WO1998020124A2 - Proteines mediatrices du diabete et leurs utilisations therapeutiques - Google Patents

Proteines mediatrices du diabete et leurs utilisations therapeutiques Download PDF

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Publication number
WO1998020124A2
WO1998020124A2 PCT/IB1997/001627 IB9701627W WO9820124A2 WO 1998020124 A2 WO1998020124 A2 WO 1998020124A2 IB 9701627 W IB9701627 W IB 9701627W WO 9820124 A2 WO9820124 A2 WO 9820124A2
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WIPO (PCT)
Prior art keywords
diabetes
protein
expression
mediating
proteins
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PCT/IB1997/001627
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English (en)
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WO1998020124A3 (fr
Inventor
Peter Mose Larsen
Stephen J. Fey
Jørn NERUP
Allan E. Karlsen
Ulla Bjerre Christensen
Flemming Pociot
Henrik U. Andersen
Original Assignee
Peter Mose Larsen
Fey Stephen J
Nerup Joern
Karlsen Allan E
Ulla Bjerre Christensen
Flemming Pociot
Andersen Henrik U
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Application filed by Peter Mose Larsen, Fey Stephen J, Nerup Joern, Karlsen Allan E, Ulla Bjerre Christensen, Flemming Pociot, Andersen Henrik U filed Critical Peter Mose Larsen
Priority to CA002269646A priority Critical patent/CA2269646A1/fr
Priority to US09/297,040 priority patent/US7078375B1/en
Priority to EP97947839A priority patent/EP0934409A2/fr
Priority to JP52118298A priority patent/JP2002504806A/ja
Priority to AU54070/98A priority patent/AU5407098A/en
Publication of WO1998020124A2 publication Critical patent/WO1998020124A2/fr
Publication of WO1998020124A3 publication Critical patent/WO1998020124A3/fr
Priority to US11/488,184 priority patent/US7531323B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/0002—Inspection of images, e.g. flaw detection
    • G06T7/0012—Biomedical image inspection
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0325—Animal model for autoimmune diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2550/00—Electrophoretic profiling, e.g. for proteome analysis

Definitions

  • This invention relates generally to diabetes-mediating proteins, methods of identifying diabetes-mediating proteins, transgenic animals useful in the assays of the invention, methods for screening for drugs which affect the expression of diabetes- mediating proteins, and therapeutic compounds for the treatment and prevention of diabetes.
  • IDM insulin-dependent diabetes mellitus
  • insulitis pancreatic islets
  • BB-DP diabetes-prone BB
  • NOD non-obese diabetic mice
  • 2-Dimensional (2D) gel maps of rat islet proteins have been constructed and used to determine qualitative and quantitative changes in protein synthesis resulting with in vitro exposure of rat islet cells to IL-l ⁇ (Andersen et al.
  • Transgenic animal models of human diseases are known.
  • one model for human diabetes is a transgenic mouse expressing a viral protein in the pancreatic ⁇ cells under control ofthe rat insulin promoter (van Herrath et ⁇ /. J Clin. Invest. 98:1324-1331 (1996)).
  • the transgenic mice develop diabetes spontaneously; however, after a 2 month challenge with the virus, IDDM occurs in more than 95% ofthe mice.
  • the invention is based, in part, on the discovery and identification of diabetes- mediating (DM) proteins.
  • DM proteins are proteins which are involved in the development of diabetes or in the prevention of diabetes development in a subject at risk for the development of diabetes, and are identified by differential expression during the presence and absence of disease development.
  • the development of diabetes includes all stages which precede the clinically detectable stage.
  • the invention features substantially purified diabetes- mediating proteins exhibiting an altered expression during development of diabetes relative to expression in the absence of diabetes development.
  • the purified diabetes-mediating proteins of the invention are selected from the proteins listed in Tables 1 and 2.
  • Novel diabetes-mediating proteins are provided characterized by molecular weight, pi, and the mass spectroscopic characteristics as shown in FIGs. 6-40. These proteins, referred to by their position on 10% IEF or NEPHGE 2-dimensional gels (FIGs. 1A-1B), are selected from the group consisting of NEPHGE 7, 9, 102, 123, 129,130, 174, 181, 182, 211, 231, 236,
  • DM proteins are further characterized as protective or deleterious DM proteins.
  • a protective diabetes-mediating protein (“protective protein”) is characterized as a protein capable of protecting against the development of diabetes and/or delaying the onset of diabetes in a subject at risk for development of diabetes, or ameliorating the symptoms of diabetes in a subject suffering from diabetes.
  • a protective protein may also be a protein which does not alter expression during development of diabetes, but exhibits an altered expression in a subject at risk for diabetes who escapes the development of diabetes.
  • a deleterious diabetes-mediating protein (“deleterious protein”) is characterized as a protein capable of enhancing the development of diabetes, increasing the risk of a subject developing diabetes, or reducing the time required for development of diabetes in a subject at risk for development of diabetes.
  • a deleterious protein may also be a protein which does not alter expression during development of diabetes, but exhibits an altered expression in a subject at risk for diabetes who escapes the development of diabetes.
  • the diabetes-mediating protein ofthe invention may be identified by any means known to the art, including gel electrophoresis, immunoblotting, mass spectrometry, or chromatography, and is characterized by an altered protein expression during development of diabetes as compared to the same protein expressed in the absence of diabetes development.
  • U.S. provisional patent application Serial No. 60/029,324 identifies proteins expressed in pancreatic islet cells identified by molecular weight and pi (FIGs. 1 A and IB). The instant application provides a selection of these proteins which have been identified as diabetes- mediating proteins, as listed in Tables 1 and 2, and FIGs. 6-40.
  • the diabetes-mediating proteins ofthe invention are useful in drug screening assays for identifying compounds capable of modulating the development of diabetes, useful as therapeutic agents for the treatment or prevention of diabetes, and useful as targets of therapeutic agents capable of preventing or ameliorating diabetes by modulating the expression ofthe diabetesmediating protein.
  • the invention features a method for diagnosing the development of diabetes by measuring an increase in protein expression in one or more proteins selected from the group consisting of the diabetes-mediating proteins listed in Table 1, and a decrease in the protein expression of one or more proteins selected from the list consisting ofthe diabetes-mediating proteins listed in Table 2.
  • Changes in protein expression are measured in a test subject suspected of developing diabetes or at risk for the development of diabetes and are expressed relative to protein expression in a normal non-diabetes control.
  • changes of combinations of one or more of the proteins of Tables 1 and 2 is indicative of the development of diabetes.
  • changes of a combination of 5 or more ofthe proteins of Tables 1 and/or 2 is indicative ofthe development of diabetes.
  • changes of a combination of 10 or more of the proteins of Tables 1 and/or 2 is indicative ofthe development of diabetes.
  • the invention features an in vivo assay method for identifying proteins which are involved in the development of disease, e.g., diabetes.
  • cells which secret insulin or are capable of developing into insulinproducing cells are transplanted to an immunologically compatible host animal which is an animal at risk for the development of diabetes.
  • Protein expression is analyzed in transplanted cells rescued at time points between the time of transplantation and disease onset, and proteins exhibiting an altered expression during disease development relative to their expression in the absence of the development of diabetes are identified.
  • the transplanted cells are neonatal islet cells which are transplanted into a animal model at risk for development of diabetes.
  • the neonatal islet cells are taken from neonatal BB-DP rats and the host animal is BB-DP rat.
  • the source of transplanted cells and host animals are NOD mice.
  • the invention provides identified diabetes-mediating proteins which may be further characterized as protective or deleterious proteins.
  • a candidate protective or deleterious protein is identified in vitro by transfecting cultured cells with a polynucleotide encoding the candidate protective or deleterious protein, and the effect of expression ofthe diabetes-mediating protein on in vitro cell functionality upon challenge with IL-l ⁇ determined.
  • the polynucleotide may be operably connected to an inducible promoter such that expression ofthe candidate protective or deleterious protein is under exogenous control. Exogenous control may be exerted by agents, e.g., interferon, such agents being determined by the promoter selected.
  • cell functionality is determined by measurement of nitric oxide (NO) production, insulin secretion, cell survival, and/or cytotoxicity upon exposure to IL-l ⁇ .
  • NO nitric oxide
  • a transgenic mammal is generated expressing the candidate protein, wherein the transgenic mammal is at risk for developing diabetes, and the effect of transgene expression on the development and timing of diabetes onset is determined.
  • a protective protein is one which prevents, inhibits, or slows the development of diabetes in a subject at risk for diabetes
  • a deleterious protein is one that causes the development of diabetes, increases the risk of development of diabetes, or decreases the time required for the development of diabetes in a subject at risk for developing diabetes.
  • a deleterious protein is also a protein that prevents or interferes with the expression of a protective protein.
  • the invention includes a substantially purified protective or deleterious diabetes- mediating protein, and polynucleotide sequence which encodes the diabetes-mediating protein of the invention.
  • the protective protein is galactin-3 (FIGs. 4 and 5) (SEQ ID NOs:l-2).
  • the deleterious protein is mortalin (FIGs. 2-3) (SEQ ID NOs:3-4).
  • the invention features a transgenic mammal having an exogenous diabetesmediating protein gene or genes inserted into its genome.
  • the transgenic mammal ofthe invention is useful in assay methods for determining the effect ofthe expression of a diabetes-mediating protein in the development of diabetes, and for identifying protective or deleterious proteins.
  • the transgene may be a natural, or partially or wholly artificial diabetes-mediating gene, and may be different from or the same as an endogenous diabetes-mediating protein gene.
  • the transgene is under control of an inducible promoter.
  • the invention features a transgenic mammal having an exogenous deleterious gene, and exhibiting an increased incidence of the spontaneous development of diabetes within a predictable period of time.
  • the transgenic mammal exhibits a greater than 50% chance, more preferably a greater than 60% chance, even more preferably a greater than 70% chance, even more preferably a greater than 80% chance, and most preferably a greater than 90% chance of developing diabetes.
  • the transgenic mammal ofthe invention is transgenic for one or more genes encoding a deleterious diabetes-mediating protein.
  • the transgenic mammal additionally has one or more endogenous diabetes-mediating protein genes ablated.
  • the transgenic mammal will have the transgenic gene under control of an insulin, CMV, interferon, or MHC promoter.
  • a transgenic mammal expresses elevated levels of an endogenous diabetes-mediating gene obtained by an enhanced promoter or a high copy number of an endogenous diabetes-mediating gene.
  • the transgenic mammal has a disrupted diabetes-mediating protein gene.
  • the invention further includes mammals in which an endogenous diabetes- mediating protein gene is exogenously altered by methods known in the art, for example, by application of gene activation technologies such as that described in U.S. Patent No. 5,641,670, entirely incorporated herein by reference. -0-
  • the invention features an assay for screening compounds which effect the expression of one or more diabetes-mediating proteins.
  • animals at risk for spontaneous development of diabetes are used in an assay for determining the ability of a test compound to effect the expression of one or more diabetes-mediating protein(s).
  • the assay animal is the transgenic mammal of the invention having a high risk of the development of diabetes.
  • effect the expression of a diabetes-mediating protein is meant a compound which induces, enhances, inhibits, or decreases the expression of an endogenous diabetes-mediating protein.
  • the invention provides an assay for identifying a compound capable of inducing or enhancing the expression of an endogenous protective protein, and thus to delay or inhibit the development of diabetes.
  • the assay method of the invention is useful for identifying a compound capable of suppressing or inhibiting the expression of a deleterious diabetes-mediating protein, thus delaying or inhibiting the development of diabetes.
  • the invention provides an assay for identifying a compound which modulates the activity of a diabetes-mediating protein, e.g., an agonist, an antagonist, or by blocking a post-translational step required for activation of a diabetes- mediating protein.
  • Changes in the expression of specific DM proteins are useful in a screening method for identifying compounds capable of modulate the expression of DM proteins.
  • a compound which modulates the expression of one or more diabetes mediating proteins is useful as a potential therapeutic in the treatment or prevention of diabetes.
  • the invention features an assay method for identifying compounds capable of modulating the expression of diabetes-mediating proteins having the steps of contacting a test compound with a cell or tissue expressing one or more diabetes-mediating proteins, and determining the effect of the test compound on the expression of one or more diabetes-mediating proteins.
  • Determination ofthe effect of a compound may be conducted by a variety of methods known to the art, including hybridization to probes or other oligonucleotides, antibody recognition, e.g., immunodiffusion, immunofluorescence, ELISA, RIA, blotting, immunoprecipitation, immunoelectrophoresis, or chromatography, and electrophoresis.
  • a compound capable of increasing the expression of one or more proteins selected from the group consisting ofthe diabetes-mediating proteins listed in Tables 1 and 2 and decreasing the expression of one or more proteins selected from the list consisting ofthe diabetesmediating proteins listed in Tables 1 and 2 is a candidate therapeutic agent for the prevention or treatment of diabetes. Changes in protein expression are determined relative to expression in the absence ofthe test compound.
  • the invention provides a therapeutic method for preventing diabetes in a subject at risk for diabetes or of ameliorating the symptoms of diabetes in a diabetic subject by administering a therapeutically effective amount of a protective diabetes-mediating protein.
  • the subject is a human.
  • gene therapy by providing a polynucleotide encoding a protective diabetes- mediating protein.
  • the invention further includes a therapeutic method for preventing and/or treating diabetes by administering an effective amount of a polynucleotide which inhibits the in vivo expression of a deleterious diabetes-mediating protein.
  • Candidate therapeutic compounds are selected from the proteins of Tables 1 and 2.
  • the invention provides a therapeutic method of preventing and/or treating diabetes in a subject at risk for diabetes by administering a therapeutically effective amount of a compound capable of suppressing or reducing the expression of an endogenous deleterious diabetes-mediating protein.
  • the invention provides a therapeutic method of preventing and/or treating diabetes by administering a therapeutically effective amount of a compound capable of inducing or enhancing the expression of an endogenous protective diabetes-mediating protein.
  • the invention provides a therapeutic method of preventing and/or treating diabetes in a subject at risk for diabetes by administering a therapeutically effective amount of a compound capable of modulating the activity of a diabetes-mediating protein, e.g., as an agonist, an antagonist, or by preventing the activation of a diabetes-mediating protein.
  • the therapeutic method of the invention includes ex vivo methods known to the art for providing the therapeutic agent to a subject in need thereof.
  • An object ofthe invention is to identify proteins which mediate diabetes onset.
  • An object of the invention is to provide an in vivo assay for identification of diabetesmediating proteins.
  • Another object ofthe invention is to provide diabetes-mediating proteins which are useful in assays for identifying test compounds capable of preventing, delaying, or ameliorating diabetes in a subject.
  • Another object ofthe invention is to provide transgenic animals useful in assays to identify protective or deleterious diabetes-mediating proteins.
  • Another object ofthe invention is to provide transgenic animals useful in assay to identify test compounds capable of affecting the expression of a diabetes-mediated protein.
  • Another object ofthe invention is to provide a transgenic host mammal (which is small, e.g., less than 1 kg when full grown, and inexpensive to maintain) such as a mouse, rat or hamster which includes a natural, or partially or wholly artificial diabetes-mediating gene.
  • transgenic mammal can be used to identify a protective protein which can prevent or inhibit disease development in a manner which is substantially faster, more efficient and cheaper than presently available assay methods.
  • transgenic mammal can be used as test animals for testing drugs for efficacy in the treatment of humans suffering from diabetes or at risk for the development of diabetes.
  • Another object of the invention is to provide an assay for identification of test compounds which effect the expression of a diabetes-mediating protein and which are capable of preventing the onset of diabetes in a subject at risk for development of the disease, or for ameliorating the symptoms of diabetes in a diabetic subject.
  • FIGs. 1 A and IB show a fluorograph of a 2-dimensional gel of proteins expressed in neonatal rat islet cells incubated for 24 h in RPMI 1640 + 0.5% normal human serum, followed by a 4 h labeling with [ 35 S]-methionine.
  • FIG. 1 A is the isoelectric focusing gel
  • FIG. IB is the non-equilibrium pH-gradient electrophoresis gel (NEPHGE; pH 6.5-10.5). Arrows mark 105 diabetes-mediating proteins.
  • FIG. 2 is the amino acid sequence of murine mortalin.
  • FIG. 3 is the amino acid sequence of human mortalin.
  • FIG. 4 is the amino acid sequence of rat galectin.
  • FIG. 5 is the amino acid sequence of human galectin.
  • FIG. 6 is the mass spectroscopy spectrum for diabetes-mediating protein GR75 (mortalin), IEF Spot No. 340, determined using the parameters indicated on the figure legend.
  • FIG.7 is the mass spectroscopy spectrum for diabetes-mediating protein, tentatively identified as lamin a, IEF Spot No. 655, determined as indicated.
  • FIGs. 8-10 are the mass spectroscopy spectrum for a diabetes-mediating, tentatively identified as pyruvate kinase, NEPHGE Spot No. 1, determined as indicated.
  • FIG. 11 is the mass spectroscopy spectrum for a diabetes-mediating, tentatively identified as 6-phosphobructose-2-kinase, NEPHGE Spot No. 169, determined as indicated.
  • FIG. 12 is the mass spectroscopy spectrum for a diabetes-mediating, tentatively identified as triose phosphate isomerase, NEPHGE Spot No. 334, determined as indicated.
  • FIG. 13 is the mass spectroscopy spectrum for a diabetes-mediating, tentatively identified as fructose-biphosphase aldolase, NEPHGE Spot No. 668, determined as indicated. -o-
  • FIGs. 14-15 are the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "IEF Spot No. 665,” having a molecular weight of 42,243 daltons and a pi of 5.82, determined as indicated.
  • FIG. 16 is the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed, "IEF Spot No. 939,” having a molecular weight of 25,851 daltons and a pi of 5.09, determined as indicated.
  • FIGs. 17-18 are the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed “IEF Spot No. 941” having a molecular weight of 22,704 daltons and a pi of 5.15, determined as indicated.
  • FIG. 19 is the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "IEF Spot No. 950,” having a molecular weight of 25,753 daltons and a pi of 4.53, determined as indicated.
  • FIGs. 20-23 are the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "IEF Spot No. 1196" having a molecular weight of 143,064 daltons and a pi of 5.41 , determined as indicated.
  • FIG. 24 is the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE Spot No. 7," having a molecular weight of 65,522 daltons and a pi of 7.28, determined as indicated.
  • FIG. 25 is the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE Spot No. 9," having a molecular weight of 115,709 daltons and a pi of
  • FIG. 26 is the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE Spot No. 102,” having a molecular weight of 63,560 daltons and a pi of 7.26, determined as indicated.
  • FIG. 27 is the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE Spot No. 123,” having a molecular weight of 57,040 daltons and a pi of 8.17, determined as indicated.
  • FIG. 28 is the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE Spot No. 129,” having a molecular weight of 57,609 daltons and a pi of 7.72, determined as indicated.
  • FIG. 29 is the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE Spot No. 130,” having a molecular weight of 55,734 daltons and a pi of 8.07, determined as indicated.
  • FIGs. 30-31 are the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE Spot No. 174,” having a molecular weight of 53,830 daltons and a pi of 7.92, determined as indicated.
  • FIGs. 32-33 are the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE Spot No.181,” having a molecular weight of 49,422 daltons and a pi of 7.40, determined as indicated.
  • FIGs. 34-35 are the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE Spot No.182,” having a molecular weight of 54,098 daltons and a pi of 7.61, determined as indicated.
  • FIG. 36 is the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE Spot No.211,” having a molecular weight of 47,925 daltons and a pi of
  • FIG. 37 is the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE Spot No.231 ,” having a molecular weight of 44,362 daltons and a pi of 8.34, determined as indicated.
  • FIG. 38 is the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE Spot No.236,” having a molecular weight of 43,162 daltons and a pi of 7.90, determined as indicated.
  • FIGs. 39-40 are the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE SpotNo.253,” having a molecular weight of 39,106 daltons and a pi of 9.05, determined as indicated.
  • FIG. 41 is a mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "IEF Spot No. 831 ,” having a molecular weight of 34,600 daltons and a pi of 4.76, determined as indicated.
  • FIGs. 42-43 are the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "IEF Spot No.949,” having a molecular weight of 26,800 daltons and a pi of 4.49, determined as indicated.
  • FIGs. 44-46 are the mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE Spot No.129,” having a molecular weight of 57,600 daltons and a pi of 7.72, determined as indicated.
  • FIG. 47 is a mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE Spot No. 310,” having a molecular weight of 35,800 daltons and a pi of 7.57, determined as indicated.
  • FIG. 48 is a mass spectroscopy spectrum for a novel diabetes-mediating protein, termed "NEPHGE Spot No. 326," having a molecular weight of 34,500 daltons and a pi of 8.62, determined as indicated.
  • references to “diabetes-mediating protein” or “a diabetes-mediating protein” include mixtures of such diabetes-mediating proteins
  • reference to “the formulation” or “the method” includes one or more formulations, methods, and/or steps ofthe type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
  • protein includes proteins, polypeptides, and peptides which are chains of amino acids, including all post-translational modifications (e.g., processing and truncations, glycosylations or phosphorylations) which often play decisive roles in modulating protein function.
  • the term also encompasses natural proteins as well as synthetic or recombinant proteins, polypeptides, and peptides.
  • diabetes includes insulin-dependent diabetes melitis (IDDM) and type
  • diabetes-related diseases includes such conditions as obesity, circulatory deficiencies, insulin-resistance, syndrome X, diabetic retinopathy, diabetic neuropathy, and the involvement of advanced glycation end products (AGE) in neuropathy and atherosclerosis.
  • AGE advanced glycation end products
  • diabetes-mediating protein means a protein which is involved in the development of diabetes.
  • a diabetes-mediating protein is a protein which exhibits an altered expression during the development of diabetes, that is, a protein which is up- or down-regulated, or whose expression is modulated up or down, during the development of diabetes, as compared to the expression of the same protein in the absence of the development of diabetes.
  • a diabetes-mediating protein also means a protein that is modified as associated with the development of diabetes or diabetes-related diseases.
  • interleukin l ⁇ IL-l ⁇
  • IL-l ⁇ interleukin l ⁇
  • Tthe treatment of islets causes the modulation of 106 proteins, up or down regulating their expression.
  • the term "diabetes mediating protein” is defined to also include all the proteins (and all of their modification products) which have been demonstrated to be modulated by IL- ⁇ in rat islets in vitro, as further described in U.S. provisional application Nos. 60/029,324 (filed October 25, 1996), 60/030,186 (filed November 5, 1996) and 60/030,088 (filed November 5, 1996), the entire contents of which are incorporated herein by reference.
  • protein modification includes any change in structure (i. e. , a qualititive change) of a protein.
  • Such modifications can include, but are not limited to, changes in the amino acid sequence, transcriptional or translational splice variation, pre- or post- translational modifications to the DNA or RNA sequence, addition of macromolecules or small molecules to the DNA, RNA or protein, such as peptides, ions, vitamins, atoms, sugar-containing molecules, lipid-containing molecules, small molecules and the like, as well-known in the art.
  • One type of protein modification according to the present invention is by one or more changes in the amino acid sequence (substitution, deltion or insertion). Such changes could include, at one or more amino acids, a change from a charged amino acid to a different charged amino acid, a non-charged to a charged amino acid, a charged amino acid to a non-charged amino acid (e.g., giving rise to difference in pi or possibly molecular weight). Any other change in amino acid sequence is also included in the invention.
  • the overall positional change in a gel of a modified protein with a changed amino acid sequence also depends on how many overall charges there are in the protein, as known in the art.
  • Changes in the resolution of the gel can allow detection of minor or major amino acid sequence changes.
  • the type of analysis can also affect how changes are detected, e.g., using sequencing, mass spectrometry, labeled antibody binding,
  • Another type of modification is by change in length, conformation or orientation in the protein-encoding DNA or RNA that affects the way the open reading frame is read in the cell, which can give large changes in position ofthe spot on the gel and which could affect the analysis ofthe protein type and position in the gel.
  • Another typd of protein modification is by changes in processing ofthe protein in the cell.
  • a non-limiting example is where some proteins have an "address label" specififying where in (or outside of) the cell they should be used.
  • a label or tag can be in the form of a peptide, a sugar or a lipid, which when added or removed from the protein, determines where the protein is located in the cell.
  • a further type of protein modification is due to the attachment of other macromolecules to a protein.
  • This group can include, but is not limited to, any addition/removal of such a macromolecule.
  • These molecules can be of many types and can be either permanent or temporary. Examples include: (i) polyribosylation, (ii) DNA/RNA (single or double stranded); (iii) lipids and phosphlipids (e.g., for membrane attachment); (iv) saccharides/polysaccharides; and (v) glycosylation (addition of a multitude of different types of sugar and sialic acids — in a variety of single and branched structures so that the number of variations possible is large).
  • Another type of protein modification is due to the attachment of other small molecules to proteins. Examples can include, but are not limited to: (i) phosphorylation; (ii) acetylation; (iii) uridylation; (iv) adenylation; (v) methylation, and (vi) capping (diverse complex modification ofthe N-terminus ofthe protein for assorted reasons). Most of these changes are often used to regulate a protein's activity, (v) and (vi) are also used to change the half-life of the protein itself. These protein changes can be detected by 2D using several methods, such as labeling, changes in pi, antibodies or other specific techniques directed to the molecules themselves, as known in the art. Molecular weight changes can be, but may not usually be detectable by 2DGE. MALD (matrix assisted laser desorption of flight mass spectrometry) is preferred to detect and characterize these modifications.
  • MALD matrix assisted laser desorption of flight mass spectrometry
  • expression is meant to include not only the physical expression of a protein, but also as a measure of the activity of an expressed protein.
  • a protein can be expressed as an inactive form, which is activated by phosphorylation. While the actual expression ofthe protein has not changed, its effective expression (activity) has been modified. On a gel, the change in activity may be measured as the change in expression of a modified form ofthe protein.
  • affected protein means a protein that is modified in expression or modified structurally.
  • An affected protein can thus be a protein in which expression is modified due to treatment with one or more compounds, a diseased or pathological state and/or an immunological change in or outside the cell from which the protein is derived.
  • An affected protein can alternatively or additionally also be a protein which exhibits an altered expression as up- or down-regulated, or whose expression is modified in structure in any way that can be detected by a method ofthe present invention, as compared to the the expression ofthe same protein (i.e., an "unaffected protein" in the absence of such treatment, disease or immunological change.
  • diabetes-mediating gene or polynucleotide means genetic material encoding a protein, peptide, or protein fragment which encodes an intact or fragment of a diabetes-mediating protein.
  • the term includes any gene from any species which encodes a diabetes-mediating protein.
  • a diabetes-mediating gene or polynucleotide may be naturally occurring or partially or wholly synthetic.
  • substantially pure when referring to a polypeptide, means a polypeptide that is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • a substantially pure diabetes- mediating protein is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, diabetes-mediating protein.
  • a substantially pure diabetes-mediating protein can be obtained, by extraction from a natural source; by expression of a recombinant nucleic acid encoding a diabetes-mediating protein, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • polynucleotide refers to a nucleic acid sequence of deoxyribonucleotides or ribonucleotides in the form of a separate fragment or a component of a larger construct.
  • DNA encoding portions or all ofthe polypeptides ofthe invention can be assembled from cDNA fragments or from oligonucleotides that provide a synthetic gene which can be expressed in a recombinant transcriptional unit.
  • Polynucleotide sequences of the invention include DNA, RNA, and cDNA sequences, and can be derived from natural sources or synthetic sequences synthesized by methods known to the art.
  • an "isolated" polynucleotide is a polynucleotide that is not immediately contiguous (i.e., covalently linked) with either ofthe coding sequences with which it is immediately contiguous (i.e., one at the 5' end and one at the 3' end) in the naturally-occurring genome ofthe organism from which the polynucleotide is derived.
  • the term therefore includes, for example, a recombinant polynucleotide which is inco ⁇ orated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequences.
  • the isolated and purified polynucleotide sequences ofthe invention also include polynucleotide sequences that hybridize under stringent conditions to the polynucleotide sequences specified herein.
  • stringent conditions means hybridization conditions that guarantee specificity between hybridizing polynucleotide sequences.
  • posthybridization washing conditions including temperature and salt concentrations, which reduce the number of nonspecific hybridizations such that only highly complementary sequences are identified (Sambrook et al. in Molecular
  • such conditions are hybridization under specified conditions, e.g. involving presoaking in 5xSSC and prehybridizing for lh at about 40°C in a solution of 20% formamide, 5xDenhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 ⁇ g of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 100 ⁇ M ATP for 18 h at about 40°C (Sambrook et al. supra (1989)).
  • the isolated and purified polynucleotide sequences ofthe invention also include sequences complementary to the polynucleotide encoding a diabetesmediating protein (antisense sequences) and ribozymes.
  • the terms "host animal” and “host mammal” are used to describe animals into which donor cells are transplanted. Convenient host animals include mice, hamsters and rats.
  • ablated diabetes-mediating protein gene means an endogenous diabetes-mediating protein gene which has been altered (e.g., add and/or remove nucleotides) in a manner so as to render the gene inoperative. It is also used to include ablation or modification of controlling sequences or regulatory genes which also render the gene inoperative (partially or completely).
  • altered risk of developing diabetes and the like mean animals which are genetically predisposed to develop diabetes, preferably having a greater than 50% chance, more preferably a greater than 60% chance, even more preferably a greater than 70% chance, even more preferably a greater than 80% chance, and most preferably a greater than 90% chance to develop diabetes.
  • altered protein or “altered protein expression” is meant proteins whose expression is increased (“up regulated”), decreased (“down regulated”), inhibited (i.e., turned off), or induced (i.e., turned on) during the development of diabetes.
  • modulating the activity is meant altering the activity of a protein to prevent or enhance its normal activity, e.g., as an agonist, antagonist, or by blocking a post-translational modification step required for protein activity.
  • an effective amount or “therapeutically effective amount” is meant an amount of a compound sufficient to obtain the desired physiological effect, e.g., suppression of or delay ofthe development of diabetes.
  • treatment means obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may by prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it;
  • the invention is directed to treating patients with or at risk for development of diabetes and related conditions mediated diabetes, insulin insufficiency, or insulin resistance. More specifically, “treatment " is intended to mean providing a therapeutically detectable and beneficial effect on a patient at risk for or suffering from diabetes.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down the development of a disease.
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in which the disorder is to be prevented.
  • synergistic “synergistic effect” and like are used herein to describe improved treatment effects obtained by combining one or more therapeutic agents. Although a synergistic effect in some field is meant an effect which is more than additive - 10-
  • IDDM insulin dependent diabetes mellitus
  • the present invention encompasses several aspects including: (1) diabetes- mediating proteins identified by differential expression in the presence and absence ofthe development of diabetes; (2) patterns and combinations of DM proteins useful for predicting the development of diabetes and for identifying a compound able to effect a combination of DM proteins in a desired manner; (3) protective diabetes-mediating proteins; (4) deleterious diabetes-mediating proteins; (5) a method to diagnose for the onset or development of diabetes based on the detection of one or more ofthe DM proteins, their post-translational modification or degradation products in a body fluid; (6) an in vivo method for identifying a diabetes-mediating protein; (7) a transgenic mammal containing an exogenous gene encoding a diabetes-mediating protein; (8) a transgenic mammal useful in an in vivo assay for identifying protective or deleterious diabetes-mediating proteins; (9) an in vitro assay using transduced cultured cells expressing a diabetes-mediating protein useful for identifying protective or deleterious diabetes-mediating proteins; (
  • diabetes-mediating proteins that is, proteins identified as involved in or effected during the development of diabetes.
  • Diabetes-mediating proteins are characterized as proteins whose expression is altered during the development of diabetes relative to their expression in the absence ofthe development of diabetes.
  • the present disclosure identifies diabetes-mediating proteins from a 2-dimensional gel database of pancreatic islet cell proteins. Diabetes-mediating proteins include protective diabetes- mediating proteins and deleterious diabetes-mediating proteins.
  • the invention provides in vitro methods for identifying diabetes-mediating proteins, including by functionally assessment by expression of cloned cDNA as sense or antisense constructs in transfected cells to establish their deleterious or protective role in cytokine-mediated cytotoxicity.
  • B cells of the islets of Langerhans are specifically sensitive to the toxic effect of cytokines. It has previously been demonstrated that lipofection of rat ⁇ cells with heat shock protein 70 (HSP70) and induction of hemeoxygenase (HO) by exposure to hemin improved in vitro survival of cells exposed to IL-1 (Karlsen et al. in: Insulin Secretion and Pancreatic B Cell Research, ed: P.R. Flatt and S.
  • HSP70 heat shock protein 70
  • HO hemeoxygenase
  • Cells may be transfected in a number of ways known to the art, for example, the adenoviral vector method. See, for example, Korbutt et /. Transplantation Proceedings 27:3414 (1995); Csete etal. 26:756- 757 (1994); Becker et al. J. Biol. Chem. 269:21234-21238 (1994).
  • the effect of the expression of a diabetes-mediating protein may be determined by functional analysis ofthe clones cultured in the absence and presence of cytokines.
  • the functional analyses include nitrous oxide production (NO) measured as nitrite (Green et al. Anal. Biochem. 126:131- 138 (1982)), insulin secretion (Id.), cytotoxicity, and 2D-gel electrophoresis.
  • NO nitrous oxide production
  • Cytotoxicity may be measured by a variety of methods known to the art, including (1) a colorimetric assay based on lactate dehydrogenase (LDH) release (CytoTox, Promega), (2) a life-death assay based on calcein uptake and fluorescence of living cells and ethidium bromide staining of the nuclei of dead cells (Molecular Probes), (3) non-radioactive cell proliferation assay (MTT, Promega), apoptosis (Nerup et al. in: IDDM, S. Baba & T. Kaneko, eds., Elsevier Science, pp. 15-21 (1994)), and/or semiquantitative multiplex PCR analysis of gene expression.
  • LDH lactate dehydrogenase
  • MTT non-radioactive cell proliferation assay
  • 2-dimensional gels can be used to compare control and cytokine stimulated islets to identify which proteins respond, identifying the proteins which play a role in the cell response.
  • Interlink analysis can be used to define functional groups of proteins and their regulation (e.g., by kinase phosphorylation or other post-translational modifications).
  • Preferred cells for use in the in such an assay are insulin-secreting cells, for example, MSL or RIN cells.
  • the MSL cell line is a pluripotent or stem-cell like metastatic rat insulinoma cell line.
  • the MSL cell can acquire all four hormone secretory phenotypes characteristic ofthe islets of Langerhans (Mandrup-Poulsen et al. Eur. J Endocrinology 133:660-671 (1995);
  • RIN cells are a cultured line of insulinoma cells (Nielsen E p. Clin. Endocrinol. 93:277-285 (1989)).
  • the invention provides substantially purified protective diabetes-mediating proteins ("protective proteins") characterized as capable of protecting against development of diabetes in a subject at risk for the development of the disease or ameliorating or reducing the symptoms of diabetes in a subject suffering from diabetes.
  • the protective protein ofthe invention may act directly to protect against diabetes, or may act indirectly by inducing or increasing the synthesis of a second protective protein or by reducing or inhibiting the synthesis of a deleterious protein.
  • the invention provides the substantially pure protective protein galectin-3.
  • the sequence of rat galectin (S ⁇ Q ID NO:3) is shown in FIG. 4, and for human galectin (S ⁇ Q ID NO:4) in FIG. 5.
  • gal-3 expressed in transfected cells increased cell survival upon challenge with IL-l ⁇ .
  • Galectins are lectins with specificity for ⁇ -galactoside sugars or glycoconjugates which are present in fetal and adult pancreatic islet cells.
  • substantially pure refers to gal-3 which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • One skilled in the art can purify gal-3 using standard techniques for protein purification.
  • the purity ofthe gal-3 polypeptide can also be determined by amino-terminal amino acid sequence analysis.
  • the gal-3 protein includes functional fragments ofthe polypeptide, as long as the protective activity remains. Smaller peptides containing the biological activity of gal-3 are included in the invention.
  • the invention further includes amino acid sequences having at least 80%, preferably 90%), more preferably 95% identity to the fully length amino acid sequence of S ⁇ Q ID NO:4. Percent homology or identity can be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin
  • the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter ofthe two sequences.
  • the preferred default parameters for the GAP program include: (1) a unitary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and
  • the invention also provides purified proteins identified or characterized by a computer system or method of the present invention, where the protein can be selected from the group consisting of
  • marker proteins having the corresponding molecular weights and pis as presented in Table 10 and wherein said at least one of said proteins is optionally further selected from the group consisting of (i) unaffected proteins having the corresponding molecular weights and pis as presented in Table 11 ; (ii) affected proteins having the corresponding molecular weights and pis as presented in Table 12; and (iii) marker proteins having the corresponding molecular weights and pis as presented in Table 13.
  • an affected or unaffected peptide includes an association of two or more polypeptide domains, such as transmembrane, cytoplasmic, hydrophobic, hydrophilic, ligand binding, or pore lining domains, or fragments thereof, corresponding to an affected or unaffected peptide, such as 1-40 domains or any range or value therein.
  • Such domains of an affected or unaffected peptide of the invention can have at least 74% homology, such as 74-100% overall homology or identity, or any range or value therein to one or more corresponding affected or unaffected protein or peptide domains as described herein.
  • the above configuration of domains are provided as part of an affected or unaffected peptide ofthe invention, such that a functional affected or unaffected protein or peptide, when expressed in a suitable cell, is capable ofthe associated biological activity found in that affected islet cell type.
  • Such activity as measured by suitable affected or unaffected protein or peptide activity assays, establishes affected or unaffected protein or peptide activity of one or more affected or unaffected proteins or peptides ofthe invention.
  • an affected or unaffected peptide of the invention alternatively includes peptides having a portion of an affected or unaffected protein or peptide amino acid sequence which substantially corresponds to at least one 20 to 10,000 amino acid fragment and/or consensus sequence of an affected or unaffected peptide, or group of affected or unaffected peptides, wherein the affected or unaffected protein or peptide has homology or identity of at least 14-99%, such as 88-99% (or any range or value therein, e.g., 87-99, 88-99, 89-99, 90-99, 91-99, 92-99, 93-99, 94-99, 95-99, 96-99, 97-99, or 98- 99%) homology or identity to at least one sequence or consensus sequence of at least one protein characterized as presented in one or more of tables 8-13, or as presented in Figures 2-7, having the mass spec characteristics of one or more of proteins according to the present invention.
  • an affected or unaffected peptide can maintain affected or unaffected protein or peptide biological activity. It is preferred that an affected or unaffected peptide ofthe invention is not naturally occurring or is naturally occurring but is in a purified or isolated form which does not occur in nature. Preferably, an affected or unaffected peptide ofthe invention substantially corresponds to any set of domains of an affected or unaffected protein or peptide of the invention, having at least 10 contiguous amino acids of proteins characterized in one or more of tables 8-13, comprising SEQ ID NOS:, having the mass spec characteristics of one or more of proteins according to the present invention.
  • an affected or unaffected peptide ofthe invention can comprise at least one domain corresponding to known protein domains, such as cytoplasmic, intracellular, transmembrane, extracellular, or other known domains, having 74-100%) overall homology or any range or value therein.
  • Alternative domains are also encoded by DNA which hybridizes under stringent conditions to at least 30 contiguous nucleotides encoding at least 10 contiguous amino acids of proteins characterized in one or more of tables 8-13, Figures 2-7, comprising SEQ ID NOS:, having the mass spec characteristics of one or more of of proteins according to the present invention, or at least 14% homology thereto, or having codons substituted therefor which encode the same amino acid as a particular codon.
  • phosphorylation domains are also considered when providing an affected or unaffected peptide or encoding nucleic acid according to the invention.
  • a non-limiting example of this is presented in proteins 672-674 of table 12, wherein the same protein is differentially phosphorylated.
  • the invention further includes polynucleotide sequences encoding the diabetes- mediating proteins ofthe invention, including DNA, cDNA, and RNA sequences. It is also understood that all polynucleotides encoding all or a portion of a diabetes-mediating protein are also included herein, as long as they encode a polypeptide with the diabetes- mediating activity.
  • polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides. For example, such a polynucleotide may be subjected to site-directed mutagenesis.
  • the polynucleotide sequences ofthe invention also include antisense sequences. Antisense sequences include sequences synthesized with modified oligonucleotides.
  • the polynucleotides of the invention include sequences that are degenerate as a result ofthe genetic code. There are 20 natural amino acids, most of which are specified by more than one **codon.
  • DNA sequences of the invention can be obtained by several methods.
  • the DNA can be isolated using hybridization techniques which are well known in the art. These include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences, 2) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the DNA sequence of interest, and 3) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features.
  • the DNA sequences of the invention is derived from a mammalian organism, and most preferably from a human. Screening procedures which rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available. Oligonucleotide probes, which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically.
  • Hybridization is particularly useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present.
  • stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA clone by the hybridization ofthe target DNA to that single probe in the mixture which is its complete complement (Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Plainview, NY (1989)).
  • the development of specific DNA sequences encoding the diabetes-mediating proteins of the invention can also be obtained by: 1) isolation of double-stranded DNA sequences from the genomic DNA; 2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest; and 3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA.
  • the isolation of genomic DNA isolates is the least common. This is especially true when it is desirable to obtain the microbial expression of mammalian polypeptides due to the presence of introns.
  • DNA sequences are frequently the method of choice when the entire sequence of amino acid residues ofthe desired polypeptide product is known.
  • the direct synthesis of DNA sequences is not possible and the method of choice is the synthesis of cDNA sequences.
  • the standard procedures for isolating cDNA sequences of interest is the formation of plasmid- or phage-carrying cDNA libraries which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of genetic expression for the protein of interest. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned.
  • the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA DNA hybridization procedures which are carried out on cloned copies ofthe cDNA which have been denatured into a single-stranded form (Jay et al. Nucl. Acid Res. , 11 :2325
  • a cDNA expression library such as lambda gtl 1
  • Such antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of the desired cDNA.
  • DNA sequences encoding a diabetes-mediating can be expressed in vitro by DNA transfer into a suitable host cell.
  • "Host cells” are cells in which a vector can be propagated and its DNA expressed.
  • the term also includes any progeny ofthe subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell” is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
  • the polynucleotide sequences encoding diabetes-mediating proteins may be inserted into a recombinant expression vector.
  • recombinant expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or inco ⁇ oration of the XI 30 genetic sequences.
  • Such expression vectors contain a promoter sequence which facilitates the efficient transcription ofthe inserted genetic sequence ofthe host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection ofthe transformed cells.
  • Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg et al. Gene 56:125 (1987)), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans J. Biol. Chem.
  • the DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or polyhedron promoters).
  • a promoter e.g., T7, metallothionein I, or polyhedron promoters.
  • Polynucleotide sequences encoding a diabetes-mediating protein can be expressed in either prokaryotes or eukaryotes.
  • Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art.
  • Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to inco ⁇ orate DNA sequences ofthe invention.
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art.
  • the host is prokaryotic, such as E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method using procedures well known in the art.
  • MgCl 2 or RbCl can be used. Transformation can also be performed after forming a protoplast ofthe host cell if desired.
  • Eukaryotic cells can also be cotransformed with DNA sequences encoding the diabetes- mediating protein of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the he ⁇ es simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to infect, transform, or transduce eukaryotic cells and express the protein (see, for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ea., 1982).
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • Isolation and purification of microbial expressed polypeptide, or fragments thereof, provided by the invention may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
  • 2DGE is a preferred method for purification of modifications variants ofthe proteins from each other.
  • Deleterious diabetes-mediating proteins are characterized as enhancing the development of or increasing the risk of a subject developing diabetes.
  • the invention includes substantially purified protective diabetes-mediating proteins, and polynucleotide sequences encoding such proteins.
  • a deleterious protein is mortalin.
  • the amino acid sequence of murine mortalin (SEQ ID NO:l) is shown in FIG. 2, and of human mortalin (SEQ ID NO:2) in FIG. 3.
  • the diabetes-mediating proteins ofthe invention can also be used to produce antibodies which are immunoreactive or bind to epitopes ofthe diabetes mediating proteins.
  • An antibody may consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations.
  • Monoclonal antibodies are made from antigen containing fragments ofthe protein by methods well known in the art (Kohler et al. Nature 256:495 (1975) ; Current Protocols in Molecular Biology, Ausubel et al, ea., 1989).
  • antibody as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab')2, and Fv which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows:
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain
  • Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion ofthe heavy chain
  • two Fab' fragments are obtained per antibody molecule
  • (Fab')2 the fragment ofthe antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;
  • Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • Single chain antibody defined as a genetically engineered molecule containing the variable region ofthe light chain, the variable region ofthe heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
  • SCA Single chain antibody
  • Methods of making these fragments are known in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), inco ⁇ orated herein by reference.
  • epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • Antibodies which bind to the diabetes-mediating polypeptides ofthe invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or a peptide used to immunize an animal can be derived from translated cDNA or chemical synthesis which can be conjugated to a carrier protein, if desired.
  • Such commonly used carriers which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • the coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or-a rabbit).
  • polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elusion from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies. See, for example, Coligan et al. Unit 9, Current Protocols in Immunology, Wiley Interscience (1994), herein specifically inco ⁇ orated by reference.
  • an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the "image" ofthe epitope bound by the first monoclonal antibody.
  • an antibody or nucleic acid probe specific for a diabetes-mediating protein may be used to detect the diabetes-mediating protein (using antibody) or encoding polynucleotide (using nucleic acid probe) in biological fluids or tissues.
  • the antibody reactive with the diabetes-mediating protein or the nucleic acid probe is preferably labeled with a compound which allows detection of binding to the diabetes-mediating protein.
  • any specimen containing a detectable amount of antigen or polynucleotide can be used.
  • specific proteins may be selected by their ligands, e.g., galectin-3 binding to lectin-binding protein), and is useful for diagnosis and/or purification of the protein of interest.
  • the cell component is nucleic acid
  • PCR polymerase chain reaction
  • other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used.
  • LCR ligase chain reaction
  • LAT ligated activated transcription
  • NASBA nucleic acid sequence-based amplification
  • the in vivo transplantation assay of the invention described herein allows the identification of proteins involved or effected.
  • insulin- secreting cells or cells capable of developing into insulin-secreting cells are transplanted into a host animal.
  • Transplanted cells are rescued at time points between transplantation and the onset of diabetes, and protein expression determined, and protein expression compared with nontransplanted islets and syngeneic transplants in animals not developing diabetes.
  • the method ofthe invention allows proteins exhibiting an altered expression during development of diabetes to be identified. Identified proteins are then isolated and tested further to identification as protective or deleterious diabetes-mediating proteins.
  • Cells capable of developing into insulin-producing cells include pancreatic islet cells and ⁇ cells.
  • Transplanted cells may be obtained from any species of interest, including human cells.
  • a host animal is preferably one which is immunologically compatible with the transplanted cells such that the transplanted cells do not undergo rejection in the host animal.
  • the host animal may be any animal which develops diabetes and is convenient for study.
  • Preferred host animals are mice, rats and hamsters, with rats and mice being most preferred.
  • the host animal is selected from a strain bred for an increased incidence of diabetes, including BB-DP rats and NOD mice.
  • the method ofthe invention may also be used with host animals engineered to develop diabetes at a predetermined time, e.g., upon exposure to a specific antigen. See, for example, Oldstone et al. APMIS 104:689-97 (1996); von Herrath et al. J. Clin. Invest. 98:1324-1331
  • mice e.g. mice
  • Rattus e.g. rats
  • Oryctolagus e.g. rabbits
  • Mesocricetus e.g. hamsters
  • Cavia e.g., guinea pigs
  • mammals with a normal full grown adult body weight of less than 1 kg which are easy to breed and maintain can be used.
  • Protein expression may be assessed by a variety of means known to the art, including one or two-dimensional gel electrophoresis and immunoblotting.
  • Two-dimensional gel electrophoresis is a particularly effective tool for separating mixtures of proteins (Andersen et al. Diabetes 44:400-407 (1995)).
  • Cell protein extracts are put onto a gel, and the individual proteins are separated first by charge and then by size.
  • the result is a characteristic picture of as many as 1,000 to 5,000 spots, each usually a single protein.
  • Resolution is improved by increasing gel size, and by enhancing the sensitivity trough the use of radiolabel methods, silver staining, and the reduction in thickness ofthe gels to 1.5 mm and less.
  • the peptides are recovered from the gel and subjected to mass spectroscopy (matrix assisted laser deso ⁇ tion/ionization mass spectrometry)(MALDI) and the resulting MS-profiles are analyzed against the computerized MS-profiles of all sequences found in the public sequence databases, as well as against propriety sequence information. If any matches to previously cloned sequences are obtained, information about the corresponding gene and encoded protein is collected.
  • mass spectroscopy matrix assisted laser deso ⁇ tion/ionization mass spectrometry
  • the protein may be microsequenced to obtain partial amino acid sequence information by methods known to the art (see Example 5 below). Based upon results obtained from database searches or amino acid sequencing, specific or degenerate primers are constructed and used to screen rat and human islets libraries or first-strand cDNA by PCR is used to clone partial sequences ofthe corresponding cDNA. The obtained sequences are then used to obtain full-length coding regions either by 5'-race PCR or by conventional hybridization screening techniques, followed by expression ofthe recombinant protein (Karlsen et al. Proc. Natl. Acad. Sci.
  • Diabetes-mediating proteins can be isolated in a variety of ways known to the art, including purification from biological material, expression from recombinant DNA (see above). Conventional method steps include extraction, precipitation, chromatography, affinity chromatography, and electrophoresis. For example, cells expressing a diabetes- mediating protein can be collected by centrifugation, or with suitable buffers, lysed, and the protein isolated by column chromatography, for example, on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, hydroxyapatite or by electrophoresis or immunoprecipitation. Diabetes-mediating proteins may alternatively be isolated by immunoprecipitation with the use of specific antibodies.
  • the invention includes a transgenic animal containing a gene encoding a diabetes-mediating protein, as well as transgenic animals which are the offspring of a transgenic animal ofthe invention.
  • the gene encoding a diabetes-mediating protein may be comprised of a naturally occurring or partially or completely of an artificial polynucleotide sequence, i.e. codon sequences not present in the native gene sequence.
  • Transgenic animals containing elevated levels of expression ofthe diabetes-mediating gene of the invention can be obtained for example, by over-expression of the gene with an enhanced promoter and/or with high copy numbers ofthe natural gene.
  • Transgenic animals also specifically include a hybrid transgenic animal produced by crossing a transgenic animal with an animal in which one or more diabetes-mediating protein gene(s) are ablated.
  • Transgenic animals ofthe invention are useful in a number of ways, including in assays for determining the effect of a candidate protective or deleterious diabetes- mediating protein on the development of diabetes.
  • a transgenic animal carrying a transgene for a candidate protective protein is useful for determining the effect of the expression of the protective transgene on the development of diabetes.
  • Preferred host animals are mice, rats and hamsters, with rats and mice being most preferred in that there exists considerable knowledge on the production of transgenic animals.
  • Other possible host animals include those belonging to a genus selected from Mus (e.g. mice), Rattus (e.g. rats), Oryctolagus (e.g. rabbits), and Mesocricetus (e.g. hamsters) and Cavia (e.g., guinea pigs).
  • Mus e.g. mice
  • Rattus e.g. rats
  • Oryctolagus e.g. rabbits
  • Mesocricetus e.g. hamsters
  • Cavia e.g., guinea pigs
  • the transgenic non-human mammal ofthe invention (preferably a rat or mouse) will have in some or all of its nucleated cells a gene encoding a diabetes-mediating protein, e.g., one or more of a protection protein or deleterious protein, which gene was introduced into the mammal, or an ancestor of that mammal, at an embryonic or germ cell stage.
  • a diabetes-mediating protein e.g., one or more of a protection protein or deleterious protein, which gene was introduced into the mammal, or an ancestor of that mammal, at an embryonic or germ cell stage.
  • embryonic stage may be any point from the moment of conception (e.g., as where the sperm or egg bears the foreign gene) throughout all of the stages of embryonic development of the fetus.
  • a "transgenic mammal” as used herein denotes a mammal bearing in some or all of its nucleated cells one or more genes derived from the same or a different species; if the cells bearing the foreign gene include cells of the animal's germline, the gene may be transmissible to the animal's offspring.
  • the transgenic mammal ofthe invention exhibits a 80% incidence of diabetes within 70 ⁇ 10 days; more preferably, a 90% incidence of diabetes within 60 ⁇ 5 days; most preferably, a 95% incidence of diabetes within 55 ⁇ 5 days; and even more preferably, a 97% incidence of diabetes within 50 ⁇ 5 days.
  • the animals ofthe invention are used in assays to test the ability of a candidate protective or deleterious diabetes-mediating protein, or a test compound to prevent, enhance, or slow the development of diabetes.
  • transgenic animals may be selected from a genetic background predisposed for development of diabetes, or may be double-transgenic animals which will develop diabetes predictable and in a short period of time.
  • the animals are used in assays to test the ability of the candidate protective protein to inhibit or reduce the incidence of disease onset.
  • the animals are used in assays to test the ability of a compound to induce expression of a protective protein and thus to protect the animal from disease development.
  • genetics constructs and methodologies of the invention are used to create animals which due to their genetic make up will develop diabetes within a predictable period of time.
  • transgenic animals are created which express a deleterious diabetes-mediated disease such as mortalin.
  • the animals of the invention are used in assays to test compounds able to prevent or delay the onset of diabetes.
  • the invention also provides a means of creating animal models for diabetes or diabetes related diseases.
  • the transgenic animals of the invention provide a way to develop and test potential therapies for the diabetes, and will eventually lead to cures for this disease.
  • ASSA YS FOR SCREENING FOR DRUGS CAPABLE OF EFFECTING THE EXPRESSION OF DIABETES-MEDIATING PROTEINS are useful for screen compounds capable of effecting the expression of a diabetes-mediating protein, and thus the development of diabetes in a mammal.
  • One model for screening drugs capable of effecting the expression of one or more diabetes-mediating proteins is the administration of compounds suspected of having beneficial effects (including antisense oligonucleotides) to cells in culture.
  • Useful cells are RIN, transfected, or islet cells. The effects ofthe test compound on protein expression may then be assayed by 2D gel electrophoresis.
  • Another screening model is an in vivo method with the use of a mammal at risk for development of diabetes. Briefly, a mammal with an increased risk for diabetes (e.g., diabetes-prone BB rat or NOD mouse) is exposed to a test compound, and the effect of exposure to the test compound on the development of diabetes determined.
  • a mammal with an increased risk for diabetes e.g., diabetes-prone BB rat or NOD mouse
  • the development of diabetes may be monitored throughout the developmental period by determining the expression of one or more diabetes-mediating proteins and comparing by comparing the time of disease onset with expression and timing in the absence of disease development. Determining the expression of one or more diabetes- mediating proteins includes the diabetes-mediating protein itself, a post-translational modification product, and/or diabetesmediating protein degradation product.
  • activation of a diabetes-mediating protein is determined by measuring the level ofthe diabetes-mediating protein expression in a test sample.
  • a suitable test sample includes a body fluid, such as blood, urine, or cerebrospinal fluid, or fluid derived from it, such as plasma or serum.
  • the level of protein expression in a test sample is measured by Western blot analysis. The proteins present in a sample are fractionated by gel electrophoresis, transferred to a membrane, and probed with labeled antibodies specific for the protein(s).
  • the level of diabetesmediating protein expression is measured by Northern blot analysis.
  • a mammal capable of developing diabetes is one selected from a strain of mammals which have been bred for an increased incidence of diabetes. Preferable, the mammal is selected from a strain which exhibits a 75% chance of developing diabetes within 69 ⁇ 25 days. More preferably, the mammal is a transgenic mammal of engineered to exhibit a high incidence of diabetes within a predictable period of time.
  • the transgenic mammal exhibits a 80% incidence of diabetes within 70 + 10 days; more preferably, a 90% incidence of diabetes within 60 ⁇ 5 days; most preferably, a 95% incidence of diabetes within 55 + 5 days; and even more preferably, a 97% incidence of diabetes within 50 + 5 days.
  • the invention provides for methods for identifying compounds capable of suppressing or reducing the expression of an endogenous deleterious protein, as well as methods for preventing and/or treating diabetes by administering a therapeutically effective among of a compound capable of suppressing or reducing the expression of an endogenous deleterious protein.
  • the diabetes-mediating proteins ofthe invention are also useful to screen reagents that modulate diabetes-mediating protein activity. Accordingly, in one aspect, the invention features methods for identifying a reagent which modulates diabetes-mediating protein activity, by incubating a cell expressing a diabetes mediating protein with the test reagent and measuring the effect of the test reagent on diabetes-mediating protein synthesis, phosphorylation, function, or activity. When activation of a diabetes-mediating protein is via phosphorylation, the test reagent is incubated with the diabetes-mediating protein and with either gamma- [labeled-ATP or [ 35 S]-methionine, and the rate of phosphorylation determined.
  • the test reagent is incubated with a cell transfected with an diabetes-mediating protein polynucleotide expression vector, and the effect of the test reagent on diabetes-mediating protein transcription is measured by Northern blot analysis.
  • the effect ofthe test reagent on diabetes- mediating protein synthesis is measured by Western blot analysis using an antibody to the diabetesmediating protein.
  • the effect of a reagent on diabetes- mediating protein activity is measured by incubating diabetes-mediating protein with the test reagent, [ 32 ]P-ATP, and a substrate in the diabetes-mediating protein pathway. All experiments would be compared against a normal labeling of cells with [ 35 S]-methionine to determine modulation of protein expression. The rate of substrate phosphorylation is determined by methods known in the art.
  • modulation of diabetes-mediating protein activity includes agonists and antagonists.
  • the invention is particularly useful for screening reagents that inhibit deleterious protein activity. Such reagents are useful for the treatment or prevention of diabetes. V. THERAPEUTIC APPLICATIONS
  • the invention provides methods for preventing and/or treating diabetes in a mammal by administering a therapeutically effective amount of a protective diabetesmediating protein.
  • a protective diabetesmediating protein Preferably the mammal is a human subject at risk for diabetes.
  • identified protective or deleterious diabetesmediating proteins are used to identify test compounds capable of effecting their expression.
  • Test compounds so identified are candidate therapeutic agents for preventing, ameliorating, or delaying the onset of diabetes in a subject at risk.
  • a test therapeutic compound which effects the expression of a diabetes-mediating proteins can be, but is not limited to, at least one selected from a nucleic acid, a compound, a protein, an element, a lipid, an antibody, a saccharide, an isotope, a carbohydrate, an imaging agent, a lipoprotein, a glycoprotein, an enzyme, a detectable probe, and antibody or fragment thereof, or any combination thereof, which can be detectably labeled as for labeling antibodies, as described herein.
  • labels include, but are not limited to, enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiluminescent compounds and bioluminescent compounds.
  • a therapeutic compound is identified in the drug screening assay ofthe invention through its ability to induce or enhance the expression of a protective protein, such that disease onset is prevented or delayed in a subject at risk for the development of diabetes.
  • a candidate therapeutic compound is also identified by its ability to prevent or decrease the expression of a deleterious protein, such that disease onset is prevented or delayed in a subject at risk for the development of diabetes.
  • a therapeutic nucleic acid as a therapeutic compound can have, but is not limited to, at least one ofthe following therapeutic effects on a target cell: inhibiting transcription of a deleterious protein DNA sequence; inhibiting translation of a deleterious protein RNA sequence; inhibiting reverse transcription of an RNA or DNA sequence corresponding to a deleterious protein; inhibiting a post-translational modification of a protein; inducing transcription of a DNA sequence corresponding to a protective protein; inducing translation of an RNA sequence corresponding to a protective protein; inducing reverse transcription of an RNA or DNA sequence corresponding to a protective protein; translation ofthe nucleic acid as a protein or enzyme; and inco ⁇ orating the nucleic acid into a chromosome of a target cell for constitutive or transient expression ofthe therapeutic nucleic acid.
  • Therapeutic effects of therapeutic nucleic acids can include, but are not limited to: tiiming off a defective gene or processing the expression thereof, such as antisense RNA or DNA; inhibiting viral replication or synthesis; gene therapy as expressing a heterologous nucleic acid encoding a therapeutic protein or correcting a defective protein; modifying a defective or underexpression of an RNA such as an hnRNA, an mRNA, a tRNA, or an rRNA; encoding a drug or prodrug, or an enzyme that generates a compound as a drug or prodrug in pathological or normal cells expressing the diabetes-mediating protein or peptide; and any other known therapeutic effects.
  • Also included in the invention is gene therapy by providing a polynucleotide encoding a protective diabetes-mediating protein.
  • the invention further includes a method for preventing diabetes by administering an effective amount of a polynucleotide which inhibits the in vivo expression of a deleterious diabetes-mediating protein.
  • a therapeutic compound is administered to a human patient chronically or acutely.
  • a protective protein is administered chronically in combination with an effective amount of a compound that acts on a different pathway than the therapeutic compound.
  • the therapeutic method ofthe invention can be combined with other treatments for diabetes or with methods for the management of diabetes.
  • Therapeutic formulations ofthe therapeutic compound for treating or preventing diabetes are prepared for storage by mixing the compound having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., 1980), in the form of lyophilized cake or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter ions such as sodium; and/or non-ionic surfactants such as Tween, Pluronics, or polyethylene glycol (PEG).
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • the compound is also suitably linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • the amount of carrier used in a formulation may range from about I to 99%, preferably from about 80 to 99%, optimally between 90 and 99% by weight.
  • the therapeutic compound to be used for in vivo administration must be sterile. This is readily accomplished by methods known in the art, for example, by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.
  • the therapeutic compound ordinarily will be stored in lyophilized form or in solution.
  • Therapeutic compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.
  • the therapeutic compound administration is in a chronic fashion using, for example, one ofthe following routes: injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, or intralesional routes, orally or using sustained-release systems as noted below.
  • the therapeutic compound is administered continuously by infusion or by periodic bolus injection if the clearance rate is sufficiently slow, or by administration into the blood stream or lymph.
  • the preferred administration mode is targeted to the tissue of interest ( ⁇ cell or pancreatic cells) so as to direct the molecule to the source and minimize side effects ofthe compound.
  • sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) as described by Langer et al. J. Biomed. Mater. Res. 15:167-277 (1981) and Langer Chem. Tech. 12:98-105 (1982), or poly(vinyl alcohol)), polylactides (U.S. Patent No.
  • the therapeutic compound also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interracial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly- [methylmethacy late] microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions.
  • interracial polymerization for example, hydroxymethylcellulose or gelatin-microcapsules and poly- [methylmethacy late] microcapsules, respectively
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • Sustained-release compositions also include liposomally entrapped therapeutic compound(s).
  • Liposomes containing therapeutic compound(s) are prepared by methods known er se: DE 3,218,121; Epstein et al. Proc. Natl. Acad. Sci. USA 82:3688-3692 (1985) ; Hwang et al.Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP 102,324.
  • the liposomes are ofthe small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol% cholesterol, the selected proportion being adjusted for the optimal agonist therapy.
  • a specific example of a suitable sustained-release formulation is in EP 647,449.
  • an effective amount of therapeutic compound(s) to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition ofthe patient. Accordingly, it will be necessary for the clinician to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect.
  • both therapeutic compounds are administered together, they need not be administered by the same route, nor in the same formulation. However, they can be combined into one formulation as desired. Both therapeutic compounds can be administered to the patient, each in effective amounts, or each in amounts that are sub- optimal but when combined are effective. In one embodiment, the administration of both therapeutic compounds is by injection using, e.g., intravenous or subcutaneous means, depending on the type of protein employed. Typically, the clinician will administer the therapeutic compound(s) until a dosage is reached that achieves the desired effect for treatment or prevention of diabetes. For example, the amount would be one which ameliorates symptoms of diabetes and restores normoglycemia. The progress of this therapy is easily monitored by conventional assays.
  • the therapeutic compound is a protective diabetes-mediating gene encodes gal-3, and/or a post-translational modification product of gal-3.
  • a typical daily dosage of a therapeutic compound used alone might range from about 1 ⁇ g/kg to up to 100 mg/kg of patient body weight or more per day, depending on the factors mentioned above, preferably about 10 ⁇ g/kg/day to 50 mg/kg/day.
  • the present invention also provides gene therapy for the treatment of diabetes and diabetes-related disorders, which are improved or ameliorated by a protective polypeptide. Such therapy would achieve its therapeutic effect by introduction of the protective polynucleotide into insulin-producing cells. Delivery of a protective polynucleotide can be achieved using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Especially preferred for therapeutic delivery of sequences is the use of targeted liposomes.
  • RNA virus such as a retrovirus
  • retroviral vector for in vitro cell transformation is a derivative of a murine or avian retrovirus adenovirus.
  • the advantage of adenovirus transduction compared to other transfection methods is the high transfection affectivity and the ability to transfect whole islets.
  • the level of expression can be adjusted by the virus concentration and transduction-time used. Even though the adeno virus-mediated expression is transient, the expression in islets is stable for at least several weeks (Becker et al. J. Biol. Chem. 269:21234 (1994); Korbutt et al. Transplantation Proc. 27:3414 (1995)).
  • retroviral vector For stable integration of a transgene into a mammal, a retroviral vector is preferred.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • GaLV gibbon ape leukemia virus
  • a number of additional retroviral vectors can inco ⁇ orate multiple genes.
  • Retroviral vectors can be made target specific by attaching, for example, a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody to target the retroviral vector.
  • telomere sequences which can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery ofthe retroviral vector containing the protective polynucleotide. Since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids encoding all ofthe structural genes ofthe retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation.
  • Helper cell lines which have deletions ofthe packaging signal include, but are not limited to ⁇ 2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced. Alternatively, NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
  • colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • the preferred colloidal system of this invention is a liposome.
  • Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LW), which range in size from 0.2-4.0 ⁇ m can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules.
  • LW large unilamellar vesicles
  • RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al. Trends Biochem. Sci. 6:77 (1981)).
  • liposomes In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells.
  • a liposome In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation ofthe genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery ofthe aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Manning et al. Biotechniques 6:682 (1988)).
  • the composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with sterols, especially cholesterol. Other phospholipids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidyl-glycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipid, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidyl-glycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
  • Illustrative phospholipids include egg phosphatidylcholine, dipalm itoylphosphatidylcholine and distearoylphosphatidylcholine.
  • the targeting of liposomes can be classified based on anatomical and mechanistic factors.
  • Anatomical classification is based on the level of selectivity, for example, organ- specific, cell-specific, and organelle-specific.
  • Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells ofthe reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries.
  • RES reticulo-endothelial system
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein
  • the surface ofthe targeted delivery system may be modified in a variety of ways.
  • lipid groups can be inco ⁇ orated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.
  • Various linking groups can be used for joining the lipid chains to the targeting ligand.
  • the polypeptide or polynucleotide of the invention provides methods for treatment of diabetes and diabetes-related disorders, which are improved or ameliorated by modulation of deleterious diabetesmediating gene expression or activity.
  • the deleterious diabetes-mediating gene encodes mortalin.
  • modulate envisions the suppression of expression of mortalin. Where suppression of a deleterious protein expression is desirable, for example, suppression of mortalin, nucleic acid sequences that interfere with mortalin expression at the translational level can be used.
  • This approach utilizes, for example, antisense nucleic acid, ribozymes, or triplex agents to block transcription or translation of a specific mortalin mRNA, either by masking that mRNA with an antisense nucleic acid or triplex agent, or by cleaving it with a ribozyme.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub Scientific American 262:40 (1990)). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation ofthe mRNA, since the cell will not translate a mRNA that is double-stranded.
  • Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target mortalin-producing cell.
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases.
  • RNA molecules which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (CechJ Amer. Med. Assn. 260:3030 (1988 ).
  • a major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • ribozymes There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff Nature 334:585 (1988)) and "hammerhead"-type. Tetrahymena- y ⁇ pe ribozymes recognize sequences which are four bases in length, while “hammerhead” -type ribozymes recognize base sequences 11 - 18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-based recognition sequences are preferable to shorter recognition sequences.
  • Blocking mortalin action either with anti-mortalin antibodies or with a mortalin antisense polynucleotide may be useful for slowing or ameliorating diabetes.
  • the above described method for delivering a protective polynucleotide are fully applicable to delivery of an mortalin antagonist for specific blocking of mortalin expression and/or activity when desirable.
  • a mortalin antagonist can be a mortalin antibody, an antisense polynucleotide sequence, or a compound which suppresses or inhibits the expression of mortalin.
  • High resolution two-dimensional (2D) gel electrophoresis may be used to separate approximately 2500 protein spots, each spot corresponding to a protein according to molecular weight and pi.
  • a computerized 2-D gel database of 1373 WF rat islet proteins has recently been reported (Andersen et al. Diabetes 44:400-407 (1995)) with a qualitative reproducibility of 81% (FIGs. 1A-1B).
  • IL-l ⁇ has been shown to induce changes in the expression of 82 protein in BB-DP islet cells incubated in vitro, in vitro incubation of BB- DP islets taken from animals after the onset of diabetes shows that 33 ofthe 82 proteins determined to respond to IL-l ⁇ show significant changes in expression at the onset of diabetes.
  • diabetes-mediating proteins Based on the results of 2D gel analysis and peptide sequencing or mass spectroscopy analysis, several candidate diabetes-mediating proteins have been identified (Table 1) and cloned from rat and human islet cells, including iNOS, mortalin, and galectin-3 (Madsen et al. in Insulin secretion and pancreatic B cell research, P.R. Flatt &
  • Manganese superoxide dismustase has been transfected into ⁇ -cells and whole islets of Langerhans with the use of adenovirus mediated gene transfer and expression. Transfection include the generation of stable or transiently transfected cells. Following transfection, subcloning, and establishment of stable RIN clones expressing the coding region or antisense-fractions of the protein of interest under the control of a CMV or insulin promoter, the diabetes-mediating protein of interest is functionally characterized. Preliminary results show a 100% transduction in RIN cells and up to 70% in isolated islets.
  • the increased or decreased expression ofthe protein in the transfected cells is characterized in vitro by measurement of NO production, insulin secretion, and/or cytotoxicity.
  • cell cycle and mitochondrial activity may be determined by FACS analysis (Mandrup-Poulsen et al. Diabetes/Metabolism Reviews 9:295-309 (1993)) and semiquantitative analysis of gene expression characterized by multiplex PCR (Nerup et al. Anales Espanoles Pediatria 58:40-41 (1994)).
  • secondary cellular effects of the over- or under-expression of the protein as well as posttranslational modification may be characterized by 2D-gel electrophoresis of the transfected cells.
  • High resolution 2DGE was also used to detect protein changes in BB-DP islet syngrafts transplanted into 30 day old BB-DP rats (Example 3). In BB-DP islet syngrafts from newly onset diabetic BB-DP rats, but not in WF islet syngrafts, 15 of these 82 proteins were found to change level of expression (Example 4).
  • IL-1 ⁇ induced changes in protein expression in vitro were compared to protein changes in the process of disease occurrence in BB-DP islet syngrafts (Example 4) or in
  • BB-DP islet allograft rejection (Example 6). In BB-DP islet syngrafts from newly onset diabetic BB-DP rats, but not in WF islet syngrafts 15 of these 82 proteins were found to change level of expression.
  • Proteins exhibiting altered expression with diabetes onset in syngrafted islets were further characterized (Example 5).
  • One of the altered protein spots was identified by amino acid sequencing to have high homology to the heat shock 70 protein (mortalin), which has been demonstrated to be involved in cellular mortality and apoptosis following translocation of this constitutively expressed protein from a perinuclear to the cytoplasmic region (Wadhwa et al. J. Biol. Chem. 268:6615 and 268:22239 (1993)).
  • mortalin cDNA was cloned from rat and human islet for further characterization of its involvement in diabetes development.
  • Gal-3 is a protein involved in islet development and inhibition of apoptosis. Using the nucleotide sequence, gal-3 was cloned from rat and human islets, subcloned and expressed in RIN cells after selection for stable clones.
  • RIN cells expressing gal-3 exhibited an increased metabolic activity and proliferative rate, and became more resistant to the negative effect of cytokines.
  • the in vivo effect of gal-3 expression was studied by transplanting 200 neonatal islets to 30 day old diabetes prone Bio-Breeding rats (BB-DP) (Example 8).
  • iNOS Inducible nitric oxide synthase
  • Manganese superoxide dismutase was identified as a down regulated protein by 2D gel analysis. The expression of MnSOD was further characterized in ⁇ and islet cells in vitro and in vivo following adenovirus transduction and transplantation.
  • Mitochondrial isocitate dehydrogenase was identified as a diabetes-mediating protein.
  • BB-DP islet cells transplanted into 30 day old WK rats were retrieved after 12 days, and protein expression determined (Example 6). It was found that BB-DP islet allografts in WK rats but not in WF islet syngrafts 9 of 82 proteins were found to change level of expression.
  • Both the syn-and allografted BB-DP islets were compared to non-grafted BB-DP islets with regard to the proteins found to change expression levels during IL-1 exposure of the BB-DP islets in vitro.
  • 200 neonatal WF islets were grafted to 30 day old WF rats.
  • Grafts were retrieved 48 days after transplantation corresponding to the mean time of onset of diabetes in the colony and in the syngrafted BB-DP rats.
  • the grafts were processed and analyzed as described for comparison with WF control islets with regard to the 105 proteins previously found to be changed during IL-1 incubation. This was done to identify protein changes inducible both by IL-1 and islet syngrafting. as well as to identify rejection- specific proteins.
  • transgenic animals carrying the gene encoding the candidate protein and able to express the candidate protein under tissue-specific promoters are generated.
  • the transgenic animals of the invention express candidate proteins under specific promoters such as the insulin, amylin, CMV, or HLA promoters.
  • adenoviral mediated transduction of islets resulted in dose-dependent efficient gene transfer with stable transgene expression in the absence of toxicity.
  • Ketamin was purchased from Park-Davis (Barcelona, Spain), xylazin from Bayer (Leverkusen, Germany), and Temgesic® from Reckitt and Colemann (Hull,
  • RPMI 1640 Hanks' balanced salt solution (HBSS), and DMEM were purchased from Gibco, Paisley, Scotland.
  • RPMI 1640 contained 11 mmol D-glucose, and was supplemented with 20 mM HEPES buffer, 100,000 IU/I penicillin and 100 mg/1 streptomycin.
  • Authentic recombinant human IL-l ⁇ was provided by Novo Nordisk Ltd. (Bagsvaerd, Denmark) having a specific activity of 400 U/ng.
  • reagents used 2-mercaptoethanol, bovine serum albumin (BSA), Tris HCl, Tris base, glycine, (Sigma, St. Louis, USA); trichloracetic acid (TCA), phosphoric acid, NaOH, glycerol, n-butanol, bromophenol blue, sodium nitroprusside (SNP), H 3 PO 4 and NaNO 2 (Merck, Darmstadt, Germany); filters (HAWP 0.25 mm pore size) (Millipore, Boston, USA); RNAse A, DNAse I (Worthington, Freehold, NJ, USA); [ 35 S]-methionine
  • BB-DP Wistar Furth
  • WK Wistar Kyoto Rats
  • BB-DP rats were housed separately in a specific pathogen-free environment. All rats were housed under controlled conditions of light (light on from 6:00 am to 6:00 pm), humidity (60 - 70%) and temperature (20 - 22C°) from five days prior to transplantation until sacrifice. The rats were offered a standard rat chow (Altrornin, Chr. Petersen A S, Ringsted, Denmark) and free access to tap water.
  • BB-DP and WF rats were picked up at M ⁇ llegarden in the morning on the day of islet isolation, and transported in animal transport boxes.
  • ketamin 8.75 mg/100 g
  • xylazin 0.7 mg/100 g
  • a pocket using a blunt instrument was gently made in the capsule.
  • the islets were placed under the capsule towards the lower kidney pole.
  • the muscle and skin were sutured, the rats allowed to recover in a cage in the operating facility and given Temgesic (0.01 mg) sc in the neck before returning to the animal facility.
  • Temgesic (0.01 mg) sc in the neck before returning to the animal facility.
  • the rats were given Temgesic twice daily for 3 days post transplantation. Blood glucose (BG) was measured every third day.
  • the rats were killed by cervical dislocation and immediately afterwards the grafted kidneys were removed.
  • the grafts were carefully dissected from the kidneys and capsules under a microscope and placed in HBSS.
  • graft and islet labeling The grafts were labeled immediately after retrieval. Cultured islets were labeled after 24 h of incubation with or without IL-1. Grafts and islets were washed twice in HBSS and labeled for 4 h at 37°C in 200 ml methionine-free Dulbecco's modified Eagle's medium (DMEM) with 10% NHS dialyzed for amino acids, and either 330 mCi [35S]-methionine for the grafts or 200 mCi [35S]-methionine for the islets. To eliminate 2-mercaptoethanol, [35S]-methionine was freeze-dried for at least 4 h before labeling. After labeling, the grafts and islets were washed three times in HBSS, the supernatant was removed and the tissue immediately frozen at -80°C.
  • DMEM Dulbecco's modified Eagle's medium
  • Sample preparation The frozen grafts were crushed in a mortar. The grafts and islets were resuspended in 100 ml DNAse I/RNAse A solution and lysed by freeze-thawing twice. After the second thawing, the samples were left on ice for 30 min for the digestion of nucleic acids and then freeze dried overnight. The samples were dissolved by shaking in 120 ml lysis buffer (8.5 M urea, 2% Nonidet P-40, 5% 2-mercaptoethanol and 2% ampholytes, pH range 7-9) for a minimum of 4 h.
  • 120 ml lysis buffer 8.5 M urea, 2% Nonidet P-40, 5% 2-mercaptoethanol and 2% ampholytes, pH range 7-9
  • first dimension gels contained 4% acrylamide, 0.25% bisacrylamide and ampholytes. Equal numbers of counts (106 cpm) of each sample were applied to the gels. In case of lower amounts of radioactivity it was necessary to regulate the exposure time ofthe gel so that comparable total optical densities were obtained.
  • the samples were analyzed on both isoelectric focusing (IEF; pH 3.5-7) and non-equilibrium pH-gradient electrophoresis (NEPHGE; pH 6.5-10.5) gels.
  • Second dimension gels contained 12.5% acrylamide and 0.063% bisacrylamide and were run overnight.
  • the gels were fixed and treated for fluorography with Amplify® before being dried.
  • the gels were placed in contact with X-ray films and exposed at -70°C for 3 to 40 days. Each gel was exposed for at least 3 time periods to compensate for the lack of dynamic range of X-ray films.
  • MW and pi Molecular weights ofthe proteins were determined by comparison with standard gels. The pis for the individual proteins on the gels were determined by the use of pi calibration kits. Landmark proteins were identified on gels by one or several ofthe following techniques: immunoblotting, immunoprecipitation, micro sequencing or peptide mapping.
  • Islet isolation and culture Islets from pancreata of four to five day old inbred BB- DP and WF rats were isolated after collagenase digestion as described by Brunstedt et al. in Methods in Diabetes Research, Vol. 1, Wiley & Sons, New York, pp. 254-288 (1984), specifically inco ⁇ orated herein by reference. After 4 days of preculture in RPMI 1640 +
  • islets were cultured as follows: 150 BB-DP islets were incubated for 24 h in 37°C humidified atmospheric air in 300 ml RPMI 1640 + 0.5% normal human serum (NHS) with or without the addition of 150 pa/ml IL-18. In separate series of experiments, 200 BB-DP or WF islets were incubated for 24 h in 300 ml RPMI 1640 + 0.5% NHS before grafting.
  • NHS normal human serum
  • Nitrite and insulin measurements Nitrite was measured by Griess reagents as previously described (Green et al. Anal. Biochem. 126:131-138 (1982)). The detection limit of the assay was 1 mmol/1, corresponding to 2 pmol/islet. The nitrite level of the corresponding medium without islets was subtracted if the value ofthe islet-free medium was above the detection limit. The experiments were run in the same assay. Intra- and interassay coefficients of variation calculated from 3 points on the standard curve were: 1 mmol/1: 1.6%, 16.3%; 10 mmol/1 : 1.6%, 15.3%; 25 mmol/1: 1.5, 17.0%. Insulin was measured by RIA (Yang et al. Proc.
  • IL-1 inhibited accumulated islet insulin release by 3.4 fold and increased NO production by 4.4 fold, similarly to results obtained using neonatal WF rat islets (IL-1 induced NO production: (WF islets) 9,29 ⁇ 1,21 versus (BB-
  • the grafted islets did not affect IDDM incidence or time of disease onset. On the day of diabetes onset (78 ⁇ 5 days) and on day 17 in the control WK animals, the grafts were excised for immunohistochemistry and [ 35 S]-methionine labeling.
  • Allogeneic transplant controls showed fibrosis, less infiltration of mononuclear cells, MHC class II positive cells (152/mm 2 ), MHC class I positive cells (48/mm 2 ), macrophages (172/mm 2 ), T-helper cells (254/rnm 2 ), cytotoxic T-cells (42/mm 2 ), and insulin content of 100% in the remaining cells.
  • Computerized analysis of 2D gels showed a greater than 2-fold increase in the expression of 18 ofthe 33 proteins altered in vitro by IL-1 exposure. 14 were specific to the syngeneic transplants in the BB-DP animals. 8 proteins specifically changed level of expression.
  • IL-1 induced proteins in vitro specifically seen in islet-grafts during disease occurrence or islet- graft rejection. Proteins that were found to change level of expression in islets after IL-1 incubation in vitro and not found to significantly change level of expression in syngrafted
  • WF islets were denoted specific for disease occurrence if found in syngrafted BB-DP islets or specific for graft-rejection if found in allografted BB-DP islets to WK rats.
  • 28 proteins and in allografted BB-DP islets 29 proteins changed level of expression (data not shown).
  • Proteins exhibiting IL-l ⁇ -induction of synthesis were analyzed by mass spectrometry and microsequencing as described below. Commercially available protein databases were searched for matches, including SWISS-PROT, PIR, NIH, and GENEBANK.
  • Microsequencing Protein spots identified by 2DGE as diabetes-mediated proteins were further characterized by being digested with trypsin in the gel, concentrated, HPLC separated, peak sequenced, and partial sequence compared to known sequences, according to known methods. Results of microsequences are as follows:
  • Protein 22 (peak 22): A Q Y E E L I A N G (D) (M) (SEQ ID NO:5)
  • PEAIKGAWGIDLG SEQ ID NO: 10.
  • This protein has high homology to 75kD glucose regulated protein (GR75, Table 2) (PBP74, P66mot, mortalin); is a constitutive member of hsp70 protein family, not heat-inducible; is ubiquitously expressed in different tissues; comprises a 46 residue leader peptide 75kD processed to 66 kD; is found in mitochondria; is associated with cellular mortality and with antigen presentation.
  • galectin-3 a 27kD protein which is present in several tissues, and known to have a role as a pre-mRNA splicing factor.
  • the amino acid sequence of human gal-3 is shown in FIG. 5 (SEQ ID NO:4).
  • Mass spectroscopy In situ digestion is performed on at least one gel plug including at least one protein spot in at least one gel according to the present invention.
  • Gels are prepared by a modification of the method of Rosenfeld et al. Anal. Biochem. 203:173-179 (1992), as described in Fey et al. Electrophoresis 18:1-12 (1997), both of which references are herein specifically incorporated by reference. Briefly, gels are quickly stained and destained. The protein of interest is obtained by cutting a gel band containing the protein with a scalpel and storing in eppendorf tubes with UHQ water at -20°C.
  • the protein is digested by washing the gel plug for at least 1 hour in 40% acetonitrile/60% digestion buffer until the coomassie stain is removed. This wash removes coomassie stain, gel buffers, SDS and salts. If necessary the wash can be repeated.
  • the gel plug is then dried in a vacuum centrifuge for 20-30 min. until the plug shrinks and becomes white on the surface. Drying time depends on the size and thickness ofthe gel plug. Trypsin (or the enzyme being used is dissolved in digestion buffer and 5 mis added to the gel plug (depending on the amount ofthe protein in the gel to be analyzed (0.1 mg)). Additional digestion buffer is added until the gel plug is almost covered by buffer in the bottom ofthe tube, approximately 10 ml.
  • the gel plug is then incubated at 37°C for 6 hours or overnight, then incubated with 70-100 ml 60% acetonitrile/40% water for 2-6 hours to extract the peptides. The extraction may be repeated to increase recovery. The extract is then lyophilized and dissolved in 30% acetonitrile/2%TFA before analyzing by MALDI-
  • FIGs. 6-48 provide the mass spectroscopic data obtain for the indicated protein of
  • the gels of the BB-DP islet allografts were compared to gels of neonatal BB-DP rat islets with regard to the 82 proteins that significantly changed level of expression in the BB-DP islets after IL-1 incubation in vitro. Sixty-six of the eighty-two proteins were re-identified.
  • neonatal rat islets were transduced in groups of 25 in triplicate with an adeno viral vector ⁇ -galactosidase (Ad ⁇ Gal) at doses of multiplicity of infection (moi) 0, 10, 100, and 1000 pfu per islet.
  • Efficiency of gene transfer was determined by gross inspection and estimated by the percentage of,6- galactosidase positive cells after islet dispersion at 1, 4, 7, and 10 days post-transduction.
  • Islet toxicity was assessed by measuring accumulated insulin levels at each time-point and by assessing insulin release in response to hyperglycemia at 3 and 10 days.
  • the insulin secretory response to glucose obtained by dividing the insulin response to high glucose incubation by the insulin response to low glucose incubation was similar in transduced and non-transduced islets at 3 days at all doses studied (mod 0; 12.7 ⁇ 4.1; moi 10, 14.9 ⁇ 7.9; moi 100, 15.7 ⁇ 0.7; and moi 1,000, 22.3 ⁇ 6.7).
  • the ratios were similar in transduced and non-transduced cells at 10 days post-transduction.
  • EXAMPLE 8 EXPRESSION OF GALECTIN-3 IN THE SPONTANEOUS DEVELOPMENT OF IDDM.
  • Neonatal BB-DP islets stimulated with IL-l ⁇ in vitro showed changes in 82 proteins. 97-98%) of these proteins were identified in all grafts at all time points.
  • Expression levels of graft proteins compared with non-stimulated normal BB-DP islets in vitro showed the following changes: Ofthe 82 proteins which exhibit increased expression when neonatal islet cells are treated with IL-l ⁇ in vitro, 42 were increased in the 7 day transplant islets, 31 were increased with diabetes onset, and 14 were increased in animals which did not develop diabetes. Ofthe proteins increased, 4 of them are only seen at onset of IDDM and not at the timepoints detailed.
  • EXAMPLE 9 CYTOKINE INDUCTION OF IL-1 CONVERTING ENZYME (ICE), INDUCIBLE NITRIC OXIDE SYNTHASE (iNOS), AND APOPTOSIS IN INSULIN- PRODUCING CELLS.
  • Rat insulinoma and pluripotent cells lines RTN-SAH and MSL-G2 were cultured 20 hours with a mixture of cytokines (IL-l ⁇ , TNFo ⁇ , and IFN ⁇ ), RNA isolated, and multiplex PCR analysis (27 cycles) with primers against ICE, iNOS, and SP-1 (a general transcription factor used as a housekeeping control gene for normalization) were formed. Results were normalized to SP-1 mRNA expression, and calculated following gel electrophoresis separation and Phosphorlmager quantification.
  • Two-dimensional (2-D) gel electrophoresis of pancreatic islet proteins can be an important tool facilitating studies of the molecular pathogenesis of insulin-dependent diabetes mellitus.
  • Insulin-dependent diabetes mellitus is caused by an autoimmune destruction ofthe ⁇ -cells in the islets of Langerhans.
  • the cytokine interleukin 1 ⁇ inhibits insulin release and is selectively cytotoxic to ⁇ -cells in isolated pancreatic rat islets.
  • the antigen(s) triggering the immune response as well as the intracellular mechanisms of action of interleukin 1 ⁇ -mediated ⁇ -cell cytotoxicity are unknown. However, previous studies have found an association with alterations in protein synthesis.
  • 2-D gel electrophoresis of islet proteins can lead to 1) the identification of primary antigen(s) initiating the immune destruction of the ⁇ -cells 2) the determination of qualitative and quantitative changes in specific islet proteins induced by cytokines and 3) the determination ofthe effects of agents modulating cytokine action. Therefore, the aim of this study was to create databases of all reproducibly detectable protein spots on 10% and 15% acrylamide 2-D gels of neonatal rat islets (10% & 15% DB), labelled under standardized culture conditions. 1792 spots were present in 5 of 5 gels in the 15% DB, whereas 1373 spots were present in 5 of 5 gels in the 10% DB, yielding a qualitative reproducibility between 75.2% and 91.7%.
  • the average coefficient of variation ofthe percentage of integrated optical density (CV% of %IOD) for spots present in all gel was between 42.4% and 45.1%.
  • the average CV% of %IOD was 35.5%-36.1%.
  • the average CV% of %IOD was 30.2% in the IEF gels, while the average CV% of %IOD was unchanged (45.7%) in the NEPHGE gels.
  • the cytokine interleukin 1 ⁇ inhibits insulin release and is selectively cytotoxic to ⁇ -cells in isolated pancreatic rat islets (Mandrup-Poulsen, T, Diabetologia, in press (1996)). Active protein synthesis is a crucial part of ⁇ -cell destruction, defense and repair after insults such as cytokines.
  • the free radical nitric oxide (NO) has been demonstrated to be an important mediator of the deleterious effects of cytokines on islet ⁇ -cells (Southern, et al., FEBS. Lett. 276:42-44 (1990); Welsh, et al., Endocrinol. 129:3161-3113 (1991); Corbett, et al., J.
  • HSP heat shock proteins
  • HSP32 heme oxygenase
  • HSP70 Helqvist, et al
  • IL-l ⁇ inhibits the synthesis of a number of unknown proteins with molecular weights of 45, 50 (Hughes, et al., J. Clin. Invest. 55:856-863 (1990)), 75, 85, 95 and 120 kDa (Welsh, et al., Autoimmunity 9:33-40 (1991)) in islets.
  • DMEM, RPMI 1640 and Hanks' balanced salt solution (HBSS) were purchased from Gibco, Paisley, Scotland.
  • RPMI 1640 was supplemented with 20 mM HEPES buffer, 100,000 IU/I penicillin and 100 mg/L streptomycin.
  • Authentic recombinant human IL-1 ⁇ was provided by Novo Nordisk Ltd. (Bagsvaerd, Denmark). The specific activity was 400 U/ng (Moelvig, et al., Scand. J. Immunol. 57:225-235 (1990).
  • the following other reagents were used: 2-mercaptoethanol, bovine serum albumin (BSA), Tris HCl, Tris base, glycine, (Sigma, St.
  • Nonidet P-40 (BDH, Poole, UK); ampholytes: pH 5-7 and sodium dodecyl sulphate (Serva, Heidelberg, Germany); agarose (Litex, Copenhagen, Denmark); ethanol (absolute 96%) (Danish Distillers, Aalborg, Denmark); methanol (Prolabo, Brione Le Blanc, France); acetic acid (technical quality, 99% glacial) (Bie & Berntsen, Arhus, Denmark) and X-ray film (Curix RP-2) (AGFA).
  • Islet isolation and culture For the database and assay variation experiments, 12 different islet isolations were performed, 10 for the databases, 1 for intraassay and 1 for interassay analysis. For the studies involving IL-l ⁇ , 3 additional islet isolations were performed. Islets from pancreata of 4 day old inbred Wistar Furth rats (M ⁇ llegard, Lille
  • Islet labelling After 24 h in culture, the 150 islets were harvested, washed twice in HBSS and labelled for 4 h in 200 ⁇ l methionine-free Dulbecco's modified Eagle's's medium (DMEM) with 10% NHS dialysed for amino acids, and 200 ⁇ Ci ( 35 S)-methionine.
  • DMEM Dulbecco's modified Eagle's's medium
  • the amount of ( 35 S)-methionine incorporation was quantitated in duplicate by adding 10 ⁇ g BSA (0.2 ⁇ g/ml H 2 0) as a carrier to 5 ⁇ l of a 1:10 dilution of each sample, followed by 0.5 ml of 10% TCA. This was left to precipitate for 30 min at 40 °C before being filtered through 0.25 ⁇ m filters.
  • the HAWP filters were dried and placed into scintillation liquid for counting. 2-D gel electrophoresis. The procedure was essentially as described by O'Farrell et al, Cell 72:1133-1142 (1977) and Fey, S.J.
  • first dimension gels contained 4% acrylamide, 0.25% bisacrylamide and ampholytes (the actual ratio depending upon the batch) and were 175 mm long and 1.55 mm in diameter. Equal numbers of counts (10 6 cpm) of each sample were applied to the gels. In case of lower amounts of radioactivity it was necessary to regulate the exposure time ofthe gel so that comparable total optical densities were obtained.
  • the samples were analyzed on both isoelectric focusing (IEF; pH 3.5-7) and non-equilibrium pH-gradient electrophoresis (NEPHGE; pH 6.5-10.5) gels.
  • IEF gels were prefocused for approximately 4 h at 140 ⁇ A/gel (limiting current), the sample was then applied and focused for 18 h at 1200 V (limiting voltage).
  • NEPHGE gels were focused for approximately 6.5 h using 140 ⁇ A/gel and 1200 V as the limiting parameters.
  • Second dimension gels 1 mm thick, 200 mm long and 185 mm wide contained either 15% acrylamide and 0.075% bisacrylamide or 10% acrylamide and 0.05% bisacrylamide and were run overnight. After electrophoresis, the gels were fixed in 45% methanol and 7.5% acetic acid for 45 min and treated for fluorography with Amplify® for 45 min before being dried. The gels were placed in contact with X-ray films and exposed at -70 °C for 1 to 40 days. Each gel was exposed for at least 3 time periods to compensate for the lack of dynamic range of X-ray films. Determination of MW and pi. Molecular weights ofthe proteins were determined by comparison with standard gels (Fey, S.J.
  • the study comprised three different series of analyses: database, intra- and interassay analysis.
  • IEF and NEPHGE gels were run using 10% and 15% acrylamide in the second dimension. This gave us four subgroups: 10% IEF; 15% IEF; 10% NEPHGE; 15% NEPHGE.
  • the approximate MW range of detection were between 20 and 250 kDa, while the approximate range of detection was between 6 and 125 kDa on 15% acrylamide gels. Consequently, proteins with a MW between 20 and 125 kDa were included in both databases, whereas proteins with lower and higher MW were particular to 15% and 10% DBs, respectively.
  • the databases were based on 10 different isolates analyzed in one set of gels. After 2-D gel electrophoresis, 5 gels with the best technical quality and with comparable optical densities were chosen for computer analysis. Before computer analysis, one gel in each subgroup was arbitrarily selected to be the "master gel” used for comparison with the other 4 database gels, the 5 intraassay gels and the 5 interassay gels. The database "master gel” was used as a master for intra- and interassay analysis to ensure that a given spot had the same match number in the three series of analyses. Data from the "master gel” are only included in the database analysis. The "master gel” was from the same isolate in all 4 subgroups, whereas the identity ofthe isolates producing the 4 other database gels varied slightly from subgroup to subgroup (Table 3).
  • NEPHGE gels of IL-l ⁇ exposed islets previously analyzed visually (Andersen, et al, Diabetes 44:400-401 (1995)), were matched to the 10% IEF and NEPHGE DBs.
  • the database spots present in all gels were ranked in increasing order of CV% of %IOD, resulting in similar sigmoid-shaped curves for spots in all four database subgroups.
  • 30% of the spots had a CV% that was lower than 29.7% -32.5%
  • 50% ofthe spots had a CV% that was lower than 37.8% - 42.8 %
  • 90% ofthe spots had a CV% that was lower than 68.4% - 80.6%.
  • the slopes ofthe curves indicate that the 5%-10% spots with the highest CV% contribute significantly to the average CV% of %IOD. This is supported by the fact that the median values ofthe database subgroups are 2.3% to 5.5% lower than the mean values ofthe subgroups (Table 7).
  • Each litter of newborn rats used for islet isolation typically consists of 8-12 pups with a varying frequency of males and females. Since comparison of Coomassie Blue- stained gels of liver proteins from male and female outbred Wistar rats revealed quantitative differences in 7 of 250 analyzed spots and since six proteins were found exclusively in males and one protein exclusively in females (Steiner, et al, Electrophoresis 75:1969-1976 (1995)), it is likely that some of the proteins in our database are gender- specific or gender-regulated. Consequently, it is possible that the high variation of some of the spots in our databases could be reduced if we had chosen to construct separate databases of islets from male and female rats.
  • Detection of islet proteins Not all spots detected in our databases will represent different protein entities, since some spots can represent modifications (e.g. acetylation, methylation, phosphorylation or carbamylation) of other proteins. However, the detected number of spots is an underestimation of the total number of islet proteins, since the protein database does not include proteins below the limit of sensitivity, proteins not containing methionine, proteins with a molecular weight below 6 kDa or above 250 kDa or proteins with a pH below 3.5 or above 10.5. Further, about 40% ofthe spots with IODs above limits of detection have previously been estimated to be missed because they are obscured by other spots (Garrels, J Biol. Chem. 254:5269-5282 (1989)). Finally, the 4h labelling period favours the labelling of proteins with high synthesis rates, whereas longer labelling periods could be required to produce databases where all proteins are in steady- state.
  • modifications e.g. acetylation, methylation, phosphoryl
  • the fraction of the CV% that is attributable to biological variation should be given by the difference in CV% between database and intraassay analysis for a given spot.
  • approximately one third ofthe average CV% of %IOD is due to biological variation. Effects of IL-1 fi on islet protein expression. IL-1 ⁇ altered the expression of 105 so far unidentified proteins.
  • IL-1 ⁇ mechanism of action on islet cells is not fully clarified, but three distinct groups of proteins might play important roles: proteins participating in signal- transduction and proteins encoded by so-called early response and late response genes (Eizirik, et al, Diabetologia 59:875-890 (1996)).
  • IL-l ⁇ -induced signal transduction in target cells is thought to involve four major signalling pathways: nuclear factor- ⁇ b, the stress-activated protein kinases (SAPK/JNK), protein kinase C and tyrosine kinase (Mandrup-Poulsen, T., Diabetologia 39:1005-1029 (1996); Eizirik, et al, Diabetologia 59:875-890 (1996)).
  • SAPK/JNK stress-activated protein kinases
  • Mandrup-Poulsen T., Diabetologia 39:1005-1029 (1996)
  • the early response genes activate specific genes with possible deleterious (iNOS, cycloxygenase-2 and lipoxygenase) and protective (HSP72, haem oxygenase, Mn superoxide dismustase) action on islets (Mandrup-Poulsen, T., Diabetologia, 59. 1005-1029 (1996); Eizirik, et al, Diabetologia 59:875-890 (1996)).
  • iNOS cycloxygenase-2 and lipoxygenase
  • HSP72 haem oxygenase
  • Mn superoxide dismustase mandrup-Poulsen, T., Diabetologia, 59. 1005-1029 (1996); Eizirik, et
  • DB DB gel DB 10 (master): 1 gel DBlO (master): 1 gel DBl: 0.947 gel DBl: 1.660 gel DB4: 0.358 gel DB7: 3.215 gel DB6: 1.167 gel DB8: 2.959 gel DB8: 1.145 gel DB9: 1.197
  • the databases were based on 10 different isolates analyzed in one set of gels, while interassay analysis consisted of 10 gels ofthe same sample analyzed in one set of gels and interassay analysis was based on the analysis ofthe same sample run in 10 consecutive sets of gels on different days.
  • one gel in each database subgroup was arbitrarily selected to be the "master gel" used for comparison with the other 4 database gels, the 5 intraassay gels and the 5 interassay gels.
  • the numbers (1-10) of the isolates/replicates chosen are indicated in the Table.
  • the correction factors between the total optical densities ofthe master and non-master gels were calculated in the Biolmage® program following analysis.
  • correction factors were calculated between an arbitrarily selected gel and the 4 other gels. Comparison cannot be made between subgroups because gels with a correction factor of 1 not necessarily have the same intensity.
  • Table 4 Reproducibility of spot detection in 15% IEF and NEPHGE 2-DGE DB of neonatal rat islet proteins. total spots in 5 of 5 gels spots in 4-5 of 5 gels spots in 3-5 of 5 gels spots in 2-5 of 5 gels no. of
  • IEF IEF analysis spots no. % analysis spots no. % gel IA2 1289 1085 84.2 gel IE3 1319 1082 82.0 gel IA3 1337 1085 81.2 gel IE4 1348 1082 80.3 gel IA4 1289 1085 84.2 gel IE8 1333 1082 81.2 gel IA6 1303 1085 83.3 gel IE9 1342 1082 83.2 gel lAlO 1326 1085 81.8 gel lElO 1300 1082 80.6 avg ⁇ SD 82.9 ⁇ 1.4 avg ⁇ SD 81.5 ⁇ 1.2
  • Table 7 Average coefficients of variance of % integrated optical density of spots detectable in 5 of 5 gels in databases and replicate 2-D gels of neonatal rat islet proteins.
  • the average coefficient of variance was calculated from the CV% of %IOD of all spots present in 5 of 5 gels in each subgroup of analysis. Results are presented as means ⁇ SD (left column) and as medians (ranges). The number of spots in 5 of 5 gels in each subgroup is shown in Tables 2-4. For details of design databases and replicate analyses, please see Materials and Methods.
  • the match numbers ofthe table correspond to the match numbers ofthe 1 0% IEF and NEPHGE DBs
  • the spot numbers given in a previous paper (l 2) to spots aftered in expression by IL-1 B are indicated in parenthesis * indicates that the spot was not previously found to be aftered m expression by IL-1 B alone but by other expe ⁇ mental conditions (1 2)
  • the analysis was based on 5 DB gels and 3 IL-LB gels and P ⁇ 001 was chosen as level of significance
  • the %IOD ratio expresses the average %IOD of IL-1 B gels/average %IOD of DB gels
  • a ratio above 1 indicates that the spot is upregulated DN indicates that the spot is synthesized de novo by IL- 1 B
  • the match numbers ofthe table correspond to the match numbers ofthe 1 0% IEF and NEPHGE DBs
  • the spot numbers given in a previous paper (l 2) to spots aftered in expression by IL-1 B are indicated in parenthesis * indicates that the spot was not previously found to be aftered in expression by IL-1 B alone but by other expe ⁇ mental conditions (1 2)
  • the analysis was based on 5 DB gels and 3 IL-LB gels and
  • the %IOD ratio expresses the average %IOD of IL-1 B gels average %IOD of DB gels Thus, a ratio above 1 indicates that the spot is upregulated DN indicates that the spot is synthesized de novo by IL-1 B
  • the match numbers ofthe table correspond to the match numbers ofthe 1 0% IEF and NEPHGE DBs
  • the spot numbers given in a previous paper (l 2) to spots aftered in expression by IL-1 B are indicated in parenthesis * indicates that the spot was not previously found to be aftered in expression by IL-1 B alone but by other experimental conditions (1 2)
  • the analysis was based on 5 DB gels and 3 IL-LB gels and P ⁇ 001 was chosen as level of signfficance
  • the %IOD ratio expresses the average %IOD of IL-1 B gels average %IOD of DB gels
  • a ratio above 1 indicates that the spot is upregulated DN indicates that the spot is synthesized de novo by IL-1 B
  • the match numbers ofthe table correspond to the match numbers of the 1 0% IEF and NEPHGE DBs
  • the spot numbers given in a previous paper (l 2) to spots aftered in expression by IL-1 B are mdicated in parenthesis * indicates that the spot was not previously found to be aftered in expression by IL- 1 B alone but by other expe ⁇ mental conditions (1 2)
  • the analysis was based on 5 DB gels and 3 IL-LB gels and P ⁇ 001 was chosen as level of signfficance
  • the %IOD ratio expresses the average %IOD of IL-1 B gels average %IOD of DB gels
  • a ratio above 1 indicates that the spot is upregulated DN indicates that the spot is synthesized de novo by IL-1 B
  • the match numbers ofthe table correspond to the match numbers ofthe 1 0% IEF and NEPHGE DBs
  • the spot numbers given in a previous paper (12) to spots aftered in expression by IL-1 B are indicated in parenthesis * indicates that the spot was not previously found to be aftered in expression by IL-1 B alone but by other expe ⁇ mental conditions (1 2)
  • the analysis was based on 5 DB gels and 3 IL-LB gels and P ⁇ 001 was chosen as level of signfficance
  • the %IOD ratio expresses the average %IOD of IL-1 B gels average %IOD of DB gels
  • a ratio above 1 indicates that the spot is upregulated DN indicates that the spot is synthesized de novo by IL-1 B
  • the match numbers ofthe table correspond to the match numbers ofthe 1 0% IEF and NEPHGE DBs
  • the spot numbers given in a previous paper (l 2) to spots aftered in expression by IL-1 B are indicated in parenthesis * indicates that the spot was not previously found to be aftered in expression by IL- 1 B alone but by other expe ⁇ mental conditions (1 2)
  • the analysis was based on 5 DB gels and 3 IL-LB gels and P ⁇ 001 was chosen as level of signfficance
  • the %IOD ratio expresses the average %IOD of IL-1 B gels/average %IOD of DB gels
  • a ratio above 1 indicates that the spot is upregulated DN indicates that the spot is synthesized de novo by IL-1 B
  • ADDRESSEE Jorge A. Goldstein, Sterne, Kessler, Goldstein and Fox, P.L.L.C.

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Abstract

L'invention concerne des protéines médiatrices du diabète, protectrices et néfastes, les polynucléotides codant pour lesdites protéines, des animaux transgéniques exprimant une protéine médiatrice du diabète, des méthodes de criblage de médicaments qui permettent d'identifier un composé test capable de modifier l'expression desdites protéines, ainsi que des méthodes qui permettent de prévenir ou d'améliorer le diabète en administrant un composé capable de modifier l'expression d'une protéine médiatrice du diabète.
PCT/IB1997/001627 1996-10-25 1997-10-24 Proteines mediatrices du diabete et leurs utilisations therapeutiques WO1998020124A2 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CA002269646A CA2269646A1 (fr) 1996-10-25 1997-10-24 Proteines mediatrices du diabete et leurs utilisations therapeutiques
US09/297,040 US7078375B1 (en) 1996-10-25 1997-10-24 Diabetes-mediating proteins and therapeutic uses thereof
EP97947839A EP0934409A2 (fr) 1996-10-25 1997-10-24 Proteines mediatrices du diabete et leurs utilisations therapeutiques
JP52118298A JP2002504806A (ja) 1996-10-25 1997-10-24 糖尿病媒介タンパク質およびその治療的使用
AU54070/98A AU5407098A (en) 1996-10-25 1997-10-24 Diabetes-mediating proteins and therapeutic uses thereof
US11/488,184 US7531323B2 (en) 1996-10-25 2006-07-18 Diabetes-mediating proteins and therapeutic uses thereof

Applications Claiming Priority (8)

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US2932496P 1996-10-25 1996-10-25
US60/029,324 1996-10-25
US3018696P 1996-11-05 1996-11-05
US3008896P 1996-11-05 1996-11-05
US60/030,186 1996-11-05
US60/030,088 1996-11-05
US89709897A 1997-07-18 1997-07-18
US08/897,098 1997-07-18

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US11/488,184 Continuation US7531323B2 (en) 1996-10-25 2006-07-18 Diabetes-mediating proteins and therapeutic uses thereof

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000024422A1 (fr) * 1998-10-27 2000-05-04 Suntory Limited Procede de production de regulateurs glycolytiques du metabolisme
WO2000040722A2 (fr) * 1999-01-07 2000-07-13 Incyte Pharmaceuticals, Inc. Genes de synthese de l'insuline
EP1179175A2 (fr) * 1999-05-14 2002-02-13 Karolinska Innovations AB Materiaux et procedes se rapportant au diagnostic de maladie
WO2002097434A1 (fr) * 2001-05-29 2002-12-05 Syddansk Universitet Proteines utiles dans l'analyse proteomique du diabete
WO2003020963A2 (fr) * 2001-09-05 2003-03-13 Pride Proteomics A/S Proteines impliquees dans le diabete de type 2
WO2003078456A2 (fr) * 2002-03-20 2003-09-25 Syddansk Universitet Proteines humaines mediatrices du diabete

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANDERSEN, H.U. ET AL.: "Two-dimensional gel electrophoresis of rat islet proteins. Interleukin 1Beta-induced changes in protein expression are reduced by L-arginine depletion and nicotinamide" DIABETES, vol. 44, no. 4, April 1995, pages 400-407, XP002062766 *
CHRISTENSEN, U.B. ET AL.: "Protein expression at diabetes onset in BB-rats differs from that seen during islet allograft rejection" DIABETOLOGIA, vol. 38, no. suppl. 1, 1995, page A85 XP002062765 & 31st annual meeting of the european association for the study of diabetes, STOCKHOLM, SWEDEN, September 12-16 1995 *
KARLSEN, A.E. ET AL.: "Identification and characterization of proteins involved in cytokine mediated beta-cell destruction and insulin-dependent diabetes mellitus" CYTOKINE, vol. 9, no. 11, November 1997, page 912 XP002062768 *
POCIOT, F. ET AL.: "A comprehensive approach to identifying new susceptibility genes to insulin-dependent diabetes mellitus: combining proteome and genome analysis" CYTOKINE, vol. 9, no. 11, November 1997, page 899 XP002062767 & Fifth annual conference of the international cytokine society, Lake Tahoe, USA. November 9-13 1997, *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6620424B1 (en) 1998-10-27 2003-09-16 Suntory Limited Process for producing glycolytic metabolism regulators
WO2000024422A1 (fr) * 1998-10-27 2000-05-04 Suntory Limited Procede de production de regulateurs glycolytiques du metabolisme
WO2000040722A2 (fr) * 1999-01-07 2000-07-13 Incyte Pharmaceuticals, Inc. Genes de synthese de l'insuline
WO2000040722A3 (fr) * 1999-01-07 2001-11-29 Incyte Pharma Inc Genes de synthese de l'insuline
EP1179175A2 (fr) * 1999-05-14 2002-02-13 Karolinska Innovations AB Materiaux et procedes se rapportant au diagnostic de maladie
AU773329B2 (en) * 1999-05-14 2004-05-20 Proteomedica Ab Materials and methods relating to disease diagnosis
WO2002097434A1 (fr) * 2001-05-29 2002-12-05 Syddansk Universitet Proteines utiles dans l'analyse proteomique du diabete
EP2161578A1 (fr) * 2001-05-29 2010-03-10 Pride Proteomics A/S Protéines dans l'analyse du protéome du diabète
WO2003020963A2 (fr) * 2001-09-05 2003-03-13 Pride Proteomics A/S Proteines impliquees dans le diabete de type 2
WO2003020963A3 (fr) * 2001-09-05 2004-03-25 Pride Proteomics As Proteines impliquees dans le diabete de type 2
CN100415899C (zh) * 2001-09-05 2008-09-03 普赖德普罗特奥米克斯公司 Ii型糖尿病蛋白质
US7470542B2 (en) 2001-09-05 2008-12-30 Pride Proteomics A/S Proteins in type 2 diabetes
WO2003078456A2 (fr) * 2002-03-20 2003-09-25 Syddansk Universitet Proteines humaines mediatrices du diabete
WO2003078456A3 (fr) * 2002-03-20 2004-01-15 Univ Syddansk Proteines humaines mediatrices du diabete
US7521193B2 (en) 2002-03-20 2009-04-21 Pride Proteomics A/S Human diabetes-mediating proteins

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EP0934409A2 (fr) 1999-08-11
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AU5407098A (en) 1998-05-29
CA2269646A1 (fr) 1998-05-14
JP2008195721A (ja) 2008-08-28

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