WO2007082757A2 - Test systems for the analysis of polypeptides and cells adhering to silicones - Google Patents
Test systems for the analysis of polypeptides and cells adhering to silicones Download PDFInfo
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- WO2007082757A2 WO2007082757A2 PCT/EP2007/000442 EP2007000442W WO2007082757A2 WO 2007082757 A2 WO2007082757 A2 WO 2007082757A2 EP 2007000442 W EP2007000442 W EP 2007000442W WO 2007082757 A2 WO2007082757 A2 WO 2007082757A2
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- Prior art keywords
- silicone
- polypeptide
- adhering
- protein
- mammal
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
Definitions
- the present invention relates in general to the field of diagnostics, namely to test systems for polypeptides in respect to silicone. More precisely, the present invention, in one embodiment thereof, concerns test systems for identifying and analyzing polypeptides adhering to silicone. Furthermore, the present invention relates to newly identified polypeptides adhering to silicone and to respective uses of such proteins, e.g., in test systems for detecting a response reaction of a mammal to a silicone.
- Dimethylsiloxane polymer also known as 'silicone', is the most widely utilized inorganic biomaterial in medical practice, because of its assumed biocompatibility. It is used in a large variety of applications, such as medical tubing systems, syringe lubricants, coating of active medical devices like bypass apparatus and cochlear implants, and for the production of passive implants ranging from joint replacements and eye lenses to ventriculo-peritoneal shunts, and mammary implants.
- the so-called local reaction of the organism against the silicone is characterized by the formation of a fibrotic connective tissue capsule that can vary in extent [Siggelkow, W., Faridi, A., Spiritus, K., Klinge, U., Rath, W., and Kleinhalfen, B. Histological analysis of silicone breast implant capsules and correlation with capsular contracture. Biomaterials 24, 1101-1109 (2003)].
- fibrosis progresses causing functional impairment of the implant.
- Local side effects are often followed by systemic problems, and there have been reports on patients with long term silicone implants that have been diagnosed with fibromyalgia [Vermeulen, R. C. and Scholte, H. R.
- activated macrophages produce TNF- ⁇ , leading to the recruitment of lymphocytes and inducing transdifferentiation, proliferation and migration of fibroblasts to the surrounding tissue.
- Fibrosis occurs as the response of the surrounding tissue to the chronic inflammatory -A-
- the chronic proliferative inflammation pattern of fibroblasts is resulting in dense collagenous fibrosis after an implant duration of more than two years [Siggelkow, W., Faridi, A., Klinge, U., Rath, W., and Borhalfen, B. Ki67, HSP70 and TUNEL for the specification of testing of silicone breast implants in vivo. J Mater. Sci. Mater. Med 15, 1355-1360 (2004)]. Additional stimulation of local fibrotic processes through secretion of Connective Tissue Growth Factor (CTGF) is mediated by ⁇ -T-cells in the capsule [Workalemahu, G., Foerster, M., Kroegel, C, and Braun, R. K.
- CTGF Connective Tissue Growth Factor
- 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)], which was also found in the peri-implant connective tissue capsule.
- macrophages are stimulated via a positive feedback loop by TNF- ⁇ produced by ⁇ -T-cells.
- US 4,048,298 discloses a solid double-antibody radioimmunoassay procedure.
- the patent concerns a radioimmunoassay (RIA) procedure for assaying body fluid content of an antigenic substance which may either be an antigen itself or a hapten capable of being converted, such as by means of reaction with a protein, to an antigenic material.
- RIA radioimmunoassay
- This invention relates to a novel and improved modification of a double-antibody RIA technique in which there is a first antibody that is specific to the antigenic substance suspected to be present in a body fluid from which the assay is intended.
- the second antibody is not specific to the antigenic substance or analyte, but is an antibody against the first antibody.
- WO 02/41000 discloses an immunoassay method for membrane-bound matrix metallopro tease.
- MTl-MMP can be quickly quantified or the enzymatic activity of MTl-MMP can be measured at a high sensitivity and a high accuracy.
- a method of immunologically quantifying MTl-MMP by using an anti-MTl-MMP antibody and a reagent to be used therewith a method of immunologically quantifying a member selected from the group consisting of (i) MTl-MMP having been released and/or solubilized from cell membrane with the use of a surfactant and/or a reducing agent and (ii) autonomously solubilized MTl-MMP and a reagent to be used therewith; and solid-phase MTl-MMP makes it possible to quantify MTl-MMP or measure the enzymatic activity of MTl-MMP.
- an MTl-MMP expression promoter or inhibitor and an MTl-MMP activity promoter or inhibitor can be screened thereby.
- useful drugs can be facilitated.
- These reagents are also useful in examining cancer and cancer metastasis.
- the gel inside the silicone implant is in a polymerized form.
- Unpolymerized silicone can bleed through an intact silicone envelope into the surrounding tissue. It is theorized that this unpolymerized silicone stimulates the fibrotic reaction responsible for capsular contraction. Silicone bleeding through an intact envelope, as well as implant rupture, is associated with granuloma formation. Nevertheless, also polymerised silicone that covers the surface of an intact implant, i.e. the "true" surface, has a potential for a granulomatous or fibrotic capsule formation, i.e. even without bleeding of the silicone.
- Tavazzani et al. developed a model of human monocyte-derived macrophages to address the mechanisms of silicone-induced inflammation. With this model it was shown that the interaction between collagen type I and a silicone gel induces IL-I secretion by the cells. However, the binding characteristics of collagen to silicone was not investigated [Tavazzani, F., Xing, S., Waddell, J. E., Smith, D., and Boynton, E. L. In vitro interaction between silicone gel and human monocyte-macrophages. J Biomed Mat. Res. - Part A, 72, 2, 161-167 (2004)].
- the present invention provides a method for identifying polypeptides adhering to silicone surface, comprising the following steps of: a) obtaining and/or providing a sample from a mammal, wherein said sample is suspected to contain silicone-adhering polypeptides and, optionally, contains silicone; b) contacting said sample with at least one silicone, c) isolating, preferably comprising eluting, said polypeptides that are adhered to said silicone from step a) and/or b); and d) identifying said isolated silicone-adhering polypeptides.
- the present invention relates to a reagent kit for identifying polypeptides adhering to a silicone surface, comprising materials, buffers and auxiliary agents and substances for performing a method according to the present invention.
- the present invention provides silicone-adhering polypeptides identified through a method or by using a kit according to the present invention.
- the present invention relates to a polypeptide identified according to the present invention or a substance that interferes with the binding of a silicone- adhering protein and/or polypeptide to a silicone identified according to the present invention, and respective uses thereof.
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising at least one silicone-adhering polypeptide or a substance that interferes with the binding of a silicone-adhering protein and/or polypeptide to a silicone identified according to the present invention, and a pharmaceutical carrier.
- 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.
- the present invention relates to a nucleic acid comprising a nucleotide sequence encoding for a polypeptide according to the present invention, in particular for a polypeptide having the amino acid sequence set forth in any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO. 4, or a functional variant thereof, or a complementary nucleotide sequence of said nucleic acid.
- the present invention relates to a nucleic acid that hybridizes under stringent conditions with the nucleic acid according to the present invention.
- the present invention provides a recombinant vector, comprising the nucleic acid according to the present invention.
- the present invention concerns a host cell comprising a nucleic acid or vector or an expression vector according to the present invention.
- the present invention relates to an antibody that immunologically recognizes a polypeptide according to the present invention.
- the present invention provides a polypeptide, a nucleic acid or vector or an expression vector according to the present invention for use in medicine.
- the present invention relates to an antibody that immunologically recognizes 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.
- the present invention concerns a pharmaceutical composition, comprising an effective dose of 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 an antibody according to the present invention, and a pharmaceutically acceptable carrier.
- the present invention relates to the use of 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 an antibody according to the present invention for detecting a response reaction of a mammal to a silicone, in particular to a medical silicone.
- the present invention concerns the use of a polypeptide for detecting a response reaction of a mammal to a silicone, wherein said polypeptide is selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO.3, SEQ ID NO.4, Myeloid related protein 8, Myeloid related protein 14, HTO 18, and PRO2619 or a functional variant thereof.
- the present invention relates to a method for detecting a response reaction of a mammal to a silicone, comprising the steps of: a) providing a sample from a mammal to be diagnosed suspected of containing at least one silicone-adhering polypeptide or a functional variant thereof; b) detecting the presence or a change in expression of said at least one silicone-adhering polypeptide or said functional variant thereof in said sample; and c) concluding from said presence or change in expression of said at least one silicone-adhering polypeptide or a functional variant thereof on a response reaction of said mammal to said silicone.
- the present invention relates to a diagnostic kit for detecting a response reaction in a mammal against silicone, in particular medical silicone, comprising materials, buffers, auxiliary agents, and substances for performing a method according to the present invention.
- the present invention relates to a method for monitoring a response reaction of a mammal to a silicone, in particular medical silicone, comprising a) implanting at least one silicone into said mammal, and b) detecting the presence or a change in expression of a silicone-adhering polypeptide according to the present invention using a method or kit according to the present invention, and optionally, repeating step b).
- the present invention provides a method for screening substances that interfere with at least one silicone-adhering polypeptide, comprising the steps of a) providing a substance, wherein said substance is suspected to interfere with the adhering properties of a silicone-adhering polypeptide; b) providing a silicone, in particular a medical silicone; c) providing a silicone-adhering polypeptide; and, d) analyzing the interfering properties of said substance through analyzing the adhesion of said silicone-adhering polypeptide to said silicone in the presence and absence of said substance, wherein a change in the adhesion of said silicone-adhering polypeptide to said silicone in the presence and absence of said substance is indicative for a substances that interferes with the binding of a silicone-adhering protein and/or polypeptide to a silicone.
- the present invention relates to a method for detecting adhesion of cells to a silicone, comprising a) providing a sample of serum or wound bed fluid of a mammal containing cells; b) providing a silicone; and c) analyzing the amount or number of cells from said serum or wound bed fluid that adhere to said silicone.
- the present invention provides a method of treating a mammal suffering from an adverse reaction to an implanted silicone, comprising administering to said mammal an effective amount of a substance interfering with the adhering properties of a silicone-adhering polypeptide as identified through a method according to the present invention.
- the present invention provides a method for monitoring the treatment of a mammal suffering from a response reaction to a silicone, comprising: a) administering to said mammal an effective amount of a substance interfering with the adhering properties of a silicone-adhering polypeptide as identified according to the present invention b) obtaining a sample from said mammal; and c) detecting the presence or a change in expression of at least one silicone-adhering polypeptide or a functional variant thereof.
- polypeptides which are involved for a response reaction of a mammal to a silicone. These polypeptides presumably play a role as key activators of the immune response, which is in turn responsible for the fibrosis leading to implant failure. Thus, a detailed analysis of their composition and the dynamics of their deposition is necessary. Furthermore, these identified polypeptides might serve as both diagnostic as well as potential therapeutic target molecules with respect to the response reaction to silicone.
- the term "response reaction” as used herein, shall mean the physiological reaction of a body or a cell to a silicone, such as a medical silicone.
- Such reaction can manifest itself as an immunological response involving the immune system of the body, and can be present as a local (i.e. at or closely around the site of the contact with the silicone) or systemic (i.e. generally found in the body, such as, for example, in the blood, serum or other fluids) side effect, for example as a response to an implanted silicone, such as to implanted SMIs [Smalley, D. L., Levine, J. J., Shanklin, D. R., Hall, M. F., and Stevens, M. V. Lymphocyte response to silica among offspring of silicone breast implant recipients. Immunobiology 196, 567-574 (1996)].
- fibrotic connective tissue capsule usually with a foreign-body granuloma formation
- fibromyalgia connective tissue disease (e.g. inflammation)
- connective tissue disease e.g. inflammation
- scleroderma-like fibrotic syndrome e.g. fibrotic syndrome.
- fibrotic process occurring in response to a silicone implant are a model for other, non-implant associated fibrotic diseases, of which four different types are known: (i) post-necrotic diseases, e.g. hepatic cirrhosis, (ii) inflammatory diseases, e.g. rheumatoid arthritis, (iii) or spontaneous forms, e.g. Dypuytren's contracture and (iv) foreign-body reactions in general (e.g. reactions against implants).
- post-necrotic diseases e.g. hepatic cirrhosis
- inflammatory diseases e.g. rheumatoid arthritis
- spontaneous forms
- hypersensitivity refers to a particular type of (an exaggerated) response reaction of a mammal to silicone, e.g. an adverse reaction of an individual in response to a particular silicone, which, in one embodiment, can only be found in this individual, whereas other individuals might not show any respective response (that is, an individual response to a certain silicone).
- the present invention provides a method for identifying polypeptides adhering to a silicone surface, comprising the following steps of: a) obtaining and/or providing a sample from a mammal, wherein said sample is suspected to contain silicone-adhering polypeptides and, optionally, contains silicone; b) contacting said sample with at least one silicone, c) isolating, preferably comprising eluting, said polypeptides that are adhered to said silicone from step a) and/or b) and d) identifying said isolated polypeptides.
- the polypeptides are first separated using gel electrophoresis, more preferably two-dimensional gel electrophoresis.
- this step is then followed by a staining procedure known to a person skilled in the art, preferably silver staining.
- the thus stained polypeptides will be detectable as a polypeptide "spot pattern".
- This pattern is matched and compared with e.g. a control pattern or with a database, e.g. with the ExPasy database of 2D gels, using a software.
- a polypeptide spot of interest is excised, more preferably the polypeptide spot of interest is excised manually.
- the excised polypeptide spot is preferably digested to obtain fragments that are suitable to be analyzed by mass spectrometry, e.g. peptide mass fingerprint (PMF) using MALDI-TOF.
- the samples are further analyzed using LIFT-TOF/ TOF MS/MS. More preferably, the results are then confirmed using Western blot analysis as known by a person skilled in the art.
- adhere refers to the firmly sticking of a compound, e.g. a polypeptide to a surface, e.g., the surface of a silicone. This sticking can be performed by both covalent and non-covalent binding between the polypeptide and the silicone.
- polypeptide in accordance with the present invention, shall mean a peptide or a protein which encompasses amino acid chains of a given length of at least 10, preferably, 20- 50, more preferred 30-100 amino acids, wherein the amino acid residues are linked by covalent peptide bonds.
- peptidomimetics of such polypeptides wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs are also encompassed by the invention.
- a most preferred polypeptide is a part of the peptide that is functionally responsible or involved for the adhesion of the polypeptide to silicone.
- silicone shall encompass all dimethylsiloxane polymers that can be used in a physiological environment, such as the human body. Usually, such silicones show a biocompatibility (i.e. do induce no or only slight immune responses of the body) and preferably have a high physiological acceptance. Such silicones are also known as “medical silicones". Such medical silicones can be used for medical implants, such as, for example, silicones mammary implants or as coatings for medical devices e.g. bypass implants. Such medical silicones suitable to be used according to the present invention are known by the person skilled in the art. Preferred examples of such medical silicones are the silicone types MED151 1, MED4860, MED4211, MED360 provided by the company Nusil (Waldbroon, Germany).
- the sample is selected from a proteinaceous film covering a silicone, urine, pleural- or peritoneal fluid, cerebrospinal fluid, wound bed fluid, or serum.
- said sample is selected from a proteinaceous film covering a silicone, serum or wound bed fluid.
- said wound bed fluid is preferably obtained with drainage.
- the sample is obtained from a mammal showing a response reaction to silicone.
- a mammal preferably is a human.
- the sample is obtained from a mammal showing a response reaction selected from the group consisting of a fibrotic disease of one of the following types: (i) post-necrotic diseases, e.g. hepatocirrhosis, (ii) inflammatory diseases, e.g. rheumatoid arthritis, (iii) spontaneous fibrotic diseases, e.g. Dupuytren's contracture, (iv) and foreign-body reaction in general.
- a fibrotic disease of one of the following types: (i) post-necrotic diseases, e.g. hepatocirrhosis, (ii) inflammatory diseases, e.g. rheumatoid arthritis, (iii) spontaneous fibrotic diseases, e.g. Dupuytren's contracture, (iv) and foreign-body reaction in general.
- Such diseases can result in the formation of a fibrotic connective tissue capsule, fibromyalgia, connective tissue diseases
- the fibrotic connective tissue capsule is formed through an immunological response.
- the wound bed fluid or serum is provided together with an additional silicone, preferably with a medical silicone.
- the silicone is provided unpolymerized.
- the term "unpolymerized” as used in accordance with the present invention relates to the state of a silicone, e.g. a medical silicone, which is essentially not polymerized. Unpolymerized silicone usually occurs as a by-product of the polymerization of silicone implants (in particular breast implants) and is believed to cause many of the problems associated with such implants.
- the silicone can be any medical silicone known by the person skilled in the art.
- the silicone is selected from the group consisting of the silicone types MED1511, MED4860, MED4211, and MED360 provided by the company Nusil (Waldbroon, Germany).
- step d) comprises any method known by the person skilled in the art.
- step d) comprises methods selected from one or two- dimensional electrophoresis, Western blot analysis, protein assays suitable to measure the amount and/or presence of said proteins, mass spectrometry, a method suitable for analyzing modifications, e.g. silver staining, advanced glycation end products (AGE) formation, oxidation, antibody arrays, ligand arrays/assays, enzyme immunoassay (EIA, e.g. ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), and Luminex assay.
- EIA enzyme immunoassay
- RIA radioimmunoassay
- FFA fluorescence immunoassay
- the method further comprises in step e) identifying polypeptides that are suitable as marker polypeptides for detecting a response reaction in a mammal to silicone.
- proteins eluted from proteinaceous film covering explanted silicone are compared to silicone-adhering proteins obtained from wound bed fluid or serum. More preferably, the proteins eluted from the two systems are compared by one and/or two- dimensional gel electrophoresis.
- proteins from the two systems are labeled with different fluorochromes that can be excited at specific wavelengths, leading to independent images from a single gel.
- the protein spot patterns are analyzed with any software suitable to analyze such patterns known by a person skilled in the art.
- the protein spot patterns are analyzed with the 'Decyder' software (Amersham Biosciences).
- step e) comprises: e') comparing polypeptide patterns derived from a separation of said proteins identified from the proteinacous film covering said silicone with polypeptide patterns derived from the separation of silicone- adhering polypeptides eluted from wound bed fluid or serum; and e") selecting and analyzing a spot of interest in said pattern.
- the amount of proteins deposited are considered different, if the spot volume ratio between samples exceeded two standard deviations of total spot distribution, most preferably, a spot is defined as present only in a system, if the volume ratio exceeded a threshold of fivefold.
- the polypeptides are furthermore labeled with different fluorochromes or are radioactive labeled.
- the fluorochromes are excitable at specific wavelengths. Any fluorochrome known by the person skilled in the art can be used.
- the fluorochromes are selected from the group consisting of 7- aminoactinomycine d (7-AAD), acridine orange, alexa dyes, amino methylcoumarine-acetate (AMCA), Cy-dyes, e.g.
- Cy2, Cy3, Cy5, Cy7, 4',6-Diamidino-2-phenylindole dihydrochloride DAPI
- fluorescein isothiocyanate FITC
- Hoechst-dyes e.g., Hoechst 33342, Hoechst 33258, "blue” fluorescence, 5,5',6,6'Tetrachloro-l,l ',3,3'-tetraethyl- benzimidazolylcarbocyanine iodide (JC-I), peridinin-chlorophyll-protein (PerCP), propidium jodide, rhodamine Red-X, phycoerythrin (R-PE), Aminomethylcoumarin acetate sulfonyl chloride rhodamine -derivate (Texas-Red), and Tetramethyl rhodamine isothiocyanate (TRITC).
- polypeptide spot patterns are analyzed by searching for all spots presented in the polypeptide patterns from polypeptides eluted from silicone and presented in the polypeptide patterns of polypeptides of separated silicone- adhering polypeptides eluted from wound bed fluid or serum.
- the mammal is human.
- the present invention relates to a reagent kit for identifying polypeptides adhering to a silicone surface, comprising materials, buffers and auxiliary agents and substances for performing the method according to the present invention.
- the present invention relates to a silicone-adhering polypeptide identified through a method or by using a kit according to the present invention.
- the present invention relates to a polypeptide identified according to the present invention or a substance that interferes with the binding of a silicone-adhering protein and/or polypetide to a silicone identified according to the present invention, and respective uses thereof.
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising at least one silicone-adhering polypeptide or a substance that interferes with the binding of a silicone-adhering protein and/or polypeptide to a silicone identified according to the present invention, and a pharmaceutical carrier.
- the present invention relates to 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.
- the term "functional variants" in the sense of the present invention means polypeptides that are functionally related to the polypeptides of the present invention, i.e. share the common structural or functional feature of the polypeptides, to adhere to silicone.
- Examples of functional variants are orthologs, i.e. the corresponding polypeptide derived from another organism as human, preferably from non-human mammals such as, for example, monkeys, pigs, mice, and rats.
- Other examples of functional variants are polypeptides that are encoded by different alleles of the gene, in different individuals or in different organ of an organism.
- polypeptides shall be encompassed by said term that have a sequence homology, in particular a sequence identity, of about 70%, preferably about 80%, in particular about 90%, more preferred about 95% to the polypeptide with the amino acid sequence according to the polypeptides of the present invention and/or DNA sequences that are derived from these polypeptide sequences. Included are additions, inversions, substitutions, deletions, insertions or chemical/physical modifications and/or exchanges or parts of the polypeptide in the range of about 1-60, preferably of about 1-30, and most preferred about 1-15 amino acids.
- functional variants preferably can encompass domain(s) of the protein that are responsible for the adhesion of said polypeptide to a silicone.
- polypeptide adheres to the surface of a silicone.
- the polypeptide is a fusion protein.
- fusion protein denotes any polypeptide consisting or comprising of at least two polypeptides not naturally forming such a polypeptide. On the DNA level, the two or more coding sequences are fused in frame. The parts of the fusion protein can be connected and/or spaced apart by linkers (usually peptide linkers or chemical linkers) that are known to the person of skill.
- the present invention relates a nucleic acid comprising a nucleotide sequence encoding for a polypeptide according to the present invention, in particular for a polypeptide having the amino acid sequence set forth in any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, or SEQ ID NO. 4, or a functional variant thereof, or a complementary nucleotide sequence of said nucleic acid.
- hybridization conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y., 6.3.1-6.3.6, 1991.
- Moderate hybridization conditions are defined as equivalent to hybridization in 2X sodium chloride/sodium citrate (SSC) at 3O 0 C, followed by a wash in 1 X SSC, 0.1% SDS at 50 0 C.
- Highly stringent conditions are defined as equivalent to hybridization in 6X sodium chloride/sodium citrate (SSC) at 45°C, followed by a wash in 0.2 X SSC, 0.1 % SDS at 65°C.
- the nucleic acid is DNA, cDNA, PNA, CNA, RNA or combinations thereof.
- the nucleic acid molecules of the present invention can be genomic DNA, synthetic DNA, or combinations thereof, and can be double-stranded or single-stranded, the sense- and/or an antisense strand. Segments of these molecules are also considered within the scope of the invention, and can be produced by, for example, the polymerase chain reaction (PCR) or generated by treatment with one or more restriction endonucleases.
- PCR polymerase chain reaction
- a ribonucleic acid (RNA) molecule can be produced by in vitro transcription.
- nucleic acid molecules of the present invention can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same peptide (for example, the peptides with SEQ ID NOs: 1, 2, 3 or 4).
- these nucleic acid molecules are not limited to coding sequences, e.g., they can include some or all of the non-coding sequences that lie upstream or downstream from a coding sequence.
- the nucleic acid molecules of the invention can be synthesized in vitro (for example, by phosphoramidite-based synthesis) or obtained from a cell, such as the cell of a bacterium or mammal.
- the nucleic acids can be those of a human but also derived from a non-human primate, mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, dog, or cat. Combinations or modifications of the nucleotides within these types of nucleic acids are also encompassed.
- the isolated nucleic acid molecules of the invention can encompass segments that are not found as such in the natural state.
- the invention encompasses recombinant nucleic acid molecules incorporated into a vector (for example, a plasmid or viral vector) or into the genome of a heterologous cell (or the genome of a homologous cell, at a position other than the natural chromosomal location). Recombinant nucleic acid molecules and uses therefore are discussed further below.
- the nucleic acid according to the present invention can 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 active groups, etc. All these markers or labels and respective technologies for attaching these are well known to the person of skill.
- the present invention provides a nucleic acid that hybridizes under stringent conditions with the nucleic acid according to the present invention.
- the present invention relates to a recombinant vector comprising a nucleic acid according to the present invention.
- the present invention relates in another embodiment to a vector comprising the nucleic acid molecule of this invention.
- a vector is capable of being introduced or of introducing the nucleic acid into a cell. It is preferred that the polypeptides encoded by the introduced nucleic acids are expressed within the cell upon introduction of the vector.
- the recombinant vector is a recombinant expression vector, including both overexpression and repression vector.
- the vector of the present invention comprises plasmids, phagemids, phages, cosmids, artificial mammalian chromosomes, knock-out or knock-in constructs, viruses, in particular adenoviruses, vaccinia viruses, attenuated vaccinia viruses, canary pox viruses, lentivirus (Chang, L.J. and Gay, E. E. (20001) Curr. Gene Therap. 1 :237- 251), herpes viruses, in particular Herpes simplex virus (HSV-I, Carlezon, W. A. et al. (2000) Crit. Rev.
- viruses in particular adenoviruses, vaccinia viruses, attenuated vaccinia viruses, canary pox viruses, lentivirus (Chang, L.J. and Gay, E. E. (20001) Curr. Gene Therap. 1 :237- 251)
- herpes viruses in particular Herpes simplex virus (HSV-I, Carlez
- baculovirus baculovirus
- retrovirus retrovirus
- adeno-associated-virus AAV, Carter, PJ. and Samulski, RJ. (2000) J. MoI. Med. 6:17-27
- rhinovirus human immune deficiency virus
- HIV human immune deficiency virus
- f ⁇ lovirus engineered versions thereof (see, for example, Cobinger G. P. et al (2001) Nat. Biotechnol. 19:225-30)
- virosomes "naked" DNA liposomes
- nucleic acid coated particles in particular gold spheres.
- viral vectors like adenoviral vectors or retroviral vectors (Lindemann et al. (1997) MoI. Med.
- Liposomes are usually small unilamellar or multilamellar vesicles made of cationic, neutral and/or anionic lipids, for example, by ultrasound treatment of liposomal suspensions.
- the DNA can, for example, be ionically bound to the surface of the liposomes or internally enclosed in the liposome.
- Suitable lipid mixtures are known in the art and comprise, for example, DOTMA (l,2-dioleyloxpropyl-3- trimethylammoniumbromide), and DPOE (dioleoylphosphatidyl-ethanolamine), which both have been used on a variety of cell lines.
- Nucleic acid coated particles are another means for the introduction of nucleic acids into cells using so called “gene guns", which allow the mechanical introduction of particles into the cells.
- the particles Preferably the particles itself are inert, and therefore, are in a preferred embodiment made out of gold spheres.
- the nucleic acid of the present invention is operatively linked to expression control sequences allowing expression in prokaryotic and/or eukaryotic host cells.
- the transcriptional/translational regulatory elements referred to above include but are not limited to inducible and non-inducible, constitutive, cell cycle regulated, metabolically regulated promoters, enhancers, operators, silencers, repressors and other elements that are known to those skilled in the art and that drive or otherwise regulate gene expression.
- regulatory elements include but are not limited to regulatory elements directing constitutive expression like, for example, promoters transcribed by RNA polymerase III like , e.g., promoters for the snRNA U6 or scRNA 7SK gene, the cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, viral promoter and activator sequences derived from, e.g., NBV, HCV, HSV, HPV, EBV, HTLV, MMTV or HIV; which allow inducible expression like, for example, CUP-I promoter, the tet-repressor as employed, for example, in the tet-on or tet-off systems, the lac system, the trp system; regulatory elements directing tissue specific expression, regulatory elements directing cell cycle specific expression like, for example, cdc2, cdc25C or cyclin A; or the TAC system, the TRC system, the major operator and promoter regions
- operatively linked means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest.
- nucleic acids of the present invention can form part of a hybrid gene encoding additional peptide sequences, for example, a sequence that functions as a marker or reporter.
- marker and reporter genes include ⁇ -lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo r , G418 r ), dihydro folate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding /-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT).
- CAT chloramphenicol acetyltransferase
- ADA adenosine deaminase
- DHFR dihydro folate reductase
- the hybrid peptide will include a first portion and a second portion; the first portion being a silicon-adhering polypeptide and the second portion being, for example, the reporter described above or an Ig constant region or part of an Ig constant region, e.g., the CH2 and CH3 domains of IgG2a heavy chain.
- Other hybrids could include an antigenic tag or His tag to facilitate purification and/or detection.
- Recombinant nucleic acid molecules can also contain a polynucleotide sequence encoding a silicon-adhering polypeptide operatively linked to a heterologous signal sequence.
- signal sequences can direct the protein to different compartments within the cell and are well known to someone of skill in the art.
- a preferred signal sequence is a sequence that facilitates secretion of the resulting protein.
- a polypeptide according to the present invention In order to express cDNAs encoding a polypeptide according to the present invention, one typically subclones a polypeptide according to the present invention cDNA into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and a ribosome-binding site for translational initiation.
- Suitable bacterial promoters are well known in the art, e.g., E. coli, Bacillus sp. , and Salmonella, and kits for such expression systems are commercially available.
- 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 may be, for example an adenoviral vector, an adeno-associated vector, or a retroviral vector.
- the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the silicon-adhering polypeptide-encoding nucleic acid in host cells.
- a typical expression cassette thus contains a promoter operatively linked to the nucleic acid sequence encoding the silicon- adhering polypeptide and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette may include, for example enhancers.
- An expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
- the particular expression vector used to transport the genetic information into the cell is not particularly critical. 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, but there are many more known in the art to the skilled person that can be usefully employed.
- Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus.
- eukaryotic vectors include pMSG, pAV009/A.sup.+, pMTO10/A.sup.+, pMAMneo-5, baculovirus pDSVE, pcDNA3.1, pIRES and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
- Some expression systems have markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase.
- markers that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase.
- high yield expression systems not involving gene amplification are also suitable.
- the elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding drug resistance to permit selection of bacteria that harbour recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences.
- the particular drug resistance gene chosen is not critical, any of the many drug resistance genes known in the art are suitable.
- the prokaryotic sequences are optionally chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary.
- Standard transfection methods can be used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of the receptor, which are then purified using standard techniques.
- Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA -2 ⁇
- the transfected cells may be cultured under conditions favoring expression of the silicon-adhering polypeptide, which is recovered from the culture using standard techniques.
- the cells may be burst open either mechanically or by osmotic shock before being subject to precipitation and chromatography steps, the nature and sequence of which will depend on the particular recombinant material to be recovered.
- the recombinant peptide may be recovered from the culture medium in which the recombinant cells had been cultured.
- the present invention concerns a host cell comprising a nucleic acid or vector, or an expression vector according to the present invention.
- the host cells that may be used for purposes of the invention include but are not limited to prokaryotic cells such as bacteria (for example, E. coli and B.
- subtilis which can be transformed with, for example, recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the polynucleotide molecules of the invention; simple eukaryotic cells like yeast (for example, Saccharomyces and Pichia), which can be transformed with, for example, recombinant yeast expression vectors containing the nucleic acid molecule of the invention; insect cell systems like, for example, Sf9 of Hi5 cells, which can be infected with, for example, recombinant virus expression vectors (for example, baculovirus) containing the nucleic acid molecules of the invention; Xenopus oocytes, which can be injected with, for example, plasmids; plant cell systems, which can be infected with, for example, recombinant virus expression vectors (for example, cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors containing a
- Also useful as host cells are primary or secondary cells obtained directly from a mammal and transfected with a plasmid vector or infected with a viral vector.
- the nucleic acid can integrate, for example, into the chromosome or the mitochondrial DNA or can be maintained extrachromosomally like, for example, episomally or can be only transiently comprised in the cells.
- the present inventions provides an antibody that immunologically recognizes a polypeptide and/or protein according to the present invention.
- antibody comprises monoclonal and polyclonal antibodies and binding fragments thereof, in particular Fc-fragments as well as so called “single-chain-antibodies” (Bird R. E. et al (1988) Science 242:423-6), chimeric, humanized, in particular CDR-grafted antibodies, and dia or tetrabodies (Holliger P. et al (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-8).
- immunoglobulin like proteins that are selected through techniques including, for example, phage display to specifically bind to the peptides of the present invention.
- Preferred antibodies bind to those parts of the peptide responsible for binding to their respective receptor.
- the present invention relates to an antibody that immunologically recognizes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO.l, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO. 4 or a functional variant thereof.
- the present inventions relates to a polypeptide, a nucleic acid or vector, or an expression vector according to the present invention for use in medicine.
- the present inventions concerns to a pharmaceutical composition, comprising an effective dose of a polypeptide according to the present invention, a nucleic acid or vector according to the present invention, or an expression vector according to the present invention, a host cell according to the present invention, or an antibody according to the present invention, and a pharmaceutically acceptable carrier and/or other suitable pharmaceutical auxiliary agents.
- compositions e.g. in form of medicaments with a content at least one of a polypeptide according to the present invention, a nucleic acid or vector according to the present invention or an expression vector according to the present invention, a host cell according to the present invention or an antibody according to the present invention (in the following simply designated as "active ingredients") and their uses according to the present invention occurs according to standard pharmaceutical technology and methods.
- active ingredients together with pharmaceutical acceptable carriers and/or other suitable pharmaceutical auxiliary agents, are produced into medical forms that suitable for the different indications, and places of administration.
- compositions can be produced in a manner that the release rate that is desired, e.g. a quick onset and/or a retard- or depot-effect is achieved.
- the pharmaceutical compositions can be an ointment, gel, patch, emulsion, lotion, foam, creme or mixed-phase or amphiphilic emulsion systems (oil/water-water/oil-mix-phase), liposome, transfersome, paste or powder.
- auxiliary agent shall mean any, non-toxic, solid or liquid filling, diluting or packaging material, as long as it does not adversely react and/or interacts with the active ingredients or the patient.
- Liquid galenical auxiliary agents for example, are sterile water, physiological saline, sugar solutions, ethanol and/or oils.
- Galenical auxiliary agents for the production of tablets and capsules for example, can contain binders and filling materials.
- the active ingredients according to the invention can be used in the form of systemically employed medicaments.
- These include parenterals belonging to which are injectables and infusions.
- injectables are either present in the form of ampoules or as so-called ready-to-use injectables, e.g. as ready-to-use syringes or disposable syringes, and, in addition, are provided in puncture-sealed bottles.
- the administration of the injectables can take place in form of subcutaneous (s.c), intramuscular (i.m.), intravenous (i.v.) or intracutaneous (i.e.) application.
- suitable forms for injection can pe produced as crystal suspensions, solutions, nanoparticular or colloidal-disperse systems, such as, for example, hydrosoles.
- the injectable compositions can further be produced as concentrates that are dissolved or dispersed with aqueous isotonic diluents.
- the infusions can also be prepared in form of isotonic solutions, fatty emulsions, liposome compositions, micro emulsions.
- infusion compositions can be prepared in form of concentrates for dilution.
- the injectable compositions can also be applied in form of continuous infusions, both in the stationary as well as in the ambulant therapy, e.g. in form of mini pumps.
- the active ingredients according to the invention can be bound to a micro carrier or nanoparticle, for example to finely dispersed particles on the basis of poly(meth)acrylates, polylactates, polyglycolates, polyaminoacids or polyetherurethanes.
- the parenteral compositions can also be modified into a depot preparation, e.g. based on the "multiple unit principle", if an active ingredient according to the invention is embedded in finely divided or dispersed, suspended form or as crystal suspension, or based on the "single unit principle", if an active ingredient according to the invention is included in a medicinal form, e.g. in a tablet or a stick that is subsequently implanted.
- these implants or depot medicaments in the case of ,,single unit"- and ,,multiple unif'-medicaments consist of so-called biodegradable polymers, such as, for example polyesters of lactic and glycolic acid, polyether urethanes, polyaminoacids, poly(meth)acrylates or polysaccharides.
- auxiliary agents for producing of parenterals, aqua sterilisata substances influencing the value of the pH, such as, for example, organic and inorganic acids and bases as well as their salts, buffer substances for adjusting the value of the pH, isotoning agent, such as, for example, sodium chloride, sodium hydrogen carbonate, glucose and fructose, tensides or surface active substances and emulgators, such as, for example, partial fatty acid esters of polyoxyethylene sorbitane (T ween®) or, for example, fatty acid esters of polyoxyethylene (Cremophor®), fatty oils, such as, for example, peanut oil, soy bean oil, and castor oil, synthetic fatty acid esters, such as, for example, ethyloleate, isopropylmyristate and neutral oil (Miglyol®), as well as polymeric auxiliary agents, such as, for example, gelatine, dextran, polyvinylpyrrolidone, solubid
- Active ingredient complexes can be achieved with different polymers, such as, for example, polyethyleneglycoles, polystyrenes, carboxymethyl cellulose, Pluronics® or polyethylene glycolsorbite fatty acid esters.
- scaffold forming agents such as, for example, mannit, dextran, sucrose, human albumin, lactose, PVP or gelatines are used.
- the medical forms that are each suitable can be produced in accordance with manuals and procedures known to the person of skill on the basis of pharmaceutical/physical technologies.
- a further aspect of the present invention then relates to the respectively produced pharmaceutical composition, comprising an effective dose of at least one of a polypeptide according to the present invention, a nucleic acid or vector according to the present invention or an expression vector according to the present invention, a host cell according to the present invention or an antibody according to the present invention, and a pharmaceutically acceptable carrier.
- This pharmaceutical composition can be characterized in that the active ingredient is present in form of a depot substance or as precursor together with a suitable, pharmaceutically acceptable diluent or carrier substance as above.
- the above pharmaceutical composition can be present in the form of tablets, dragees, capsules, droplets, suppositories, compositions for injection or infusion for peroral, rectal or parenteral use.
- Such administration forms and their production are known to the person of skill.
- the present inventions relates to the use of a polypeptide according to the present invention, a nucleic acid or vector according to the present invention, or an expression vector according to the present invention, a host cell according to the present invention, or an antibody the present invention for detecting a response reaction of a mammal to a silicone, in particular to a medical silicone.
- a response reaction of a human to a silicone Preferably a response reaction of a human to a silicone.
- the present inventions concerns to the use of a polypeptide for detecting a response reaction as described below of a mammal to a silicone, wherein said polypeptide is selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO.3, SEQ ID NO.4, Myeloid related protein 8, Myeloid related protein 14, HTOl 8, and PRO2619 or a functional variant thereof.
- a diagnostic strategy can be established by identifying polypeptides prone to response reactions and related complications or adverse reactions.
- a test system for the detection of these polypeptides and/or nucleic acids can be constructed, e.g. by attaching such polypeptides to the surface of silicone from e.g. serum or through amplification methods, such as, for example rt-PCR.
- polypeptides according to the present invention can be detected with methods known in the art, which comprises, immunological methods like ELISA or Western blotting.
- test system is based on an ELISA system, a method well- known by a person skilled in the art.
- Production of ELISA system for the measurement of silicone-adhering polypeptides is easy to perform and stable for a long time at room temperature. This would facilitate shipping to end-users in order to use these tests as "point- of-care" tests, e.g. in the doctor's office.
- such test system uses serum.
- an ELISA system is a standard procedure in many laboratories. This combination would allow for easy integration into current clinical routine.
- the present inventions provides a method of diagnosis for detecting a response reaction of a mammal to a silicone, comprising the steps of: a) providing a sample from a mammal to be diagnosed suspected of containing at least one silicone-adhering polypeptide or a functional variant thereof; b) detecting the presence or a change in expression of said at least one silicone-adhering polypeptide or a functional variant thereof in said sample; and c) concluding from said presence or change in presence or change in expression of at least one silicone-adhering polypeptide or a functional variant thereof on a response reaction of said mammal to said silicone.
- the silicone-adhering polypeptide is selected from the group consisting of a polypeptide according to the present invention, Myeloid related protein 8, Myeloid related protein 14, HTOl 8, and PRO2619 or a polypeptide identified according by a for identifying polypeptides adhering to silicone surface according to the present invention, or functional variants thereof.
- the silicone-adhering polypeptide is detected together with at least one additional silicone-adhering protein and/or polypeptide selected from the group consisting of Isotype control, IgG-HRP, IgE-HRP, and IGA-HRP, AMBP protein precursor, Plasma-retinol binding protein, Kininogen, Eosinophil peroxidase, Fibroblast growth factor 11, TGF- ⁇ , Integrin- ⁇ 4, T-cell receptor, NADH dehydrogenase, Coagulation factor V, ⁇ -spectrin, Monocyte chemotactic protein-2, ⁇ - glutamyltransferase, Immunoglobulin G, Immunoglobulin A, Complement C3, Complement C2, Complement CIs, C reactive protein, Alpha 1-microglobulin/bikunin precursor, Von Willebrandt Factor, Fibronectin, Vitronect
- the presence or a change in expression of the silicone-adhering polypeptide(s) Immunoglobulin G, C-reactive protein, Fibronectin, Collagen I, Procollagen III, Complement C3 precursor, and Myeloid related protein 14 or functional variants thereof is/are detected.
- the presence or a change in expression of the silicone-adhering polypeptides Immunoglobulin G, C-reactive protein, Fibronectin, Collagen I, Procollagen III, Complement C3 precursor, and Myeloid related protein 14 or functional variants thereof are detected.
- the combination in detection the presence or a change in expression of the silicone-adhering polypeptide Immunoglobulin G, C-reactive protein, Fibronectin, Collagen I, Procollagen III, Complement C3 precursor, and Myeloid related protein 14 increases the sensitivity, specificity, positive and negative predictive value for detecting a response reaction of a mammal to a silicone.
- the sample from said mammal is serum and/or wound bed fluid.
- the sample is collected before or after implantation of a silicone that is suitable for long-term implantations and coating of medical devices.
- the method further comprises a second sample as a control, wherein said control is either obtained from the same mammal before or after implantation, or from a different mammal without any implantations of a medical silicone.
- the polypeptide or functional variant thereof are detected by one-or two-dimensional gel electrophoresis and/or ELISA.
- the method further comprises detecting the presence or amount of at least one nucleic acid having a nucleotide sequence encoding for a silicone-adhering polypeptide according to the present invention.
- said nucleic acid is detected by PCR, RT-PCR or gel electrophoresis.
- said method further comprises step a') between step a) and b) which comprises contacting said sample with a silicone under conditions that allow for adhering of said polypeptides to said silicone.
- the present inventions relates to a diagnostic kit for detecting a response reaction of a mammal to a silicone comprising materials, buffers, auxiliary agents, and substances for performing a method according to the present invention.
- diagnostic kit shall mean a reagent kit comprising a polypeptide, a nucleic acid molecule, a vector, a host cell, or an antibody of the present invention or as identified or characterized by a method of the present invention.
- the reagent kit further comprises reaction buffer(s), storage solutions and/or remaining reagents or materials required for the conduct of scientific or diagnostic assays or the like.
- parts of the kit of the invention can be packed individually in vials or bottles or in combination in container or multi-container units.
- the present inventions relates to a method for monitoring a response reaction of a mammal to a silicone, in particular medical silicone, comprising a) implanting at least one silicone into said mammal, and b) detecting the presence or a change in expression of a silicone-adhering polypeptide according to the present invention using a method or kit according to the present invention, and optionally, repeating step b).
- the present inventions concerns a method for screening substances that interfere with at least one silicone-adhering polypeptide, comprising the steps of a) providing a substance, wherein said substance is suspected to interfere with the adhering properties of a silicone-adhering polypeptide; b) providing a silicone, in particular a medical silicone; c) providing a silicone-adhering polypeptide; and d) analyzing the interfering properties of said substance through analyzing the adhesion of said silicone-adhering polypeptide to said silicone in the presence or absence of said substance, wherein a change in the adhesion of said silicone-adhering polypeptide to said silicone in the presence and absence of said substance is indicative for a substance that interferes with the binding of a silicone- adhering protein and/or polypeptide to a silicone.
- the silicone-adhering polypeptide is selected of a group consisting of a polypeptide according to the present invention, Myeloid related protein 8, Myeloid related protein 14, HTOl 8, PRO2619, a polypeptide identified according to the present invention, Isotype control, IgG-HRP, IgE- HRP, and IGA-HRP, AMBP protein precursor, Plasma-retinol binding protein, Kininogen, Eosinophil peroxidase, Fibroblast growth factor 11, TGF- ⁇ , Integrin- ⁇ 4, T-cell receptor, NADH dehydrogenase, Coagulation factor V, ⁇ -spectrin, Monocyte chemotactic protein-2, ⁇ - glutamyltransferase, Immunoglobulin G, Immunoglobulin A, Complement C3, Complement C2, Complement CIs, C reactive protein, Alpha 1 -microglobul
- the present invention provides a method for detecting adhesion of cells to a silicone, comprising a) providing a sample of serum or wound bed fluid of a mammal containing cells; b) providing a silicone; and c) analyzing the amount or number of cells from said serum or wound bed fluid that adhere to said silicone.
- the mammal is human.
- the silicone can be any medical silicone known by the person skilled in the art.
- the silicone is selected from the group comprising medical silicone types MED1511, MED4860, MED4211, and MED360 provided by the company Nusil (Waldbroon, Germany).
- the present invention relates to a substance that interferes with the binding of a silicone-adhering protein and/or polypeptide to a silicone, identified according to a method according to the present invention.
- the present invention provides a method for producing a pharmaceutical composition, comprising a method according to a method according to the present invention, and formulating said substance into a pharmaceutical composition.
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising a substance according to the present invention or that is produced according to the present invention.
- the present inventions provide a method of treating a mammal suffering from an adverse reaction to an implanted silicone, comprising administering to said mammal an effective amount of a substance interfering with the adhering properties of a silicone-adhering polypeptide as identified through a method according to the present invention.
- an effective amount of a substance interfering with a silicone-adhering polypeptide is administered in the form of a pharmaceutical composition, those compositions are well known by a person skilled in the art.
- the adverse reaction is a silicosis and/or silicone-related fibrosis.
- the present inventions relates to a method for monitoring the treatment of a mammal suffering from a response reaction to a silicone, comprising: a) administering to said mammal an effective amount of a substance interfering with the adhering properties of a silicone-adhering polypeptide as identified according to the present invention, b) obtaining a sample from said mammal; c) detecting the presence or a change in expression of at least one silicone-adhering polypeptide or a functional variant thereof.
- the sample is serum or wound bed fluid of a mammal, preferably of a human.
- the silicone-adhering polypeptide is selected of a group consisting of a polypeptide according to the present invention, Myeloid related protein 8, Myeloid related protein 14, HTOl 8, PRO2619, a polypeptide identified according to the present invention, Isotype control, IgG- HRP, IgE-HRP, and IGA-HRP, AMBP protein precursor, Plasma-retinol binding protein, Kininogen, Eosinophil peroxidase, Fibroblast growth factor 11, TGF- ⁇ , Integrin- ⁇ 4, T-cell receptor, NADH dehydrogenase, Coagulation factor V, ot-spectrin
- FIG. 1 Construction scheme of the ELISA system for protein adhesion to silicone.
- a 96- well plate was coated with silicone and sterilized as described. It was then incubated with serum samples (complemented with protease inhibitors) for 4 days and washed with PBS and water.
- Adhering proteins were detected using specific primary antibodies and appropriate secondary antibodies labeled with enzyme (e.g. horseradish peroxidase) converting uncolored substrate into a colored product that was measured at a specific wavelength. The amount of protein was directly proportional to the intensity of the developed dye.
- the assay can be performed in various other formats.
- FIG. 1 Proof of principle screen of patients with fibrotic complications upon implantation of SMIs. Following the ELISA principle as shown in Figure 1, 12 protein candidates in 10 patients (5 with and 5 without fibrotic complications), 7 controls (age and sex matched) and one patient with fluid silicone injection causing dramatic generalized fibrotic complications were tested.
- A Photo of a plate with protein candidates tested. Green coloration indicated high abundance of protein. The variations in protein deposition could generally be distinguished macroscopically. However, absorption measurements of each well were performed for exact quantification and statistical analysis of results.
- B Statistical analysis of different patient groups. After the absorption was measured, values were expressed as means ⁇ standard deviations for each group.
- the Kolmogorov-Smirnoff test was used to test the normality of the distribution, and t-test was used to calculate p-values. A significant positive correlation of the occurrence of a fibrotic complication with the deposition of fibronectin 1, C-reactive protein (CRP) and IgA, and significant negative correlation with the deposition of complement component C3 and IgG was found.
- CRP C-reactive protein
- Figure 3 Algorithm of ex vivo and in vitro protein adhesion analysis.
- ex vivo analysis saline-filled explanted SMIs were cut in pieces, washed, and adhering proteins eluted in a buffer appropriate for downstream analysis (either for Western blot or for 2D electrophoresis/MS).
- a buffer appropriate for downstream analysis either for Western blot or for 2D electrophoresis/MS.
- standardized pieces of silicone cut out of new, intact, sterile saline-filled SMIs were incubated for 4 days with WBF or serum. They were then washed in exactly the same way as ex vivo prepared SMIs, and proteins eluted in an appropriate buffer for downstream analysis (either for Western blot or for 2D electrophoresis/MS). For comparison, all protein amounts were normalized per unit of surface.
- FIG. 4 Adhesion of foreskin fibroblasts to silicone coated with serum proteins.
- Human primary fibroblasts were grown out from foreskin preparations. After their phenotype homogeneity had been determined (data not shown), they were cultured under standard conditions. 96-well plates were coated with silicone, while uncoated polystyrene plates were taken as control. Both were incubated with serum for 4 days and extensively washed with PBS and water. Fibroblasts were then seeded onto both silicone-coated and uncoated plastic wells and eluted at various time points between 15 and 300 minutes.
- FIG. 5 Retrospective case control study, based on a preferred embodiment of the method and kit of the present invention.
- Example 1 Construction of an in vitro system for screening of proteins adhering to various silicone types in vivo
- the inventors have analyzed proteins deposited on SMIs explanted from women suffering from complications after breast augmentation for cosmetic reasons. Thirteen explanted SMIs were obtained from the Clinic for Plastic and Reconstructive Surgery, Medical University Innsbruck (MUI, Innsbruck, Austria). Nine SMIs were gel and 4 saline filled. Eleven SMIs were rough and 2 smooth surfaced. Only intact implants, i.e. without silicone 'gel bleeding 1 , were considered for analysis. They were taken under sterile conditions immediately after surgery. The fibrotic capsule was removed in the surgical theatre, and SMIs were transported to the laboratory in phosphate buffered saline (PBS, pH 7.2) supplemented with protease inhibitors.
- PBS phosphate buffered saline
- silicone types MED151 1, MED4860, MED4211, MED360 do not contain or require organic solvents or high temperatures for polymerization, they were tested first. Their physical characteristics made them compatible with polystyrene flat bottom plates routinely used in laboratory practice that we employed in order to have controlled and standardized conditions. All silicone types tested were unrestricted types, i.e. they were certified from producer for long-term implantation by the silicone manufacturer (Nusil; Waldbronn, Germany). They also represented silicone types that are widely used in SMI manufacturing and coating of medical devices. Other silicone types are planned by the inventors to be tested in the future, and the design of the test will be adjusted according to their chemical composition.
- FIG. 3 shows the model for in vitro protein adsorption and analysis.
- the inventors compared proteins eluted from the two systems by one and two-dimensional gel electrophoresis. For differential analysis, the inventors needed a sensitive analytical tool and that is why the DIGETM (Amersham Biosciences, Upsala Sweden) system was employed. Proteins from the two systems were labelled with different fluorochromes that can be excited at specific wavelengths. This experimental setup allowed the inventors to obtain three 69-
- Protein spot patterns were analyzed with the 'Decyder' software (Amersham Biosciences). The amount of proteins deposited was considered different, if the spot volume ratio between samples exceeded two standard deviations of total spot distribution. More stringently, it was defined as present only in either the ex vivo or the in vitro system, if the volume ratio exceeded a threshold of fivefold. Using this approach, the inventors detected so far a total of 184 protein spots. Of these, 163 spots were present in the same amounts in both systems, 13 proteins bound stronger in the ex vivo and 5 in the in vitro system, and only 5 spots were unique for either of the systems. In other words, 89% of protein deposited in vivo are also deposited in our in vitro system. Using mass spectrometry, the
- Proteins eluted in vitro and ex vivo were primarily analyzed after separation by two- dimensional gel electrophoresis (2D electrophoresis) [Anderson, L. and Anderson, N. G. High resolution two-dimensional electrophoresis of human plasma proteins. Proc. Natl. Acad. Sci. U S A 74, 5421-5425 (1977)].
- 2D electrophoresis two- dimensional gel electrophoresis
- rehydration buffer 7M urea, 2M thiourea, 4% CHAPS, 18mM DTE
- the inventors then added resolyte buffer and loaded proteins on to 18 cm IPG strips pi 3-11. Proteins were focused for a total of 55000 Vhrs (IPGphor, Amersham Biosciences). After isoelectric focusing, proteins on strips were reduced (2% DTT, 15 min) and subsequently alkylated (2.5 % IAA, 15 min). Second dimension electrophoresis was performed under reducing conditions, in 20x24 cm gradient 9-16% polyacrylamide gels at 16 0 C.
- Pattern matching and mass spectrometry Silver stained protein spot patterns were matched and compared to the ExPasy database of 2D gels (http://us.expasy.org/ch2d/) using the 'Melanie' software (version 4, Swiss Institute of Bioinformatics, Basel, Switzerland) [Hughes, G. J., Frutiger, S., Paquet, N., Ravier, F., Pasquali, C, Sanchez, J. C, James, R., Tissot, J. D., Bjellqvist, B., and Hochstrasser, D. F. Plasma protein map: an update by microsequencing. Electrophoresis 13, 707-714 (1992)].
- the inventors performed Western blots of proteins eluted from silicone.
- the inventors used proteins eluted in SDS buffer (2%SDS, 10OmM DTT, 5OmM TrisCl, 10% glycerol, pH 9.0) that were loaded to small (7x10 cm) homogenous 12% polyacrylamide gels. After the run, proteins were blotted to on the nitrocellulose membrane for 1200 mAh in a wet blotting chamber, and blocked with 5% fat free milk.
- proteins as identified according to the invention can be classified as follows with respect to their biological functions:
- NADH dehydrogenase unknown protein 1 (SEQ ID NO. 1) unknown protein2 (SEQ ID NO. 2) unknown protein3 (SEQ ID NO. 3) unknown protein4 (SEQ ID NO. 4) 5. Proteins suitable to discriminate between normal and fibrotic tissue
- Proteins could be classified into different categories: anchor proteins, a primary protein set, and a secondary protein set. Only the first two categories have been reconfirmed by alternative techniques (Western Blot, ELISA).
- anchor proteins Proteins of equal, strong adhesion (anchor proteins) These proteins were defined as at least 'abundant', i.e. ++, and presented as equally adhesive towards all silicone types tested. Seven (7) candidates were included into this group. They are marked with the symbol "*"
- PRO2619 5. Ace. no.: gi
- test protocol is based on ELISA micro well plates. Production of this test system is easy to perform and stable for a long time at room temperature. This would facilitate shipping to end-users. 2) The test system uses sera. Moreover, an ELISA system is a standard procedure in many laboratories. This combination allows an easy integration into current clinical routine.
- Polystyrene 96 well flat bottom microtiter plates were coated with medical silicone types 1511, 4860 and 4211. Fifty ⁇ l of unpolymerized silicone was added per well of 96-well polystyrene plates (Greiner, Frickenhausen, Germany), and plates were immediately centrifuged for various times, depending on the silicone type (MED 151 1 : 10 min at 30Og; MED 4860: 60 min 100Og; MED 4211 : 10 min- 150g) in order to distribute silicone evenly on the bottom of the plate. After silicone curing (3 days at 37 0 C and 98% humidity), plates were sterilized for 120 seconds under 200 mJoules of UV light (GsGene UV chamber, Biorad, Hercules, CA).
- silicone coated 96 well plates were incubated with serum from patients before and after undergoing primary breast augmentation or with serum of patients having their SMIs exchanged. All sera are supplemented with 1 ⁇ g/ml of leupeptin, pepstatin and aprotinin (protease inhibitors, Sigma-Aldrich, Vienna, Austria), f ⁇ ltered through 0.22 ⁇ m filters and 100 ⁇ l is applied to each well under aseptic conditions. In some experiments, sera were diluted with PBS prior to application. After 50 hours, excess proteins were washed away with 4x 5 min washing with PBS, and further binding sites on the plates blocked with 2.5% BSA/0,l%tween 20 in PBS for 100 minutes at room temperature.
- the inventors managed to significantly reduce the background level.
- the inventors defined the cut-off for presence of a protein as 2 absorbance values of isotype controls. Background values were less than 1% of probe measurements, the linear range spanning over two orders of magnitude.
- the whole test system was highly reproducible and allowed for semi-quantitative analysis of the deposited proteins.
- a major advantage of this system is that simultaneous analysis of large number of patients and various proteins respectively deposited onto silicone is possible in a relatively short time.
- the inventors have noticed different pattern of protein deposition from various patients as a proof of principle.
- the inventors would like to extend the analysis and correlate the results with implant duration and local or systemic complications on a large cohort of patients.
- the inventors have adjusted the test system to a 384-well format, which allows for rapid screening and larger clinical studies.
- the amount of sera which often is a limiting factor for the analysis, can be reduced, saving costs as well as enhancing reproducibility due to the possibility of replication.
- the detection system with primary antibodies against specific proteins and appropriate HRP conjugated secondary antibodies is the same as above, but in lower volume (50 ⁇ l).
- BS Baker Score
Abstract
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CA002636353A CA2636353A1 (en) | 2006-01-18 | 2007-01-18 | Test systems for the analysis of polypeptides and cells adhering to silicones |
BRPI0706553-1A BRPI0706553A2 (en) | 2006-01-18 | 2007-01-18 | testing systems for the analysis of polypeptides and cells that adhere to silicones |
US12/160,996 US20110135606A1 (en) | 2006-01-18 | 2007-01-18 | Test systems for the analysis of polypeptides and cells adhering to silicones |
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US5834215A (en) * | 1994-10-05 | 1998-11-10 | The Administrators Of The Tulane Educational Fund | Method for detecting antipolymer antibodies and diagnosing silicone related disease (SRD) fibromyalgia and chronic fatigue syndrome (CFS) |
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JP2009524031A (en) | 2009-06-25 |
AU2007207089A1 (en) | 2007-07-26 |
WO2007082757A3 (en) | 2007-09-20 |
CA2636353A1 (en) | 2007-07-26 |
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