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EP1620567A1 - Methode de diagnostic de la polyarthrite rhumatoide ou de l' osteoarthrite - Google Patents

Methode de diagnostic de la polyarthrite rhumatoide ou de l' osteoarthrite

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Publication number
EP1620567A1
EP1620567A1 EP03722486A EP03722486A EP1620567A1 EP 1620567 A1 EP1620567 A1 EP 1620567A1 EP 03722486 A EP03722486 A EP 03722486A EP 03722486 A EP03722486 A EP 03722486A EP 1620567 A1 EP1620567 A1 EP 1620567A1
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Prior art keywords
rheumatoid arthritis
osteoarthritis
genes
protein
expression profiling
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EP03722486A
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German (de)
English (en)
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Hans-Jürgen Thiesen
Peter Lorenz
Peter Stiehl
Dirk Koczan
Peter Ruschpler
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to a method for diagnosing rheumatoid arthritis or osteoarthritis, or of a predisposition therefor, using expression profiling data of differentially expressed genes and a biosensor chip and medical or diagnostic instrument suitable for carrying out this method, and a method for monitoring the therapeutical effect of an anti-rheumatoid arthritis or anti-osteoarthritis drug or for screening for a potential anti-rheumatoid arthritis or anti-osteoarthritis drug, and a method for the production of an anti-rheumatoid arthritis or anti-osteoarthritis drug, and the anti-rheumatoid arthritis or anti-osteoarthritis drugs obtainable by this method.
  • RA rheumatoid arthritis
  • OA osteoarthritis
  • MnSOD manganese superoxide dismutase
  • the new enabling technologies in functional genomics and proteomics are powerful tools to study complex multifactorial diseases.
  • a molecular inventory of diseased tissues provides a first step to gain insight into the pathophysiology of these diseases.
  • Differentially expressed genes and their products have to be elucidated in relation to specific cell types/tissues and with respect to their expression patterns in space and time.
  • the functional states of the gene products have to be described. All these approaches will provide candidate targets for in-depth functional studies that foster drug discovery processes.
  • Microarrays have proven very useful to acquire large data sets of differential gene expression on the transcriptional level [1-3].
  • transcript levels do not necessarily reflect the amount of protein that represents the actual functional entity of a gene [4-9].
  • some compartments of an organism that are of interest do not contain RNA at all, e.g. body fluids.
  • Technologies in proteomics aim at the cataloguing of the whole protein complements of biological fluids, cells and tis- sues and at the description of specific functional states of the proteins.
  • the complexity of protein profiles exceeds that of the RNA profiles manifold due to the posttranscriptional and posttrans- lalional generation of different protein species [10].
  • Two-dimensional gel eleclrophoresis coupled to MALDI-TOF mass spectrometry yields valuable insights into the molecular composition of cells and tissues.
  • technical difficulties impede or even prevent the display of certain proteins and the limited dynamic range results in a bias towards abundant proteins [11-13].
  • These limitations have sparked efforts to find alternatives.
  • One possibility is to make use of antibodies for protein profiling, e.g. in multi-Western blot formats or on protein microarrays [14-16].
  • Rheumatoid arthritis with a prevalence of about 1 % is a chronic systemic disease that involves mechanisms of autoimmunity [17, 18]. Characteristic is a severe inflammation of the joints with infiltration of activated T cells, macrophages, plasma cells and other immunocompetent cells that leads to progressive destruction of cartilage and bone. Synovial cells show signs of hyperpro- liferation and hypertrophy and the synovial tissue is transformed into a pathologic mass of cells called pannus tissue that also undergoes neovascularization. The etiology of the disease remains unresolved. Hypotheses on the ultimate cause of RA include antigen-specific autoimmune reactions and the existence of an infectious agent [19].
  • RA osteoarthritis
  • GenBank's URL is http://www.ncbi.nlm.nih.gov/Genbank/GenbankSearch.html.
  • the inventors conducted a comparative molecular characterization of synovial tissues of patients suffering from RA versus OA on the RNA and protein levels.
  • selected genes found to be differentially expressed on the protein levels were validated by Western blotting with a higher sample number and assigned to particular cell types within the synovial tissue.
  • RNA profiling screen 44928 transcripts were compared for 6 RA and 6 OA syno- vial tissue samples by DNA oligonucleotide microarrays.
  • the data exemplify approaches to improve our understanding of diseases that target complex tissues using global profiling technologies.
  • rheumatoid and osteoarthritic synovial tissue were obtained as part of surgical therapy of diseased joints during synovectomy or implantation of an endoprothesis. Collection of samples was approved by the ethical board of the University of Leipzig. Patients with rheumatoid arthritis (RA) had classical late-stage disease and met the American College of Rheumatology classification criteria. The synovial tissue was dissected, separated from associated fat, snap-frozen in liquid nitrogen and stored either in liquid nitrogen or at -80° C. All tissues were assessed by histopathology. All RA patients were under medication including steroids, disease-modifying anti-rheumatic drugs (DMARDs) and other non-steroidal anti-inflammatory drugs. Diagnosis of osteoarthritis (OA) was based on clinical examination and histopathology. OA patients typically received non-steroidal antiinflammatory drugs (NSAIDs).
  • NSAIDs non-steroidal antiinflammatory drugs
  • TS buffer preheated TS buffer was added and the homogenate was immediately mixed and heated for 5min in a 90° C water bath with occassional further heavy vortexing.
  • the heating procedure with TS buffer followed recommendations of the supplier of the PowerBlotTM service (see below).
  • tissue ho- mogenates were centrifuged for 10 min at 10° C at 20 000 x g.
  • the supematants contained the protein fractions to be analyzed.
  • the protein content of the extracts were meas- ured using the BCA method (Perbio, Bonn, Germany).
  • the PowerblotTM is a commercial Western blot format of BD Biosciences (through BD Transduction Laboratories, Lexington, KY, USA; http://bioinfo.clontech.com/powerblot/). Samples of different tissues or conditions are subjected to semi-quantitative comparative immunostaining with a panel of at that time 791 mouse monoclonal antibodies in the company's portfolio. Samples are loaded across the whole width of the gel. A manifold is then used to separate 45 lanes on the blots. Immunostaining is performed in such a way that several antibodies recognizing clearly distinguishable proteins with respect to their mobility on SDS-PAGE are added together to one lane formed by the manifold.
  • Triplicates of each antibody reaction visualized by chemiluminiscence are evaluated by densitometry and compared between two samples.
  • the output displays ratios of protein levels between two samples in several confidence groups depending on the quality and strength of the signal.
  • Signals are normalized by dividing the optical density obtained for a signal through the total intensity value of all pixels in an image and multiplied with 1 ,000,000. These normalized quantities are used to compute the changes between two samples.
  • Lammli-type 10 or 12 % SDS polyacrylamide mini-gels were run according to standard procedures. They were blotted to PVDF membrane (Polyscreen, NEN Life Sciences, Zaventem, Belgium ) by semi-dry electroblotting [24]. The blot quality was assessed by staining the membrane for 5 min- utes in 0.3% Ponceau S in 3% trichloroacetic acid and subsequent washing with H20. Blocking of the membrane was usually done overnight at 4° C with 50 mM TRIS/HCI pH 7.4, 150 mM NaCI (TBS) plus 5 % nonfat dry milk powder and 1 % bovine serum albumin.
  • TRIS/HCI pH 7.4 150 mM NaCI (TBS) plus 5 % nonfat dry milk powder and 1 % bovine serum albumin.
  • Immunological reagents were usually diluted in blocking buffer and incubated at room temperature for 2 hours (primary antibodies) or 1 h (secondary antibody and tertiary reagent).
  • the primary antibodies included rabbit anti-Statl (sc-346; Santa Cruz Biotechnology, Heidelberg, Germany; used at 1 :2000), goat anti-CD3 ⁇ (sc-1127; Santa Cruz Biotechnology, Heidelberg, Germany; used at 1 :500), mouse monoclonal anti-p47phox, anti-manganese superoxide dismutase and anti-cathepsin D (P33720, M99920, and C47620 respectively; BD Transduction Laboratories, Heidelberg Germany; used at 1 :1000, 1 :1000 and 1 :3000, respectively). All secondary antibodies and Streptavidin-Peroxidase were from Jackson/Dianova (Hamburg, Germany) and were used at 50 and 100 ng/ml, respectively.
  • Blots intended to be measured by densitometry were developed with 4-chloro-1-naphthol as described [24].
  • the blot signals were scanned using a HP desktop scanner in color mode at 150 dpi, 8bit resolution.
  • Raw bitmap images were converted into grayscale Tiff-files and the integrated optical density, i.e. the volume of the signals, was quantitatively assessed by Phoretix 6.0.1 Advanced (Nonlinear Dynamics Ltd) without any image adjustments. Boundaries of signals were drawn by hand and background was removed by subtraction of the "average on boundary" volume. Differences between patient groups were statistically assessed by Student's t-test after calculation of the mean integrated optical density ⁇ SD.
  • RNA was isolated from snap-frozen synovial tissue pieces by grinding the tissue together with frozen RLT buffer (RNeasy Kit, Qiagen, Hilden, Germany) supplemented with 1 % ⁇ -mercaptoethanol in the frozen state. After thawing and rigorous vortexing RNA was isolated from tissue lysates with the RNeasy kit as recommended by the manufacturer. 5 ⁇ g total RNA was processed and labeled as recommended by the chip manufacturer.
  • RLT buffer RNeasy Kit, Qiagen, Hilden, Germany
  • Hybridizations of HG-U95A or HG-U133A and HG-U133B DNA oligonucleotide microarrays were performed according to the instructions of the manufacturer. Data were processed using Affymetrix Microarray Suite 4.0 and 5.0 and Affymetrix Data Mining Tool 3.0. The default parameter settings of the software were used. 3 Results
  • RNA profiling results showed 6630 (53 %) and 6963 (56 %) gene sets to be detected in the RA and OA synovial tissues, respectively (Table 1 ). These values indicate that the corresponding transcripts of 47 or 44 % of gene sets are either not expressed in synovial tissues or have not been detected due to sensitivity limits of the chip technology employed.
  • Transcripts of 1062 genes were differentially expressed in the RA versus the OA synovial tissue, 416 overexpressed and 645 underexpressed.
  • the overexpressed gene cluster contained genes indicative of inflammation and the activated state of immunocompe- tent cells like cytokines/chemokines and their receptors or immunoglobulins.
  • Prominent examples of the former were interleukin 15, CXCL9/Mig, CXCL10/IP10, and CCL5/RANTES, CXCL13/B-cell chemoattractant, CCL19/Ebi1 ligand and the interleukin receptors 2 ⁇ , 7, and 15 or the chemokine receptor CCR5 (data not shown).
  • cartilage oligomeric matrix protein that is synthesized by synovial cells and believed to be associated with OA disease [20].
  • examples for other genes in this class are the adhesion molecule thrombospondin 4, growth arrest protein GAS1 and the C2H2 zinc finger protein ZIC1.
  • This set of antibodies provided an opportunity to i) detect high and low abundant proteins side by side and ii) to analyze different proteins in a number that can usually not been accomplished in a single laboratory because of time and resource constraints. From the panel of 791 antibodies 260 (33 %) detected their corresponding protein.
  • RNA and protein level changes in the RA compared to the OA synovial tissue are grouped and displayed in table 1 with annotation of their known function. The inventors selected some of these genes for further analysis since concordance on the RNA and protein levels underscored the changes.
  • the aspartic protease cathepsin D was included. Cathepsin D was found to be among the most prominent proteins underrepresented in RA compared to OA synovial tissues in the PowerBlotTM screen. This was observed with two different antibodies. On the mRNA level the expression of this gene was detectable in tissues derived from both diseases. However, cathepsin D mRNA levels were not significantly different as judged by the Affymetrix microarray analysis software.
  • RNA and protein profiling screens One prominent gene from table 1 is the signal transducer and activator of transcription 1 (Statl ). On the RNA level, all the chip's probe sets specific for Statl are scored “increased” with a highest change of 13.3 fold. On the PowerBlotTM two different antibodies scored the Statl protein "increased”.
  • the p47phox and p67phox genes in table 1 both overexpressed in RA, gained immedi- ate attention since their gene products cooperate in the NADPH oxidase complex. This complex is involved in superoxide production in phagocytic cells as a means of pathogen defense [26].
  • MnSOD manganese superoxide dismutase
  • RNA and protein levels described above resulted from a case study using one patient sample of each disease (RA vs. OA).
  • RA vs. OA
  • Wesl- ern blots of selected gene products were performed on a panel of 16 synovial tissue samples, representing 8 cases of each disease (RA and OA) and the specific signals on the blot were quanti- tated by densitometry (Fig 1).
  • Statl -specific antibodies detected double bands of signals in all OA and RA samples. These bands most likely represent the two Stall isoforms, Statl ⁇ and Statl ⁇ , that are known to result from alternative splicing. Densitometry was used to quantitatively compare the RA and OA conditions.
  • the mean difference in Statl protein content was an about 3.7fold higher expression in synovial tissues from RA patients (p ⁇ 0.01 ). In contrast to Statl , cathepsin D was more abundantly expressed in OA synovial tissue. Here the mean difference was about 2.7fold (p ⁇ 0.05). Noticeably, the OA patient samples showed divergent protein levels with five extracts standing clearly out against the other three OA and the eight RA samples. The clinical data of re- spective patients did not hint at obvious reasons for these variations.
  • the signals for p47phox consisted of an expected band at around 44/45 kD plus a faster migrating band at about 32kD that might represent a degradation product. Both bands were measured by densitometry and their optical densities added.
  • RA synovial tissue contained about 2.8fold more p47phox than the respective OA tissue (p ⁇ 0.01 ).
  • the higher abundance of MnSOD in the RA synovial tissue could be validated in the Western blot as well. Clear signals at about the expected size of 25 kD were obtained in all RA and most OA samples.
  • the mean increase in protein content in RA versus OA tissue was 2.7fold (p ⁇ 0.01 ).
  • immunostaining with antibodies against the T cell marker CD3 ⁇ was performed.
  • As expected RA tissue had significantly more CD3 ⁇ (4.3fold mean increase, p ⁇ 0.05) reflecting the higher infiltration of T cells in RA compared to the OA disease.
  • the blot showed heterogeneity of signals and thus of respective protein contents in different patients of one disease group, most pronounced in the cathepsin D stain as described above. These deviations are likely due to differences in individual patients, different disease manifestations and/or different therapeutic treatment responses.
  • transcriptome case study was extended by subjecting synovial tissue from 6 more RA and OA patients, each, to RNA profiling on DNA oligonucleotide microarrays. Differential analysis of the 6
  • RA samples against the 6 OA samples resulted in 36 cross-comparisons with each OA sample as baseline for each RA sample. Concordant expression changes in 25 out of 36 cross-comparisons were taken as significant.
  • the analysis resulted in 547 genes typically overexpressed and 773 genes typically underexpressed in the RA versus the OA synovial tissue. The genes provide additional expression signatures to distinguish RA and OA disease.
  • RNA/protein abundance were in many cases not reliably indicated on the transcript level. E.g., out of 58 unambiguous changes on the protein level only 16 were found on the transcript level as well (28%). Discrepancies between protein and transcript abundances reflect mechanisms of gene expression regulation that do not act at the mRNA level. Such mechanisms include translational control, post-translational processing and regulation of protein stability [27, 28]. A relatively poor overall correlation of mRNA/protein levels was pointed out before in yeast [6, 7], human liver [4] and more recently in human tumor samples [9]. In the latter case correlations of RNA/protein abundance were low not only when determined within one sample but also when the data for a particular gene were averaged across all samples.
  • the inventors strategy was to validate candidates for differential expression between RA/OA by Western blotting using 8 RA against 8 OA samples. Furthermore, additional 6 RA and OA samples were analyzed on the transcript levels leading to characteristic RNA expression signatures for the two diseases.
  • the differences in gene expression between RA and OA synovial tissues likely reflect i) the differ- ences in the pathological phenotypes of resident synovial cells ii) the different extent of infiltration of in particular lymphoid cells into the synovium and iii) the different disease-specific interplay between all cell types in the arthritic microenvironment.
  • a good example for case i) are the synovial fibroblasts.
  • Such fibroblasts from RA patients display an activated/transformed phenotype, secrete a distinct pattern of cytokines and are mainly responsible for the massive synovial hyperplasia [31].
  • the activated RA synovial fibroblasts are e.g.
  • RA synovium is infiltrated to a higher extent by mononuclear cells like T and B lymphocytes or macrophages.
  • mononuclear cells like T and B lymphocytes or macrophages.
  • differences in cellular composition will certainly contribute to expression differences when complex tissues were compared.
  • infiltrating cells do also change their phenotype in the arthritic joint. Immigrating monocytes, e.g, can differentiate into mature macrophages [33]. Such changes will also contribute to differences in gene expression.
  • Statl was identified to be significantly more abundant in RA compared to OA synovial tissue on RNA as well as protein levels.
  • Stat proteins are important mediators of cyto- kine signalling and Statl is at the center of interferon signal transduction [35].
  • the pro-inflammatory chemokine RANTES that is overexpressed in RA synovial tissue ( [23], see also the inventors data in the Results section and below), is known to activate Statl in T cells [36].
  • Statl is also considered to be an important transcription factor in macrophages, especially in response to bacterial lipopolysaccharide [37, 38]. So far studies of Statl in the subject of arthritis are rudimentary. Two reports analysed Statl activation in synovial fluid cells, but came to different conclusions [39, 40]. In preliminary experiments analyzed the activation state of Statl protein immunoprecipi- tated from RA and OA synovial tissues through determination of its phosphorylation status. In both diseases found tyrosine 701 and serine 727 phosphorylated Statl species (data not shown). The investigation of quantitative differences in phosphorylation states between both diseases awaits further experimentation.
  • RNA profiling results identified a number of interferon-inducible genes with more abundant transcript levels in RA than OA synovial tissue.
  • Prominent examples are cytokines/chemokines interleukin 15, CXCL9/Mig, CXCL10/IP10, and CCL5/RANTES that are also well-established genes abundantly expressed in inflammatory disorders including rheumatoid arthritis (fore reviews see [41 , 42]).
  • the Statl gene itself can also be induced by interferons [43].
  • the suppressor of cytokine signaling (SOCS)1 gene was also among the interferon-inducible genes whose transcripts were overexpressed in RA versus OA in the inventors screen.
  • SOCS proteins are important negative regulators of cytokine sig- naling [44].
  • the mRNA expression of the related SOCS3 was more abundant in OA (data not shown). This might be of importance since a recent report highlighted a strategy to treat RA disease by upregulation of SOCS3 protein [45].
  • p47phox and p67phox proteins are part of the NADPH oxidase system of phagocytic cells that is essential for the innate immune response to pathogens [26].
  • the NADPH oxidase system is further considered an important mediator of inflammatory disorders like ischemia-reperfusion injury in the brain, heart and liver [46].
  • the upregulation of p47phox and p67phox in synovial tissue of RA pa- tients described in this invention pointed to a possible role of NADPH oxidase produced superoxide in RA in humans.
  • RNA profiling data also contained expression information on other genes whose products can be found in the NADPH oxidase complex: Both, p40phox and the small GTPase Rac2 were found to be upregulated (1.9fold and 11fold, respectively) in RA compared to OA tissue. In contrast, gp91 phox was neither detected on the RNA level nor on the protein level in the PowerblotTM of both tissues (data not shown). Strikingly, the p47phox gene has recently been shown to be an arthritis susceptibility gene in the rat, regulating the severity of the disease [47]. A natural occuring polymorphism was found that resulted in decreased NADPH oxidase activity and promoted activation of arthritogenic T cells.
  • MnSOD manganese superoxide dismutase
  • the superoxide dismutase enzyme family is essential for the elimination of tissue damaging superoxide. Thus, this enzyme family can be considered a counter-player to the superoxide producing NADPH oxidase system.
  • MnSOD a mitochondrial matrix protein, is the principal scavenger of superoxide that is leaking from mito- chondrial respiratory chains [51].
  • the anti- inflammatory effects of increased superoxide dismutase have been observed after gene transfer of the extracellular enzyme in the collagen-induced arthritis mouse model where it improved the clinical and histological score [52].
  • protease cathepsin D has not been studied in much detail with respect to arthritis in contrast to e.g. cathepsins B and L.
  • Cathepsin D was mainly observed in tissue macrophages by immunohistochemistry (data not shown).
  • elevated cathepsin D transcripts were described in interstitial and in part also in perivascular synovial regions in RA [55]. The protein abundance was not examined.
  • differentially expressed genes in RA versus OA synovial tissues by expression profiling on the RNA and protein levels.
  • Prominent examples included Statl , p47phox, MnSOD and cathepsin D.
  • Discordant gene expression of respective transcripts and proteins emphasize the need of proteome analysis to un- derstand the pathological phenotype.
  • the search for differentially expressed genes is an important means to find markers of disease for diagnosis and starting points for evaluation of pathophysi- ological pathways.
  • Such differentially expressed genes are also candidates for studies on the post- translational level, functional investigations in animal models, e.g. employing knock-out or trans- genic strategies, and for searches for genetic polymorphisms.
  • Table 1 Compilation of concordant expression changes between K55/RA and K42/OA synovial tissues on protein and transcript levels
  • Western blots with specific antibodies against respective proteins were analyzed by densitometry. Plotted is the mean volume of the signals ⁇ S.D. for each patient group. The significance of the differences between the two patient groups is indicated by the as- teriks * (p ⁇ 0.05) and ** (p ⁇ 0.01 ).
  • the global scale molecular profiling of diseased tissues is an important first step to unravel candidate target molecules that are involved in the pathogenesis of a disease.
  • the inventors have performed a comparative molecular characterization on the transcriptome (microarray with 12526 gene specificities) and proteome level (multi-Western blot PowerblotTM with 791 antibodies) of synovial tissue from rheumatoid arthritis (RA) compared to osteoarthritis (OA) patients. From the panel of 791 antibodies 260 (33 %) detected their corresponding protein. Out of 58 unambigu- ous changes on the protein level only 16 coincided on the transcript level (28%>).
  • the 773 gene transcripts numbered 1 to 773 represented by GeneBank accession numbers AA017245 to Z24725 are expressed at a reduced level for RA in comparison to OA and are marked "D" ("decreased")
  • the 546 gene transcripts numbered 774 to 1320 with GeneBank accession numbers AA005023 to Z83819 are expressed at increased levels for RA in comparison to OA and are marked "I” ("increased”)
  • the 88 gene products numbered 1321 to 1408 represented by GeneBank accession numbers AA888001 to Z18859 are expressed either at a reduced protein level for RA and then marked “D" ("decreased") or at an elevated level compared to OA and then marked “I” ("increased”). 5 References

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WO2006061430A2 (fr) * 2004-12-10 2006-06-15 Proskelia Itgbl-1 utile comme cible regulant la metastase osseuse et le developpement osseux
WO2007082352A1 (fr) * 2006-01-20 2007-07-26 Child Health Research Institute Inc Méthode de traitement, de prophylaxie et de diagnostic de pathologies osseuses
CA2643744A1 (fr) 2006-02-27 2007-08-30 Technische Universitaet Muenchen Imagerie et traitement du cancer
CA2680339C (fr) 2007-03-08 2017-02-28 F. Hoffmann-La Roche Ag Utilisation de slim-1 dans l'evaluation d'une insuffisance cardiaque
EP2379096B1 (fr) 2008-12-19 2019-10-30 Baxalta GmbH Inhibiteurs de tfpi et procédés d utilisation
ES2621809T3 (es) 2010-03-19 2017-07-05 Baxalta GmbH Inhibidores de TFPI y procedimientos de uso
ES2733958T3 (es) 2012-03-21 2019-12-03 Baxalta GmbH Inhibidores de TFPI y métodos de uso
TWI648273B (zh) 2013-02-15 2019-01-21 英商葛蘭素史克智慧財產發展有限公司 作為激酶抑制劑之雜環醯胺類（三）
EP3198023B1 (fr) 2014-09-26 2020-04-22 Somalogic, Inc. Prédiction d'évènement de risque cardio-vasculaire et leurs utilisations
CN109061192B (zh) * 2018-08-24 2021-07-16 中国医学科学院北京协和医院 一种与骨关节炎相关的尿液蛋白及其应用
CN112094846B (zh) * 2020-05-20 2022-02-18 中山大学孙逸仙纪念医院 特异性靶向骨性关节炎滑膜细胞的修饰碱基适配体及其应用

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