml in the non-immune block and applied overnight (18–20 h) at 4°C. A non-immune goat/rabbit/mouse IgG (R&D Systems Incorporated, Minneapolis, MN, USA) was used at a matching concentration to the primary antibody and was included for each tissue. After stringent washing with high salt TBS and 0.6% Tween 20 (BioRad Laboratories, Hercules, CA, USA), detection of positive binding was performed by the sequential application of the appropriate secondary antibody diluted in non-immune block and avidin–biotin–peroxidase conjugate (Dako, Glostrup, Denmark). This was followed by the substrate diaminobenzidine (Dako) for between 3 and 5 min. Sections were counterstained with Harris' hematoxylin (Sigma Chemical Company, St Louis, MO, USA), dehydrated, and mounted from Histosol with DPX mounting medium (BDH Laboratory Supplies, Poole, UK). Immunostaining was analysed by two independent observers for staining intensity and heterogeneity in each of the endometrial compartments (glandular and luminal epithelium; stroma, including decidualized stromal cells; vasculature).Stromal cell isolation and cultureTissue biopsies ranging from Days 10–21 were used for in vitro decidualization studies. HESC were isolated from tissue by enzymatic digestion and filtration as described previously (Dimitriadis et al., 2002). Briefly, tissue was finely minced with scissors and digested by bacterial collagenase type III (Worthington Biochemical Corporation, Freehold, NJ, USA) at a concentration of 45 IU/ml, in the presence of 3.5 µg/ml deoxyribonuclease (DNase; Boehringer Mannheim Biochemica, Mannheim, Germany). After 30–45 min of agitation at 37°C, the digested tissue was filtered sequentially through 45 and 10 µm nylon filters to remove glands. Cell suspensions were centrifuged at 1500 rpm for 5 min. Cells were plated at a density of 5 × 106 in a 75 cm2 flask and grown to confluency in DMEM and Ham's F12 medium (1:1) (DMEM/F12) (Trace Biosciences, Sydney, Australia), supplemented with 1% PSF (penicillin, streptomycin and fungizone) (Commonwealth Serum Laboratories, Melbourne, Australia) and 10% charcoal stripped FCS (csFCS) (Thermo Scientific, Maple Plain, USA). Approximately 30 min after plating, medium containing non-attached epithelial cells was removed and replaced with fresh medium. This results in a >97% pure stromal cell culture (Dimitriadis et al., 2002).In vitro decidualizationConfluent HESC were rinsed with phosphate-buffered saline, trypsinized and replated into 24-well plates using DMEM/F12 and 10% csFCS. Once confluent, HESC were washed with DMEM/F12 and the medium replaced with a serum-free medium containing DMEM/F12 and a serum-free mix (SFM) including transferrin (10 µg/ml; Sigma), sodium selenite (25 ng/ml; Sigma), linoleic acid (10 nmol/L; Sigma), bovine serum albumin (0.1%; Sigma) and insulin (5 µg/ml; Actrapid, Novo-Nordisk Pharmaceuticals Pty Ltd, Sydney, Australia) for 48 h prior to treatment addition. HESC were decidualized by two distinct methods as previously described (Dimitriadis et al., 2005). For the inhibitor, mRNA expression and protein secretion studies, 0.5 mM cAMP (Sigma) was added to medium for 4–6 days. Alternatively, when exogenous proteins were added, cells were decidualized with E2 (10−8 M; Sigma) and MPA (10−7M; Sigma) for 8 days. All media were replaced every 48–72 h.RNA extraction and cDNA synthesisTotal RNA was extracted from non-decidualized and decidualized HESC from two separate biopsies at Day 4–6 using the RNeasy Minikit (Qiagen Sciences, Germantown, MD, USA), according to the manufacturer's instructions. RNA from positive control tissues was extracted by homogenization in Trizol reagent (Qiagen Sciences, Clifton Hill, Victoria, Australia), according to the manufacturer's instructions, with the exception of an additional chloroform extraction step to minimize carryover of phenol into the precipitate. The control tissues used were: term placenta (activin βA, βB, GDF11, TGFβ1, -2, -3); pregnant mouse endometrium (BMP2); mature rat ovary (BMP4, GDF5); cycling mouse endometrium (BMP7); mouse heart (GDF8); JEG3 cells (Nodal). All samples were treated with RNase-free DNase (Ambion, Austin, TX, USA) to remove the possibility of genomic DNA contamination. RNA samples were then analysed by spectrophotometry to determine RNA concentration, yield and purity. Total RNA (1 µg) was reverse transcribed at 46°C for 1.5 h in 20 µl reaction mixture using 100 ng random hexanucleotide primers and 6 IU AMV reverse transcriptase (Roche, Castle Hill, Australia) in the presence of cDNA synthesis buffer (Roche), 1 mmol/l dNTPs (Roche), 10 mmol/l dithiothreitol (Roche), 10 IU ribonuclease inhibitor (RNasin; Promega, Annandale, Australia). The resultant cDNA mixtures were heated at 95°C for 3 min before storage at −20°C. Negative controls were performed by omission of reverse transcriptase. Triplicate RNA samples were reverse transcribed in triplicate for each condition (non-decidualized and decidualized) with the successful conversion to cDNA monitored by 18S expression (data not shown).RT–PCRMessenger RNA expression for all named TGFβ superfamily ligand members was determined using a conventional PCR block cycler (Hybaid, Middlesex, UK). All ligands (excluding Nodal), used a 1 µl aliquot of RT product, to be amplified in a total volume of 40 µl using 4 µl of RT single strength PCR buffer (10 mmol/l Tris–HCl, 1.5 mmol/l MgCl2, 50 mmol/l KCl, pH 8.3; Roche), 2.5 mmol/l dNTPs (Gibco, Melbourne, Australia), 0.5 pmol/µl sense and antisense primers (Sigma Genosys Australia Pty Ltd, Castle Hill, Australia) and 2.5 IU Taq DNA polymerase (Roche). For Nodal, 1 µl of RT was amplified in a total of 50 µl using the KOD-Taq PCR kit (Bioron, Germany), which included 10× PCR KOD Hot Start buffer, 2 mM dNTPs, 0.5 pmol/μl primers, 2 mM MgSO4 and 2.5 IU Taq DNA polymerase (Roche). For all ligands, the PCR was performed in three stages as follows: the first stage involved 94°C for 5 min, x C for 1 min, where x is the annealing temperature for the individual primer pairs (see Supplementary data) and 72°C for 3 min; the second stage involved 35–40 cycles of 94°C for 1 min, x C for 1 min, and 72°C for 1 min; and the final stage was 72°C for 7 min. PCR products including positive controls were analysed by electrophoresis on a 2% agarose gel (Roche) and stained with ethidium bromide. Bands of interest were excised from the gel, purified (DNA purification kit, Qiagen) and directly sequenced to confirm their identity.Inhibitor experimentsFor examination of the effect of inhibitors on decidualization, cells (from n = 10 biopsies) were plated at a density of 2.5 × 105/well in 24-well plates until confluent, and then equilibrated in serum-free medium containing SFM for 48 h. This was designated Day 0 and first day of treatments. HESC were exposed to varying doses of either the inhibitor Activin-M108A (M108A) (0.39 1.56, 6.25 and 25 nM) (Harrison et al., 2004, 2006) or the inhibitor SB431542 (1.25, 2.5, 5 and 10 µM) (TOCRIS Bioscience, Northpoint, UK). Following 1 h incubation with either inhibitor, 0.5 mM cAMP (Sigma) was added as decidualizing stimulus. Each treatment was performed in triplicate wells of a 24-well plate and the experiments ran for 4–6 days with media collection and replenishment (including inhibitors) every 48–72 h. In each case, the final medium collection represented the final 48 h. Each experiment included a medium-only and a cAMP-only control. Cells were photographed for morphological analysis and cells from triplicate wells were pooled for trypan blue cell exclusion to check cell viability at the end of each experiment. Five separate cultures were conducted for each inhibitor.TGFβ superfamily assaysCulture medium from triplicate wells from three tissue biopsies (n = 3 separate decidualization experiments) were collected, pooled and concentrated 5-fold to measure BMP2 (ELISA; R&D Systems), BMP4, BMP7, TGFβ1 and TGFβ2 (ELISA; Ray BioTech., Norcross, GA, USA) according to the manufacturer's instructions. Mean sensitivities of the assays were: BMP4, 15 pg/ml, BMP7, 10 pg/ml, TGFβ1, 80 pg/ml, TGFβ2, 15 pg/ml, with intra-assay CV <10% and inter-assay CV <12% for each assay. Mean sensitivity for BMP2 was 11 pg/ml and intra- and inter-assay variabilities were 2.6 and 6.3%, respectively. TGFβ1 and TGFβ2 ELISAs measured only the activated protein form and required activation steps prior to assay. Dimeric activin A secretion from stromal cells was measured by Activin A Immunofluorometric Assay (IFMA) (Harrison et al., 2006). The working range of the assay is 0.03–3 ng/well, with a sensitivity of 0.03 ng/well. All assays were read at 450 nm.Addition of exogenous BMP2 and TGFβ1 during decidualizationHESC from three separate cultures were decidualized using E2 and MPA as described above, in the presence of varying doses of either recombinant human (rh) BMP2 (5, 50, 500 ng/ml; R&D Systems) or TGFβ1 0.5, 5, 50 ng/ml; PeproTech Inc., NJ, USA) added every 48 h with media and decidualizing stimulus replenishment. At Day 8, cells were morphologically assessed and conditioned media were collected for PRL measurement.PRL and protein assaysPRL production by HESC was assayed in duplicate by ELISA (Bioclone Australia Pty Ltd, Sydney, Australia) to determine the extent of decidualization (Dimitriadis et al., 2002). Media collected at the end of the experiment were concentrated 5-fold for PRL measurement. A quality control sample (culture medium from a single endometrial cell culture) was included in every assay. The lower detection limit of the assay was 50 mIU/l. The inter- and intra-assay variablilities were 5.3 and 3.0%, respectively. PRL concentrations (mIU/l) were corrected for the amount of protein (μg/μl) determined using the Bradford reagent.Statistical analysisData were expressed as mean ± SEM. Statistical significance for inhibitor studies was determined following confirmation of normal distribution, by one-way ANOVA followed by Dunnett's multiple comparisons test. Data from ELISA and recombinant protein studies were analysed by Student's t-test. A P-value of <0.05 was considered statistically significant.
ResultsLocalization of TGFβ superfamily ligands in human endometrium and first trimester deciduaAll TGFβ superfamily members studied, except Nodal, were detected in mid-late secretory endometrium and first trimester decidua. As previously described (Jones et al., 2000), activin A and activin B were localized to the morphologically distinct decidual cells (Fig. 1A and B), glandular and luminal epithelial cells and some leukocytes (data not shown). Some glands had punctate staining for BMP2 (Fig. 1C), but BMP2 was most strongly stained in decidual cells in mid-late secretory endometrium (Fig. 1D). In first trimester decidua, BMP2 protein was detected in both the decidual cells (Fig. 1E, inset) and glandular epithelium, while the vessels were devoid of stain (Fig. 1E). GDF5 protein was observed in the decidualized stromal cells (Fig. 1F and G) and was very low in the glands (Fig. 1F) and luminal epithelium (data not shown). Similarly, GDF5 was very strongly expressed by the decidua of first trimester placenta, as well as the glandular epithelium (Fig. 1H, inset). TGFβ1 was present in decidualized stromal cells (Fig. 1I and J), with minimal staining evident in the glandular epithelium (Fig. 1I). As previously described (Graham et al., 1992), TGFβ1 was detected in decidual cells and extravillous trophoblasts of first trimester placenta (Fig. 1K). BMP4 was not detected in the glandular or luminal epithelium, but was present in the cytoplasm of both non-decidualized and decidualized stromal cells (Fig. 1L and M). However, in first trimester placenta, BMP4 was reduced in the decidual cells, but strongly produced by the glandular epithelium (Fig. 1N, inset). BMP7 was detected in the stroma and not in the glands in mid-late secretory endometrium (Fig. 1O). Stromal staining for BMP7 did not appear to be decidual cell specific, however, in highly decidualized tissue stronger ‘vesicle staining’ was evident (Fig. 1P). Similar but faint punctate staining for BMP7 was detected around spiral arterioles of first trimester placenta, but not specifically in decidual cells (Fig. 1Q, inset). BMP7 was also strongly localized to glandular and luminal epithelium (Fig. 1Q). GDF8 immunostaining in mid-late secretory endometrium showed minimal glandular and luminal epithelial staining, and intense stromal cell cytoplasmic staining, in both non-decidualized and decidualized cells (Fig. 1R). GDF11 immunolocalized to glandular epithelium and endothelial cells, but was very low in the stroma (Fig. 1S). GDF8 and GDF11 in first trimester placenta showed similar staining patterns to BMP4 (data not shown). No immunoreactive Nodal was detected in late secretory endometrium (Fig. 1T), although the immunostaining protocol was verified by positive staining in term placenta (not shown).Figure 1:Localization of TGFβ superfamily members during decidualization in mid-late secretory endometrium and first trimester decidua.Photomicrographs are representatives of immunostaining for activin A (A), activin B (B), BMP2 (C–E), GDF5 (F–H), TGFβ1 (I–K), BMP4 (L–N), BMP7 (O–Q), GDF8 (R), GDF11 (S), Nodal (T). In mid-late secretory endometrium; scale bar = 100 µm (C, F, I, L, O, R, S); scale bar = 10 µm (D, G, J, M, P, T). In first trimester decidua; scale bar = 200 µm (E, H, K, N, Q); inserts show higher power images of same tissue (E, H, K, N, Q). Negative controls for each antibody are shown in inserts (C, F, I, L, O, R, S, T). Arrows highlight decidualized (D), non-decidualized (ND) stromal cells and glands (G).TGFβ superfamily mRNA expression in non-decidualized and decidualized HESCBy RT–PCR, mRNA expression was evident for activin βA, activin βB, BMP2, BMP4, BMP7, GDF5, GDF8, GDF11, TGFβ1, TGFβ2 and TGFβ3 in both non-decidualized and decidualized HESC (Fig. 2). Nodal mRNA was undetectable in both. As conventional RT–PCR was used, only the presence/absence of each gene was assessed. Positive control PCR products (Fig. 2; see supplementary data) confirmed sequence identity and no signals were detected when reverse transcriptase was omitted (Fig. 2).Figure 2:mRNA expression for TGFβ superfamily members by decidualized and non-decidualized HESC.HESC were either non-decidualized or decidualized with cAMP for 7 days. Total RNA was extracted from cells for RT–PCR. Representative products demonstrating Activin βA, Activin βB, BMP2, BMP4, BMP7, GDF5, GDF8, GDF11, TGFβ1, TGFβ2, TGFβ3 and Nodal mRNA are shown in non-decidualized (ND) and cAMP-decidualized (D) HESC; +ve=positive control; −RT=negative control. Data shown is from a single experiment, which is representative of 2 independent culture experiments.Activin-M108A and SB431542 significantly decreases PRL secretion during HESC decidualizationTo establish whether HESC decidualization could be blocked or reduced, the newly developed activin type II receptor antagonist, Activin-M108A or the type I receptor kinase inhibitor, SB431542 were used in an in vitro decidualization model with PRL secretion, a marker of decidualization, as the end-point. These inhibitors affect activin and various other TGFβ family members (see Table I). Cells maintained in medium alone (non-decidualized) showed non-detectable PRL levels over the final 48 h of culture (Fig. 3). In contrast, cells treated with cAMP secreted PRL (mean 372.6 ± 145.5 mIU/l), and morphologically changed from elongated spindle-shaped cells to typical enlarged polygonal cells demonstrating successful decidualization, as previously described (Dimitriadis et al., 2002). Stromal cells cultured with cAMP and Activin-M108A showed a significant dose-dependent decrease in PRL secretion at 0.39, 1.56 and 6.25 nM with no further decrease at 25 nM (Fig. 3A). At the 25 nM dose, a 60% reduction in PRL secretion was seen compared with cAMP-treated cells without inhibitor, which was similar to decidualization reduction seen with follistatin. Similarly cells co-cultured with cAMP and SB431542 showed a dose-dependent decrease in PRL secretion. Addition of SB431542 at a concentration of 1.25 µM significantly reduced PRL secretion compared with cAMP-only treated cells (Fig. 3B). Further significant decreases were observed with doses ranging from 2.5–10 µM (Fig. 3B). At 10 µM, SB431542 reduced PRL secretion by 77% compared with cAMP alone (Fig. 3B). Higher doses were not tested. When either inhibitor (Activin-M108A or SB431542) was administered to cells in the absence of cAMP, PRL was not detected in culture medium. In addition, cell viability was tested using the trypan blue exclusion method at the end of the experiment and was found to not differ between treatments with medium-only, cAMP and either inhibitor. Microscopic visualization indicated cell morphology was not altered by inhibitor use, suggesting this effect was not due to toxicity to cells.Figure 3:Activin-M108A and SB43 154 inhibitors significantly decrease HESC decidualization.Confluent stromal cells were cultured for 4–5 days with cAMP, with medium changes every 2–3 days. (A) M108A was added at doses 0.39, 1.5, 6.25 and 25 nM to cAMP-treated cultures. (B) SB431542 was added at doses 1.25, 2.5, 5 and 10 µM. Controls (not decidualized; ND) and cAMP (decidualized; D). PRL secretion from cells was used as a marker of decidualization, and values were corrected for total protein. All data (mean ± SEM) is expressed as % change from control (100%). Results are combined data from triplicate wells of five independent experiments for each inhibitor. *P < 0.05 and **P < 0.01 compared with cAMP alone.Secretion of activin A, TGFβ1, BMP2 and BMP4, but not TGFβ2 or BMP7 from non-decidualized and decidualized HESC in vitroTGFβ ligands are active as secreted proteins. Therefore we measured BMP2, BMP4, BMP7, TGFβ1, TGFβ2 from three different cultures by ELISA and activin A by IFMA (n = 1) in serum-free culture medium. There was a 4-fold increase in dimeric activin A secreted from decidualized (1.9 ng/106cells) compared with non-decidualized cells (0.43 ng/106cells), supporting previous published data from our laboratory (Jones et al., 2002). Non-decidualized and decidualized HESC secreted BMP2, TGFβ1 and BMP4, each in the range of 5–20 pg/106 cells (Fig. 4). BMP7 and TGFβ2 levels were below the sensitivity of the assays (data not shown). Decidualized stromal cells secreted significantly higher amounts of BMP2 and TGFβ1 compared with non-decidualized cells (TGFβ1 20.6 ± 3.8 versus 14 ± 3.1 pg/106cells; BMP2 17.2 ± 2.32 versus 8.44 ± 1.5 pg/106cells) (Fig. 4). BMP4 secretion was increased slightly with decidualization (6.9 ± 2.17 versus 10.1 ± 3.45 pg/106cells), but this was not significant (Fig. 4). Western blot analysis was performed for GDF5, GDF8, GDF11 and TGFβ3 using 100–150 µg of cell lysates from non-decidualized and decidualized HESC; however, these proteins were below the level of detection. Positive controls (GDF5—rat heart and human term placenta; TGFβ3—human term placenta; GDF8 and GDF11—recombinant human myostatin/GDF8 and recombinant human GDF11, respectively; data not shown) validated the technique.Figure 4:BMP2, TGFβ1 and BMP4 protein secretion by HESC during decidualization.BMP2, BMP4 and TGFβ1 protein were measured in culture medium from non-decidualized (dark) and decidualized (grey) cells at Day 4 of culture. Results are combined data from n = 3 independent culture experiments and data (mean ± SEM) is represented as % difference from control (non-decidualized cells; defined as 100%). *P < 0.05 and **P < 0.01 compared to control.Exogenous BMP2 and TGFβ1 increases HESC decidualization in vitroTo assess whether addition of rhBMP2 and/or rhTGFβ1 to HESC enhances decidualization in vitro, cells were decidualized with E2 and MPA under serum-free conditions for 8 days and PRL secretion measured. At Day 8, PRL secretion from non-decidualized cells was minimal but was increased when cells were decidualized (Fig. 5). When rhBMP2 (5, 50 or 500 ng/ml) was added to decidualizing HESC, PRL secretion was significantly increased at the 50 ng/ml dose compared with the decidualized control (P < 0.05). The addition of rhTGFβ1 at 0.5, 5 or 50 ng/ml to decidualizing HESC, however this was only significant at the 0.5 ng/ml dose (P < 0.01) compared with decidualized cells alone.Due to the variability of endometrial biopsies only one dose for each BMP2 and TGFβ1 has resulted in a significant promotion of decidualisation. Also as the rhTGFβ1 is known to be quite potent, this result could be explained by the fact that at the higher doses it might be negatively affecting the response, for example receptor and downstream signalling disturbances.Figure 5:Effect of exogenous BMP2 and TGFβ1 on PRL secretion from HESC.Cells were untreated or treated with E2 (10–8 M), MPA (10–7 M) alone or combined with recombinant human (rh) BMP2 (5–500 ng/ml) (A), rhTGFβ1 (0.5–50 ng/ml) (B) for 8 day. PRL was measured in cultured medium from the last 48 h of treatment. Data are represented as percent fold change from control (decidualized; D) defined as 100%. ND, non-decidualized HESC. Results are combined data from triplicate wells of three independent cultures. *P < 0.05 and **P < 0.01 compared with D.
DiscussionThis is the first study to identify BMP2, BMP4, BMP7, GDF5, GDF8 and GDF11 protein in secretory phase human endometrium and show the mRNA expression of these ligands in cultured HESC. In addition, the current study has immunolocalized BMP2, TGFβ1 and GDF5 protein to decidualized stromal cells, and provided further evidence for a role for secreted activin, BMP2 and TGFβ1 in decidualization through the use of novel inhibitors and a well-established ex vivo decidualization model.Decidualization of endometrial stromal cells is a pivotal event in the preparation for blastocyst implantation. Although this process is governed by hormonal regulation, it is becomingly increasingly evident that a multitude of factors, including cytokines and growth factors, are essential in ensuring this process occurs successfully such that endometrial stromal cells are differentiated into the implantation-favourable decidual cells. The identification of BMP2, TGFβ1 and GDF5 protein in mid-secretory human endometrial tissue, together with the increased secretion of both BMP2 and TGFβ1 from stromal cells following decidualization, suggests these TGFβ family members are hormonally regulated and important for decidualization.The finding that BMP2 secretion from HESC increases as they decidualize in vitro and that addition of rhBMP2 further drives decidualization as determined by secretion of PRL, extends the recent study (Li et al., 2007), which showed an increase of BMP2 mRNA with decidualization in a similar model and of PRL mRNA following treatment of HESC with rhBMP2. This is important given that translation and secretion do not necessarily follow increases in mRNA. The inhibitor M108A which acts primarily on those ligands acting through the activin type 11 receptor (Table I) is not known to block the action of BMP2 which acts through the BMP type II receptor (BMPRII). Therefore, the inability of M108A to fully block the decidualization process suggests that factors other than activins and GDFs are likely to be important to this process: BMP2 is a likely candidate.Interestingly in mice, uterine-specific conditional ablation of BMP2 resulted in uterine stromal disruption and an inability to undergo normal decidual reactions and pregnancy progression (Lee et al., 2007). Our demonstration of BMP2 in human endometrium in vivo with the strongest immunostaining in decidualized cells in the mid-late secretory phase and in the decidua of early pregnancy, confirms the biological relevance in women. Given this data in conjunction with our functional studies of human decidualization, there appears to be little doubt that BMP2 is an important mediator of both mouse and human decidualization.This study also demonstrates, for the first time, the presence of GDF5 in human endometrium and first trimester placenta where it is intensely and specifically stained in the decidual cells. GDF5 is best known for its role in early chondrogenesis and joint formation, being involved in inducing cartilage differentiation, growth and maturation (Mikic, 2004), as well as bone formation and angiogenesis (Yamashita et al., 1997). Differentiation and angiogenesis are the main events of decidualization. Unfortunately, assays for GDF5 were not available to measure the secretion from decidualized cells. The decrease in decidualization seen following administration of M108A was not likely to be due to GDF5 blockade, as this ligand has a relatively low, if any, affinity for the activin type II receptor. Future studies should address whether GDF5 has a functional role in decidualization, whether it be in promoting differentiation or in the formation of associated blood vessels to support the blastocyst in early pregnancy and throughout gestation.Although the TGFβs have been previously detected in the human endometrium, the information regarding their role during decidualization is contentious, as studies report contrasting effects on PRL production by endometrial stroma and during decidualization in vitro (Kubota et al., 1997; Kim et al., 2005). We showed here that under serum-free conditions, TGFβ1 secretion increases during cAMP-induced decidualization in vitro. Microarray studies have likewise shown increased TGFβ1 and TGFβ2 mRNA when cAMP or progesterone is used as decidual stimuli (Popovici et al., 2000; Tierney et al., 2003). The protein for both isoforms localizes to stromal cells in human endometrium throughout the menstrual cycle (Gold et al., 1994; Godkin and Dore, 1998), although decidualized stromal cells were not shown. In contrast, the present study identified TGFβ1 protein in decidualized stromal cells in human endometrium, and its secretion increased in cAMP-decidualized HESC. Administration of SB431542, which effectively blocks the TGFβs as well as activins by blocking their structurally similar ALK5 and ALK4 receptors (Inman et al., 2002), resulted in a 77% reduction in decidualization, a greater decrease than that seen with M108A (this study) and follistatin (Tierney and Giudice, 2004). This, along with our demonstration that addition of TGFβ1 during progesterone-induced decidualization enhances decidualization, provides convincing evidence to suggest TGFβ1 is important during decidualization. The level of activated TGFβ2 was undetectable by ELISA in cAMP-decidualized endometrial stromal cells, and therefore this ligand is unlikely to contribute to the decidual response.The identification of BMP4, BMP7, GDF8 and GDF11 during the mid-secretory phase is also an important finding. In addition to decidualization, there are many remodelling processes occurring in the endometrium during this time. Both BMP4 and GDF8 protein showed intense immunolocalization to the cytoplasm of the stroma, irrespective of whether or not the cells were decidualized. This was verified for BMP4 by the minimal difference in secretion levels between non-decidualized and decidualized cells in vitro. BMP4 mRNA expression has been localized to the vascular endothelial cells in pregnant mice (Ying and Zhao, 2000), and in the non-pregnant rat uterus, where it is constitutively expressed throughout the cycle (Erickson et al., 2004). This differs to the localization of BMP4 protein shown here in human endometrium. It may be that the mRNA localized to blood vessels in the rodent studies is not translated into functional protein.GDF8 (more commonly referred to as Myostatin) is a negative regulator of skeletal muscle, but more recently has been shown to be involved in glucose metabolism (McPherron and Lee, 2002), particularly in placenta (Mitchell et al., 2006). This study verified the presence of GDF8 protein in first trimester placenta, and showed for the first time its presence in secretory phase endometrium and in HESC. Given the lack of specific decidual staining it is unlikely that the response from M108A inhibitor was due to blocking GDF8 in HESC, even though the predominant receptor for GDF8 is the activin type II receptor. The presence of GDF8 in non-muscular tissue and organs is rare and future experiments should determine whether it has a role in endometrial biology as it does throughout gestation in the placenta.Vesicular staining for BMP7 (or Osteogenic protein-1; OP-1) was evident in highly decidualized cells during the secretory phase of human endometrium with minimal staining in selected glandular and luminal epithelial cells. OP-1 has been identified in the uterine epithelium of non-pregnant mice and rat uterus (Ozkaynak et al., 1997; Erickson et al., 2004), with localization remaining unchanged across the cycle. In pregnant mice, however, BMP7 mRNA was detected in the decidualizing stromal cells surrounding the blastocyst with no staining in the epithelial cells (Ying and Zhao, 2000). In the present study, BMP7 secretion from HESC was undetectable by ELISA, making it difficult to assess whether it may have a functional role during decidualization.In conclusion, this study has identified that a wide range of TGFβ ligands are present in mid-late secretory human endometrium but of these, in addition to activins, only BMP2, GDF5 and TGFβ1 increase in stromal cells as they undergo decidualization. BMP2 and TGFβ1, along with activins, are important in driving the decidualization process. Functions for the remaining ligands remain to be determined but they are likely to participate in the extensive tissue remodelling that occurs in this highly dynamic tissue as it prepares for blastocyst implantation and pregnancy.
Supplementary materialSupplementary material is available at HUMREP Journal online
FundingThis work was funded by the National Health and Medical Research Council of Australia (#241000, #388901) and a Prince Henry's Institute Postgraduate Scholarship.
Supplementary Material
[Supplementary Data]

human endometrium
decidualization
implantation
growth factors (activins, bmp, tgfβ)


The analgesic effect of inhalational Entonox for extracorporeal shock wave lithotripsy

Extracorporeal shock wave lithotripsy (ESWL) is a non-invasive procedure that allows urinary stones to be fragmented using acoustic shock waves. The impact of the shock waves causes transient stinging pain at the entry site as well as deep visceral discomfort, requiring analgesia during the procedure. The objective of this study was to compare the clinical efficacy of Entonox and pethidine for pain relief during outpatient ESWL. We randomized 150 outpatients undergoing elective ESWL into three groups of 50 patients, each group receiving inhalational Entonox, intravenous pethidine, or inhalational compressed air during ESWL. Quantitative evaluation of pain was performed according to a visual analogue scale (VAS), before and after the intervention. Analysis of variance (ANOVA) and paired t tests were used to compare VAS scores in the three groups, before and after the intervention. Entonox and pethidine decreased the pain score significantly, while compressed air did not. There was no significant difference between pain relief by Entonox and pethidine. This study demonstrates for the first time that inhalational Entonox is an effective analgesic regimen for ESWL. Entonox can be regarded as an appropriate alternative to analgesics like opioids in relieving pain during ESWL.
Introduction
Since 1980, extracorporeal shock wave lithotripsy (ESWL) has become the first-line treatment for most urinary stones in adults and children [1]. However, the vast majority of patients do not tolerate the procedure without analgesia or sedation. Several monitored anesthesia care techniques have been used to provide sedation and analgesia [2–5]. Some of the analgesic drugs administered for ESWL, however, carry the risk of respiratory depression, delayed discharge, and/or unplanned hospital admission [6].
Entonox is a mixture of 50% nitrous oxide and 50% oxygen. Inhalation produces analgesia without loss of consciousness [7]. Self-administration of Entonox as an analgesic has been widely used for many years, particularly in obstetric practice [8], and by paramedics for prehospital care [9]. Entonox has a good safety record, with no serious side effects recorded for intermittent use [7], and is rapidly cleared from the circulation by exhalation [10].
The aim of this study was to compare the clinical efficacy of inhalational Entonox and pethidine for pain relief during outpatient ESWL.
Methods
A total of 150 patients (97 men and 53 women) with stones located in the renal pelvicalyceal system were prospectively randomized to receive Entonox (prefixed equimolar nitrous oxide and oxygen mixture), pethidine, or compressed air for sedoanalgesia during lithotripsy with a third generation electromagnetic lithotripter (Dornier Compact Delta magneto lithotripter).
After institutional review board approval, written informed consent was obtained during the anesthesia consultation, which was performed at least 48 h before ESWL. During this consultation, the method of pain assessment was explained to the patients. Patients were asked to rate their pain on a scale from 0 (no pain) to 10 (worst possible pain).
Patients were included in the study if they were older than 15 years of age, had pelvicalyceal stones, and understood the pain scoring. Patients were excluded from the trial if they had any of the following: (1) serum creatinine > 200 μmol/l; (2) history of chronic use of analgesics and/or sedatives; (3) allergy to any of the study medications; or (4) history of middle ear surgery within the previous month.
Just before entering the lithotripsy room, the patients were randomly assigned to one of the three treatment groups (Entonox, pethidine, 1 mg/kg intravenously over 10 min, or compressed air, each to be provided if the patient asked for pain relief during ESWL) by opening a sealed envelope. All patients were assessed by a single physician who was blinded to the patient group assignment. All patients received no premedication and they were told not to eat or drink for at least 4 h prior to the procedure.
The control group received compressed air, followed by Entonox gas if pain relief was considered inadequate by the patient.
Pain was assessed before and 2 min after receiving pain medication by using a visual analogue scale (VAS) with two anchor points, zero denoting no pain and ten for the worst pain the patient had ever experienced.
Statistical analysis
Data are expressed as mean (±SD) and numbers (percentages). The paired t test was used to analyze VAS score before and after medication. Comparison of means (VAS decrease) in the three groups was performed using one-way ANOVA. Comparison of percentages was performed using Chi-square analysis.
All statistical analyses were performed using SPSS® software, version 9.0, for Windows. Results throughout the text and tables are presented as mean ± SD unless otherwise specified, and statistical significance was defined as P < 0.05.
Results
The three study groups were comparable with respect to demographic data, history of urinary calculi and ESWL, and location of calculi. There were no statistically significant differences between the three groups (Table 1). All patients in the three groups required analgesia. Rescue medication in the form of pethidine was administered in only one case in the Entonox group after recording VAS scores because the analgesia was inadequate.
Table 1Demographic data and ESWL variables in three different groupsGroupsEntonoxPethidineControlAge (years)43.76 ± 11.8945 ± 13.4643.9 ± 14.21Weight (kg)75 ± 278 ± 379 ± 2Height (cm)165 ± 10167 ± 12164 ± 8Gender (M/F) (n)30/2034/1633/17Stone location (%)  Renal807884  Ureteral202015  Both021Number of shocks delivered2,010 ± 3202,000 ± 5602,500 ± 450Maximum voltage (kV) used4.04 ± 1.154.14 ± 0.754.51 ± 1.00Past history of urinary calculi (%)707276Past history of ESWL (%)424636Data are expressed as mean ± SD, numbers (n), or percentages (%)
No ESWL procedure had to be prematurely terminated because of inadequate analgesia. A patient in the Entonox group had mild nausea that subsided spontaneously. The other patients did not develop any complications.
Mean ± SD of VAS scores before and after intervention are shown in Table 2. Statistical analysis showed significant decrease (Table 2) in pain severity in patients receiving Entonox or pethidine. In contrast, patients receiving compressed air had no such change.
Table 2Mean ± SD of VAS scores and P-value of comparing scores before and after Entonox, pethidine, and compressed air administration in different groupsVAS scoresEntonoxPethidineCompressed airEntonox in the compressed air groupBefore administration5.76 ± 1.926.04 ± 1.543.6 ± 1.14.96 ± 1.42After administration3.73 ± 2.134.11 ± 1.693.48 ± 1.33.8 ± 1.74P-value0.0010.0010.4260.001In control group, after testing with compressed air, we used Entonox. As can be seen in this group, like Entonox group, VAS score decreased significantly
While VAS scores after receiving either Entonox or pethidine were significantly different from those in the control group (P = 0.001) after receiving compressed air, statistical comparison showed no significant difference between post-Entonox or post-pethidine VAS scores (P = 0.5), suggesting that inhalational Entonox may be as effective as intravenous pethidine in alleviating ESWL-associated pain.
No significant difference was found in VAS scores of male and female patients in each group (Fig. 1).
Fig. 1VAS scores in different groups before and after administration of Entonox, pethidine, and compressed air
Discussion
Extracorporeal shock wave lithotripsy (ESWL) has become a valuable asset to the urologist and greatly benefits stone patients. In a short period of time, ESWL has completely changed the management of urinary stone disease and has almost entirely supplanted open surgical and most endourologic approaches [11]. Today, third-generation mobile electromagnetic lithotripters yield an average of 80% stone-free rate for kidney and ureteral calculi [12].
As ESWL is frequently carried out on an outpatient basis, it is crucial to provide adequate analgesia with minimal adverse effects [13].
Chaussy and Thuroff [14] demonstrated that analgesia requirements in ESWL depend on lithotripter, stone location, age, gender, and number of shock waves. The pathogenesis of pain in ESWL is still not clearly known. Whether it is due to cutaneous or deep visceral afferent stimulation is debatable [15]. The pain is presumably due to cavitation-mediated stimulation of nerve fibers. The intensity of pain perceived during ESWL depends on the energy level of shock waves passing through the tissues [16]. Although the development of new generation lithotripters has decreased pain during ESWL, some form of sedation and anesthesia may still be required to provide adequate patient comfort and effective treatment [15].
Any technique for alleviating ESWL-associated discomfort/anxiety should ideally be reliable in its effect, of rapid onset and short duration of action, free of adverse events, cost-effective, and easily administered [17].
Opioids are the most commonly used analgesics during ESWL [18]. Despite their effectiveness, their use may be complicated by central nervous system (CNS) or respiratory depression, circulatory failure, or gastrointestinal problems [19].
Several studies have compared different local and systemic analgesia regimens for pain relief during ESWL, but this, to our knowledge, is the first randomized clinical trial to evaluate the efficacy of Entonox in the treatment of ESWL-associated pain.
Nitrous oxide has been used for pain relief during childbirth since the 1930s and was initially delivered at a concentration of 50% in air, producing an effective analgesic mixture. In 1961, Mike Tunstall premixed 50% nitrous oxide with oxygen, and called it Entonox, which was stored as a compressed gas mixture in cylinders. Since then, the convenience of Entonox has made it a successful and popular analgesic for labour [20]. Entonox has also proved effective in providing pain relief in other specialties [21, 22].
Our randomized clinical trial demonstrated that Entonox and pethidine were equally effective in providing analgesia during ESWL and that they were superior to compressed air. Entonox fulfills many of the criteria for an ideal analgesic during ESWL [17] and, compared to intravenously administered pethidine, provided comparable analgesia with less CNS, circulatory, and respiratory depression or nausea and vomiting.
Entonox provides rapid and effective analgesia without heavy sedation and leads to adequate patient relaxation and cooperation. The effect of Entonox was of short duration, allowing the patients to leave the ESWL unit without the need for a long recovery period [23]. Entonox appeared to be associated with few, minor, and short-lived adverse effects such as nausea and vomiting, dizziness, dry mouth (breathing dry gas), buzzing in the ears, and rarely, pins and needles or numbness, dreams or drowsiness, ranging from 0 to 30% [24]. Nitrous oxide–oxygen inhalation may thus provide a valuable alternative to conventional analgesia regimens during ESWL.
In addition to short-acting parenteral sedative narcotics, e.g., alfentanil, midazolam, and propofol, topical agents such as EMLA cream, lidocaine, prilocaine and piroxicam have been used to minimize pain during SWL [25]. Further studies are, therefore, warranted to compare the efficacy and safety of Entonox with those of other analgesic regimens.
Conclusions
Entonox can provide easy, rapid, and adequate pain relief for patients undergoing ESWL, and is associated with few and minimal side effects.

entonox
extracorporeal shock wave lithotripsy
pethidine
pain relief


IMGT, the international ImMunoGeneTics information system®: a standardized approach for immunogenetics and immunoinformatics

IMGT, the international ImMunoGeneTics information system®, was created in 1989 by the Laboratoire d'ImmunoGénétique Moléculaire (LIGM) (Université Montpellier II and CNRS) at Montpellier, France. IMGT is a high quality integrated knowledge resource specialized in immunoglobulins (IG), T cell receptors (TR), major histocompatibility complex (MHC) of human and other vertebrates, and related proteins of the immune system (RPI) of any species which belong to the immunoglobulin superfamily (IgSF) and to the MHC superfamily (MhcSF). IMGT consists of five databases, ten on-line tools and more than 8,000 HTML pages of Web resources. IMGT provides a common access to standardized data from genome, genetics, proteome and three-dimensional structures. The accuracy and the consistency of IMGT data are based on IMGT-ONTOLOGY, a semantic specification of terms to be used in immunogenetics and immunoinformatics. IMGT-ONTOLOGY comprises six main concepts: IDENTIFICATION, CLASSIFICATION, DESCRIPTION, NUMEROTATION, ORIENTATION and OBTENTION. Based on these concepts, the controlled vocabulary and the annotation rules necessary for the immunogenetics data identification, classification, description and numbering and for the management of IMGT knowledge are defined in the IMGT Scientific chart. IMGT is the international reference in immunogenetics and immunoinformatics for medical research (repertoire analysis of the IG antibody sites and of the TR recognition sites in autoimmune and infectious diseases, AIDS, leukemias, lymphomas, myelomas), veterinary research (IG and TR repertoires in farm and wild life species), genome diversity and genome evolution studies of the adaptive immune responses, biotechnology related to antibody engineering (single chain Fragment variable (scFv), phage displays, combinatorial libraries, chimeric, humanized and human antibodies), diagnostics (detection and follow up of residual diseases) and therapeutical approaches (grafts, immunotherapy, vaccinology). IMGT is freely available at .
Introduction
IMGT, the international ImMunoGeneTics information system®[1,2], was created in 1989, by Marie-Paule Lefranc, at the Laboratoire d'ImmunoGénétique Moléculaire (LIGM) (Université Montpellier II and CNRS) at Montpellier, France, in order to standardize and manage the complexity of the immunogenetics data. Fifteen years later, IMGT is the international reference in immunogenetics and immunoinformatics, and provides a high quality integrated knowledge resource, specialized in the immunoglobulins (IG) and T cell receptors (TR), major histocompatibility complex (MHC) of human and other vertebrates, and related proteins of the immune systems (RPI) of any species which belong to the immunoglobulin superfamily (IgSF) and to the MHC superfamily (MhcSF)[3-13]. The number of potential protein forms of the antigen receptors, IG and TR, is almost unlimited. The potential repertoire of each individual is estimated to comprise about 1012 different IG (or antibodies) and TR, and the limiting factor is only the number of B and T cells that an organism is genetically programmed to produce. This huge diversity is inherent to the particularly complex and unique molecular synthesis and genetics of the antigen receptor chains. This includes biological mechanisms such as DNA molecular rearrangements in multiple loci (three for IG and four for TR in humans) located on different chromosomes (four in humans), nucleotide deletions and insertions at the rearrangement junctions (or N-diversity), and somatic hypermutations in the IG loci (see FactsBooks[3,4] for review). Although IMGT was initially implemented for the IG, TR and MHC of human and other vertebrates [6], data and knowledge management standardization, based on the IMGT unique numbering [14-19], has now been extended to the IgSF [15-17,20-22] and MhcSF [18,23,24] of any species. Thus, standardization in IMGT contributed to data enhancement of the system and new expertised data concepts were readily incorporated.
IMGT, the international ImMunoGeneTics information system® consists of five databases, ten on-line tools and Web resources [1,2]. Databases include sequence databases (IMGT/LIGM-DB, IMGT/PRIMER-DB and IMGT/MHC-DB), one genome database (IMGT/GENE-DB) and one three-dimensional (3D) structure database (IMGT/3Dstructure-DB) [1,2] (Figure 1). Interactive tools are provided for sequence analysis (IMGT/V-QUEST, IMGT/JunctionAnalysis, IMGT/Allele-Align, IMGT/PhyloGene), genome analysis (IMGT/LocusView, IMGT/GeneView, IMGT/GeneSearch, IMGT/CloneSearch and IMGT/GeneInfo) and 3D structure analysis (IMGT/StructuralQuery) [1,2] (Figure 1). Web resources ("IMGT Marie-Paule page") comprise more than 8,000 HTML pages of synthesis [IMGT Repertoire (for IG and TR, MHC, RPI)], knowledge [IMGT Scientific chart, IMGT Education (IMGT Lexique, Aide-mémoire, Tutorials, Questions and answers), IMGT Medical page, IMGT Veterinary page, IMGT Biotechnology page, IMGT Index], and external links [IMGT Immunoinformatics page, IMGT Bloc-notes (Interesting links, etc.) and IMGT other accesses (SRS, BLAST, etc.)] [2]. Despite the heterogeneity of these different components, all data in the IMGT information system are expertly annotated. The accuracy, the consistency and the integration of the IMGT data, as well as the coherence between the different IMGT components (databases, tools and Web resources) are based on IMGT-ONTOLOGY[5], which provides a semantic specification of the terms to be used in immunogenetics and immunoinformatics. IMGT-ONTOLOGY, the first ontology in the domain, has allowed the management of knowledge in immunogenetics [2,25] and provided standardization for immunogenetics data from genome, genetics, proteome and 3D structures [3-13]. IMGT-ONTOLOGY concepts are available, for the biologists and IMGT users, in the IMGT Scientific chart[2], and for the computing scientists, in IMGT-ML which uses XML (eXtensible Markup Language) Schema [26].
Figure 1
IMGT, the international ImMunoGeneTics information system® . Databases and tools for sequences, genes and structures are in green, yellow and blue, respectively. The IMGT Repertoire and other Web resources are not shown. Interactions in the genetics, genomics and structural approaches are represented with dotted, continuous and broken lines, respectively.
IMGT-ONTOLOGY concepts and IMGT Scientific chart rules
The IMGT Scientific chart[2] comprises the controlled vocabulary and the annotation rules necessary for the immunogenetics data identification, description, classification and numbering and for knowledge management in the IMGT information system. Standardized keywords, labels and annotation rules, standardized IG and TR gene nomenclature, the IMGT unique numbering, and standardized origin/methodology were defined, respectively, based on the six main concepts of IMGT-ONTOLOGY: IDENTIFICATION, CLASSIFICATION, DESCRIPTION, NUMEROTATION, ORIENTATION and OBTENTION[2,5] (Table 1). The IMGT Scientific chart is available as a section of the IMGT Web resources (IMGT Marie-Paule page). Examples of IMGT expertised data concepts derived from the IMGT Scientific chart rules are shown in Table 1.
Table 1
IMGT-ONTOLOGY concepts, IMGT Scientific chart rules and examples of IMGT expertised data concepts.
IMGT-ONTOLOGY main concepts 5
IMGT Scientific chart rules [2]
Examples of IMGT expertised data concepts [2]
IDENTIFICATION
Standardized keywords [5]
Species, molecule type, receptor type, chain type, gene type, structure, functionality, specificity
CLASSIFICATION
Reference sequencesStandardized IG and TR gene nomenclature (group, subgroup, gene, allele) [5]
Nomenclature of the human IG and TR genes (entry in 1999 in GDB, HGNC [27] and LocusLink at NCBI) [3, 4]Alignment of alleles [3, 4]Nomenclature of the IG and TR genes of all vertebrate species
DESCRIPTION
Standardized labels and annotations [5]
Core (V-, D-, J-, C-REGION) Prototypes [5]Labels for sequencesLabels for 2D and 3D structures
NUMEROTATION
IMGT unique numbering [14-18] for: V- and V-LIKE-DOMAINs [16]C- and C-LIKE-DOMAINs [17]G- and G-LIKE-DOMAINs [18]
Protein displaysIMGT Colliers de Perles [19]FR-IMGT and CDR-IMGT delimitations [16]Structural loops and beta strands delimitations [16, 17]
ORIENTATION
Orientation of genomic instances relative to each other
Chromosome orientation
Locus orientation
Gene orientation
DNA strand orientation
OBTENTION
Standardized originStandardized methodology [2]
The IMGT Scientific chart rules, based on the IMGT-ONTOLOGY concepts [5], are used in the three major IMGT biological approaches, genomics, genetics and structural approaches [2], and corresponding data (Genes, Sequences, 3D structures) are available in the IMGT components (databases, tools and Web resources) [1,7-13].
IMGT sequence databases, tools and Web resources
IMGT sequence databases, tools and Web resources correspond to the IMGT genetics approach that refers to the study of genes in relation with their polymorphisms, mutations, expression, specificity and evolution (Table 2). The IMGT sequence knowledge management and the IMGT genetics approach heavily rely on the DESCRIPTION concept (and particularly on the V-REGION, D-REGION, J-REGION and C-REGION core concepts for the IG and TR), on the CLASSIFICATION concept (gene and allele concepts) and on the NUMEROTATION concept (IMGT unique numbering [14-18]).
Table 2
The IMGT sequence databases, sequence analysis tools and Web resources
IMGT sequence databases [1]
IMGT sequence analysis tools [1]
IMGT Repertoire"Proteins and alleles" section [2](2)
IMGT/LIGM-DB [7]IMGT/PRIMER-DB [1]IMGT/MHC-DB [28]
IMGT/V-QUEST [10]IMGT/JunctionAnalysis [11]IMGT/Allele-AlignIMGT/PhyloGene [12]IMGT/Automat [29, 30] (1)
Alignments of alleles IG and TR [3, 4]Alignments of alleles RPI [22]Protein displays IG and TR [3, 4, 16, 17]Protein displays MHC [18]Protein displays RPI [16-18, 21]Tables of alleles IG and TRTables of alleles RPI [22, 24]Allotypes Isotypes, etc.
(1) IMGT/Automat [29, 30] is an integrated internal IMGT Java tool which automatically performs the annotation of rearranged cDNA sequences that represent the half of the IMGT/LIGM-DB content. So far 7,418 human and mouse IG and TR cDNA sequences have been automatically annotated by the IMGT/Automat tool, with annotations being as reliable and accurate as those provided by a human annotator.
(2) IMGT publications from the IMGT Repertoire "Proteins and alleles" section are available as pdf in IMGT Locus in Focus , in IMGT Index (see also [2]).
IMGT sequence databases
IMGT/LIGM-DB
IMGT/LIGM-DB is the comprehensive IMGT database of IG and TR nucleotide sequences from human and other vertebrate species, with translation for fully annotated sequences [7]. It was created in 1989 by LIGM (Montpellier, France), and is on the Web since July 1995 [6]. In August 2005, IMGT/LIGM-DB contained more than 96,500 sequences of 150 vertebrate species [7]. The unique source of data for IMGT/LIGM-DB is EMBL, which shares data with the other two generalist databases GenBank and DNA DataBank of Japan (DDBJ). Based on expert analysis, specific detailed annotations are added to IMGT flat files. The annotation procedure includes the IDENTIFICATION of the sequences, the CLASSIFICATION of the IG and TR genes and alleles, and the DESCRIPTION of all IG and TR specific and constitutive motifs within the nucleotide sequences. The Web interface allows searches according to immunogenetic specific criteria and is easy to use without any knowledge in a computing language. Selection is displayed at the top of the resulting sequences pages, so the users can check their own queries. Users have the possibility to modify their request or consult the results with a choice of nine possibilities. The IMGT/LIGM-DB annotations (gene and allele name assignment, labels) allow data retrieval not only from IMGT/LIGM-DB, but also from other IMGT databases. Thus, the IMGT/LIGM-DB accession numbers of the cDNA expressed sequences for each human and mouse IG and TR gene are available, with direct links to IMGT/LIGM-DB, in the IMGT/GENE-DB entries. IMGT/LIGM-DB data are also distributed by anonymous FTP servers at CINES  and EBI  and from many Sequence Retrieval System (SRS) sites . IMGT/LIGM-DB can be searched by BLAST or FASTA on different servers (EBI, IGH, INFOBIOGEN, Institut Pasteur, etc.).
IMGT/PRIMER-DB
IMGT/PRIMER-DB[1] is the IMGT oligonucleotide primer database for IG and TR, created by LIGM, Montpellier in collaboration with EUROGENTEC S.A., Belgium, on the Web since February 2002. In August 2005, IMGT/PRIMER-DB contained 1,827 entries. IMGT/PRIMER-DB provides standardized information on oligonucleotides (or Primers) and combinations of primers (Sets, Couples) for IG and TR. These primers are useful for combinatorial library constructions, scFv, phage display or microarray technologies. The IMGT Primer cards are linked to the IMGT/LIGM-DB flat files, IMGT Colliers de Perles and IMGT Alignments of alleles (IMGT Repertoire) of the IMGT/LIGM-DB reference sequence used for the primer description.
IMGT/MHC-DB
IMGT/MHC-DB[28] comprises databases hosted at EBI and includes a database of human MHC allele sequences or IMGT/MHC-HLA, developed by Cancer Research UK and maintained by ANRI, London, UK, on the Web since December 1998, and a database of MHC sequences from non human primates IMGT/MHC-NHP, curated by BPRC, The Netherlands, on the Web since April 2002.
IMGT sequence analysis tools
The IMGT sequence analysis tools comprise IMGT/V-QUEST[10], for the identification of the V, D and J genes and of their mutations, IMGT/JunctionAnalysis[11] for the analysis of the V-J and V-D-J junctions which confer the antigen receptor specificity, IMGT/Allele-Align for the detection of polymorphisms, and IMGT/PhyloGene[12] for gene evolution analyses.
IMGT/V-QUEST
IMGT/V-QUEST (V-QUEry and STandardization) is an integrated software for IG and TR [10]. This tool, easy to use, analyses an input IG or TR germline or rearranged variable nucleotide sequence. The IMGT/V-QUEST results comprise the identification of the V, D and J genes and alleles and the nucleotide alignments by comparison with sequences from the IMGT reference directory, the FR-IMGT and CDR-IMGT delimitations based on the IMGT unique numbering, the translation of the input sequence, the display of nucleotide and amino acid mutations compared to the closest IMGT reference sequence, the identification of the JUNCTION and results from IMGT/JunctionAnalysis (default option), and the two-dimensional (2D) IMGT Collier de Perles representation of the V-REGION [10] ("IMGT/V-QUEST output" in IMGT/V-QUEST Documentation).
IMGT/JunctionAnalysis
IMGT/JunctionAnalysis[11] is a tool, complementary to IMGT/V-QUEST, which provides a thorough analysis of the V-J and V-D-J junction of IG and TR rearranged genes. IMGT/JunctionAnalysis identifies the D-GENEs and alleles involved in the IGH, TRB and TRD V-D-J rearrangements by comparison with the IMGT reference directory, and delimits precisely the P, N and D regions [11] ("IMGT/JunctionAnalysis output results" in IMGT/JunctionAnalysis Documentation). Several hundreds of junction sequences can be analysed simultaneously.
IMGT/Allele-Align
IMGT/Allele-Align is used for the detection of polymorphisms. It allows the comparison of two alleles highlighting the nucleotide and amino acid differences.
IMGT/PhyloGene
IMGT/PhyloGene[12] is an easy to use tool for phylogenetic analysis of variable region (V-REGION) and constant domain (C-DOMAIN) sequences. This tool is particularly useful in developmental and comparative immunology. The users can analyse their own sequences by comparing with the IMGT standardized reference sequences for human and mouse IG and TR [12] (IMGT/PhyloGene Documentation).
IMGT sequence Web resources
The IMGT sequence Web resources are compiled in the IMGT Repertoire "Proteins and alleles" section that include Alignments of alleles, Proteins displays, Tables of alleles, Allotypes, Isotypes, etc. (Table 2). Standardized IMGT criteria for amino acid sequence analysis are described in [31].
IMGT gene databases, tools and Web resources
IMGT gene databases, tools and Web resources correspond to the IMGT genomics approach that refers to the studies of the genes within their loci and on their chromosome [2] (Table 3).
Table 3
The IMGT gene database, genome analysis tools and Web resources
IMGT genome database [1]
IMGT genome analysis tools [1]
IMGT Repertoire"Locus and genes" section [2] (1)
IMGT/GENE-DB [8]
IMGT/LocusViewIMGT/GeneViewIMGT/GeneSearchIMGT/CloneSearchIMGT/GeneInfo [13]
Chromosomal localizations [3, 4]Locus representations [3, 4]Locus descriptionGene exon/intron organizationGene exon/intron splicing sitesGene tablesPotential germline repertoiresLists of genesCorrespondence between nomenclatures [3, 4]
(1) IMGT Web resources (IMGT Marie-Paule page) also include IMGT Index, IMGT Education (IMGT Lexique, Aide-mémoire, Tutorials, Questions and answers), The IMGT Medical page, The IMGT Veterinary page, The IMGT Biotechnology page, The IMGT Immunoinformatics page, IMGT Bloc-notes (Interesting links, etc.) [2] which are not detailed in this paper.
(2) IMGT publications from the IMGT Repertoire "Locus and genes" section are available as pdf in IMGT Locus in Focus , in IMGT Index (see also [2]).
IMGT/GENE-DB, the IMGT gene database
Genomic data are managed in IMGT/GENE-DB, which is the comprehensive IMGT genome database [8]. IMGT/GENE-DB, created by LIGM (Montpellier, France) is on the Web since January 2003. In August 2005, IMGT/GENE-DB contained 1,377 genes and 2,207 alleles (673 IG and TR genes and 1,209 alleles from Homo sapiens, and 704 IG and TR genes and 998 alleles from Mus musculus, Mus cookii, Mus pahari, Mus spretus, Mus saxicola, Mus minutoïdes). All the human and mouse IG and TR genes are available in IMGT/GENE-DB. Based on the IMGT CLASSIFICATION concept, all the human IMGT gene names [3,4] were approved by the Human Genome Organisation (HUGO) Nomenclature Committee HGNC in 1999 [27], and entered in IMGT/GENE-DB [8], Genome DataBase GDB (Canada) [32], LocusLink and Entrez Gene at NCBI (USA) [33], and GeneCards [34]. Reciprocal links exist between IMGT/GENE-DB, and the generalist nomenclature (HGNC Genew) and genome databases (GDB, LocusLink and Entrez at NCBI, and GeneCards). All the mouse IG and TR gene names with IMGT reference sequences were provided by IMGT to HGNC and to the Mouse Genome Database (MGD) [35] in July 2002. Queries in IMGT/GENE-DB can be performed according to IG and TR gene classification criteria and IMGT reference sequences have been defined for each allele of each gene based on one or, whenever possible, several of the following criteria: germline sequence, first sequence published, longest sequence, mapped sequence [2]. IMGT/GENE-DB interacts dynamically with IMGT/LIGM-DB [7] to download and display gene-related sequence data. As an example ans as mentioned earlier, the IMGT/GENE-DB entries provide the IMGT/LIGM-DB accession numbers of the IG and TR cDNA sequences which contain a given V, D, J or C gene. This is the first example of an interaction between IMGT databases using the CLASSIFICATION concept.
IMGT gene analysis tools
The IMGT gene analysis tools comprise IMGT/LocusView, IMGT/GeneView, IMGT/GeneSearch, IMGT/CloneSearch and IMGT/GeneInfo. IMGT/LocusView and IMGT/GeneView manage the locus organization and the gene location and provide the display of physical maps for the human IG, TR and MHC loci and for the mouse TRA/TRD locus. IMGT/LocusView allows to view genes in a locus and to zoom on a given area. IMGT/GeneView allows to view a given gene in a locus. IMGT/GeneSearch allows to search for genes in a locus based on IMGT gene names, functionality or localization on the chromosome. IMGT/CloneSearch provides information on the clones that were used to build the locus contigs displayed in IMGT/LocusView (accession numbers are from IMGT/LIGM-DB, gene names from IMGT/GENE-DB, and clone position and orientation, and overlapping clones from IMGT/LocusView). IMGT/GeneInfo[13] provides and displays information on the potential TR rearrangements in human and mouse.
IMGT gene Web resources
The IMGT gene Web resources are compiled in the IMGT Repertoire "Locus and genes" section that includes Chromosomal localizations, Locus representations, Locus description, Gene exon/intron organization, Gene exon/intron splicing sites, Gene tables, Potential germline repertoires, the complete lists of human and mouse IG and TR genes, and the correspondences between nomenclatures [3,4] (Table 3). The IMGT Repertoire "Probes and RFLP" section provides additional data on gene insertion/deletion.
IMGT structure database, tool and Web resources
The IMGT structural approach refers to the study of the 2D and 3D structures of the IG, TR, MHC and RPI, and to the antigen or ligand binding characteristics in relation with the protein functions, polymorphisms and evolution (Table 4). The structural approach relies on the CLASSIFICATION concept (IMGT gene and allele names), DESCRIPTION concept (receptor and chain description, domain delimitations), and NUMEROTATION concept (amino acid positions according to the IMGT unique numbering [14-18]).
Table 4
IMGT structure database, analysis tool and Web resources
IMGT structural database [1]
IMGT structural analysis tool [1]
IMGT Repertoire"2D and 3D structures" section [2]
IMGT/3D structure-DB [15]
IMGT/StructuralQuery [15]
2D Colliers de Perles IG and TR [3, 4, 16, 17, 19] (1)2D Colliers de Perles MHC [18, 36]2D Colliers de Perles RPI [16-18, 21, 22, 24, 37]IMGT classes for amino acid characteristics [31]IMGT Colliers de Perles reference profiles [31]3D representations (1)
(1) Cover of the Nucleic Acids Research 1999 database issue
Structural and functional domains of the IG and TR chains comprise the variable domain or V-DOMAIN (9-strand beta-sandwich) which corresponds to the V-J-REGION or V-D-J-REGION and is encoded by two or three genes [3,4], the constant domain or C-DOMAIN (7-strand beta-sandwich), and, for the MHC chains, the groove domain or G-DOMAIN (4 beta-strand and one alpha-helix). A uniform numbering system for IG and TR V-DOMAINs of all vertebrate species has been established to facilitate sequence comparison and cross-referencing between experiments from different laboratories whatever the antigen receptor (IG or TR), the chain type, or the species [14-16]. In the IMGT unique numbering, conserved amino acids from frameworks always have the same number whatever the IG or TR variable sequence, and whatever the species they come from. As examples: Cysteine 23 (in FR1-IMGT), Tryptophan 41 (in FR2-IMGT), hydrophobic amino acid 89 and Cysteine 104 (in FR3-IMGT) (Figure 2). This numbering has been applied with success to all the sequences belonging to the V-set of the IgSF [20], including non-rearranging sequences in vertebrates (human CD4, Xenopus CTXg1, etc.) and in invertebrates (drosophila amalgam, drosophila fasciclin II, etc.) [15,16,21]. The IMGT unique numbering, initially defined for the V-DOMAINs of the IG and TR and for the V-LIKE-DOMAINs of IgSF proteins other than IG and TR, has been extended to the C-DOMAINs of the IG and TR (Figure 2B), and to the C-LIKE-DOMAINs of IgSF proteins other than IG and TR [17]. An IMGT unique numbering has also been implemented for the groove domain (G-DOMAIN) of the MHC class I and II chains (Figure 3), and for the G-LIKE-DOMAINs of MhcSF proteins other than MHC [18].
Figure 2
IMGT Colliers de Perles of a V-DOMAIN (A) and of a C-DOMAIN (B) (code PDB 1mcd in IMGT/3Dstructure-DB [9]). IMGT Colliers de Perles are shown on one layer (on the left hand side) and on two layers with hydrogen bonds (on the right hand side). (A) The IMGT Collier de Perles of a V-DOMAIN is based on the IMGT unique numbering for V-DOMAIN and V-LIKE-DOMAIN [16]. The CDR-IMGT are limited by amino acids shown in squares, which belong to the neighbouring FR-IMGT. The CDR3-IMGT extends from position 105 to position 117. CDR-IMGT regions are colored as follows on the IMGT site: CDR1-IMGT (blue), CDR2-IMGT (bright green), CDR3-IMGT (dark green) and hydrogen bonds are shown as green lines. (B) The IMGT Collier de Perles of a C-DOMAIN is based on the IMGT unique numbering for C-DOMAIN and C-LIKE-DOMAIN [17]. Amino acids are shown in the one-letter abbreviation. Arrows indicate the direction of the beta strands that form the two beta sheets of the immunoglobulin fold [3, 4]. Hatched circles correspond to missing positions according to the IMGT unique numbering [16, 17]. In the IMGT Collier de Perles on the IMGT Web site  hydrophobic amino acids (hydropathy index with positive value) and Tryptophan (W) found at a given position in more than 50 % of analysed IG and TR sequences are shown in blue, and all Proline (P) are shown in yellow.
Figure 3
IMGT Colliers de Perles of the two G-DOMAINs of MHC class I (A) and of MHC class II (B) proteins (codes PDB 1bd2 and 1aqd, respectively, in IMGT/3Dstructure-DB [9]). The IMGT Collier de Perles of a G-DOMAIN is based on the IMGT unique numbering for G-DOMAIN and G-LIKE-DOMAIN [18]. (A) The two MHC-I G-DOMAINs, G-ALPHA1 (top) and G-ALPHA2 (bottom), form the groove of the MHC class I chain (I-ALPHA). (B) The two MHC-II G-DOMAINs, G-ALPHA (top) of the MHC class II alpha chain (II-ALPHA) and G-BETA (bottom) of the MHC class II beta chain (II-BETA), form the groove of the MHC class II protein [36]. Amino acids are shown in the one-letter abbreviation. Hatched circles correspond to missing positions according to the IMGT unique numbering [18]. Positions in colour correspond to the IMGT contact sites provided, for each peptide/MHC 3D structure, in IMGT/3Dstructure-DB [36].
IMGT/3Dstructure-DB, the IMGT 3D structure database
IMGT/3Dstructure-DB is the IMGT 3D structure database, created by LIGM, and on the Web since November 2001 [9]. In August 2005, IMGT/3Dstructure-DB contained 946 atomic coordinate files. IMGT/3Dstructure-DB comprises IG, TR, MHC and RPI with known 3D structures [9,36,37]. Coordinate files extracted from the Protein Data Bank (PDB) [38] are renumbered according to the standardized IMGT unique numbering [16-18]. The IMGT/3Dstructure-DB card provides, on-line, the complete information for each IMGT/3Dstructure-DB entry. The IMGT/3Dstructure-DB card shows a summary table and a menu that gives access to five sections: "Chain details", "Contact analysis", "Visualization with Jmol", "Renumbered file" and "References and links". The "Chain details" section provides chain description, IMGT gene and allele names, IMGT chain and domain labels, domain delimitations, amino acid positions according to the IMGT unique numbering, IMGT Colliers de Perles [16-19]. The "Contact analysis" section provides contact types and categories between domains (in IMGT/3Dstructure-DB Domain contacts) and atom contacts at the residue and position level (in IMGT/3Dstructure-DB Residue@Position contacts) [37]. (IMGT/3Dstructure-DB Documentation). The "Renumbered file" section downloadable provides renumbered IMGT/3Dstructure-DB flat files.
IMGT/StructuralQuery tool
The IMGT/StructuralQuery tool [9] analyses the interactions of the residues of the antigen receptors IG and TR, MHC, RPI, antigens and ligands. The contacts are described per domain (intra- and inter-domain contacts) and annotated in term of IMGT labels (chains, domain), positions (IMGT unique numbering), backbone or side-chain implication [37]. IMGT/StructuralQuery allows to retrieve the IMGT/3Dstructure-DB entries, based on specific structural characteristics: phi and psi angles, accessible surface area (ASA), amino acid type, distance in angstrom between amino acids, CDR-IMGT lengths.
IMGT structure Web resources
The IMGT stucture Web resources are compiled in the IMGT Repertoire "2D and 3D structures" section which includes 2D representations or IMGT Colliers de Perles [16-19], 3D representations, FR-IMGT and CDR-IMGT lengths [16], amino acid chemical characteristics profiles [31], etc. In order to appropriately analyse the amino acid resemblances and differences between IG, TR, MHC and RPI chains, eleven IMGT classes were defined for the 'chemical characteristics' amino acid properties and used to set up IMGT Colliers de Perles reference profiles [31]. The IMGT Colliers de Perles reference profiles allow to easily compare amino acid properties at each position whatever the domain, the chain, the receptor or the species. The IG and TR variable and constant domains represent a privileged situation for the analysis of amino acid properties in relation with 3D structures, by the conservation of their 3D structure despite divergent amino acid sequences, and by the considerable amount of genomic (IMGT Repertoire), structural (IMGT/3Dstructure-DB) and functional data available. These data are not only useful to study mutations and allele polymorphisms, but are also needed to establish correlations between amino acids in the protein sequences and 3D structures and to determine amino acids potentially involved in the immunogenicity.
Conclusion
In order to allow any IMGT component to be automatically queried and to achieve a higher level of interoperability inside the IMGT information system and with other information systems, our current objectives include the modelling of the three major IMGT biological approaches, genomics, genetics and structural approaches, the analysis of the IMGT components (databases, tools and Web resources) in relation with the concepts, and the development of Web services [2]. They are the first steps towards the implementation of IMGT-Choreography [2], which corresponds to the process of complex immunogenetics knowledge [25] and to the connection of treatments performed by the IMGT component Web services. IMGT-Choreography has for goal to combine and join the IMGT database queries and analysis tools. In order to keep only significant approaches, a rigorous analysis of the scientific standards [3,4], of the biologist requests and of the clinician needs [39-42] has been undertaken in the three main biological approaches: genomics, genetics and structural approaches. The design of IMGT-Choreography and the creation of dynamic interactions between the IMGT databases and tools, using the Web services and IMGT-ML, represent novel and major developments of IMGT, the international reference in immunogenetics and immunoinformatics. IMGT-Choreography enhances the dynamic interactions between the IMGT components to answer complex biological and clinical requests.
Since July 1995, IMGT has been available on the Web at . IMGT has an exceptional response with more than 140,000 requests a month. The information is of much value to clinicians and biological scientists in general. IMGT databases, tools and Web resources are extensively queried and used by scientists from both academic and industrial laboratories, from very diverse research domains: (i) fundamental and medical research (repertoire analysis of the IG antibody sites and of the TR recognition sites in normal and pathological situations such as autoimmune diseases, infectious diseases, AIDS, leukemias, lymphomas, myelomas), (ii) veterinary research (IG and TR repertoires in farm and wild life species), (iii) genome diversity and genome evolution studies of the adaptive immune responses, (iv) structural evolution of the IgSF and MhcSF proteins, (v) biotechnology related to antibody engineering (single chain Fragment variable (scFv), phage displays, combinatorial libraries, chimeric, humanized and human antibodies), (vi) diagnostics (clonalities, detection and follow up of residual diseases) and (vii) therapeutical approaches (grafts, immunotherapy, vaccinology).
Citing IMGT
If you use IMGT databases, tools and/or Web resources, please cite [1] and this paper as references, and quote the IMGT Home page URL address, .

imgt
immunogenetics
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immunoinformatics
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immunoglobulin
t cell receptor
mhc
superfamily
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three-dimensional
ontology
annotation
antibody
3d structure
collier de perles
polymorphism
hla