JP2009524031A - Test system for analyzing polypeptides and cells that adhere to silicon - Google Patents

Abstract

  The present invention relates generally to the field of diagnostics, ie test systems in polypeptides related to silicon. More precisely, the present invention relates to a test system for identifying and analyzing polypeptides that in one embodiment adhere to silicon. Furthermore, the present invention relates to newly identified polypeptides that adhere to silicon and the use of each such protein in a test system for detecting response responses to, for example, mammalian silicon.

Description

  The present invention relates generally to the field of diagnostics, ie, test systems in silicon related polypeptides. More particularly, the invention relates to a test system for identifying and analyzing polypeptides that in one embodiment adhere to silicon. In addition, the invention relates to newly identified polypeptides that adhere to silicon and the use of each such protein in a test system for detecting response responses to, for example, mammalian silicon.

  Dimethylsiloxane polymers, also known as “silicon”, are the most widely used inorganic biomaterials in the medical industry because of their assumed biocompatibility. It has a wide variety of applications such as medical tubing systems, syringe lubricants, active medical device coatings such as bypass devices and transplanted cochlear stimulators, as well as joint replacement and eye lenses to ventricular abdominal shunts and manmaries. Used in the creation of passive injections that span injections.

  However, various local and systemic side effects have been reported as response responses to silicon, especially after prolonged infusion. These phenomena have been observed, for example, in women with silicon mammary injections (SMIs) [Smoray D. L. (Smallley D.L.), Levin J.H. J. et al. (Levine JJ), Shanklin D. R. (Shanklin DR), Hall M. F. (Hall MF) and Stevens M.F. V. (Stevens MV) “Lymphocyte response to silica ammon offspring of silicon breast imprint recipients.” Immunobiology 196, 567-574. Goldblum R. M.M. (Goldblum RM), Perry R.M. P. (Pelley RP), O'Donnell A.M. A. (O'Donell AA), Pylon D. (Pyron D.) and Hejars J.M. P. (Heggers JP) “Antibodies to silicon elastomers and reactions to ventricular peritonal shunts.” Lancet 340, 510-513 (1992). J. et al. (Kinnari T.J.), Salonen E. M.M. (Salonen EM) and Jero J.M. (Jero J.) “Durability of the binding in albuming of albuminating tubes.” International Journal of Pediatric Organic 3), 1967.

  The so-called local response of living organisms to silicon is characterized by the formation of a fibrous connective tissue capsule that can vary in size [Shiguro W. et al. (Siggelow W.), Faridi A. (Faridi A.), Spiritus K. (Spiritus K.), Klinge U. (Klinge U.), Lass. (Rath W.) and Klosterhalfen B.I. (Klostalfen B.) “Histological Analysis of silicon breast implant components and correlation with capstract structure.” Biomaterials 24, 1103-1109 (2003). The progression of fibrosis over many years induces dysfunction associated with infusion. Systemic problems often occur following local side effects, and fibromyalgia [Vermeulen R. C. (Vermeulen RC) and Scholte H. R. (Scholte HR) “Rupture of silicon gel breast implants and symptoms of pain and fatigue.” The Journal of Rheumatology 30, 2263-2267; R. (Shanklin DR), Stevens M. V. (Stevens M.V.), Hall M.V. F. (Hall M.F.) and Smalley D.M. L. (Smalley D.L.) "Environmental immunogens and T-cell-mediated responses in fibromyalgia:. Evidence for immune dysregulation and determinants of granuloma formation" Experimental and Molecular Pathology 69,102-118, pp. (2000)] and, rheumatic Diseases and other types of connective tissue disease (CTD) [Bridges A. J. et al. (Bridges AJ), Conley C.I. (Conley C.), One G. (Wang G.), Burns D. E. (Burns DE) and V. B. (Vasey FB) “A clinical and immunological evaluation of women with silicon breast implants and systems of rheumetic diseases.” Syndrome [Valga J. (Varga J.), Schumacher H .; R. (Schumacher HR) and Jimenez S. A. (Jimenez SA) "Systemic sclerosis after augmentation mammoplasty with silicon implants." Cancer Research 111, 377-383 (1989); E. (Appleton BE) and Lee P. (Lee P.) "The development of systemic sclerosis (sclerderma) following a augmentation augmentation mammoplasty." ing.

  In general, systemic side effects are thought to reflect changes to local levels, but crosstalk and the like have not been clarified. Even more importantly, in a prospective cohort study in both Europe and the United States, the effect of silicon injection could not be confirmed or denied as a risk factor in the development of CTD or other autoimmune diseases [Yanowski E . C. (Janowski E.C.), Kuppa L. L. (Kupper L.L.) and Haruka B.I. S. (Hulka B.S.) “Meta-analyses of the relation between silicone breast imprints and the risk of connective-tissue second.” (Sanchez-Guerrero J.), Colditz G. A. (Colditz GA), Carlson E.C. W. (Karlson EW), Hunter D. J. et al. (Hunter DJ), Spyzer F. E. (Speizer FE) and Liang M. H. (Liang MH) "Silicon Breast Implants and the Risk of Connective-Tissue Diseases and Symptoms." N Engl J Med 332, pp. 1666-1670 (1995)].

  In contrast, it has been shown that connective tissue disease-like symptoms in SMI holders disappeared after removal of the injection [Zazugornik J. et al. (Zazgornik J.), Pizza H. (Piza H.), Kaiser W. (Kaiser W.), Vettelheim P.M. (Bettelheim P.), Steiner G. (Steiner G.), Smolen J. et al. (Smolen J.), Biesenbach G. (Biesenbach G.) and Machek W. (Maschek W.) “Autoimmune reactions in patents with silicon breast implants.” Wien. Klin. Wochenschr. 108, 781-787 (1996)]. Furthermore, we were able to show that immunological mechanisms contribute to the development of the coating structure around SMI [Wolfram D. et al. (Wolfram D.), liner C.I. (Rainer C.), Nider Egger (Niederegger H.), Pizza H. (Piza H.) and Wick G. (Wick G.) "Cellular and molecular composition of fibrous capsules formed around silicon breast imprints with special focus on local immunity. 23, 81-91 (2004)]. The latter report showed that delayed type hypersensitivity reactions occur within these coatings.

  Most experts agree that the reaction to silicon is one of persistent and weak foreign body reactions, but the mechanisms underlying those processes are not precisely known . Although it has been clarified about the activation of the immune response against silicon, (i) whether the silicon itself has antigenic properties, (ii) acts as a hapten or carrier, (iii) exhibits an immune adjuvant, (iv) It is unclear whether it is a surface that promotes exposure of potential antigens and “altered self” or (v) induces a stress response in the surrounding tissue.

  Even though it has been identified as a major factor in the immune response to silicon, there is a poor understanding of the composition of proteinaceous films on the surface of silicon. It is obtained on the surface of the implanted silicon almost immediately after implantation [Ellwing H. et al. (Elwing H.) “Protein abstraction and ellipsis in biomaterial research.” Biomaterials 19, pp. 397-406 (1998)]. The proteins that form the film are recognized by the phagocytes and fibroblasts, the first cells that interact with the injection.

  In biological systems, it is emphasized that flow conditions, protein conformational changes, competitive protein adsorption, and initial effects on blood clotting and complement activation strongly influence the formation of protein films on artificial surfaces. It is important to do (“Vroman effect”) [Flowman L. (Vroman L.), Adams A.M. L. (Adams AL), Fisher G. C. (Fischer GC) and Munos P. C. (Munoz PC) "Interaction of High Molecular-Weight Kininogen, Factor-Xii, and Fibrinogen in Plasma at Interfaces." Blood 55, 156-159 (1980). Due to the complexity of the film, potential immunogenic epitopes of γ190-202 in the fibrinogen D domain can activate macrophages [through MAC-1 (CD11b / CD18) on their surface] Has only been shown so far [Foo W. J. et al. (Hu WJ), Eaton J.M. W. (Eaton JW), Ugarova T. P. (Ugarova TP) and Tan L. (Tang L.) "Molecular basis of biomaterial-foreign body body reactions." Blood 98, 1231-1238 (2001)]. This epitope of fibrinogen is only exposed upon adhesion to the silicon surface and plays an important role in the local side effects of SMI. In response, activated macrophages produce TNF-α, which recruits lymphocytes and induces transdifferentiation of fibroblasts to surrounding tissues, proliferation and migration. Fibrosis develops as a response of surrounding tissue to a chronic inflammatory response to silicon that is always sustained by lymphocytes, fibroblasts and macrophages that interact with proteins attached to the silicon surface.

  Further evidence about the immune hypothesis regarding fibrotic capsule formation can be found in the composition of the connective tissue capsule around the infusion. The immunohistological level in film formation has been examined in a large number of studies with similar findings [Wick G. et al. (Wick G.), Wagner R. (Wagner R.), Klima G. (Klima G.) and Wilfring Seder P. (Wilflingder P.) “Cellular, Molecular and Genetic Approaches to Immunodiagnosis and Immunotherapy” Kano K. (Kano K.), Mori S. (Mori S.), Sugisaki T. (Sugisaki T.) and Tris M. (Torisu M.), pages 231-241 (University of Tokyo Press, Tokyo; 1987) and Gabbani G. (Gabbiani G.), Hillshell B. J. et al. (Hirschel B.J.), Lian G. B. (Ryan GB), Statokov P.M. R. (Statkov PR) and Majuno G. (Majno G.) “Granulation tissue as a construct organ. A study of structure and function.” The Journal of Experimental Medicine, p.

  It has been shown that synovial-like multilayered dysplasia consisting of cells with macrophage and fibroblast phenotypes is formed towards the injectate. Interestingly, these cells also express large amounts of stress proteins such as heat shock protein 60 (HSP60) on the surface of SMI [Wolfram D. et al. (Wolfram D.), liner C.I. (Rainer C.), Nider Egger (Niederegger H.), Pizza H. (Piza H.) and Wick G. (Wick G.) "Cellular and molecular composition of fibrous capsules formed around silicon breast imprints with special focus on local immunity. 23, 81-91 (2004)]. Due to the chronic proliferative inflammation pattern of fibroblasts, dense collagen fibrosis occurs after an infusion period of more than 2 years [Shiguro W. et al. (Siggelow W.), Faridi A. (Faridi A.), Klinge U. (Klinge U.), Lass. (Rath W.) and Klosterhalfen B.I. (Klosterhalfen B.) “Ki67, HSP70 and TUNEL for the specification of testing of silicon breast implants in vivo.” J Mater. Sci. Mater. Med 15, 1355-1360 (2004)]. Further stimulation of the local fibrosis process through secretion of connective tissue growth factor (CTGF) is mediated by γδ-T-cells in the capsule [Workermaf G. (Workalemahu G.), Foerster M. (Forster M.), clay gel C.I. (Kroegel C.) and Brown R.C. K. (Braun R.K.) "Human gamma delta-T lymphocytes express and synthesize connective tissue growth factor:. Effect of IL-15 and TGF-beta 1 and comparison with alpha beta-T lymphocytes," The Journal of Biological Chemistry 170,153 -157 (2003)], it was also found in the connective tissue capsule around the implant. In addition, macrophages are stimulated through a positive feedback loop with TNF-α produced by γδ-T-cells.

  Evaluating host responses to injected ingredients requires systematic and objective studies of responses at both the cellular and molecular levels. [Hunt J. et al. A. (Hunt JA), Macrolin P.M. J. et al. (McLaughlin P.J.) and Flanagan B.E. F. (Flanagan BF) “Techniques to investigate cellular and molecular interactions in the host response to implied biomaterials. And current applications intended for the expression of antibodies as a tool for testing cellular markers or molecules, including DNA-based techniques where continuous application is considered critical to assist existing approaches It describes the technology.

  U.S. Pat. No. 4,048,298 discloses a solid double antibody radioimmunoassay method. The patent relates to a radioimmunoassay (RIA) method for assaying the body fluid content of an antigenic substance, which can be either the antigen itself or a hapten that can be converted to an antigenic substance, for example by reaction with proteins. The present invention relates to a new and improved modification of the double antibody RIA technique in the presence of a first antibody specific for an antigenic substance that is likely to be present in a body fluid to be assayed. However, the secondary antibody is not specific for the antigenic substance or analyte, but is specific for the first antibody.

  WO 02/41000 discloses an immunoassay for membrane-bound matrix metalloproteases. By using simple procedures and reagents, MT1-MMP can be rapidly quantified or the enzyme activity of MT1-MMP can be measured with high sensitivity and high accuracy. That is, use of a method for immunologically quantifying MT1-MMP with an anti-MT1-MMP antibody and a reagent to be used therewith, ie, (i) released from the cell membrane with the use of a surfactant and / or a reducing agent. And / or a member selected from the group consisting of MTl-MMP solubilized and (ii) self-solubilized MTl-MMP and reagents to be used with it and solid phase MTl-MMP The use of a quantifying method makes it possible to quantify MTl-MMP or measure the enzymatic activity of MTl-MMP. In addition, MT1-MMP expression promoters or inhibitors and MT1-MMP active promoters or inhibitors can thereby be screened. Therefore, research and development on useful drugs can be facilitated. These reagents are also useful in the examination of cancer and cancer metastasis.

  US Pat. No. 5,389,518 describes monoclonal antibodies specific for specific domains of fibronectin and vitronectin that can be deposited on certain polymers, such as silicon. These antibodies can bind to the proteins fibronectin and vitronectin and serve as biological material. However, no direct binding of fibronectin and vitronectin to the silicon surface has been shown.

  Silicon injection into the chest has a controversial history and is no longer approved for use by the Food and Drug Administration. They consisted of an outer coating of soft silicone rubber filled with silicone gel. There are numerous case reports of complications associated with chest silicon injection, including association with connective tissue disorders such as scleroderma and rheumatoid syndrome.

  The obvious complications for any type of chest injection include those associated with surgery such as bleeding and infection. In the second half of the infusion life, the major complications include capsule contractures and implant rupture. However, similar complications exist for the outer membrane of intact silicon implants.

  Several interesting things have been shown, but the cause of these complications remains unknown. For example, the gel inside the silicon implant is in a polymerized form. Unpolymerized silicon can flow through the intact silicon film into the surrounding tissue. It has been theorized that this unpolymerized silicon stimulates the fibrotic reaction responsible for the contracture of the film. The outflow of silicon through an intact membrane as well as the rupture of the implant is accompanied by the formation of granuloma. Nevertheless, polymerized silicon that coats the surface of the intact implant, or “true” surface, also has the potential to form a granulomatous or fibrous capsule without silicon spillage.

Currently, little is understood about the interaction between protein and silicon. A specific silicon, octamethylcyclotetrasiloxane (D ₄ ), has been shown to induce denaturation and conformational changes in fibrinogen and fibronectin, but there is no evidence for their relevance in immunogenicity [San L. (Sun L.), Alexander H. (Alexander H.), Rattaloro N. (Lattataro N.), Blumenthal N. C. (Blummental NC), Rich J. L. (Ricci JL) and Chen G. (Chen G.) “Protein generation induced by cyclic silicon.” Biomaterials 18, pp. 1593-1597 (1997)].

  Tavazzani et al. Developed a model of human monocyte-derived macrophages and investigated the mechanism of silicon-induced inflammation. Using this model, it was shown that the interaction between collagen type I and silicon gel induces IL-I secretion by cells. However, the binding properties of collagen to silicon were not examined [Tavazzani F. et al. (Tavazzani F.), Shin S. (Xing S.), Wadel J. et al. E. (Waddell JE), Smith D. (Smith D.) and Boynton E. L. (Boynton E.L.) “In vitro interaction between silicon gel and human monocycle-macrophages.” Biomed. Mat. Res. -Part A, 72, 2, 161-167 (2004)].

  In another study, Wolfram et al. [Wolfram D. et al. (Wolfram D.), liner C.I. (Rainer C.), Nider Egger (Niederegger H.), Pizza H. (Piza H.) and Wick G. (Wick G.) "Cellular and molecular composition of fibrous capsules formed around silicon breast imprints with special focus on local immunity. 23, 81-91 (2004) showed that silicon induces a stringent local T cell immune response and there are several proteins located within the infusate capsule. These proteins include (a) the collagenous proteins (pro) collagen types I and III, collagen type IV, (b) the non-collagenous proteins tenascin, fibronectin and laminin, and (c) heat shock protein 60. However, no data showing the binding of these proteins to silicon is provided. In contrast, coatings that were isolated from the silicon surface were studied. The authors consider only the function of the resulting protein as an immunogen, for example after exposure to a previously shown epitope.

  Backovic et al. Examined the immunological basis for film formation around the infusion [A. Oberiter B. (Oberreiter B.), Del Frari B. (Del Frari B.), Fan H. L. (Huang HL), Pizza H.L. (Piza H.), Wick G. (Wick G.) “Joint Annual Meeting of the German and Scandinavian Society of Immunology”, pages 21-24, September 2005, Kiel, Germany, 2006, No. 210-6. In this study, proteinaceous films covering SI in vivo were analyzed and a protein set consisting of 183 spots was identified, and then three proteins, fibronectin, vitronectin and heat shock protein 60 were mentioned. No further details are provided.

  Despite the above advances, the prior art provides methods and devices to further exploit the role of silicon adhesion proteins and proteinaceous films, particularly the interaction of cells and extracellular matrix (ECM) proteins with silicon. However, there is still a need to identify new proteins that can adhere to silicon surfaces. Mechanisms inherent in the subject's response to silicon when trying to identify individuals who are likely to develop local or systemic side effects such as those described above and can be the basis for methods suitable for monitoring, prevention and treatment of these outcomes It is essential to understand more fully about. An even better understanding also plays an important role in providing silicon with higher biocompatibility.

  The present invention, in its first aspect, is a method for identifying a polypeptide that adheres to a silicon surface, comprising: a) a mammal-derived, silicon-adhering polypeptide, and optionally silicon. Obtaining and / or providing a containing sample; b) contacting the sample with at least one silicon; and c) adhering to the silicon from steps a) and / or b). Isolating, preferably eluting the polypeptide, and d) identifying the isolated silicon adhesion polypeptide.

  The invention, in a further aspect thereof, is a reagent kit for identifying a polypeptide that adheres to a silicon surface, comprising a raw material, a buffer and an auxiliary agent and a substance for carrying out the method according to the invention, The present invention relates to a reagent kit.

  The invention, in a further aspect thereof, provides a silicon adhesion polypeptide identified through a method or by use of a kit according to the invention.

  In a preferred embodiment, the present invention relates to substances that interfere with the binding of the polypeptides identified by the present invention or silicon adhesion proteins and / or polypeptides to the silicon identified by the present invention, and their respective uses.

  In another preferred embodiment, the present invention contains at least one silicon adhesion polypeptide or substance that interferes with the binding of silicon adhesion proteins and / or polypeptides to the silicon identified by the present invention, and a pharmaceutical carrier. The present invention relates to a pharmaceutical composition.

  In another preferred embodiment, the present invention provides a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, or a functional variant thereof.

  In yet another preferred embodiment, the present invention provides a polypeptide according to the present invention, in particular a polypeptide having the amino acid sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. The present invention relates to a nucleic acid comprising a nucleotide sequence encoding a peptide, or a functional variant thereof, or a complementary nucleotide sequence of the nucleic acid.

  In yet another preferred embodiment, the present invention relates to nucleic acids that hybridize under stringent conditions with the nucleic acids described in the present invention. In another preferred embodiment, the present invention provides a recombinant vector comprising a nucleic acid according to the present invention. In a preferred further embodiment, the invention relates to a host cell comprising a nucleic acid or vector or expression vector according to the invention. In a further preferred embodiment, the invention relates to an antibody that immunologically recognizes a polypeptide according to the invention. In another preferred embodiment, the present invention provides a polypeptide, nucleic acid or vector or expression vector according to the present invention for use in medicine.

  In another preferred embodiment, the present invention immunologically converts a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, or a functional variant thereof. It relates to an antibody to be recognized.

  In a further preferred embodiment, the invention comprises an effective dose of a polypeptide according to the invention, a nucleic acid or vector according to the invention, an expression vector according to the invention, a host cell according to the invention, or the invention. And a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

  In a further preferred embodiment, the invention provides a polypeptide according to the invention, a nucleic acid or vector according to the invention, an expression vector according to the invention, a host cell according to the invention, or mammalian silicon, in particular medical The present invention relates to the use of the antibody according to the present invention for detecting a response to silicon.

  In yet another preferred embodiment, the present invention relates to the use of a polypeptide for detecting a response response to mammalian silicon, wherein said polypeptide is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, selected from the group consisting of Myeloid related protein 8, Spinal cord related protein 14, HT018, and PRO2619 or functional variants thereof.

  In another preferred embodiment, the present invention is a method for detecting a response response to mammalian silicon, comprising: a) a diagnostic subject that appears to contain at least one silicon adhesion polypeptide or functional variant thereof Providing a sample from a mammal of: b) detecting a change in the presence or expression of the at least one silicon adhesion polypeptide or the functional variant thereof in the sample; c) the mammal Determining a response response to said silicon from a change in the presence or expression of said at least one silicon adhesion polypeptide or functional variant thereof.

  In a further preferred embodiment, the present invention is for detecting a response response to mammalian silicon, in particular medical silicon, comprising raw materials, buffers, auxiliaries and substances for carrying out the method according to the invention. It relates to a diagnostic kit.

  In another preferred embodiment, the present invention is a method for monitoring a response response to silicon, particularly medical silicon, in a mammal, comprising a) injecting at least one silicon into said mammal; b) Detecting a change in the presence or expression of a silicon adhesion polypeptide according to the present invention using the method or kit according to the present invention, and optionally repeating step b).

  The invention, in another preferred embodiment thereof, provides a method for screening for substances that interfere with at least one silicon adhesion polypeptide, comprising: a) substances that are believed to interfere with the adhesion properties of the silicon adhesion polypeptide. B) providing silicon, in particular medical silicon, c) providing a silicon adhesion polypeptide, and d) the silicon adhesion polypeptide to the silicon in the presence and absence of the substance. Analyzing the interference properties of the substance through an analysis of adhesion, wherein a change in the adhesion of the silicon adhesion polypeptide to the silicon in the presence and absence of the substance is For the binding of silicon adhesion proteins and / or polypeptides to silicon Suggesting a substance that Wataru.

  The invention, in another preferred embodiment thereof, is a method for detecting adhesion of cells to silicon comprising the steps of: a) providing a sample of mammalian serum or wound fluid having cells; b) And c) analyzing the amount or number of cells derived from the serum or wound fluid that adheres to the silicon.

  In another preferred embodiment, the present invention is a method of treating a mammal that suffers an adverse reaction to injected silicon, wherein the effective amount of the antibody interferes with the adhesive properties of the silicon adhesion polypeptide identified by the method of the present invention. A method is provided comprising the step of administering a substance to said mammal.

  In another preferred embodiment, the present invention is a method for monitoring the treatment of a mammal undergoing a responsive response to silicon, a) an effective amount that interferes with the adhesive properties of the silicon adhesion polypeptide identified by the present invention. B) obtaining a sample from the mammal; c) detecting a change in the presence or expression of at least one silicon adhesion polypeptide or functional variant thereof; Providing a method.

  Before the present invention is described in more detail below, it is to be understood that the invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, but is only limited by the scope of the appended claims. Should also be understood. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, all references cited herein are incorporated by reference in their entirety.

  As already outlined, there is a need in the prior art to identify polypeptides involved in the response response of mammalian silicon. These polypeptides are presumed to play a role as major activators of the immune response, here involved in fibrosis leading to infusion failures. Therefore, a detailed analysis of their compositions and their deposition dynamics is necessary. In addition, these identified polypeptides can serve as diagnostic and therapeutic target molecules associated with the response to silicon.

  The term “response response” as used herein is intended to mean a physiological response of body or cells to silicon, such as medical silicon. Such a reaction can appear as an immune response involving the body's immune system, and can be local (ie, at or near the site of contact with the silicon) or systemic (ie, generally the body, such as blood, serum or other fluids) Side effects, such as injected silicon, eg, injected SMI [Smoray D. L. (Smallley D.L.), Levin J.H. J. et al. (Levine JJ), Shanklin D. R. (Shanklin DR), Hall M. F. (Hall MF), and Stevens M.F. V. (Stevens M.V.) “Lymphocyte response to silico ammon offspring of silicon breast imprint recipients.” Immunobiology 196, 567-574 (1996). Local and systemic side effects are presumed to be connective tissue fibrous capsules (usually associated with granuloma formation as a foreign body), fibromyalgia, connective tissue diseases (eg inflammation), and scleroderma-like Leading to the formation of fibrosis syndrome. It should be noted that the fibrotic process that occurs in response to silicon injection is a model in fibrotic diseases unrelated to other injections, including (i) post-necrotic diseases such as cirrhosis, (ii) inflammation Four different types are known, such as sexually transmitted diseases, such as rheumatoid arthritis, or (iii) idiopathic forms, such as Dypuytren's contracture and (iv) general foreign body reaction (eg, response to infusion) It has been.

  As used herein, the term “hypersensitivity” refers to a specific type of (excessive) response response to mammalian silicon, such as an adverse reaction in response to an individual's particular silicon, and in one embodiment in this individual Other individuals will not be able to show any response (ie, an individual response to a particular silicon) at all.

  The present invention, in its preferred embodiment, is a method for identifying a polypeptide that adheres to a silicon surface comprising: a) obtaining a sample containing a silicon-adhesive polypeptide from a mammal and optionally containing silicon. And / or providing; b) contacting the sample with at least one silicon; and c) isolating the polypeptide attached to the silicon from steps a) and / or b). And preferably eluting; and d) identifying the isolated polypeptide. Preferably, the polypeptides are first separated using gel electrophoresis, more preferably two-dimensional gel electrophoresis. Preferably, this step is followed by a staining procedure known to those skilled in the art, preferably silver staining. Thus, the stained polypeptide can be detected as a “spot pattern” of the polypeptide. This pattern is matched and compared using certain software, for example, with a control pattern or database, such as an ExParsy database of 2D gels. Preferably the polypeptide spot of interest is removed, more preferably the polypeptide spot of interest is removed manually. The removed polypeptide spots are then preferably digested to obtain fragments suitable for analysis by mass spectrometry, eg, peptide mass fingerprint (PMF) using MALDI-TOF. Preferably, the sample is further analyzed using LIFT-TOF / TOF MS / MS. More preferably, the results are then confirmed using Western blot analysis known to those skilled in the art.

  As used herein, the term “adhere” or “adhesion” indicates that a compound, eg, a polypeptide, adheres strongly to a surface, eg, a silicon surface. This attachment can be made both covalently and non-covalently between the polypeptide and silicon.

  The term “polypeptide” according to the present invention means a peptide or protein comprising amino acids of a predetermined length amino acid chain, such as at least 10, preferably 20-50, more preferably 30-100. It is assumed that amino acid residues are linked by covalent peptide bonds. However, peptidomimetics of such polypeptides in which amino acids and / or peptide bonds are replaced by functional analogs are also encompassed by the present invention. Most preferred polypeptides are part of peptides that are functionally involved or included in the adhesion of the polypeptide to silicon.

  As used herein, the term “silicone” is intended to encompass all dimethylsiloxane polymers that can be used in a physiological environment such as the human body. Typically, such silicon exhibits biocompatibility (i.e., does not elicit any or little of the body's immune response) and preferably has a high physiological tolerance. Such silicon is also known as “medical silicon”. Such medical silicon can be used in medical injections, such as silicon marymary injections, or as a coating for medical devices, such as bypass injections. Such medical silicones suitable for use according to the present invention are known to those skilled in the art. Preferred examples of medical silicon include the silicon types MED1511, MED4860, MED4211, and MED360 provided by Nusil (Waldbron, Germany).

  In a preferred embodiment of the method for identifying a polypeptide that adheres to a silicon surface according to one aspect of the invention, the sample covers silicon, urine, pleural fluid or ascites, cerebrospinal fluid, wound fluid or serum. Selected from proteinaceous films. Preferably, the sample is selected from proteinaceous films covering silicon, serum or wound fluid. Preferably, the wound fluid is obtained by drainage.

  In a preferred embodiment of the method for identifying polypeptides that adhere to a silicon surface according to the present invention, the sample is obtained from a mammal exhibiting a response response to silicon.

  In the context of the present invention, the mammal is preferably a human.

  In another preferred embodiment in the method for identifying a polypeptide that adheres to a silicon surface according to the present invention, the sample is generally of the following type: (i) post-necrotic disease, eg cirrhosis, (ii) Selected from the group consisting of inflammatory diseases such as rheumatoid arthritis, (iii) idiopathic fibrotic diseases such as Dupuytren's contracture, (iv) one fibrotic disease of foreign body reaction Obtained from mammals that exhibit a response. Such diseases can lead to the formation of a fibrous capsule of connective tissue, fibromyalgia, connective tissue disease, and scleroderma-like fibrosis syndrome.

  In a further preferred embodiment of the method for identifying a polypeptide that adheres to a silicon surface according to the present invention, a fibrous capsule of connective tissue is formed by an immune reaction.

  In a preferred embodiment of the method for identifying polypeptides that adhere to a silicon surface according to the present invention, wound fluid or serum is provided with additional silicon, preferably medical silicon.

  In a preferred embodiment of the method for identifying polypeptides that adhere to a silicon surface according to the present invention, the silicon is provided in an unpolymerized state. The term “unpolymerized” as used by the present invention relates to the state of silicon that is essentially unpolymerized, such as medical silicon. Unpolymerized silicon usually occurs as a by-product of polymerization in silicon implants (especially breast implants) and is believed to cause many of the problems associated with such implants.

  In another preferred embodiment in the method for identifying a polypeptide that adheres to a silicon surface according to the present invention, the silicon may be any medical silicon known to those skilled in the art. Preferably, the silicon is selected from the group consisting of silicon types MED1511, MED4860, MED4211, and MED360 provided by the company Nusil (Waldbron, Germany).

  In another preferred embodiment of the method for identifying a polypeptide that adheres to a silicon surface according to the present invention, step d) comprises any method known to those skilled in the art. Preferably, step d) comprises one or two-dimensional electrophoresis, Western blot analysis, protein assay suitable for measuring protein quantity and / or presence, mass spectrometry, modification, eg analysis of silver staining, advanced glycation end products ( Suitable for formation of advanced glycation end products (AGE), oxidation, antibody array, ligand array / assay, enzyme immunoassay (EIA, eg, ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), and Luminex assay A method selected from methods.

  In a preferred embodiment of the method for identifying a polypeptide that adheres to a silicon surface according to the present invention, the method comprises in step e) a polypeptide suitable as a marker polypeptide for detecting a response response to silicon in a mammal. And further comprising an identifying step. Preferably, the protein eluted from the proteinaceous film covering the explanted silicon is compared to the silicon adhesion protein obtained from wound fluid or serum. More preferably, the proteins eluted from the two systems are compared by one-dimensional and / or two-dimensional gel electrophoresis. Preferably, separate images are obtained from a single gel by labeling proteins from the two systems with different fluorescent dyes that can be excited at specific wavelengths. Nevertheless, radioactive detection may also be performed. Each label is well known to those skilled in the art. Preferably, the protein spot pattern is analyzed with any software suitable for the analysis of such patterns known to those skilled in the art. Preferably, the protein spot pattern is analyzed with "Decyder" software (Amersham Biosciences).

  In another preferred embodiment of the method for identifying a polypeptide that adheres to a silicon surface according to the present invention, the response response is a hypersensitive response to silicon in a mammal.

  In a preferred embodiment of the method for identifying a polypeptide that adheres to a silicon surface according to the present invention, step e) is derived from the separation of the protein identified from e ′) a proteinaceous film covering the silicon. Comparing the resulting polypeptide pattern with a polypeptide pattern derived from the separation of a silicon adhesion polypeptide eluted from wound fluid or serum, and e '') selecting and analyzing a spot of interest within the pattern Steps. Preferably, if the volume ratio of spots between samples exceeds 2 standard deviations of the overall spot distribution, the amount of protein deposition will be different, most preferably if the volume ratio exceeds a threshold of 5 times, It is defined as existing only within.

  In another preferred embodiment in the method for identifying polypeptides that adhere to the silicon surface according to the invention in step a), the polypeptides are further labeled with different fluorescent dyes or radiolabeled. Preferably, the fluorescent dye is excitable at a specific wavelength. Any fluorescent dye known to those skilled in the art is used. Preferably, the fluorescent dye is 7-aminoactinomycin d (7-AAD), acridine orange, Alexa dye, aminomethylcoumarin-acetate (AMCA), Cy-dye such as Cy2, Cy3, Cy5, Cy7, 4 ′, 6-diamidino-2-phenylindole dihydrochloride (DAPI), fluorescein isothiocyanate (FITC), Hoechst-dye, eg Hoechst 33342, Hoechst 33258, “blue” fluorescence, 5,5 ′, 6,6 ′ Tetrachloro-1,1 ′, 3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-I), peridinin-chlorophyll-protein (PerCP), propidium iodide, rhodamine red-X, phycoerythrin (R-PE) ),amino Chill coumarin acetate chloride rhodamine - derivatives (Texas Red (Texas-Red)), and is selected from the group consisting of tetramethylrhodamine isothiocyanate (TRITC).

  In a preferred embodiment of the method for identifying a polypeptide that adheres to a silicon surface according to the present invention according to the invention in step e ''), the polypeptide spot pattern is within the polypeptide pattern derived from the polypeptide eluted from the silicon and Analyzed by searching for all spots present in the polypeptide pattern in the polypeptide of the isolated silicon adhesion polypeptide eluted from the wound fluid or serum.

  In certain preferred embodiments of the methods described in this invention, the mammal is a human.

  The invention, in a further aspect thereof, relates to a reagent kit for identifying a polypeptide that adheres to a silicon surface, comprising materials, buffers and auxiliaries and substances for carrying out the method according to the invention.

  The invention, in a further aspect thereof, relates to a silicon adhesion polypeptide identified through a method or by using a kit according to the invention.

  The invention, in a further aspect, relates to substances that interfere with the binding of the polypeptides or silicon adhesion proteins and / or polypeptides identified according to the invention to the silicon identified according to the invention, and their respective uses.

  The invention, in a further aspect thereof, comprises a substance that interferes with the binding of at least one silicon adhesion polypeptide or silicon adhesion protein and / or polypeptide to the silicon identified according to the invention, and a pharmaceutical composition comprising a pharmaceutical carrier About.

  In a further aspect thereof, the present invention relates to a polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, or a functional variant thereof.

  In the sense of the present invention, the term “functional variant” means a polypeptide that is functionally related to the polypeptide of the present invention, ie, shares common structural or functional characteristics of polypeptides that adhere to silicon. . Examples of functional variants include orthologs, ie corresponding polypeptides derived from non-human organisms, preferably non-human mammals such as monkeys, pigs, mice and rats. Other examples of functional variants include polypeptides encoded by different alleles of genes in different individuals or in different organs of an organism. Furthermore, the polypeptide is also about 70%, preferably about 80%, in particular about 90% of the polypeptide having the amino acid sequence described in the polypeptide of the present invention and / or the DNA sequence derived from these polypeptide sequences. %, More preferably about 95% sequence homology, in particular by the term having sequence identity. Additions, transpositions, substitutions, deletions, insertions or chemical / physical modifications and / or exchanges or in the range of about 1-60, preferably about 1-30, and most preferably about 1-15 amino acids A portion of the polypeptide Thus, the functional variant preferably can include a domain of a protein responsible for adhesion of the polypeptide to silicon.

  In a preferred embodiment of the polypeptide according to the present invention, the polypeptide or functional variant thereof adheres to the surface of silicon.

  In another preferred embodiment for the polypeptide according to the invention, the polypeptide is a fusion protein.

  As used herein, “fusion protein” means any polypeptide that consists of or contains at least two polypeptides that do not naturally form a polypeptide. For the DNA level, two or more coding sequences are fused in frame. The portions of the fusion protein can be linked and / or isolated by linkers known to those skilled in the art (usually peptide linkers or chemical linkers).

  In another aspect thereof, the present invention relates to a polypeptide according to the present invention, in particular a polypeptide having the amino acid sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or The present invention relates to a nucleic acid comprising a nucleotide sequence encoding the functional variant or a complementary nucleotide sequence of the nucleic acid.

  The term “functional variant” refers to a DNA sequence that hybridizes under stringent conditions to a derived reference sequence or a portion thereof and that exhibits similar or identical activity against each peptide of the invention. All complementary DNA sequences. Hybridization conditions are known to those skilled in the art and are described in "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 6.3.1-6.3.6, 1991. Can be found in Moderate hybridization conditions are equivalent to hybridization in 30 ° C., 2 × sodium chloride / sodium citrate (SSC), followed by washing in 50 ° C., 1 × SSC, 0.1% SDS. Defined as a thing. Highly stringent conditions are for hybridization in 45 ° C., 6 × sodium chloride / sodium citrate (SSC), followed by washing in 65 ° C., 0.2 × SSC, 0.1% SDS. Defined as equivalent.

  In a preferred embodiment of the nucleic acid according to the present invention, the nucleic acid is DNA, cDNA, PNA, CNA, RNA or a combination thereof. Furthermore, the nucleic acid molecules of the invention can be genomic DNA, synthetic DNA, or combinations thereof and can be double-stranded or single-stranded, sense strand and / or antisense strand. Segments of these molecules are also considered to be within the scope of the present invention and can be generated, for example, by polymerase chain reaction (PCR) or by treatment with one or more restriction endonucleases. Ribonucleic acid (RNA) molecules can be generated by in vitro transcription.

  The nucleic acid molecules of the present invention can have naturally occurring sequences, or sequences that are naturally occurring but differ from sequences that result from the degeneracy of the genetic code are the same peptides (eg, peptides having SEQ ID NOs: 1, 2, 3, or 4). ). Furthermore, these nucleic acid molecules are not limited to coding sequences, and can include, for example, some or all of the non-coding sequences located upstream or downstream from the coding sequence.

  The nucleic acid molecules of the invention can be synthesized in vitro (eg, by synthesis based on phosphoramidites) or can be obtained from cells such as bacterial or mammalian cells. The nucleic acid can be human, but can also be derived from a non-human primate, mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, dog, or cat. Combinations or modifications of nucleotides within these types of nucleic acids are also encompassed.

  Furthermore, an isolated nucleic acid molecule of the invention can include segments that are not found, for example, in the natural state. Accordingly, the present invention encompasses recombinant nucleic acid molecules that are integrated into a vector (eg, a plasmid or viral vector) or a heterologous cell genome (or a homologous cell genome at a location different from the natural chromosomal location). Accordingly, recombinant nucleic acid molecules and uses are further discussed below.

  In addition, the nucleic acids described in the present invention may comprise markers or labels, such as affinity tags, antigen tags, fluorescent groups, quenchers, radioactive groups, dyes, heavy atoms (such as nitrogen), enzymatically detectable groups, enzymatically. It may have an active group or the like. All these markers or labels and the respective techniques for attaching them are well known to those skilled in the art.

  In another of its aspects, the present invention provides a nucleic acid that hybridizes under stringent conditions to the nucleic acid described in the present invention.

  In another aspect thereof, the present invention relates to a recombinant vector comprising the nucleic acid according to the present invention.

  Accordingly, the invention relates in another embodiment to a vector comprising a nucleic acid molecule of the invention. Such vectors have the ability to be introduced or to introduce nucleic acids into cells. It is preferred that the polypeptide encoded by the introduced nucleic acid is expressed in the cell upon introduction of the vector.

  In a preferred embodiment of the recombinant vector according to the invention, the recombinant vector is a recombinant expression vector comprising both overexpression and repression vectors.

  In a preferred embodiment, the vector of the invention comprises a plasmid, phagemid, phage, cosmid, mammalian artificial chromosome, knockout or knock-in construct, virus, in particular adenovirus, vaccination virus, attenuated vaccinia virus, canarypox virus, lentivirus (Chan LJ (Chang LJ) and Gay EE (20001) Curr. GeneTherap. 1: 237-251), herpes viruses, particularly herpes simplex viruses (HSV- I, Carlson WA, et al. (2000) Crit. Rev. Neurobiol.), Baculovirus, retrovirus, adeno-associated virus (AAV, Carter PJ (Carte). PJ) and Samulsky RJ (Samulski RJ) (2000) J. MoI. Med. 6: 17-27), linovirus, human immunodeficiency virus (HIV), filovirus and Recombinant versions thereof (see, for example, Cobinger GP, et al. (2001) Nat. Biotechnol. 19: 225-30), virosomes, “naked” DNA liposomes, As well as nucleic acid coated particles, particularly gold spheres. Particularly preferred are viral vector-like adenovirus vectors or retroviral vectors (Lindmann et al. (1997) MoI. Med. 3: 466-76 and Springer et al. (1998) Mol. Cell. 2: 549-58). Liposomes are small unilamellar or multilamellar vesicles usually composed of cationic lipids, neutral lipids and / or anionic lipids, for example by sonication of liposome suspensions. For example, the DNA can be ionically bound to the surface of the liposome or encapsulated inside the liposome. Suitable lipid mixtures are known in the art and contain, for example, DOTMA (1,2-dioleoylpropyl-3-trimethylammonium bromide) and DPOE (dioleoylphosphatidyl-ethanolamine), both Both are used in various cell systems.

  Nucleic acid coated particles are another means for introducing nucleic acids into cells using so-called “gene guns” that allow the mechanical introduction of particles into cells. Preferably, the particles themselves are inert and therefore in a preferred embodiment consist of gold spheres.

  In a further aspect of the invention, the nucleic acids of the invention are operably linked to expression control sequences that allow expression in prokaryotic and / or eukaryotic host cells. The transcription / translation regulators described above are known, but not limited to, inducible and non-inducible promoters, constitutive promoters, cell cycle regulated promoters, metabolic regulated promoters, enhancers, operators, silencers, repressors and gene expression Including other factors that drive or otherwise adjust. Such regulators include, but are not limited to, promoters transcribed by, for example, those similar to RNA polymerase III, such as promoters for the snRNA U6 or scRNA 7SK gene, cytomegalovirus hCMV immediate early gene, SV40 adenovirus early or late promoter, Viral promoters and regulatory elements that induce constitutive expression such as, for example, activation sequences derived from NBV, HCV, HSV, HPV, EBV, HTLV, MMTV or HIV, such as the CUP-1 promoter, tet- Repressors, such as those used in the tet-on or tet-off, lac, trp systems; regulators that induce tissue-specific expression, such as cdc2, cdc25C or A regulator that induces cell cycle specific expression, such as cyclin A; or TAC system, TRC system, major operator and promoter region of phage A, regulatory region of fd coat protein, promoter for 3-phosphoglycerate kinase, Allows inducible expression such as the promoter of acid phosphatase, as well as the promoter of yeast α- or a-mating factor.

  As used herein, “operably linked” means that an expression control sequence is incorporated into a gene construct so as to effectively regulate the expression of the coding sequence of interest.

Similarly, the nucleic acids of the invention can form part of a hybrid gene that encodes additional peptide sequences, such as sequences that function as markers or reporters. Examples of marker and reporter genes, beta-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase ^{(neo r,} G418 r), dihydrofolate reductase (DHFR), hygromycin -B -Phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). Those skilled in the art will appreciate useful additional reagents, such as markers or reporter sequences that may serve the function of the reporter, as well as many standard procedures related to the practice of the present invention. In general, a hybrid peptide will comprise a first portion and a second portion, where the first portion is a silicon adhesion polypeptide and the second portion is, for example, a reporter or Ig constant region or Ig constant as described above. Part of the region, for example the CH2 and CH3 domains of the IgG2a heavy chain. Other hybrids may include an antigen tag or His tag that facilitates purification and / or detection. The recombinant nucleic acid molecule can also have a polynucleotide sequence encoding a silicon adhesion polypeptide operably linked to a heterologous signal sequence. Such signal sequences can direct proteins to different compartments within the cell and are well known to those skilled in the art. Preferred signal sequences are those that facilitate secretion of the protein produced.

  In order to express cDNA encoding the polypeptide according to the present invention, the cDNA of the polypeptide according to the present invention typically initiates a strong promoter, transcription / translation terminator, and translation to induce transcription. And subcloning into an expression vector having a ribosome binding site. Suitable bacterial promoters such as E. coli, Bacillus sp. And Salmonella are well known in the art and kits for such expression systems are commercially available. Similarly, eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. The eukaryotic expression vector can be, for example, an adenoviral vector, an adeno-associated vector, or a retroviral vector.

  In addition to a promoter, an expression vector typically has a transcription unit or expression cassette that has all the additional components necessary for the expression of a nucleic acid encoding a silicon adhesion polypeptide in a host cell. Thus, a typical expression cassette has a promoter operably linked to a nucleic acid sequence encoding a silicon adhesion polypeptide, and signals necessary for efficient polyadenylation of the transcript, ribosome binding site, and translation termination. . As well as additional components of the cassette may include, for example, an enhancer. An expression cassette is also one that has a transcription termination region downstream of the constitutive gene that results in efficient termination. The termination region can be obtained from the same gene as the promoter sequence or from a different gene.

  The particular expression vector used to transport the genetic information into the cell is not particularly important. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322-based plasmids, pSKF, pET23D, and fusion expression systems such as GST and LacZ, although many are known by those skilled in the art to be useful. Exists.

  Expression vectors having eukaryotic virus-derived regulatory elements are typically used in eukaryotic expression vectors, such as SV40 vectors, papillomavirus vectors, and Epstein-Barr virus-derived vectors. Other typical eukaryotic vectors may be effective for expression in the SV40 early promoter, SV40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedron promoter, or eukaryotic cells. Under the induction of the other promoters indicated, pMSG, pAV009 / A. sup. +, PMTO10 / A. sup. +, PMAMneo-5, baculovirus pDSVE, pcDNA3.1, pIRES and any other vector that allows expression of the protein. Some expression systems have markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. Alternatively, high yield expression systems that do not involve gene amplification are also suitable.

  Factors typically included in expression vectors include replicons that function in E. coli, genes encoding drug resistance that allow selection of bacteria harboring the recombinant plasmid, and eukaryotics within non-essential regions of the plasmid. It also contains unique restriction sites that allow the insertion of the organism's sequences. The particular drug resistance gene selected is not critical and any of a number of drug resistance genes known in the art are appropriate. Prokaryotic sequences are optionally selected such that they do not interfere with DNA replication in eukaryotic cells as required.

  Using standard transfection methods, it is possible to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of the receptor, which is then purified using standard techniques.

  Any of the well-known methods for introducing foreign nucleotide sequences into host cells can be used. These include transfection of calcium phosphate, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors, and cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into host cells. Including the use of any of the other well-known methods for introducing. It is only necessary that at least one gene be sufficiently transducible into a host cell capable of expressing the silicon adhesion polypeptide according to the invention, depending on the particular genetic engineering method used.

  Transfected cells can be cultured under conditions that promote expression of the silicon adhesion polypeptide after the expression vector has been introduced into the cell, which is recovered from the culture using standard techniques. For example, cells can be opened up mechanically or by osmotic shock prior to being subjected to precipitation and chromatography steps, the nature and sequence of which will depend on the particular recombinant material to be recovered. . Alternatively, the recombinant peptide can be recovered from the medium in which the recombinant cells are cultured.

  The invention, in another aspect thereof, relates to a host cell comprising a nucleic acid or vector or expression vector according to the invention. Host cells that can be used for the purposes of the present invention include, but are not limited to, bacteria that can be transformed with, for example, a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vector having a polynucleotide molecule of the present invention (eg, , Prokaryotic cells such as E. coli and B. subtilis, eg, simple eukaryotic cell-like yeasts (eg, Saccharomyces) that can be transformed with a recombinant yeast expression vector having a nucleic acid molecule of the invention and Pichia), eg, Xenopus oocytes that can be infected with an insect cell line, eg, Sf9 of Hi5 cells, eg, a plasmid, that can be infected with a recombinant viral expression vector (eg, baculovirus) having a nucleic acid molecule of the invention. Cells, for example A plant cell line that can be infected with a recombinant viral expression vector (eg, cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or transformed with a recombinant plasmid expression vector having a nucleic acid sequence of a silicon adhesion polypeptide, or For example, a promoter derived from the genome of a mammalian cell (eg, a metallothionein promoter), a mammalian virus (eg, an adenovirus late promoter and a variola virus 7.5K promoter) or a bacterial cell (eg, tet-on and tet Mammalian cell lines (eg COS, CHO, BHK, HEK29) that can be transformed with a recombinant expression construct having a tet-repressor that binds to it used in the -off system. Include VERO, HeLa, MDCK, WI38, and NIH3T3 cells). Primary or secondary cells obtained directly from mammals and transfected with plasmid vectors or infected with viral vectors are also useful as host cells. Depending on the host cell and each vector used to introduce the nucleic acid of the invention, the nucleic acid can be incorporated into, for example, chromosomal or mitochondrial DNA, or can be maintained extrachromosomally, such as episomes, or cells. Can be included only temporarily.

  In another aspect thereof, the present invention provides an antibody that immunologically recognizes the polypeptides and / or proteins described in the present invention. The term “antibody” refers to monoclonal and polyclonal antibodies and their binding fragments, in particular Fc fragments, as well as so-called “single chain antibodies” (Bird RE, et al. (1988) Science 242. : 423-6), chimeric antibodies, humanized antibodies, particularly CDR-grafted antibodies, and bispecific or tetraspecific antibodies (Hollinger P. et al. (1993). Proc. Natl. Acad. Sci. USA 90: 6444-8). Also included are immunoglobulin-like proteins selected, for example, by techniques including phage display that specifically bind to the peptides of the invention. Preferred antibodies bind to the moiety responsible for binding of the peptide to its respective receptor.

  In another preferred embodiment, the present invention immunologically converts a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, or a functional variant thereof. Recognizing antibodies.

  The invention, in another aspect thereof, relates to a polypeptide, nucleic acid or vector or expression vector according to the invention in medical use.

  In another aspect thereof, the present invention provides a polypeptide according to the present invention, a nucleic acid or vector according to the present invention, an expression vector according to the present invention, a host cell according to the present invention, or a cell according to the present invention. It relates to a pharmaceutical composition comprising an effective dose of the antibody and a pharmaceutically acceptable carrier and / or other suitable pharmaceutical auxiliaries.

  For example, the polypeptide according to the present invention, the nucleic acid or vector according to the present invention or the expression vector according to the present invention, the host cell according to the present invention or the antibody according to the present invention (hereinafter simply referred to as “active ingredient”). The preparation of pharmaceutical compositions in the form of a medicament having the content of at least one of the above and their use according to the present invention is performed according to standard pharmaceutical techniques and methods. Thus, the active ingredient is made up in a medical form suitable for different indications and places of administration with a pharmaceutically acceptable carrier and / or other suitable pharmaceutical auxiliaries.

  Thus, the pharmaceutical composition can be made in a manner that provides the desired release rate, eg, rapid onset and / or delayed or sustained effects. Accordingly, the pharmaceutical composition can be an ointment, gel, patch, emulsion, lotion, foam, cream or mixed phase or amphiphilic emulsion system (oil / water-water / oil-mixed phase), liposome, transfersome, paste Or it may be powder.

  According to the present invention, the term “auxiliary” means any non-toxic solid or liquid filler, diluent or package as long as it does not adversely react and / or interact with the active ingredient or patient. It shall mean the material. For example, liquid herbal aids are sterile water, saline, sugar water, ethanol and / or oil. For example, herbal aids for making tablets and capsules can contain binders and fillers.

  Further, the active ingredient described in the present invention may be used in the form of a drug used systemically. These include parenterals belonging to injectable and infused substances. The injectable material exists in the form of an ampoule or as a so-called ready-made injectable material, for example a ready-made syringe or a disposable syringe, and is provided in a bottle with a sealed hole. Administration of the injectable substance may be in the form of subcutaneous (sc), intramuscular (im), intravenous (iv) or intradermal (ic) application. Forms particularly suitable for injection may be made as crystal suspensions, solutions, nanoparticles or colloidal dispersions such as hydrosols.

  Additionally, injectable compositions may be made up as a concentrate that is dissolved or dispersed in an isotonic aqueous diluent. Injectables may be prepared in the form of isotonic solutions, fat emulsions, liposome compositions, microemulsions. Like injectables, infusion compositions may also be prepared in the form of concentrates for dilution. Injectable compositions may also be applied in the form of continuous infusion, for example in the form of a minipump, for both stationary and mobile treatments.

  The active ingredients according to the invention can be bound to finely dispersed particles based on microcarriers or nanoparticles, such as poly (meth) acrylates, polylactates, polyglycolates, polyamino acids or polyether urethanes. Parenteral compositions can also be incorporated when the active ingredients according to the invention are incorporated into finely divided or dispersed suspension forms or as crystalline suspensions, for example based on the principle of “multiple units”. Alternatively, if the active ingredient according to the present invention is encapsulated in a pharmaceutical form, for example a tablet or stick to be injected as a result, on the basis of the “single unit principle”, it can be modified into a depot preparation. These injections or depots in the case of "single unit" and "multi-unit" drugs are so-called biodegradable polymers such as polyesters of lactic and glycolic acids, polyether urethanes, polyamino acids, poly (meth) Often consists of acrylates or polysaccharides.

  Suitable auxiliaries for making parenterals, adjusting the pH value of aqua sterilisata, substances that affect the pH value, such as organic and inorganic acids and bases and their salts Buffer substances, tonicity agents such as sodium chloride, sodium bicarbonate, glucose and fructose, surfactants or surfactants and emulsators such as partial fatty acids of polyoxyethylene sorbitan Synthetic fatty acid esters such as esters (Tween®) or fatty acids such as polyoxyethylene (Cremophor®), fatty oils such as peanut oil, soybean oil, and castor oil Polymeric auxiliaries such as ethyl oleate, isopropyl myristate and neutral oil (Miglyol®) such as gelatin, dextran, polyvinylpyrrolidone, solubility enhancing additives, organic solvents such as propylene glycol, ethanol, N , N-dimethylacetamide, propylene glycol and the like, or complexing substances such as citrate and urea, preservatives such as benzoic acid hydroxypropyl ester and benzoic acid methyl ester, benzyl alcohol and other antioxidants such as sodium sulfite As well as stabilizers such as EDTA may be considered.

  Add thickeners to avoid hardening of the active ingredient according to the invention in suspension or add tensides to ensure mixing of the deposits or complex formers such as eg EDTA Can be added. The active ingredient complex can be obtained using different polymers such as, for example, polyethylene glycol, polystyrene, carboxymethylcellulose, Pluronics® or polyethylene glycol sorbitol fatty acid esters. Scaffold forming agents such as mannitol, dextran, sucrose, human albumin, lactose, PVP or gelatin are used to produce lyophilisates.

  Suitable medical forms for each can be made according to manuals and procedures known to those skilled in the art based on pharmaceutical / physical techniques.

  A further aspect of the invention then comprises a polypeptide according to the invention, a nucleic acid or vector according to the invention or an expression vector according to the invention, a host cell according to the invention or an antibody according to the invention, and a medicament. Relates to individually prepared pharmaceutical compositions containing an effective dose of at least one of the pharmaceutically acceptable carriers. This pharmaceutical composition may be characterized in that the active ingredient is present in the form of a long-acting substance or as a precursor together with a suitable pharmaceutically acceptable diluent or carrier substance as described above.

  According to the present invention, the above pharmaceutical composition is in the form of a tablet, dragees, capsule, droplet, suppository, composition for injection or infusion intended for oral, rectal or parenteral use. Can exist. Such dosage forms and their production are known to those skilled in the art.

  In another aspect thereof, the present invention provides a polypeptide according to the present invention, a nucleic acid according to the present invention for detecting a response to mammalian silicon, particularly medical silicon, preferably to human silicon. Or the use of a vector, or an expression vector according to the invention, a host cell according to the invention, or an antibody according to the invention.

  The present invention, in another aspect thereof, relates to the use of a polypeptide for detecting the following response response to silicon in a mammal, wherein said polypeptide is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, selected from the group consisting of spinal cord-related protein 8, spinal cord-related protein 14, HT018, and PRO2619 or functional variants thereof.

  A diagnostic method is established by identifying polypeptides that are susceptible to response reactions and associated complications or adverse reactions because the adhesive properties of polypeptides that adhere to silicon in different mammals are indicators of possible response responses Can be done. Thus, for selection of several candidates, for example, silicon adhesion polypeptides or nucleic acids encoding silicon adhesion polypeptides, test systems for detecting these polypeptides and / or nucleic acids, such polypeptides are derived, for example, from serum It can be constructed by adhering to the surface of silicon or through an amplification method such as rt-PCR.

  Accordingly, the polypeptides described in the present invention can be detected using methods known in the art including immunological methods such as ELISA or Western blotting. Preferably, in one embodiment, such a test system is based on an ELISA system, a method well known to those skilled in the art. An ELISA system made to measure silicon adhesion polypeptides is easy to perform and is stable for a long time at room temperature. Since these tests are used as “point-of-care” tests at clinics, for example, this makes it easier to ship to end users. Preferably, serum is used in such a test system. Furthermore, the ELISA system is a standard method in many laboratories. This combination allows each integration into the current clinical routine.

  The invention, in another aspect thereof, is a diagnostic method for detecting a response response to mammalian silicon, comprising: a) a diagnostic that appears to contain at least one silicon adhesion polypeptide or functional variant thereof Providing a sample derived from a mammal of interest; b) detecting a change in the presence or expression of said at least one silicon adhesion polypeptide or functional variant thereof in said sample; c) of said mammal Determining a response to the silicon from a change in the presence or expression of at least one silicon adhesion polypeptide or functional variant thereof.

  In another preferred embodiment of the diagnostic method for detecting a response response to silicon according to the present invention in mammals, the silicon adhesion polypeptide is a polypeptide according to the present invention, spinal cord related protein 8, spinal cord related protein 14 , HT018, and PRO2619, or a polypeptide identified by the method for identifying a polypeptide that adheres to a silicon surface according to the present invention or a functional variant thereof.

  In another preferred embodiment in the diagnostic method for detecting a response response to silicon according to the present invention of a mammal, the silicon adhesion polypeptide is an isotype control, IgG-HRP, IgE-HRP, IGA-HRP, AMBP protein. Precursor, plasma-retinol binding protein, kininogen, eosinophil peroxidase, fibroblast growth factor 11, TGF-β, integrin-β4, T cell receptor, NADH dehydrogenase, coagulation factor V, α-spectrin, monocytes Chemotaxis protein-2, γ-glutamyltransferase, immunoglobulin G, immunoglobulin A, complement C3, complement C2, complement C1s, C-reactive protein, α1-microglobulin / bikunin precursor, von Willebrand ( Von Willebrandt factor, Ibronectin, vitronectin, fibrinogen, collagen I, collagen III, collagen IV, collagen VII, procollagen III, laminin, matrix metalloproteinase 2, apolipoprotein 4, apolipoprotein A1, ceruloplasmin, thyreotyrein, albumin, α2-macroglobulin precursor , Haptoglobin 1, α and β globin chains, actin, HSP60, apolipoprotein E3, complement C2 precursor, complement C3 precursor, hemoglobin, transthyretin, at least one additional silicon adhesion Detected with proteins and / or polypeptides or functional variants thereof.

  In a preferred embodiment of the method of diagnosis for detecting a response response to mammalian silicon according to the present invention, the immunoglobulin G, C-reactive protein, fibronectin, collagen I, procollagen III, complement of the silicon adhesion polypeptide Changes in the presence or expression of C3 precursor and spinal cord related protein 14 or functional variants thereof are detected. Preferably, there is a change in the presence or expression of the silicon adhesion polypeptide immunoglobulin G, C-reactive protein, fibronectin, collagen I, procollagen III, complement C3 precursor, and spinal cord related protein 14 or functional variants thereof. Detected. Silicone adhesion polypeptides against mammalian silicon by combining in detection of changes in the presence or expression of immunoglobulin G, C-reactive protein, fibronectin, collagen I, procollagen III, complement C3 precursor, and spinal cord related protein 14 Sensitivity, specificity, positive and negative predictive values for detecting response reactions are increased.

  In a further preferred embodiment of the diagnostic method for detecting a response response of a mammal according to the invention to silicon according to the invention, the sample from the mammal is serum and / or wound fluid.

  In another preferred embodiment of the diagnostic method for detecting a response response to silicon according to the present invention of a mammal, the sample is collected before or after long-term injection of medical devices and injection of silicon suitable for coating.

  In a preferred embodiment in the method of diagnosis for detecting a response response to mammalian silicon according to the present invention, the method further comprises a second sample as a control, wherein said control is the same before or after injection. Obtained from a mammal or from a different mammal without any medical silicon injection.

  In another preferred embodiment in a diagnostic method for detecting a response response to a silicon according to the invention of a mammal intended to detect a silicon adhesion polypeptide or a functional variant thereof, said polypeptide or its function An antibody suitable for immunological recognition of the variant is used.

  In a further preferred embodiment in the method of diagnosis for detecting a response response to mammalian silicon according to the invention, the polypeptide or a functional variant thereof is detected by one-dimensional or two-dimensional gel electrophoresis and / or ELISA. .

  In a preferred embodiment in a method of diagnosis for detecting a response response to a silicon according to the present invention in mammals, the method comprises at least one nucleic acid having a nucleotide sequence encoding a silicon adhesion polypeptide according to the present invention. The method further includes detecting the presence or amount.

  In another preferred embodiment of the diagnostic method for detecting a response response to silicon according to the present invention of the mammal, the nucleic acid is detected by PCR, RT-PCR or gel electrophoresis.

  In a further preferred embodiment of the diagnostic method for detecting a response response to silicon according to the invention of the mammal, the method allows the adhesion of the polypeptide to the silicon during steps a) and b). Step a ′) comprising the step of contacting said sample with silicon under conditions to

  The present invention, in another aspect thereof, relates to a diagnostic kit for detecting a response response to mammalian silicon comprising a raw material, a buffer, an auxiliary agent, and a substance for carrying out the method according to the present invention.

  As used herein, the term “diagnostic kit” refers to a reagent kit comprising a polypeptide, nucleic acid molecule, vector, host cell, or antibody of the invention or identified or characterized by the methods of the invention. Shall mean. Preferably, the reagent kit further comprises reaction buffers, storage solutions and / or residual reagents or raw materials that are required for the performance of scientific or diagnostic assays or the like. Furthermore, the parts of the kit of the present invention may be packed separately in vials or bottles, or combined in containers or multi-container units.

  The present invention, in another aspect thereof, is a method for monitoring response responses to mammalian silicon, particularly medical silicon, comprising the steps of a) injecting at least one silicon into said mammal; Detecting a change in the presence or expression of a silicon adhesion polypeptide according to the invention using a method or kit according to the present invention and optionally repeating step b).

  The present invention, in yet another aspect, provides a method for screening a substance that interferes with at least one silicon adhesion polypeptide, comprising: a) a substance that appears to interfere with the adhesive properties of the silicon adhesion polypeptide. B) providing silicon, in particular medical silicon, c) providing a silicon adhesion polypeptide, d) the silicon adhesion polypeptide in the presence or absence of the substance to the silicon Analyzing the interference properties of the substance through an analysis of adhesion, wherein a change in the adhesion of the silicon adhesion polypeptide to the silicon in the presence or absence of the substance is a silicon adhesion protein And / or anything that interferes with the binding of polypeptides to silicon It suggests.

  In one embodiment of the method for screening for an active substance that interferes with at least one silicon adhesion protein polypeptide according to the present invention, the silicon adhesion polypeptide comprises a polypeptide according to the present invention, spinal cord related protein 8, Spinal cord related protein 14, HT018, PRO2619, polypeptide identified by the present invention, isotype control, IgG-HRP, IgE-HRP, IGA-HRP, AMBP protein precursor, plasma-retinol binding protein, kininogen, eosinophil peroxidase , Fibroblast growth factor 11, TGF-β, integrin-β4, T cell receptor, NADH dehydrogenase, coagulation factor V, α-spectrin, monocyte chemotactic protein-2, γ-glutamyltransferase, immunoglobulin G Immunity Robrin A, complement C3, complement C2, complement C1s, C-reactive protein, α1-microglobulin / bikunin precursor, von Willebrand factor, fibronectin, vitronectin, fibrinogen, collagen I, collagen III, collagen IV, collagen VII, procollagen III, laminin, matrix metalloproteinase 2, apolipoprotein 4, apolipoprotein A1, ceruloplasmin, thyreotyrein, albumin, α2-macroglobulin precursor, haptoglobin 1, α and β globin chain, actin, HSP60, apolipoprotein It is selected from the group consisting of E3, complement C2 precursor, complement C3 precursor, hemoglobin, transthyretin or functional variants thereof.

  The invention, in another aspect thereof, is a method for detecting adhesion of cells to silicon comprising the steps of: a) providing a sample of mammalian serum or wound fluid containing the cells; b) providing the silicon And c) analyzing the amount or number of cells derived from the serum or wound fluid that adheres to the silicon.

  In a preferred embodiment of the method for detecting adhesion of cells to silicon according to the present invention, the mammal is a human.

  In a preferred embodiment of said method for detecting adhesion of cells to the silicon according to the invention, the silicon can be any medical silicon known to those skilled in the art. Preferably, the silicon is selected from the group consisting of medical silicon types MED1511, MED4860, MED4211, and MED360 provided by Nusil (Waldbron, Germany).

  The invention, in another aspect thereof, relates to substances that interfere with the binding of silicon adhesion proteins and / or polypeptides to silicon identified by the method according to the invention.

  The invention, in a further aspect thereof, provides a method for producing a pharmaceutical composition comprising the method according to the invention and a method for formulating said substance into a pharmaceutical composition.

  The invention, in a further aspect thereof, relates to a pharmaceutical composition comprising a substance described in the invention or produced by the invention.

  The present invention, in another aspect thereof, is a method of treating a mammal suffering an adverse reaction to injected silicon, wherein the effective amount of the substance interferes with the adhesive properties of the silicon adhesive polypeptide identified through the method of the present invention. Is provided to the mammal. Preferably, the effective amount of substance that interferes with the silicon adhesion polypeptide is administered in the form of pharmaceutical compositions well known to those skilled in the art.

  In a preferred embodiment of the method of treating a mammal subject to a response response to injected silicon according to the present invention, the adverse reaction is silicosis and / or fibrosis associated with silicon.

  The invention, in another aspect thereof, is a method for monitoring the treatment of a mammal undergoing a responsive response to silicon, comprising a) an effective amount of interfering with the adhesive properties of a silicon adhesion polypeptide identified by the invention. Administering a substance to the mammal; b) obtaining a sample from the mammal; and c) detecting a change in the presence or expression of at least one silicon adhesion polypeptide or functional variant thereof. Including methods.

  In a preferred embodiment in the method for monitoring the treatment of a mammal undergoing a response response to injected silicon according to the present invention, the sample is mammalian, preferably human serum or wound fluid.

  In a preferred embodiment in the method for monitoring the treatment of a mammal undergoing a response response to injected silicon according to the present invention, the silicon adhesion polypeptide is a polypeptide according to the present invention, spinal cord related protein 8, spinal cord related protein 14 HT018, PRO2619, polypeptides identified by the present invention, isotype control, IgG-HRP, IgE-HRP, and IGA-HRP, AMBP protein precursor, plasma-retinol binding protein, kininogen, eosinophil peroxidase, fibroblast Cell growth factor 11, TGF-β, integrin-β4, T cell receptor, NADH dehydrogenase, coagulation factor V, α-spectrin, monocyte chemotaxis protein-2, γ-glutamyltransferase, immunoglobulin G, immunoglobulin , Complement C3, complement C2, complement C1s, C-reactive protein, α1-microglobulin / bikunin precursor, von Willebrand factor, fibronectin, vitronectin, fibrinogen, collagen I, collagen III, collagen IV, collagen VII, Procollagen III, laminin, matrix metalloproteinase 2, apolipoprotein 4, apolipoprotein A1, ceruloplasmin, Thyreotyrein, albumin, α2-macroglobulin precursor, haptoglobin 1, α and β globin chain, actin, HSP60, apolipoprotein E3, It is selected from the group consisting of complement C2 precursor, complement C3 precursor, hemoglobin, transthyretin or functional variants thereof.

  The invention will now be further described in the following examples, without being limited thereto, in conjunction with the accompanying drawings. For the purposes of the present invention, all references mentioned herein are incorporated by reference in their entirety.

Example 1: Creation of an in vitro system for in vivo screening of protein adhesion to various silicon types The principle of protein preparation and analysis is shown in FIG.

1. Proteins eluted in vitro The inventors have analyzed proteins deposited on SMIs explanted from women suffering from complications after breast augmentation for cosmetic reasons. Thirteen explanted SMIs were collected from Medical University Insbruck (MUI), Clinic for Plastic and Reconstructive Surgery (Insbruck), Clinic for Plastic and Reconstructive Surgery. Obtained from Nine SMIs were filled with gel and four with saline. The surface was undulating with 11 SMIs and 2 smooth. In the analysis, only intact implants, i.e. those without silicon "gel spills" were considered. They were obtained under aseptic conditions immediately after surgery. Fibrous capsules were removed in the operating room and SMI was transferred to experimental subjects in phosphate buffered saline (PBS, pH 7.2) supplemented with protease inhibitors. Explanted SMI was washed at 4 ° C. with PBS (3 × 15 min) and distilled water (5 × 10 min). After washing, if any of the proteins are to be analyzed by 2D electrophoresis, urea lysis buffer [7M urea, 2M thiourea (both obtained from Calbiochem, Bad Soden, Germany), Elution in 30 mM Tris, 4% CHAPS, pH 8.5]. For Western blot analysis, they were prepared in SDS buffer (2% SDS, 100 mM DTT, 50 mM Tris Cl, 10% glycerin, pH 9.0). The protein concentration was measured by the bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, Ill.) And the protein solution was stored frozen at -20 ° C. until analysis.

2. Proteins eluted in vitro Silicon types MED1511, MED4860, MED4211, MED360 (Waldbron Nusil, Germany) do not have or need organic solvents or high temperatures in the polymerization Tested. Because of their physical characteristics, they fit into polystyrene flat-bottom plates commonly used in laboratory studies that are used for the purpose of having regulated and standardized conditions. All silicon types tested were of the unrestricted type, i.e. they were certified by the manufacturer of silicon (NUSIL, Waldbron, Germany) from manufacturers in long-term implants. They are also. Represented a silicon type widely used in SMI manufacturing and medical device coating. Other silicon types are designed for future testing by the inventor, and the test design will be tailored based on their chemical composition.

Immediately afterwards, 5 ml of unpolymerized silicon is added per well of a 6-well polystyrene plate (Greiner, Frickenhausen, Germany) to distribute the silicon evenly on the bottom of the plate. Was centrifuged for various times depending on the silicon type (MED 1511: 10 minutes at 300 × g; MED 4860: 60 minutes at 1000 g; MED 4211: 10 minutes at 150 × g). After curing of the silicon (3 days at 37 ° C. and 98% humidity), the plates were sterilized for 120 seconds under 200 millijoules of UV light (GsGene UV chamber, Biorad, Hercules, Calif.) And placed in a tube. Incubated with wound fluid (WBF) collected 2 days after breast augmentation to allow saturation of protein binding sites. WBF was supplemented with protease inhibitors (pepstatin, leupeptin, aprotinin, final concentration 1 μg / ml obtained from Sigma) and incubated with silicon coated plates for 4 days. Within this time, protein deposition reached equilibrium on the surface of the silicon while no spontaneous protein degradation occurred. The plate was then washed in PBS (10 × 15 min), H ₂ O was distilled at 4 ° C. (5 × 10 min), and the protein was eluted in the same manner as the in vitro protein elution described above. . FIG. 3 shows a model for protein adsorption and analysis in vitro.

3. Differential analysis of in vitro and in vitro eluted proteins In order to determine whether an in vitro model corresponds to an in vitro situation, the inventors have analyzed proteins eluted from two systems by one-dimensional and two-dimensional gel electrophoresis Compared. In differential analysis, the inventor needs a sensitive analytical tool, which is why they use the DIGE ™ (Amersham Biosciences, Uppsala, Sweden) system. Proteins from the two systems were labeled with different fluorescent dyes that can be excited at specific wavelengths. This experimental setup allowed the inventor to acquire three separate images from a single gel, thereby greatly reducing variability between gels and improving the statistical analysis of spot patterns. Protein spot patterns were analyzed with "Decyder" software (Amersham Biosciences). When the volume ratio of spots between samples exceeded 2 standard deviations of the overall spot distribution, protein deposition was considered different. More stringently, if the volume ratio exceeded the 5-fold threshold, it was defined as existing only in either the in vitro system or the in vitro system. Using this approach, the inventor has so far detected a total of 184 protein spots. Of these, 163 spots are present in equal amounts in both systems, 13 proteins bind more strongly in vitro, 5 are present in the in vitro system, and 5 spots Only was unique in any of the systems. In other words, 89% of the protein deposited in vivo is also deposited in our in vitro system. The inventor has identified the majority of the remaining 11% of the protein using mass spectrometry and found that they mainly represent intracellular proteins. These experimental results suggested that additional proteins found in vivo arise from the surrounding damaged tissue as a result of the surgical procedure.

4). Protein Identification 2D Electrophoresis Proteins eluted in vitro and in vitro were analyzed primarily after separation by two-dimensional gel electrophoresis (2D electrophoresis) [Anderson L. et al. (Anderson L.) and Anderson N. G. (Anderson N.G.) "High resolution two-dimensional electrophoresis of human plasma proteins." Proc. Natl. Acad. Sci. USA 74, 5421-5425 (1977)]. In a one-dimensional separation, the protein was dissolved in urea lysis buffer [7 M urea, 2 M thiourea (both obtained from Calbiochem, Bad Soden, Germany), 30 mM Tris, 4% CHAPS, pH 8.5]. Dissolved in. Prior to loading, 200 μg of total protein was supplemented with rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 18 mM DTE) to a final volume of 350 μl. The inventors then added lysis buffer and loaded the protein against 18 cm IPG strips pI3-11. Proteins were run at a total of 55000 Vhrs (IPGphor, Amersham Biosciences). After isoelectric focusing, the protein on the strip was reduced (2% DTT, 15 minutes) and consequently alkylated (2.5% IAA, 15 minutes). Two-dimensional electrophoresis was performed under reducing conditions in a 9-16% polyacrylamide gel at 16 ° C. and a 20 × 24 cm gradient. After 2D electrophoresis, the gel was fixed and silver stained according to the method of Farmer [Ray W. (Wray W.), Bouricus T. et al. (Boulikas T.), Ray V. P. (Wray, VP), and Hancock R. (Hancock R. "Silver staining of proteins in polyacrylamide gels." Analytical Biochemistry 118, 197-203 (1981)].

Pattern Matching and Mass Spectrometry Matching spot patterns of silver-stained proteins, “Melanie” software (version 4, Swiss Institute of Bioinformatics, Basel, Switzerland) Was used to compare with the 2D gel ExPassy database (http://us.expasy.org/ch2d/) [Hugh G. J. et al. (Hughes GJ), Flutiger S. (Frutiger S.), packet N. (Paquet N.), Ravier F. (Ravier F.), Pascali C.I. (Pasquali C.), Sanchez J .; C. (Sanchez JC), James R.C. (James R.), Chisot J. et al. D. (Tissot JD), Bjerkvist B.B. (Bjellqvist B.) and Hostalaya D. F. (Hochstrasser DF) "Plasma protein map: an update by microsequencing." Electrophoresis 13, 707-714 (1992)]. The spot of interest is cut out manually, the protein is digested with trypsin (Promega, Mannheim, Germany), and the peptide is mass analyzed (Ultraflex ™ TOF / TOF, Bruker Daltonics (Billerica, Mass.) ) Bruker Daltonics)).

  In-gel digestion was performed as described by Shevchenko et al. (Shevchenko A.), Wilm M. et al. (Wilm M.), Volm O. (Vorm O.), and Mann M. (Mann M.) "Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels." Analytical Chemistry 68, 850-858 (1996)]. These samples analyzed by peptide mass fingerprint (PMF) from MALDI-TOF were further analyzed from the same target using LIFT-TOF / TOF MS / MS. A maximum of three precursor ions per sample were selected for MS / MS analysis. After measuring the PMF in the reflection mode, MALDI-TOF / TOF measurement was performed in the LIFT mode. PMF spectra were interpreted with “Mascot” and “Profound” software. As cut-off criteria for accurate identification, a probability score higher than 64 was used for “Mascot” and a probability score of 1.65 for “Profound”.

Confirmation of results by Western blot The inventor performed Western blots of proteins eluted from silicon to supplement and confirm the data obtained from mass spectrometry and database search. For this reason, the inventors eluted in SDS buffer (2% SDS, 100 mM DTT, 50 mM Tris Cl, 10% glycerin, pH 9.0) loaded on a small (7 × 10 cm) homogeneous 12% polyacrylamide gel. Protein was used. After the run, the protein was blotted onto a 1200 mAh nitrocellulose membrane in a wet blotting chamber and blocked with 5% skimmed milk. Primary antibodies against the majority of proteins and appropriate conjugates are obtained from DAKO, and polyclonal rabbit antibodies against spinal cord associated protein (MRP) 8 and MRP14 are kindly available from Dr. Clone II, from Dr. Claus Kerkhoff (Institute of Experimental Dermatology, Münster, Germany) -13 produced a monoclonal antibody against human HSP60 purified by affinity chromatography [Xu Q., Shet G. (Schett G.), Zeitz C.I. S. (Seitz CS), Fu Y. (Hu Y.), Gupta R. S. (Gupta RS), and Wick G. (Wick G.) “Surface stating and cytotoxic activity of heat-shock protein 60 antibody in stressed endothelial cells.” Circ. Res. 75, 1078-1085 (1994)]. All antibody dilutions were prepared in 5% non-fat milk in PBS. Following secondary antibody incubation, the membrane was washed with PBS and a chemiluminescent substrate for horseradish peroxidase (Amersham Biosciences) was added for 1 minute. The membrane was exposed to AGFAX ray film for various times and developed in a Curix developing machine.

5. Proteins identified Until recently, the following 47 proteins have been identified as adhering to silicon in the context of the present invention.
Protein name actin albumin α1-microglobulin / bikunin precursor α2-macroglobulin precursor AMBP protein precursor apolipoprotein A1
Apolipoprotein E3
C-reactive protein coagulation factor V
Collagen I
Collagen IV
Collagen VII
Complement C1s
Complement C2 precursor complement C3 precursor eosinophil peroxidase fibrinogen fibroblast growth factor 11
Fibronectin haptoglobin 1
Hemoglobin HSP60
HT018
IgA
Immunoglobulin G
Integrin β4
Kininogen laminin matrix metalloprotease 2
Monocyte chemotaxis protein-2
Spinal cord related protein 14
Spinal cord related protein 8
NADH dehydrogenase plasma-retinol binding protein PR02619
Procollagen III
T cell receptor TGF-β
Transthyretin vitronectin von Willebrand factor α-spectrin γ-glutamyltransferase 4 unknown proteins

In addition, the following proteins identified by 2D in vivo, 2D in vitro and ELISA (in vitro) are listed along with their GeneBank accession numbers ( ^* indicates confirmed by Western blot (WB)).
Protein name Registration number Immunoglobulin G ^* gi | 4321905
C-reactive protein ^* p02741
Spinal cord related protein 8 ^* p05109
Fibronectin ^* p02751
Vitronectin ^* p04004
Collagen I ^* gi | 4502961
Procollagen III q541P8
Laminin ^* gi | 2784346
Actin ^* gi | 15277503
Immunoglobulin A ^* gi | 8699307
Complement C3 precursor p01024

The following proteins identified by 2D in vivo and ELISA (in vitro) are listed along with their GeneBank accession numbers ( ^* indicates confirmed by Western blot (WB)).
Protein name Registration number Spinal cord related protein 14 p06702
Spinal cord related protein 14 p06702
Collagen IV gi | 180715

The following proteins identified by 2D in vitro and ELISA (in vitro) are listed along with their GeneBank accession numbers ( ^* indicates confirmed by Western blot (WB)).
Protein name Registration number Collagen VII ^* gi | 627406
Matrix metalloprotease 2 ^* p08253
Von Willebrand factor ^* p04275
Heat shock protein 60 p10809

List the following proteins that were identified by 2D in vivo and / or 2D in vitro but not detected by ELISA with the use of antibodies, along with their GeneBank accession numbers ( ^* confirmed by Western blot (WB)) Showing).
Protein name Registration number Complement C1s p09871
Complement C2 precursor ^* gi | 67611
TGF-β p01137
T cell receptor gi | 624465548
Albumin gi | 28592
Integrin β4 ^* gi | 21361207
Unknown protein 1 gi | 345533774
Unknown protein 2 gi | 2173298
Unknown protein 3 gi | 21754778
Unknown protein 4 gi | 7020569

The following proteins identified by 2D in vivo and / or 2D in vitro when no antibodies are available are listed along with their GeneBank accession numbers ( ^* indicates confirmed by Western blot (WB)).
Protein name Registration number Haptoglobin 1 p00738
Apolipoprotein A1 gi | 178777
Transthyretin p02766
α2-macroglobulin precursor p01023
Hemoglobin gi | 556635219
α1-microglobulin / bikunin precursor p02760
Fibrinogen ^* p02671
NADH dehydrogenase gi | 5138999
HT018 gi | 9966383
AMBP protein precursor gi | 1799519
Kininogen p01042
Plasma-retinol binding protein gi | 190870
Coagulation factor V p12259
PR02619 gi | 11493459
Eosinophil peroxidase gi | 31183
Fibroblast growth factor 11 gi | 20160215
α-spectrin gi | 1899071
Monocyte chemotaxis protein-2 gi | 1905801
γ-glutamyltransferase gi | 3282014
Apolipoprotein E3 gi | 1480096

Classification of Identified Silicon Adhesion Proteins The proteins identified by the present invention can be classified as follows in relation to their biological function.
1) Functional group, inflammation C-reactive protein complement C1s
Complement C2 precursor immunoglobulin G
Spinal cord related protein 14
Spinal cord related protein 8
Von Willebrand factor IgA
Integrin β4
Complement C3 precursor apolipoprotein A1
Apolipoprotein E3
α1-microglobulin / bikunin precursor HSP60
HT018
PR02619
AMBP protein precursor T cell receptor 2) functional group, fibrosis coagulation factor V
Collagen I
Collagen IV
Fibronectin laminin collagen VII
Fibrinogen fibroblast growth factor 11
Kininogen matrix metalloprotease 2
Procollagen III
Vitronectin 3) functional groups, inflammation and fibrosis TGF-β
4) Functional group, other eosinophil peroxidase actin albumin α2-macroglobulin precursor haptoglobin 1
Hemoglobin transthyretin γ-glutamyltransferase monocyte chemotaxis protein-2
NADH dehydrogenase unknown protein 1 (SEQ ID NO: 1)
Unknown protein 2 (SEQ ID NO: 2)
Unknown protein 3 (SEQ ID NO: 3)
Unknown protein 4 (SEQ ID NO: 4)
5. Protein immunoglobulin G suitable for discrimination between normal and fibrotic tissue
C-reactive protein fibronectin collagen I
Procollagen III
Complement C3 precursor spinal cord related protein 14

Properties of proteins that specifically adhere to silicon Table 1 summarizes the proteins found to adhere to different selected medical silicon types both in vitro and in vitro. What are their relative abundances? : Not tested, 0: absent, +: present, ++: abundant, +++: expressed as extremely abundant.

  Proteins can be divided into different categories: anchor proteins, primary protein sets, and secondary protein sets. Only the first two categories have been reconfirmed by other techniques (Western blot, ELISA).

1. Equally strong adhesive protein (anchor protein)
These proteins were defined as at least “abundant” or ++ and existed as being equally adherent to all silicon types tested. Seven candidates were included in this group. The symbol “ ^* ” is given to them.

2. Primary protein set This set consists of 21 proteins. They needed to be detected in all silicon types tested for inclusion. Three subclasses can be defined as follows.
Fourteen proteins with equal but poor adhesion constitute this subclass and are defined as “present” or +. The symbol “▽” is given to them.
Proteins with adhesiveness to different silicon types Seven proteins showed significant differences in adhesion to silicon types MEDl511, 4860, 4211, and 360 (Nusil). They covered all adhesion strengths, ie 0, +, ++ and ++ molecules. The symbol “■” is given to them.
Proteins with unclassified adhesive properties In this group of 4 proteins, only 2 silicon types were tested. The success of the experiment showed the adhesion class “present” (+). To date, they appear to be most likely members of subclass 1 (equal but less adhesive proteins). The symbol “□” is given to them.

3. Secondary protein set This set consisted of 17 proteins. They are detected on only one silicon type (MED 1511) and have not been confirmed by techniques other than mass spectrometry. The following 13 proteins were found in this set.
AMBP protein precursor plasma-retinol binding protein kininogen eosinophil peroxidase fibroblast growth factor 11
TGF-β
Integrin-β4
T cell receptor NADH dehydrogenase coagulation factor V
α-spectrin monocyte chemotaxis protein-2
γ-glutamyltransferase

In addition, there were four types of hypothetical proteins identified by Homo sapiens unknown protein products and “mascots”.
Registration number: gi | 345533774; mass: 113642; unknown protein product (SEQ ID NO: 1)
Accession number: gi | 21379298, emb | CAD386696.1; mass: 117490; unknown protein product (SEQ ID NO: 2)
Registry Number: gi | 21754778; Mass: 38453; Unknown Protein Product (SEQ ID NO: 3)
Accession number: gi | 7020569; mass: 14004; unknown protein product (SEQ ID NO: 4)

Novel protein candidates There were several of the above proteins not previously mentioned in the context of hypersensitivity to silicon and secondary fibrosis.
1. Spinal cord related protein 8
2. Spinal cord related protein 14
3. HT018
4). PRO2619
5. Registration number: gi | 345533774; mass: 113642; unknown protein product (SEQ ID NO: 1)
6). Accession number: gi | 21379298, emb | CAD386696.1; mass: 117490; unknown protein product (SEQ ID NO: 2)
7). Registry Number: gi | 21754778; Mass: 38453; Unknown Protein Product (SEQ ID NO: 3)
8). Accession number: gi | 7020569; mass: 14004; unknown protein product (SEQ ID NO: 4)

Diagnostic test system It is hypothesized that the adhesion properties of protein to silicon adhesion in different patients is an indicator of the expected immune response and subsequent fibrosis response that the patient may develop. If this assumption was correct, the inventor was able to establish a diagnostic method, thereby identifying proteins prone to fibrotic complications. Thus, after performing protein analysis (see Examples 2 and 3), the inventor selects several candidates and builds a test system to detect these proteins recruited from the serum to the surface of the silicon did.

Actual advantages 1) The test protocol is based on ELISA microwell plates. This test system is easy to make and is stable at room temperature for a long time. This facilitates shipment to the end user.
2) Serum is used in the test system. Furthermore, the ELISA system is a standard method in many laboratories. This combination allows easy integration into current clinical routines.

Technical Details Polystyrene 96-well flat bottom microtiter plates were coated with medical silicon types 1511, 4860 and 4211. Immediately afterwards, 50 μl of unpolymerized silicon is added per well of a 96-well polystyrene plate (Greiner, Frickenhausen, Germany) to distribute the silicon evenly on the bottom of the plate. Was centrifuged for various times depending on the silicon type (MED 1511: 10 minutes at 300 g; MED 4860: 60 minutes at 1000 g; MED 4211: 10 minutes at 150 g). After curing of the silicon (3 days at 37 ° C. and 98% humidity), the plates were sterilized for 120 seconds under 200 millijoules of UV light (GsGene UV chamber, Biorad, Hercules, Calif.). After sterilization, silicon-coated 96-well plates were incubated with serum from patients before or after undergoing primary breast augmentation or from patients with SMI exchanged. All sera are supplemented with 1 μg / ml of leupeptin, pepstatin and aprotinin (protease inhibitors, Sigma-Aldrich, Vienna, Austria), filtered through a 0.22 μm filter, 100 μl Was applied to each well under aseptic conditions. In some experiments, serum was diluted with PBS prior to application. After 50 hours, excess protein was washed away with a 4 × 5 minute wash with PBS, and the binding sites on the plate were blocked with 2.5% BSA / 0.1% Tween 20 in PBS for 100 minutes at room temperature. 150 μl of blocking solution was applied per well. After blocking, various antibodies were applied in a predetermined optimal dilution in blocking solution. Our initial screen consisted of 24 antibodies, but we reduced our current list to 12, applied in the following optimal working dilution (Table 2).

  Of course, it is important to mention here that this list of antibodies is not a final list. It will be augmented by new experimental results that the inventors have obtained in protein analysis on films formed on the surface of silicon. After incubation with the primary antibody, the plate was washed 3 × 5 minutes with PBS and the appropriate secondary antibody conjugated with HRP was applied for 120 minutes in a volume of 100 μl / well with shaking at room temperature. Wells incubated with directly labeled primary antibody were kept in PBS until the ABTS color development step. Secondary antibodies such as rabbit anti-mouse IgG HRP, DAKO P260, 1: 1000 and porcine anti-rabbit IgG HRP, DAKO P399, 1: 1000 were used. Every dilution was made in blocking solution. After incubation of the secondary antibody at room temperature, the plates were washed 3 × 5 minutes in PBS / 0.02% Tween and 2 × 5 minutes in pure PBS. The results were imaged using a standard ABTS ELISA detection solution for HRP. Color was developed for 40 minutes, the solution was transferred to a new 96-well plate (to eliminate silicon coating interference during spectroscopic measurements), and absorbance was measured at 405 nm. Therefore, we have constructed an ELISA system for measuring the adhesion of proteins to silicon (Figure 1).

  The inventor managed to significantly reduce background levels by titrating different blocking solutions. The inventor defined the cutoff in the presence of protein as an absorbance value twice that of the isotype control. The background value was a linear range spanning two orders of magnitude, less than 1% of the probe measurements. The entire test system was highly reproducible, allowing semi-quantitative analysis of the deposited protein. The main advantage of this system is that simultaneous analysis of a large number of patients and various proteins deposited separately on silicon is possible in a relatively short time.

  In proof of principle (FIG. 2), the inventor found that 10 patients (5 with and without fibrotic complications), 7 controls (matched age and gender) and dramatic systemic fibers Twelve protein candidates were tested in one patient with liquid silicon injection (!) That induced sexual complications. We found a significant positive correlation between fibronectin, CRP and IgA deposition and fibrosis and a negative correlation with complement components C3 and IgG deposition.

  Thus, the inventor has focused on different patterns of protein deposition from various patients as proof of principle. In fact, the inventor wanted to perform further analysis and correlate the results with the duration of the infusion in the patient's large cohort and local or systemic complications. For this reason, the inventor has adjusted the test system to a 384-well format that allows for rapid screening and larger clinical trials. Furthermore, reducing the amount of serum that is often a limiting factor in the analysis may save costs and improve reproducibility due to the possibility of replication. A detection system with a primary antibody against a specific protein and a secondary antibody conjugated with the appropriate HRP is similar to the above, but with a smaller volume (50 μl). Develop for 40 minutes, transfer the solution to a new 384 well plate (to exclude silicon coating interference during spectroscopic measurements) and spectrophotometer in “Victor” 384 well plate (Perkin Elmer, Boston, USA) (Perkin Elmer)) was measured for absorption at 405 nm.

  There are about 20 additional types of unlimited (certified as long-term injection) types, but this classification covers all common types of silicon used for medical purposes.

1. Differences in the adhesion properties of a defined protein array consisting of candidates selected for background silicon could contribute to the observed variability in the resultant adverse effects of silicon devices. This system allowed simultaneous analysis of the deposition of seven proteins in a patient cohort. They were selected from the proteome screening on the silicon surface described in Example 1.

  In retrospective studies, the deposition pattern of this protein array made a good distinction between patients who developed fibrosis and those without complications (Figure 5). Interestingly, a subgroup was also identified among the control patients that had a deposition pattern similar to those who developed fibrosis in response to silicon. Follow-up prospective trials are currently being conducted to determine whether this group is at risk, i.e., a risk of a strong fibrotic adverse reaction to silicon.

2. Methods The trial included 90 women who had breast augmentation surgery especially for cosmetic purposes. They were compared to patients with fibrosis of the breast connective tissue with fibrosis (BS ≧ 3; N = 40; median age 51 years) and those without extensive fibrosis (BS) according to Baker Score (BS). <3; N = 50; median age 47 years).

  The seven different protein deposition patterns listed below were compared in this cohort study. In order to normalize the data, the absorption values were converted according to the formula x '= log (x + 1). According to the test by the Kolmogorov-Smirnov test, the conversion values had a normal distribution. In these experiments, the background value was less than 1% of the probe measurement and the linear range of detection extended to two orders of magnitude. During the analysis, it was noted that a small group (18%, n = 9) of the study branches with weak fibrosis had something similar to the protein deposition characteristics of patients with fibrosis. . They were classified as an independent group of “modified controls”. This observation was confirmed by data clustering using “Treeview” clustering software. The software clustered these 18% of the control patients in the independent group with those suffering from fibrosis.

System for testing drugs Selective adhesion of selected protein candidates to silicon suggests its functional relevance in the development of hypersensitivity reactions and the associated fibrotic complications. As a result, the use of drugs can be intended to interfere with protein adhesion, thereby coating or capturing candidates for adhesion sites for these proteins prior to binding to the silicon surface. Accordingly, the inventors recommend using the following test system for future drug screening applications. It is based on a polystyrene 384-microwell plate. The principle of silicon coating is almost the same as 2.4.1.2. The only difference is that for each unpolymerized silicon, 10 μl was applied instead of 50 μl per well. The amount of serum or any other protein solution applied per well is also significantly lower, reducing to 20 μl per well. All incubation times remain the same.

Cell adhesion to silicon in vitro For cell adhesion experiments, the same system of silicon coated plates was used. As described above, the plates were coated with various silicone types and incubated with or without WBF or serum for 4 days. After this period, the plates were washed and 2 ml per well of a suspension of 5 × 10 ⁴ cells / ml in DMEM medium without serum was added. At various time points, non-adherent cells were removed by light pipetting and the remaining cells were fixed in 4% formaldehyde. The relative number of adherent cells was determined by crystal violet assay [Guilit J. et al. (Gailit J.), Clarke C.I. (Clark C.), Newman D. (Newman D.), Tunnelnes M. et al. G. (Tonnesen MG), Mosesson M.G. W. (Mosesson MW), and Clark R. A. (Clark RA) “Human fibroblasts bind directly to fibrinogen at RGD sites through integral alpha (v) beta 3.” Experimental Cell Research 232, 118-126. Crystal violet solution was added (0.2% w / v in 2% ethanol) and incubated for 10 minutes at room temperature. The plate was washed twice in fresh water by immersion in a larger beaker. They were then discharged upside down on a paper towel and 1% SDS was added to solubilize the stain. The plates were agitated on an orbital shaker until there was no dark colored area at the bottom of the wells and the color was uniform, and the absorbance of 1% SDS solution from each well was read at 562 nm using 1% SDS as a blank. . The solution was measured by classical spectrophotometry. The entire test was performed on a 6-well plate and could be miniaturized to a 96-well format for high throughput applications. FIG. 4 shows the adhesion of fibroblasts to silicon coated plastic wells.

Configuration scheme of ELISA system for adhesion of protein to silicon. 96 well plates were coated with silicon and sterilized as described. It was then incubated for 4 days with serum samples (supplemented with protease inhibitors) and washed with PBS and water. Adhesion proteins were detected using a specific primary antibody and an appropriate secondary antibody labeled with an enzyme (eg, horseradish peroxidase) that converts a non-colored substrate into a colored product measured at a specific wavelength. . The amount of protein was directly proportional to the intensity of the chromogenic dye. The assay can be performed in a variety of other formats. Evidence of the principle in screening patients with fibrotic complications at the time of SMI infusion. In accordance with the ELISA principle shown in FIG. 1, 10 patients (5 with and without fibrotic complications), 7 controls (matched age and gender) and dramatic systemic fibrotic complications Twelve protein candidates were tested in one patient with an injection of liquid silicon that induces. (A) Photograph of a plate with protein candidates tested. Green coloration indicates high protein abundance. Any variation in protein deposition is generally distinguishable visually. However, the absorption of each well was measured for accurate quantification and statistical analysis of the results. (B) Statistical analysis of different patient groups. After absorption was measured, the values were expressed as mean ± standard deviation for each group. The normality of the distribution was tested using the Kolmogolf Smirnov test and the p-value was calculated using t-test. A significant positive correlation between the onset of fibrotic complications and the deposition of fibronectin 1, C-reactive protein (CRP) and IgA and a significant negative correlation with the deposition of complement components C3 and IgG were found. Algorithms for protein adhesion analysis in vitro and in vitro. For in vitro analysis, the explanted SMI filled with saline is cut into fragments, washed, and the adhesion protein is in a buffer suitable for downstream analysis (either in Western blot or 2D electrophoresis / MS). Was eluted. For in vitro analysis, a standardized piece of silicon cleaved from SMI filled with fresh intact sterile saline was incubated with WBF or serum for 4 days. They were then washed in exactly the same way as SMI prepared in vitro and the protein was eluted in a buffer suitable for downstream analysis (in either Western blot or 2D electrophoresis / MS). For comparison, all protein amounts were normalized per surface unit. Adhesion of foreskin fibroblasts to silicon coated with serum proteins. Human primary fibroblasts were made from a foreskin formulation. Once their phenotypic homogeneity was determined (data not shown), they were cultured under standard conditions. A 96 well plate was coated with silicon, while an uncoated polystyrene plate was taken as a control. All were incubated with serum for 4 days and further washed with PBS and water. The fibroblasts were then seeded on both silicon-coated and uncoated plastic wells and eluted at various time points of 15-300 minutes. The adhesion of fibroblasts to silicon-coated wells is stronger than that of uncoated wells (statistically significant after 240 minutes ( ^* p <0.05), after 300 minutes Was very significant ( ^** p <0.001). This indicated that selective adhesion of serum-derived proteins to silicon promotes fibroblast adhesion. A controlled trial of retrospective cases based on a preferred embodiment of the method and the kit of the invention. Patients with dilated fibrosis (group 1, BS ≧ 3; N = 40; median age 51 years, left bar) and patients without dilated fibrosis (group 2, BS <3; N = 50) According to the ELISA for median age 47 years, of which 9 patients (= responders, 18%; “corrected control”, right bar) show similar characteristics as group 1 and It is shown that 41 of them (= non-responders, middle bar) did not show.

Claims (28)

A method for identifying a polypeptide that adheres to a silicon surface, comprising:
a) obtaining a sample from a mammal suspected of containing a silicon adhesion polypeptide and optionally containing silicon;
b) contacting the sample with at least one silicon;
c) isolating the polypeptide attached to the silicon from steps a) and / or b);
d) identifying the isolated silicon adhesion polypeptide;
Including a method.   2. The method of claim 1, wherein the sample is selected from a proteinaceous film covering silicon, wound fluid, and serum derived from a mammal that responds to injected silicon. 3. The method of claim 1 or 2, further comprising the step of: e) identifying a polypeptide suitable as a marker polypeptide for detecting a response response to silicon in a mammal. Step e)
e ′) a polypeptide pattern derived from the separation of the protein identified from the proteinaceous film covering the silicon and a polypeptide pattern derived from the separation of a silicon adhesion polypeptide eluted from wound fluid or serum; A step of comparing;
e '') selecting and analyzing a target spot in the pattern;
The method of claim 3 comprising:   A reagent kit for identifying a polypeptide that adheres to a silicon surface, comprising a raw material, a buffer and an auxiliary agent, and a substance for performing the method according to any one of claims 1 to 4. kit.   A silicon adhesion polypeptide identified by the method according to any one of claims 1-4.   A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, or a functional variant thereof, wherein the polypeptide or the functional variant thereof is A polypeptide that adheres to the surface of silicon.   A nucleic acid comprising a nucleotide sequence encoding the polypeptide according to claim 6 or 7 or a functional variant thereof, or a nucleotide sequence complementary to the nucleic acid.   The group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, myeloid related protein 8, spinal cord related protein 14, HT018, and PRO2619 for detecting a response response to mammalian silicon Use of a polypeptide selected from or a functional variant thereof. A method for detecting a response response to mammalian silicon comprising:
a) providing a sample from a mammal to be diagnosed that is thought to contain at least one silicon adhesion polypeptide or a functional variant thereof;
b) detecting a change in the presence or expression of the at least one silicon adhesion polypeptide or the functional variant thereof in the sample;
c) determining a response response of the mammal to the silicon from a change in the presence or expression of the at least one silicon adhesion polypeptide or functional variant thereof;
Including a method.   The silicon adhesion polypeptide is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, spinal cord related protein 8, spinal cord related protein 14, HT018, and PRO2619 or The method according to claim 9, which is a polypeptide identified by the method according to any one of 4 or a functional variant thereof.   The silicon adhesion polypeptide is an isotype control, IgG-HRP, IgE-HRP, IGA-HRP, AMBP protein precursor, plasma-retinol binding protein, kininogen, eosinophil peroxidase, fibroblast growth factor 11, TGF-β , Integrin-β4, T cell receptor, NADH dehydrogenase, coagulation factor V, α-spectrin, monocyte chemotactic protein-2, γ-glutamyltransferase, immunoglobulin G, immunoglobulin A, complement C3, complement C2, complement C1s, C-reactive protein, α1-microglobulin / bikunin precursor, Von Willebrandt factor, fibronectin, vitronectin, fibrinogen, collagen I, collagen III, collagen IV, co -Gen VII, procollagen III, laminin, matrix metalloproteinase 2, apolipoprotein 4, apolipoprotein Al, ceruloplasmin, thyreotyrein, albumin, α2-macroglobulin precursor, haptoglobin 1, α and β globin chain, actin, HSP60, apolipo Detected with at least one additional silicon adhesion protein and / or polypeptide or functional variant thereof selected from the group consisting of protein E3, complement C2 precursor, complement C3 precursor, hemoglobin, transthyretin The method according to claim 10 or 11.   Detection of changes in the presence or expression of said silicon adhesion polypeptides, namely immunoglobulin G, C-reactive protein, fibronectin, collagen I, procollagen III, complement C3 precursor, and spinal cord related protein 14 or functional variants thereof 13. The method according to any one of claims 10 to 12, wherein:   14. The sample of any one of claims 10 to 13, wherein the sample from the mammal is serum and / or wound fluid and the sample is collected before or after injection of silicon suitable for long term injection and medical device coating. The method according to item.   15. The method of any one of claims 10-14, further comprising providing a second sample as a control obtained from the same mammal before or after injection, or from a different mammal without any medical silicon injection. The method described in 1.   16. The method of any one of claims 10-15, further comprising detecting the presence and amount of at least one nucleic acid having a nucleotide sequence encoding a silicon adhesion polypeptide according to claim 6 or 7. .   11. A step a ′) between step a) and step b), further comprising contacting the sample with silicon under conditions that allow adhesion of the polypeptide to the silicon. The method according to any one of 16.   A diagnostic kit for detecting a response response to mammalian silicon comprising a raw material, a buffer, an auxiliary agent, and a substance for performing the method according to any one of claims 10 to 17. A method for monitoring the response of a mammal to silicon,
a) injecting at least one silicon into the mammal;
b) detecting a change in the presence or expression of the silicon adhesion polypeptide of claim 6 or claim 7 using the method of any one of claims 10 to 17;
c) optionally repeating step b);
Including a method. A method for screening for substances that interfere with the binding of silicon adhesion proteins and / or polypeptides to silicon comprising:
a) providing a material that appears to interfere with the adhesive properties of the silicon adhesive polypeptide;
b) providing silicon;
c) providing a silicon adhesion polypeptide;
d) analyzing the interference properties of the substance by analyzing the adhesion of the silicon adhesion polypeptide to the silicon in the presence or absence of the substance;
Including
Wherein the change in adhesion of the silicon adhesion polypeptide to the silicon in the presence or absence of the substance suggests a substance that interferes with the binding of silicon adhesion proteins and / or polypeptides to silicon.   A polypeptide, isotype identified by the polypeptide of claim 6 or 7, spinal cord related protein 8, spinal cord related protein 14, HT018, PRO2619, or the silicon adhesion polypeptide. Control, IgG-HRP, IgE-HRP, IGA-HRP, AMBP protein precursor, plasma-retinol binding protein, kininogen, eosinophil peroxidase, fibroblast growth factor 11, TGF-β, integrin-β4, T cell receptor Body, NADH dehydrogenase, coagulation factor V, α-spectrin, monocyte chemotaxis protein-2, γ-glutamyltransferase, immunoglobulin G, immunoglobulin A, complement C3, complement C2, complement CIs, C reaction Protein, α1-microglobulin / bikunin Precursor, von Willebrand factor, fibronectin, vitronectin, fibrinogen, collagen I, collagen III, collagen IV, collagen VII, procollagen III, laminin, matrix metalloprotease 2, apolipoprotein 4, apolipoprotein Al, ceruloplasmin, Thereotyrein, From albumin, α2-macroglobulin precursor, haptoglobin 1, α and β globin chain, actin, HSP60, apolipoprotein E3, complement C2 precursor, complement C3 precursor, hemoglobin, transthyretin or functional variants thereof 21. The method of claim 20, wherein the method is selected from the group consisting of: A method for detecting cell adhesion to silicon comprising:
a) providing a sample of serum or wound fluid of cells possessed by a mammal;
b) providing silicon;
c) analyzing the amount and number of cells from the serum or wound fluid adhering to the silicone;
Including a method.   22. A substance that interferes with the binding of silicon adhesion proteins and / or polypeptides to silicon, identified by the method of claim 20 or 21.   22. A method for making a pharmaceutical composition, comprising a method based on the method of claim 20 or 21 and the step of formulating said substance into a pharmaceutical composition.   25. A pharmaceutical composition comprising the substance of claim 23 or made on the basis of claim 24.   26. A method of treating a mammal experiencing an adverse reaction to injected silicon, comprising administering to said mammal an effective amount of the substance of claim 23 or the pharmaceutical composition of claim 25.   27. The method of claim 26, wherein the adverse reaction is silicosis and / or fibrosis associated with silicon.   28. The method of any one of claims 1-4, 10-17, 22, and 26 or 27, wherein the mammal is a human.

Images