WO2007110230A2 - Proteines secretees en tant que marqueurs precoces et cibles de medicaments pour auto-immunite, tumorigenese et infections - Google Patents

Proteines secretees en tant que marqueurs precoces et cibles de medicaments pour auto-immunite, tumorigenese et infections Download PDF

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WO2007110230A2
WO2007110230A2 PCT/EP2007/002699 EP2007002699W WO2007110230A2 WO 2007110230 A2 WO2007110230 A2 WO 2007110230A2 EP 2007002699 W EP2007002699 W EP 2007002699W WO 2007110230 A2 WO2007110230 A2 WO 2007110230A2
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protein
pancreatic
disease
chosen
lipase
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PCT/EP2007/002699
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WO2007110230A3 (fr
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Evie Melanitou-Mcclymont
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Institut Pasteur
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Priority claimed from US11/389,261 external-priority patent/US20070224638A1/en
Priority claimed from EP06290485A external-priority patent/EP1840573A1/fr
Application filed by Institut Pasteur filed Critical Institut Pasteur
Publication of WO2007110230A2 publication Critical patent/WO2007110230A2/fr
Publication of WO2007110230A3 publication Critical patent/WO2007110230A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • the present invention is in the domain of diagnosis, especially in the domain of human diagnosis, of autoimmune diseases, cancers and infections.
  • T1D type 1 diabetes
  • Autoimmune diabetes can occur at any age, but is most commonly diagnosed in childhood In humans it is characterized by absolute insulin-dependency and the association of disease with the presence of certain alleles of the Human Leukocyte Antigen (HLA) class Il genes, within the major histocompatibility complex (MHC).
  • HLA Human Leukocyte Antigen
  • MHC major histocompatibility complex
  • the autoimmune etiology is characterized by infiltration of the islets of Langerhans by immune cells (insulitis) and serum autoantibodies ⁇ Bottazzo, 1986 ⁇ .
  • the genetic transmission of T1D corresponds to a multigenic and multifactorial inheritance, which is characteristic of complex characters where environmental factors play a role.
  • T1D in monozygotic (MZ) twins shows a cumulative risk from birth to age 35 years reaching 70% This degree of familial clustering is consistent with a significant genetic input to the disease
  • susceptibility genes have incomplete penetrance, i e an individual who is genetically programmed for T1D does not always develop the clinical condition.
  • This latter observation shows that environmental factors influence the genetic component of the disease Interestingly a high percentage of subjects with T1D have also other autoimmune diseases Fifteen to 30% of diabetics develop also thyroid disease, 4-9% develops celiac disease and 0.5% has Addison's disease ⁇ Reviewed by Barker, 2006 ⁇ .
  • T1 D Recessive, dominant and multifactorial hypotheses have been advanced in the past, however, it is now commonly accepted that environmental factors are also involved. It has been suggested that the rise in the incidence of T1 D worldwide may be due to environmental influences such as viruses. Viruses have been considered as an important factor triggering T1 D in genetically susceptible humans and in many animal models. The mechanism of viral infection may be due to its direct or indirect effect on the ⁇ cells of the pancreas or directly on the lymphoid cells. Rotavirus for example, has been suspected as the cause of a clinically silent pancreatic infection in patients with rapid disease onset that may have led to a rapid T cell mediated loss of ⁇ cells in the islets. Rotaviruses can infect islets in tissue culture, but these viruses are not infectious unless they are activated by trypsin secreted by the exocrine pancreas.
  • islet ⁇ cells produce a wide range of antiviral responses for host protection that may stimulate immune mediated islet destruction.
  • viral induction of the cytokine, interferon alpha (IFN ⁇ ) is strongly implicated in the disease process.
  • IFN ⁇ interferon alpha
  • IFN ⁇ can accelerate and induce autoimmune diabetes when the B7.1 human co-stimulatory molecule, under the rat insulin promoter, is expressed as a transgene in islets, in mice.
  • the present invention is based on the identification of genes implicated in the early steps of autoimmune destruction. These genes are also of interest in order to unravel the mechanisms responsible for the autoimmune condition. It is proposed that genes involved at an early stage might be responsible for immune events leading to the inflammatory autoimmune response and might also respond to environmental factors such as infections.
  • the inventor has established a first early phenotypic marker ⁇ Melanitou, 2004, 2005 ⁇ , early insulin auto-antibody, allowing to select autoimmune prone individual mice and to perform a systematic differential gene expression search by high throughput analysis of the transcriptome.
  • the data obtained by these experiments have demonstrated the existence of genes that are differentially regulated at an early stage, prior to clinical signs, among disease prone animals, in comparison with non disease prone individuals.
  • the inventor has now identified these differentially expressed genes. These genes seem to be earlier markers of a disease-prone state than the previously known markers and they are specific to the pre-inflammatory stage of the disease. Whereas insulin auto-antibodies reflect an immune state of the patient, the markers now identified by the inventor have the potential to reflect the molecular mechanisms initiating the pre-inflammatory process.
  • genes identified code for proteins located in the extra cellular space or for proteins which, according to functional analysis, are potentially secreted. They present the potential to be used as diagnostic biomarkers for type 1 diabetes or other autoimmune diseases during the pre-inflammatory stages. Due to their early differential expression they have also a potential role as therapeutic targets for autoimmunity and other diseases that are preceded by a pre-inflammatory stage (certain cancers and infections for example).
  • Secreted proteins have indeed properties that lend themselves to use as therapeutic agents or targets. They are accessible to various drug delivery mechanisms, because they are within the extracellular space.
  • This invention concerns 32 genes coding for proteins located in the extracellular space or proteins potentially secreted and suitable for use as diagnostic biomarkers for type 1 diabetes or other autoimmune diseases during the pre-inflammatory stages. These proteins are also suitable therapeutic targets since the corresponding coding genes are differentially expressed in early pre- inflammatory stages prior to autoimmune destruction observed in type 1 diabetes. Some of these genes are also implicated in certain cancers and infectious diseases at the pre-inflammatory stages.
  • a particularly preferred gene according to the present invention is the gene encoding Osteopontin.
  • the present invention relates to an in vitro method for the early diagnosis of a disease having a pre-inflammatory phase, in a mammal, prior to any clinical signs in connection with said disease.
  • the method comprises the steps of: a) measuring the level of a marker in a body fluid or a tissue sample obtained from said mammal, b) comparing the measured level to a reference level for said marker, and c) diagnosing the later onset of said disease if the measured level is significantly divergent from the reference level.
  • the invention also relates to an in vitro method for diagnosing a disease-prone state in a mammal, prior to any clinical signs of said disease, wherein the disease has a pre-inflammatory phase.
  • a disease-prone state reflects a risk of developing the disease.
  • Said method comprises the steps of: a) measuring the level of a marker in a body fluid or a tissue sample obtained from said mammal, b) comparing the measured level to a reference level for said marker, and c) diagnosing a disease-prone state if the measured level is significantly divergent from the reference level.
  • divergent it is meant that the measured level is greater than or less than the reference level.
  • significantly divergent it is to be understood that the difference between the measured level and the reference level is significant from a statistical point of view, that is the difference is greater that the standard deviation of the measured levels. The difference is thus preferably greater that the error inherent in the measurement and greater than the variations observed between individuals, independently of the disease.
  • the marker used in the diagnostic methods of the invention is preferably a marker protein, which is chosen amongst the following murine proteins, encoded by the 32 genes of the invention:
  • Tff2 Tff2 (trefoil factor 2, also known as SP; mSP (spasmolytic protein 1), encoded by U78770),
  • Klk6 (kallikrein 6 also known as KaI; KIkI , mGk-6 and 0610007D04Rik, encoded by M13500),
  • Rib1 pancreatic ribonuclease also known as Rib-1 and AI574248, encoded by X60103
  • - Klk5 (kallikrein 5 or mGK-5, encoded by Y00500), - Mud (also known as Mucin or episialin, encoded by M84683),
  • nucb2 nucleobindin 2, encoded by AJ222586
  • Pnliprp2 pancreatic lipase-related protein 2, encoded by M30687
  • Pla2g1 b phospholipase A2, group IB, pancreas, encoded by AI327450
  • Amy1 (amylase 1 , salivary, encoded by J00356)
  • chymotrypsinogen B1 also known as 2200008D09Rik; encoded by AA590358
  • markers are chosen amongst the following murine proteins: Spp1 , Pap, Reg3a, Reg2, CeI, Reg1 , Tff2, Clps, Klk9, Klk6, Rib1 , KIkS, Mud , Cckar, Pnliprp2, Pla2g1 b, EIaI , Ela2, 2210010C04Rik, Pnliprpi , Itmapi , Sycn, Amy1 and Ctrbi , and their respective mammalian orthologs.
  • a particularly preferred marker is the murine protein Spp1 or one of its mammalian orthologs.
  • the marker chosen may also be DNA or RNA transcript corresponding to one of these proteins.
  • Ortholog genes are defined as genes in different species that evolved from a common ancestral gene by speciation. They can be traced evolutionarily through different species. By comparing the ortholog sequences of a specific gene between many species, the amino acid sequences which are conserved can be determined. These highly conserved sequences provide information on which amino acids are essential to the protein structure and function. Normally, orthologs retain the same function in the course of evolution.
  • the measured level of the marker is superior to the level of the reference. The difference between the measured level and reference level may significantly vary depending on the marker, the level in a disease-prone individual is however greater than the limits of detection, and above the cut-off line. For some of the markers, the measured level may be for example at least 5% superior to the reference level, preferably at least 10% or 20% superior, depending on the reference level.
  • the measured level is at least 3 times the reference level, generally at least 6 times, and in the majority of the cases at least 10 times higher than the reference level.
  • the reference level of a marker in a body fluid or tissue of a given mammal is the mean level of said marker in the same fluid or tissue, measured in healthy individuals having no known predisposition to the disease.
  • the reference level of a RNA or DNA marker in a body fluid or a tissue is also measured in healthy individuals having no known predisposition to the disease.
  • the level of a protein may be expressed as a concentration of said protein in a body fluid or a tissue; it may also be expressed as a relative abundance of said protein, with respect to another compound of the fluid or tissue.
  • the level of the protein may for example be expressed as a number of mole per mole of albumin (especially for expressing the level of a marker in peripheral blood), or as a number of mole per mole of creatinin (especially for expressing the level of a marker in urine).
  • the sole detection of the marker in a body fluid or a tissue is indicative of a disease-prone state or indicative of a disease, without comparison of this level to a reference level.
  • some of these markers might be very low or not expressed at a normal state; therefore, their sole presence in a body fluid (or in a tissue) is an indication of a pre-inflammatory stage.
  • Such markers are for example Pap, Reg3a, Cckar, Ang and Spp1 and their respective mammalian orthologs, especially their human orthologs.
  • a preferred marker is Spp1 or its human ortholog or another mammalian ortholog.
  • the reference level in a furter embodiment is the level of the chosen marker previously measured in the same mammal, or the mean of several levels previously measured.
  • a modification, either increase or decrease, of the level of at least 5%, preferably at least 10, 20, 50, 100 percent or more with respect to the previously measured level(s), is indicative of a disease having a pre-inflammatory phase and/or indicative of a disease-prone state.
  • the previous measure of the level is preferably at least one week before, preferably at least one month, six months or one year.
  • the methods of the invention also encompass the detection in a body fluid of a mammal to be diagnosed, of auto-antibody directed against one of the following murine proteins: Spp1 , Pap, Reg3a, Reg2, CeI, Reg1 , Tff2, Clps, Klk9, Klk6, Rib1 , Klk5, Mud , Cckar, Ggh, Ang, Nucb2, Pnliprp2, Pla2g1 b, EIa 1 , Ela2, 2210010C04Rik, Pnliprpi , Itmapi , Vtn, C1qb, Sycn, Amy1 , Ctrbi , 1110002O23Rik, 1810014L12Rik or their respective mammalian orthologs.
  • Such autoantibodies are generally absent in healthy mammals.
  • Example 5 illustrates this embodiment, with the particularly preferred protein Spp1 (Osteopontin).
  • Spp1 Ostopontin
  • Preferred mammals to be diagnosed according to the methods of the invention are mammal suspected of a predisposition to the disease.
  • the predisposition may also be suspected from a genetic analysis.
  • Autoimmune diseases are characterized by a pre-inflammatory phase, these diseases are thus particularly preferred in the context of the present invention.
  • Examples of such diseases which can advantageously be diagnosed early by the methods of the invention are inter alia type 1 diabetes mellitus, multiple sclerosis, rheumatoid arthritis, collagen induced arthritis and autoimmune hepatitis.
  • a predisposition to one of these diseases may also be diagnosed or prognosed according to the methods of the invention.
  • markers to be used in the methods of the invention are proteins chosen in the group consisting of Spp1 , Reg 1, Reg2, CeI and Cckar and their mammalian orthologs, especially their human orthologs: Osteopontin (OPN), Lithostathine (REG1A), Regenerating protein I beta, Carboxyl ester lipase (bile salt-stimulated lipase) (CEL) and Cholecystokinin A receptor.
  • a particularly preferred marker to be used in the methods of the invention is Spp1 or its mammal orthologs, especially its human ortholog Osteopontin (OPN).
  • a particularly preferred marker to be used in the methods of the invention is Spp1 or its mammal orthologs, especially its human ortholog Osteopontin (OPN).
  • a particularly preferred marker to be used in the methods of the invention is Spp1 or its mammal orthologs, especially its human ortholog Osteopontin (OPN).
  • Type 1 diabetes mellitus is a particularly preferred embodiment of the diagnostic methods of the invention. According to said methods, it is indeed possible to define the propensity to the further onset of type 1 diabetes before any clinical sign of insulitis or pre-insulitis. Therefore, this disease may be diagnosed very early according to the invention and thus the treatment can be undertaken before any damage of the islet ⁇ cells of the patient. This early treatment delays the onset of the disease and improves the quality of life of the patient.
  • the diagnostic methods of the invention may also be carried out in association with the detection of the presence of insulin auto-antibodies in the peripheral blood of the diagnosed mammal. Indeed, the methods of the invention may be combined with the detection of said auto-antibody.
  • the mammal to be diagnosed is primarily tested for the presence of insulin auto-antibody before being subjected to the diagnostic methods of the invention.
  • the mammals subjected to a method of the invention are preferably mammals which have been previously been tested as negative for the presence of insulin auto-antibody. They can alternatively be positive for insulin auto-antibody.
  • Another possibility is to carry out the detection of the presence of insulin auto-antibody after having carried out the diagnostic methods of the invention.
  • the markers of the invention indeed appear as earlier markers of the disease than insulin auto-antibodies.
  • the markers of the invention are capable of distinguishing the beginning of a pre-inflammatory phase in individuals who are negative for the presence of insulin auto-antibodies.
  • the pre-inflammatory phase corresponds to the time of possible tissue disorganization, in certain cancers, such as for example pancreatic cancer, breast cancer, renal cancer, colorectal cancer, ovarian cancer and gastric cancers.
  • the pre-inflammatory stage of these cancers is usually difficult to detect, rendering the diagnostic methods of the invention particularly advantageous. It is indeed well known that most cancers, if detected sufficiently early, may be treated with a high chance of recovery.
  • the disease which is to be diagnosed is an infectious disease. Indeed, several important functions during an infectious challenge (e.g.
  • phagocytosis intracellular killing of ingested micro organisms, and immunoregulatory activities
  • phagocytosis are performed by cells of the mononuclear phagocytes system, generating a pre-inflammatory phase.
  • infectious diseases with a pre-inflammatory phase are otitis due to Gram negative bacilli such as Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus species, Streptococcus species and other. Viral infections are another example.
  • the methods may also be carried out in order to diagnose a risk of cystic fibrosis, to diagnose the pre-inflammatory phase of cystic fibrosis.
  • a particularly preferred marker to be used in the methods of the invention is EIaI or its mammalian orthologs, especially its human ortholog elastase 1 (ELA1 ).
  • EVA1 human ortholog elastase 1
  • the methods of the invention allow to diagnose a disease or a predisposition to a disease having a pre-inflammatory phase very early, before any clinical symptom of said disease.
  • the diagnostic methods of the invention may be performed as early as on an infant of 2 years old, or even before. The diagnosis is thus achieved before the disease becomes symptomatic, preferably at the pre-inflammatory stage before any injury generated by the disease.
  • the marker used in the diagnostic methods of the invention is preferably a protein chosen in the group consisting of the following murine proteins: Spp1 , Pap, Reg3a, Reg2, CeI, Reg1 , Tff2, Clps, Klk9, Klk6, Rib1 , Klk5, Mud , Cckar, Ggh, Ang, Nucb2, Pnliprp2, Pla2g1 b, EIaI , Ela2, 2210010C04Rik, Pnliprpi , Itmapi , Vtn, C1qb, Sycn, Amy1 , Ctrbi , 1110002O23Rik, 1810014L12Rik and their respective mammalian orthologs.
  • the marker is to be chosen amongst the following murine proteins Spp1 , Pap, Reg3a, Reg2, CeI, Reg1 , Tff2 and Clps, and their respective orthologs in other mammals.
  • the chosen marker is thus a protein implicated in tissue regeneration or integrity.
  • a particularly preferred marker is Spp1 or one of its mammalian orthologs.
  • the marker is to be chosen amongst the following murine proteins Spp1 , Klk9, Klk6, Rib1 , Klk5, Mud , Cckar, Ggh, Ang and Nucb2 and their respective orthologs in other mammals.
  • the chosen marker is thus a protein implicated in tumorigenesis.
  • a particularly preferred marker is Spp1 or one of its mammalian orthologs.
  • the marker is to be chosen amongst the following murine proteins: Spp1 , Pnliprp2, Pla2g1 b, EIaI 1 Ela2, 2210010C04Rik, Pnliprpi , Itmapi , Vtn and C1qb, and their respective orthologs in other mammals.
  • the chosen marker is thus a protein having an immune function.
  • a particularly preferred marker is Spp1 or one of its mammalian orthologs.
  • the marker is to be chosen amongst the following murine proteins: Sycn, Amy1 , Ctrbi , 1110002O23Rik and 1810014L12Rik, and their respective orthologs in other mammals.
  • a particularly preferred marker according to the present invention is the murine protein elastase 1 and its respective orthologs in other species of mammal.
  • Elastase 1 is indeed expressed in neutrophils and neutrophils have been implicated in the development of vascular diseases.
  • this protein has been shown regulated in different pathologies, comprising cancer, infection and autoimmunity.
  • Another particularly preferred marker according to the present invention is the murine protein osteopontin and its respective orthologs in other species of mammal, especially human
  • said fluid may be inter alia blood, serum, plasma, urine, saliva, sweat or synovial fluid.
  • the fluid in which the concentration or level of the marker is to be determined is serum or peripheral blood.
  • the marker used in the methods is the transcript of the gene coding for one of the murine proteins previously mentioned or for a mammalian ortholog.
  • the level of such a marker is preferably determined in a sample of a tissue, for example obtained after a biopsy, e.g. taken from the pancreatic lymph node of the mammal to be diagnosed.
  • This embodiment is particularly suited for the diagnosis of a cancer having a pre-inflammatory phase.
  • the tissue or organ in which the level of the marker is to be determined is preferably the tissue or organ suspected to contain tumour cells.
  • the mammal which is to be diagnosed is a human being.
  • This patient may be a young person, preferably a child, most preferably a young or very young child, especially a child less than five or less than two years old.
  • the patient may alternatively be an adult.
  • the diagnostic methods are preferably repeated regularly, for example every 6 months, especially for patients initially negatively diagnosed but coming from a family comprising diseased members.
  • the marker is to be chosen amongst the human orthologs of the murine proteins previously mentioned.
  • the marker is thus to be chosen in the group comprising the following human proteins: Osteopontin (OPN), Regenerating islet-derived 3 alpha (REG3A), Pancreatitis associated protein 1 (PAP), Regenerating protein I beta, prostrate Kallikrein 2 (KLK2), Syncollin (SYCN), Pancreatic lipase-related protein 1 (PNLIPRP1), Kallikrein ⁇ (KLK6), pancreatic Ribonuclease, RNase A family, 1 (RNASE1), Spasmolytic polypeptide (SP), Cholecystokinin A receptor, CUB and zona pellucida-like domains 1 (CUZD1 or ERG-1), pancreatic Colipase, (CLPS), pancreatic lipase-related protein 2, Lithostathine (REG 1A), Amylase 1 , Phospholipase A2, group IB (PLA2G1B), Carboxyl ester lipase (bile salt-stimulated
  • Example 2 details the human orthologs of the murine proteins previously mentioned, as well as their chromosomal location.
  • the marker protein is preferably chosen in the group comprising: Osteopontin, Regenerating islet-derived 3 alpha (REG3A), Pancreatitis associated protein 1 (PAP), Regenerating protein I beta, Spasmolytic polypeptide (SP), pancreatic Colipase, (CLPS), Lithostathine (REG1A) and Carboxyl ester lipase (bile salt-stimulated lipase) (CEL).
  • This marker protein is advantageously a protein known for its implication in tissue regeneration or integrity.
  • a particularly preferred marker protein is Osteopontin.
  • the marker protein is preferably chosen in the group comprising Osteopontin, prostrate Kallikrein 2 (KLK2), Kallikrein ⁇ (KLK6), pancreatic Ribonuclease, RNase A family, 1 (RNASE1), Cholecystokinin A receptor, transmembrane Mucin 1 (MUC1), gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamyl hydrolase) (GGH), Angiogenin and Nucleobindin 2 (NUCB2).
  • This marker protein is advantageously a protein known for its implication in tumorigenesis.
  • a particularly preferred marker protein is Osteopontin.
  • the marker protein is preferably chosen in the group comprising Osteopontin, Pancreatic lipase-related protein 1 (PNLIPRP1 ), CUB and zona pellucida-like domains 1 (CUZD1 or ERG-1), pancreatic lipase-related protein 2, Phospholipase A2, group IB (PLA2G1 B), Elastase 1 or Elastase 1 precursor (ELA1), Pancreatic elastase MA, Protease, serine, 3 (mesotrypsin) (PRSS3), Complement component 1 , q subcomponent, beta polypeptide (C 1QB) and Vitronectin (serum spreading factor, somatomedin B.complement S- protein) (VTN).
  • This marker protein is advantageously a protein known for having an immune function.
  • a particularly preferred marker protein is Osteopontin.
  • the marker protein is preferably chosen in the group comprising Syncollin (SYCN), Amylase 1 , Chymotrypsinogen B1 (CTRB1 ), FK506 binding protein 11 (FKBP11) and NT2RP2.
  • the marker protein is Elastase 1 or Elastase 1 precursor
  • the marker protein is Osteopontin.
  • the mammals likely to be diagnosed are farm animals or pets, for example cats, dogs, horses, cattle or mice.
  • the marker is to be chosen amongst the ortholgs, when orthologs exist, in said species, of the following murine proteins Spp1 , Pap, Reg3a, Reg2, CeI, Reg1 , Tff2, Clps, Klk9, Klk6, Rib1 , Klk5, Mud , Cckar, Ggh, Ang, Nucb2, Pnliprp2, Pla2g1 b, EIaI , Ela2, 2210010C04Rik, Pnliprpi , Itmapi , Vtn, C1qb, Sycn, Amy1, Ctrbi , 1110002O23Rik, 1810014L12Rik, or amongst the orthologs, in said species, of the following human proteins: Osteopontin,
  • a method according to the present invention comprises the step of measuring the level of a marker protein and the comparison of the measured level to a reference level for the marker protein.
  • a method of the invention may also advantageously comprise the measure of the level of more than one marker protein.
  • the methods comprise the measure of the level of two different marker proteins and their comparison to their respective reference levels.
  • Both markers are to be chosen in the group comprising the following murine proteins Spp1 , Pap, Reg3a, Reg2, CeI, Reg1 , Tff2, Clps, Klk9, Klk6, Rib1 , Klk5, Mud , Cckar, Ggh, Ang, Nucb2, Pnliprp2, Pla2g1 b, EIaI , Ela2, 2210010C04Rik, Pnliprpi, Itmapi , Vtn, C1qb, Sycn, Amy1 , Ctrbi , 1110002O23Rik, 1810014L12Rik, and the orthologs of said proteins in other mammalian species.
  • Both measures may be done simultaneously, for example by an automat, or sequentially, the comparison steps may be carried out after both measures, or alternatively, the measure of the level of a first marker is followed by the comparison to the reference level before the measure step for the second marker is carried out.
  • steps a) and b) of the methods of the invention are repeated twice, for two different markers. It may also be envisaged that the steps are repeated twice with the same marker, in order to minimise the possibility of false diagnosis.
  • the measures are advantageously carried out on two different samples.
  • steps a) and b) of the methods of the invention are repeated twice, the later onset of said disease, or disease-prone state, is preferably diagnosed only if both measured levels are significantly divergent from the respective reference levels.
  • the later onset of said disease, or disease-prone state is diagnosed if only one measured level is divergent from its reference level.
  • the method may advantageously be repeated with a third marker, comprised in the markers recited above.
  • steps a) and b) of the methods are repeated three times, simultaneously or sequentially as mentioned above, with three markers.
  • Said markers are to be chosen in the group comprising the following murine proteins Spp1 , Pap, Reg3a, Reg2, CeI, Reg1 , Tff2, Clps, Klk9, Klk6, Rib1 , Klk5, Mud , Cckar, Ggh, Ang, Nucb2, Pnliprp2, Pla2g1 b, EIaI , Ela2, 2210010C04Rik, Pnliprpi , Itmapi , Vtn, C1qb, Sycn, Amy1 , Ctrbi , 1110002O23Rik, 1810014L12Rik, and the orthologs of said proteins in other mammalian species.
  • said three marker proteins are distinct from each other.
  • the later onset of said disease, or disease-prone state is diagnosed only if all measured levels are significantly divergent from their respective reference levels, or at least 2 of 3.
  • the present invention also encompasses methods wherein steps a) and b) as defined above are repeated four times, five times or even more, for example 7 times or 10 times.
  • the markers may be chosen in the same group as defined above, they are preferably distinct from each other.
  • the later onset of said disease, or disease-prone state is preferably diagnosed only if all measured levels are significantly divergent from their respective reference levels, or if a majority of the levels are significantly divergent from their respective reference levels, or at least two levels are significantly divergent from their respective reference levels.
  • this association comprises at least two different markers, preferably at least 3, 4 or 5, for example 5, 7 or 10.
  • Such an association comprises preferably at least two markers in at least two differents groups, the groups being defined as followed: Group 1 : the following murine proteins Spp1, Pap, Reg3a, Reg2, CeI, Reg1 , Tff2 and Clps, and their respective orthologs in other mammals, Group 2: the following murine proteins Spp1 , Klk9, Klk6, Rib1 , Klk5, Mud , Cckar, Ggh, Ang and Nucb2 and their respective orthologs in other mammals,
  • Group 3 the following murine proteins: Spp1 , Pnliprp2, Pla2g1 b, EIaI , Ela2, 2210010C04Rik, Pnliprpi , Itmapi , Vtn and C1qb, and their respective orthologs in other mammals
  • Group 4 the following murine proteins: Sycn, Amy1 , Ctrbi , 1110002O23Rik and 1810014L12Rik, and their respective orthologs in other mammals.
  • An association of markers may comprise at least a marker in each of the four groups, or at least a marker in each of the first 3 groups.
  • an association of markers may comprise proteins encoded by genes located at the same locus on the chromosome.
  • the localization of the genes corresponding to the murine and human markers of the invention is detailed in example 2.
  • association of markers may associate marker proteins having similar function, for example Reg and Pap, or the kallikrein proteins, or the lipase proteins.
  • an association of markers comprises at least spp1 , or its human ortholog Osteopontin.
  • marker proteins comprises or consists in Spp1 and EIa 1 , for example Spp1 with EIaI and EIa 2 and Sycn, or their respective human orthologs.
  • Other preferred associations of marker proteins comprise or consist in: - Spp1 , Mud , Pla2g1 b and Pap, or
  • any method well known from the skilled person in the domain can be used.
  • the marker be an RNA 1 different methods can be used, as for example the use of chip with RNA probes.
  • the marker be a protein
  • use can advantageously be made of a labelled ligand, said ligand specifically binding the chosen protein.
  • a ligand is for example a natural ligand, for example a target of the protein.
  • a particularly preferred type of ligand is antibody. Labelled antibodies specifically directed against the chosen marker protein are indeed suited for determining the level of said marker protein in a sample of a body fluid.
  • a preferred antibody is a monoclonal antibody, although polyclonal antibodies may also be used.
  • an antibody against a marker protein When used in order to determine the level of said marker protein in a sample, said antibody is not necessarily labelled. If said antibody is for example from another species than the mammal which is diagnosed, the antibody may be detected by a further antibody. Fluorescent labels are particularly preferred in the context of the present invention for the ligand allowing the detection of the chosen marker protein, and the determination of its level.
  • a method which can be used for measuring the level of a marker protein of the invention is the so- called ELISA (Enzyme Linked Immunosorbent Assay), as exemplified in Koopmann et al (2004).
  • ELISA Enzyme Linked Immunosorbent Assay
  • the ligand is preferably chosen amongst the antibodies mentioned in example 3. These antibodies are commercially available antibodies, which can be identified and obtained without difficulty by a skilled person in the domain of the present invention.
  • the ligand may be chosen inter alia amongst the following list of antibodies: anti-pap antibody (Rec Human REG1A: Cat No. RP-10087-P1ABX), anti-kallikrein 9 antibody (KLK9 (C-15): sc- 20385 Santa Cruz), anti-kallikrein-6 antibody (KLK6 (N-16): sc-20376 Santa Cruz), anti-Trefoil factor 2 spasmolytic proteini antibody (SP (P-19): sc-23558 Santa Cruz), anti-Cholecystokinin receptor antibody (CCK-AR (G-17): sc-16173 Santa Cruz), anti-kallikrein 5 antibody (KLK5 (N-14): sc-20375 Santa Cruz), anti-Islet Regenerating antibody (Rec Human REG1A: Cat No.
  • antibodies against the marker proteins of the invention can also easily been obtained by well-known methods, either polyclonal or monoclonal antibodies. Technologies for obtaining humanized antibodies are also routine work for the skilled person in this domain.
  • the present invention also encompasses the use of a protein chosen amongst the following murine proteins Spp1 , Pap, Reg3a, Reg2, CeI, Reg1 , Tff2, Clps, Klk9, Klk6, Rib1, Klk5, Mud , Cckar, Ggh, Ang, Nucb2, Pnliprp2, Pla2g1b, EIaI , Ela2, 2210010C04Rik, Pnliprpi , Itmapi , Vtn, C1qb, Sycn, Amy1 , Ctrbi , 1110002O23Rik and 1810014L12Rik, and their respective orthologs in different mammalian species, as a seric marker for the predisposition of a mam
  • this seric marker is used to define a predisposition to the disease before any clinical signs can be detected.
  • a preferred seric marker is the murine protein Spp1 or one of its mammalian orthologs.
  • the invention also concerns the use of a protein chosen amongst the following murine proteins Spp1 , Pap, Reg3a, Reg2, CeI, Reg1 , Tff2, Clps, Klk9, Klk6, Rib1 , Klk5, Mud , Cckar, Ggh, Ang, Nucb2, Pnliprp2, Pla2g1 b, EIaI , Ela2, 2210010C04Rik, Pnliprpi , Itmapi , Vtn, C1qb, Sycn, Amy1 , Ctrbi , 1110002O23Rik and 1810014L12Rik, and their respective orthologs in different mammalian species, as a seric marker for the early pre-inflammatory condition of
  • seric markers for the uses of the invention are chosen amongst the following murine proteins: Spp1 , Pap, Reg3a, Reg2, CeI, Reg1, Tff2, Clps, Klk9, Klk6, Rib1 , Klk5, Mud , Cckar, Pnliprp2, Pla2g1 b, EIaI , Ela2, 2210010C04Rik, Pnliprpi , Itmapi , Sycn, Amy1 and Ctrbi , and their respective mammalian orthologs.
  • murine proteins Spp1 , Pap, Reg3a, Reg2, CeI, Reg1, Tff2, Clps, Klk9, Klk6, Rib1 , Klk5, Mud , Cckar, Pnliprp2, Pla2g1 b, EIaI , Ela2, 2210010C04Rik, Pnliprpi , Itmapi , Sycn, Amy1 and Ctr
  • the protein is chosen amongst the following murine proteins Spp1 , Pap, Reg3a, Reg2, CeI, Reg1 , Tff2 and Clps, and their respective orthologs in other mammals.
  • the chosen protein used as a seric marker is advantageously implicated in tissue regeneration or integrity.
  • the protein is chosen amongst the following murine proteins Spp1 , Klk9, Klk6, Rib1 , Klk5, Mud , Cckar, Ggh, Ang and Nucb2 and their respective orthologs in other mammalian species.
  • the chosen protein used as a seric marker is advantageously implicated in tumorigenesis.
  • the protein is chosen amongst the following murine proteins Spp1 , Pnliprp2, Pla2g1 b, EIaI , Ela2, 2210010C04Rik, Pnliprpi , Itmapi , Vtn and C1qb and their respective orthologs in other mammalian species.
  • the chosen protein used as a seric marker has advantageously an immune function.
  • the protein is chosen amongst the following murine proteins Sycn, Amy1 , Ctrbi , 1110002O23Rik and 1810014L12Rik and their respective orthologs in other mammals.
  • a preferred protein for use as a seric marker according to this invention is Elastase 1 and its orthologs in the other mammalian species, especially human Elastase 1.
  • Another preferred protein for use as a seric marker according to the invention is Spp1 and its orthologs in the other mammalian species, especially human Osteopontin.
  • the seric markers of the invention are particularly suited for the detection of a predisposition to a pathology having a pre-inflammatory phase in human beings.
  • the seric marker is thus to be chosen amongst the human orthologs of the murine proteins disclosed above.
  • the seric marker is thus to be chosen from the group comprising: Osteopontin (OPN), Regenerating islet-derived 3 alpha (REG3A), Pancreatitis associated protein 1 (PAP), Regenerating protein I beta, prostrate Kallikrein 2 (KLK2), Syncollin (SYCN), Pancreatic lipase-related protein 1 (PNLIPRP1), Kallikrein ⁇ (KLK6), pancreatic Ribonuclease, RNase A family, 1 (RNASE1), Spasmolytic polypeptide (SP), Cholecystokinin A receptor, CUB and zona pellucida-like domains 1 (CUZD1 or ERG-1), pancreatic Colipase, (CLPS), pancreatic lipase-related protein 2, Lithostathine (REG1A), Amylase 1 , Phospholipase A2, group IB (PLA2G1 B), Carboxyl ester lipase (bile salt-stimulated lipa
  • the protein is chosen amongst the following human proteins: Osteopontin, Regenerating islet-derived 3 alpha (REG3A), Pancreatitis associated protein 1 (PAP), Regenerating protein I beta, Spasmolytic polypeptide (SP), pancreatic Colipase, (CLPS), Lithostathine (REG1A) and Carboxyl ester lipase (bile salt-stimulated lipase) (CEL).
  • This chosen protein used as a seric marker is advantageously implicated in tissue regeneration or integrity. Osteopontin is particularly preferred.
  • the protein is chosen amongst the following human proteins: Osteopontin, prostrate Kallikrein 2 (KLK2), Kallikrein ⁇ (KLK6), pancreatic Ribonuclease, RNase A family, 1 (RNASE1), Cholecystokinin A receptor, transmembrane Mucin 1 (MUC1), gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamyl hydrolase) (GGH), Angiogenin and Nucleobindin 2 (NUCB2).
  • This chosen protein used as a seric marker is advantageously implicated in tumorigenesis. Osteopontin is particularly preferred.
  • the protein is chosen amongst the following human proteins: Osteopontin, Pancreatic lipase-related protein 1 (PNLIPRP1), CUB and zona pellucida-like domains 1 (CUZD1 or ERG-1), pancreatic lipase-related protein 2, Phospholipase A2, group IB (PLA2G1 B), Elastase 1 or Elastase 1 precursor (ELA1), Pancreatic elastase MA, Protease, serine, 3 (mesotrypsin) (PRSS3), Complement component 1 , q subcomponent, beta polypeptide (C1QB) and Vitronectin (serum spreading factor, somatomedin B.complement S-protein) (VTN).
  • This chosen protein used as a seric marker has advantageously an immune function. Osteopontin is particularly preferred.
  • the protein is chosen amongst the following human proteins: Syncollin (SYCN), Amylase 1 , Chymotrypsinogen B1 (CTRB1 ), FK506 binding protein 11 (FKBP11 ) and NT2RP2.
  • a marker protein is preferably used as a diagnostic marker of type 1 diabetes in human, especially for children as young as 5 years, preferably even younger, for example 18 or 24 months.
  • the invention also relates to the use of a ligand, which specifically binds a protein chosen from the group comprising the following murine proteins Spp1 , Pap, Reg3a, Reg2, CeI, Reg1 , Tff2, Clps, Klk9, Klk6, Rib1 , Klk5, Mud , Cckar, Ggh, Ang, Nucb2, Pnliprp2, Pla2g1b, EIaI 1 Ela2, 2210010C04Rik, Pnliprpi , Itmapi , Vtn, C1qb, Sycn, Amy1 , Ctrbi , 1110002O23Rik and 1810014L12Rik, and their respective orthologs in different mammalian species, in a method for diagnosing, before any clinical signs, a predisposition to a disease having a pre-inflammatory phase in a mammal, or in a method for diagnosing said disease before any clinical signs.
  • the preferred mammals and preferred diseases according to this invention have been previously disclosed.
  • the ligand used is preferably an antibody, particularly a monoclonal antibody.
  • the ligand used can also be the receptor of the marker.
  • Said ligand is advantageously labelled, for example by a fluorescent label.
  • the invention thus also relates to the use of a ligand, which specifically binds a protein chosen from the group consisting of: Osteopontin (OPN), Regenerating islet-derived 3 alpha (REG3A), Pancreatitis associated protein 1 (PAP), Regenerating protein I beta, prostrate Kallikrein 2 (KLK2), Syncollin (SYCN), Pancreatic lipase-related protein 1 (PNLIPRP1), Kallikrein ⁇ (KLK6), pancreatic Ribonuclease, RNase A family, 1 (RNASE1 ), Spasmolytic polypeptide (SP), Cholecystokinin A receptor, CUB and zona pellucida-like domains 1 (CUZD1 or ERG-1), pancreatic Colipase, (CLPS), pancreatic lipase-related protein 2, Lithostathine (REG1A), Amylase 1 , Phospholipase A2, group I
  • OPN Osteopontin
  • diseases having a pre-inflammatory phase comprise autoimmune diseases, cancers and infection.
  • Autoimmune diseases are particularly preferred in the context of the present invention, especially type 1 diabetes mellitus, multiple sclerosis, rheumatoid arthritis, collagen induced arthritis and autoimmune hepatitis.
  • a preferred ligand for use in a method of diagnosing is a ligand specifically binding Elastase 1.
  • Another particularly preferred ligand for use in such a method is a ligand specifically binding Osteopontin.
  • the present invention also encompasses therapeutic applications using at least one of the following murine proteins Spp1 , Pap, Reg3a, Reg2, CeI, Reg1 , Tff2, Clps, Klk9, Klk6, Rib1 , Klk5, Mud , Cckar, Ggh, Ang, Nucb2, Pnliprp2, Pla2g1 b, EIaI, Ela2, 2210010C04Rik, Pnliprpi , Itmapi , Vtn, C1qb, Sycn, Amy1 , Ctrbi , 1 110002O23Rik and 1810014L12Rik, or their respective orthologs in different mammalian species, as a therapeutic target.
  • Said therapeutic applications are preferably for human therapy, the target protein is thus to be chosen in the human orthologs of the murine proteins recited above.
  • Preferred embodiments are a protein chosen from Osteopontin, Regenerating islet-derived 3 alpha (REG3A), Pancreatitis associated protein 1 (PAP), Regenerating protein I beta, Syncollin (SYCN), Pancreatic lipase-related protein 1 (PNLIPRP1), pancreatic Ribonuclease, RNase A family, 1 (RNASE1 ), Spasmolytic polypeptide (SP), Cholecystokinin A receptor, CUB and zona pellucida-like domains 1 (CUZD1 or ERG-1), pancreatic Colipase, (CLPS), pancreatic lipase- related protein 2, Lithostathine (REG1A), Amylase 1 , Phospholipase A2, group IB (PLA2G1 B), Carboxyl ester lipase (bile salt-stimulated lipase) (CEL), Elastase 1 (or Elastase 1 precursor) (ELA1), Chymot
  • the present invention concerns an agent capable of modulating the activity of a protein chosen from the group comprising: Osteopontin, Regenerating islet-derived 3 alpha (REG3A), Pancreatitis associated protein 1 (PAP), Regenerating protein I beta, prostrate Kallikrein 2 (KLK2), Syncollin (SYCN), Pancreatic lipase-related protein 1 (PNLIPRP1), Kallikrein ⁇ (KLK6), pancreatic Ribonuclease, RNase A family, 1 (RNASE1), Spasmolytic polypeptide (SP), Cholecystokinin A receptor, CUB and zona pellucida-like domains 1 (CUZD1 or ERG-1 ), pancreatic Colipase, (CLPS), pancreatic lipase-related protein 2, Lithostathine (REG1A), Amylase 1 , Phospholipase A2, group IB (PLA2G1 B), Carboxyl ester lipase (bile salt
  • the invention also encompasses such an agent for a prophylactic use for disease-prone patients.
  • the invention also concerns the use of an agent capable of modulating the activity of a protein chosen from the group consisting of: Osteopontin, Regenerating islet-derived 3 alpha (REG3A), Pancreatitis associated protein 1 (PAP), Regenerating protein I beta, prostrate Kallikrein 2 (KLK2), Syncollin (SYCN), Pancreatic lipase-related protein 1 (PNLIPRP1 ), Kallikrein ⁇ (KLK6), pancreatic Ribonuclease, RNase A family, 1 (RNASE1), Spasmolytic polypeptide (SP), Cholecystokinin A receptor, CUB and zona pellucida-like domains 1 (CUZD1 or ERG-1), pancreatic Colipase, (CLPS), pancreatic lipase-related protein 2, Lithostathine (REG1A), Amylase 1 , Phospholipas
  • the invention also concerns the use of an agent capable of modulating the activity of a protein chosen amongst the following human proteins: Osteopontin, Regenerating islet-derived 3 alpha (REG3A), Pancreatitis associated protein 1 (PAP), Regenerating protein I beta, prostrate kallikrein 2 (KLK2), Syncollin (SYCN), Pancreatic lipase-related protein 1 (PNLIPRP1), Kallikrein ⁇ (KLK6), pancreatic Ribonuclease, RNase A family, 1 (RNASE1), Spasmolytic polypeptide (SP), Cholecystokinin A receptor, CUB and zona pellucida-like domains 1 (CUZD1 or ERG-1), pancreatic Colipase, (CLPS), pancreatic lipase-related protein 2, Lithostathine (REG1A), Amylase 1 , Phospholipase A2, group IB (PLA2G1 B), Carboxyl ester lipas
  • the protein is chosen from the group consisting of the following human proteins: Osteopontin, Regenerating islet- derived 3 alpha (REG3A), Pancreatitis associated protein 1 (PAP), Regenerating protein I beta, Spasmolytic polypeptide (SP), pancreatic Colipase, (CLPS), Lithostathine (REG1A) and Carboxyl ester lipase (bile salt-stimulated lipase) (CEL).
  • the chosen protein is advantageously known for its implication in tissue regeneration or integrity.
  • a particularly preferred protein is Osteopontin.
  • the protein is chosen from the group consisting of the following human proteins: Osteopontin, prostrate Kallikrein 2 (KLK2), Kallikrein ⁇ (KLK6), pancreatic Ribonuclease, RNase A family, 1 (RNASE1), Cholecystokinin A receptor, transmembrane Mucin 1 (MUC1), gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamyl hydrolase) (GGH), Angiogenin, Nucleobindin 2 (NUCB2) and Osteopontin, and most preferably chosen amongst the group consisting of pancreatic Ribonuclease, RNase A family, 1 (RNASE1), Cholecystokinin A receptor, transmembrane Mucin 1 (MUC1 ), gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamyl hydrolase) (GGH), Angio
  • the protein is chosen from the group consisting of the following human proteins: Osteopontin, Pancreatic lipase-related protein 1 (PNLIPRP1 ), CUB and zona pellucida-like domains 1 (CUZD1 or ERG-1), pancreatic lipase- related protein 2, Phospholipase A2, group IB (PLA2G1 B), Elastase 1 (or Elastase 1 precursor) (ELA1), Pancreatic elastase MA, Protease, serine, 3 (mesotrypsin) (PRSS3), Complement component 1 , q subcomponent, beta polypeptide (C1QB) and Vitronectin (serum spreading factor, somatomedin B.complement S-protein) (VTN).
  • human proteins Osteopontin, Pancreatic lipase-related protein 1 (PNLIPRP1 ), CUB and zona pellucida-like domains 1 (CUZD1 or ERG-1), pan
  • the protein is advantageously known as having an immune function. Osteopontin is particularly preferred.
  • the protein is chosen from the group of Syncollin (SYCN), Amylase 1 , Chymotrypsinogen B1 (CTRB1 ), FK506 binding protein 11 (FKBP11) and NT2RP2.
  • a particularly preferred protein is the human Elastase 1.
  • Another particularly preferred protein is the human Osteopontin.
  • the modulation induced by the agent is an inhibition of the protein activity or an enhancement of said activity, but preferably inhibition. This agent may modulate the activity of the chosen protein either by direct or indirect interaction with the protein, or by interaction with the DNA or RNA transcript of the protein.
  • a suitable agent capable of inhibiting the protein activity is for example a ligand which is a competitor of the natural ligands of the protein.
  • Such an agent is for example an antibody, preferably a monoclonal antibody.
  • the agent may intervene at the translation level by inhibiting the translation of the RNA.
  • Such an agent is for example a ribozyme or an RNAi.
  • the agent may also intervene at the DNA level, for example by inhibiting the initiation of the transcription.
  • a preferred agent is an antibody, especially a monoclonal antibody.
  • a suitable agent which can be used in the therapeutic applications of the present invention is preferably an antibody chosen amongst the commercially availabe antibodies directed against the above-mentioned human proteins.
  • An agent can be chosen for example from the following list of antibodies: Antibody anti-pap (Rec Human REG1A: Cat No. RP-10087-P1ABX), antibody anti-kallikrein 9 (KLK9 (C-15): sc-20385 Santa Cruz), antibody anti-kallikrein-6 (KLK6 (N-16): sc-20376 Santa Cruz), antibody anti-Trefoil factor 2 spasmolytic proteini (SP (P-19): sc-23558 Santa Cruz), antibody anti-Cholecystokinin receptor (CCK-AR (G-17): sc-16173 Santa Cruz), antibody anti-kallikrein 5 (KLK5 (N-14): sc- 20375 Santa Cruz), antibody anti-Islet Regenerating (Rec Human REG1A: Cat No.
  • the agent may enhance the activity of said protein.
  • the enhancement may also be an enhancement at the DNA level, at the translation level or at the protein level.
  • An agent capable of enhancing the activity of a protein of the invention may be the same protein, the activity is thus enhanced by increasing the concentration of the protein.
  • the patient which is to be treated by an agent as described has not yet evident sign of disease.
  • the patient which is to be treated by an agent as described has not yet evident sign of insulitis or mild-insulitis, therefore the patient is still in the early stages of type 1 diabetes.
  • the patient which is to be treated according to the invention is preferably under 10 years, more preferably under 5 years, the patient may also be as young as 18 or 24 months.
  • kits for diagnosing a disease or a predisposition to a disease having a pre-inflammatory phase The kit is to be used for diagnosing such a disease in a patient before any clinical signs of the disease.
  • a kit according to the invention comprises: - a means for dosing (determining the level of) a protein chosen from the group comprising:
  • PAP Regenerating protein I beta
  • KLK2 prostrate kallikrein 2
  • SYCN Syncollin
  • Pancreatic lipase-related protein 1 PNLIPRP1
  • Kallikrein ⁇ KLK6
  • RNASE1 Ribonuclease, RNase A family, 1 (RNASE1), Spasmolytic polypeptide (SP), Cholecystokinin A receptor, CUB and zona pellucida-like domains 1 (CUZD1 or ERG-1), pancreatic Colipase,
  • CPS pancreatic lipase-related protein 2
  • RAG1A Lithostathine
  • Amylase 1 Amylase 1
  • Phospholipase A2, group IB Phospholipase A2, group IB (PLA2G1 B), Carboxyl ester lipase (bile salt-stimulated lipase)
  • CEL Elastase 1
  • ELA1 Elastase 1
  • CRB1 Chymotrypsinogen B1
  • Pancreatic elastase HA Protease, serine, 3 (mesotrypsin) (PRSS3), transmembrane Mucin 1 (MUC1), gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamyl hydrolase) (GGH), Angiogenic FK506 binding protein 11 (FKBP11), NT2RP2, Complement component 1 , q subcomponent, beta polypeptide (C1QB), Nucleobindin 2 (NUCB2) and Vitronectin (serum spreading factor, somatomedin B, complement S-protein) (VTN), or a mammalian ortholg thereof and - a reference level for said protein.
  • MUC1 transmembrane Mucin 1
  • GGH gamma-glutamyl hydrolase
  • FKBP11 Angiogenic FK506 binding protein 11
  • NT2RP2 NT2RP2
  • Complement component 1 q subcom
  • the reference level is as defined previously.
  • a kit of the invention may be based on the measure of the level of a protein of the invention, or of the level of the RNA corresponding to said protein.
  • the preferred embodiments already mentioned for the various methods and uses of the invention are also applicable to the kit.
  • a kit according to the invention is preferably used to diagnose a predisposition to an autoimmune disease, a cancer or an infection.
  • Preferred autoimmune diseases are type 1 diabetes mellitus, multiple sclerosis, rheumatoid arthritis, collagen induced arthritis and autoimmune hepatitis.
  • the chosen protein is preferably Osteopontin.
  • the patient to be diagnosed by said kit is preferably a person from a family presenting high risk of potential autoimmune diseases or cancer.
  • a patient is preferably a child, for example less than 10 years.
  • the kit may advantageously be used for diagnosing child as young as 5 years or even less, for example for diagnosing children with 18 or 24 months.
  • the preclinical stage may begin early in the life of patients, as early as 2 years and even before. It is thus very important to be able, thanks to the kit of the invention, to diagnose the pathology at this preclinical stage, in order to avoid, delay or attenuate the further symptoms.
  • the means for dosing (determining the lelvel of) the protein chosen, in a kit of the invention is preferably a ligand which specifically binds the chosen protein.
  • Said ligand may advantageously be labelled, for example by a fluorescent label.
  • preferred ligands are antibodies, especially monoclonal antibodies.
  • An antibody comprised in a kit of the invention can be chosen inter alia from the antibodies detailed in example 3 of the present application. It can be chosen for example from the following list of antibodies: Antibody anti-pap (Rec Human REG1A: Cat No.
  • RP-10087-P1ABX antibody anti-amylase 1 (Amylase (G-10): sc-46657), antibody anti-secreted phosphoprotein 1 (Rabit polyclonal RB-9097), antibody anti-mucin 1 (CD24 (GPI-linked surface mucin) Ab-1 ) and anti-osteopontin antibody (human Osteopontin Quantikine DOSTOO or SOSTOO).
  • the reference level provided in the kit may be materialized by a line on the test tube of the kit or may be indicated in the instructions for users.
  • the invention is also directed to a method for the early diagnosis of a disease having a pre- inflammatory phase and/or of a disease-prone state, in a mammal, prior to any clinical signs, comprising a) measuring the level of at least a marker protein chosen amongst the following murine proteins Spp1 , Pap, Reg3a, Reg2, CeI, Reg1 , Tff2, Clps, Klk9, Klk6, Rib1 , Klk5, Mud ,
  • the previous measure of the chosen marker is carried out at least one week before, preferably at least one month, three or six months before, especially if the mammal to be diagnosed is a human patient.
  • the rapid change of the level of a marker protein of the invention may also be indicative of a pre-inflammatory phase.
  • the level of the preferred marker osteopontin varies dramatically during the pre- inflammatory phase, firstly by a sharp increase and then by a rapid decrease. Therefore, in a preferred embodiment, the second measured level of the chosen protein is increased with respect to the previously first measured level.
  • three successive measures are made, wherein the second measure is increased with respect to the first one and the third one is increased with respect to the second one in order to diagnose a disease having a pre-inflammatory phase and/or of a disease-prone state.
  • the second measured level of the chosen marker protein is decreased with respect to the previously measured level.
  • a third measure carried out after the second one, confirms the decrease of the level of the marker protein.
  • Intervals between the measures are preferably between one month and one year for human diagnosis, for example 3 months or 6 months.
  • the variation in intensity between two measures is at least 5% (positive or negative) of the measured levels, and most preferably at least 10%, even most preferably 20, 50 or 100%.
  • W-I Phenotypic Window I between 3 to 5 weeks;
  • W-Il Phenotypic Window Il between 8 to 12 weeks and
  • W-III Phenotytpic Window III after 10 weeks of age ⁇ Adapted from Melanitou, 2004 ⁇ .
  • Figure 2 Data Analysis- Genespring GS6.0-II
  • Figure 3 Distribution of gene expression patterns in PaLN of IAA+ and IAA- NOD mice. All genes spotted on the Affymetrix Arrays MU74A version 2 are taken in consideration. To note the points placed by the program according to their gene expression differences out of the median line. These genes correspond to the differentially expressed genes between the two sets of samples. To be noted also the smaller differences found in the differentially expressed genes in the IAA negative samples.
  • Figure 4 Fold increase of genes potentially implicated in tissue integrity and regeneration (Group I) and differentially expressed between IAA pos and IAA neg NOD mice in the PaLN.
  • Figure 5 Fold increase of genes potentially implicated in tumorigenesis (Group II) and found to be differentially expressed between IAA pos and IAA neg NOD mice in the PaLN.
  • Figure 6 Fold increase of genes potentially implicated in immune function (Group III) and found to be differentially expressed between IAA pos and IAA neg NOD mice in the PaLN.
  • Figure 7 Fold increase of genes in the unclassified (Group IV) and found to be differentially expressed between IAA pos and IAA neg NOD mice in the PaLN.
  • Figure 8A Standard Curve of OPN and tendency curve.
  • Figure 8B Longitudinal analysis of OPN concentrations in the NOD mouse serum.
  • Figure 11 11 A: OPN concentrations in sera of Control and Diabetic patients.
  • Figure 13 Fold change of OPN concentrations in the sera of Human control and diabetes patients.
  • the reported genes have been identified by analysis of expression patterns between NOD (Non- Obese Diabetes) diabetes prone mice positive or negative for the presence of Insulin Auto Antibodies (IAA) at an early age (at 5 weeks).
  • IAA Insulin Auto Antibodies
  • the aim of these experiments has been to gain a better understanding of the molecular mechanisms initiating the autoimmune process in type 1 diabetes. It has previously been established by the inventor that the early appearance of IAA (E- IAA) is a prediabetes marker.
  • the age of 5 weeks in the NOD mouse corresponds to prior or around the first phase of the disease i.e. prior the presence of peri-insulitis or non-destructive insulitis.
  • Additional applications include diagnostic and prognostic markers for certain cancers and infection-mediated immune responses.
  • encoded proteins In addition to the potential of encoded proteins to be used as predictive biomarkers for early proinflammatory condition in autoimmune diseases (type 1 diabetes), they probably represent functional properties for pancreatic tissue damage and regeneration (Reg genes products and Spp1) and they are therefore valuable therapeutic targets.
  • mice have been purchased from Taconic farms (NOD/fac), Germantown, NY, and housed in specific pathogen-free conditions. Experimental protocols were conformed to guidelines from the Institutional Animal Care and Use Committee. Pregnant females are tested one week before delivery for the presence of IAA. Animals were tested for the presence of IAA as previously described ⁇ Melanitou, 2004 ⁇ . Briefly, sera from 3 weeks old (at weaning) NOD mice and at 4 and 5 weeks have been assessed for the presence of IAA. The last sera for IAA detection are taken at 5 weeks of age on the moment of sacrificing the animals for the RNA preparation and represent another checkpoint for the presence or absence of IAA.
  • IAA assay IAA are measured in a previously established radioimmunoassay incorporating competition with unlabeled insulin and precipitation with Protein A/G sepharose in a 96 well filtration plate, as previously described ⁇ Melanitou, 2004 ⁇ .
  • RNA preparation for microarrays PaLN have been immediately placed in RNA later (Qiagen) and processed for total RNA preparation on the same day.
  • Total RNAs were prepared with the Qiagen mini- or midi-RNA kit following the manufacturer's protocol. After quantification by spectrophotometry, 500 ng of Total-RNA was assessed for quality using an Agilent Bioanalyzer (Agilent Technologies). Integrity is evaluated by the presence of the ribosomal RNAs that should not be degraded.
  • RNA samples (4.5 ⁇ g) from E-IAA positive (4 mice) and E-IAA negative (5 mice) were reversed transcribed and after biotinylated labeling of cRNA were used to hybridize to Affymetrix (Santa Clara, CA) first array MG-U74Av2 GeneChip according to standard Affymetrix protocols.
  • the first array in the set (MG-U74Av2) represents the majority of sequences ( ⁇ 6,000) in the Mouse UniGene database that have been functionally characterized. In addition, approximately 6,000 EST clusters are also represented on this single array.
  • MG-U74AV2 GeneChip contains probe sets representing 12,488 transcripts. Microchip scanning and data capture were also according to standard Affymetrix methods:
  • IAA can be detected as early as at 3 weeks of age in the NOD mice, before insulitis appears and it is a quantal phenotype as it runs by litters ( Figure 1A). This indicates that the mechanisms of autoimmune destruction put in place early, are not only dependent upon genetic factors but also upon factors inherent to the individual animal/litter.
  • the maternal autoimmune-prone environment influences the IAA levels of the litters as reflected by the role played by maternal autoantibodies at ante partum. Indeed maternal autoantibodies influence the appearance of early autoimmunity in the litters ( Figure 1 B).
  • Figure 1A & 1 B show that the presence of E-IAA predisposes to early T1 D and emphasize the biological significance of this sub phenotype as an early marker of autoimmunity ⁇ Melanitou, 2004 ⁇ .
  • the early insulin autoantibodies sub phenotype has been used as an early marker of autoimmune destruction ⁇ Melanitou, 2004 ⁇ to select individual mice, E-IAA positive or negative and apply genomic approaches.
  • E-IAA positive or negative mice In total 9 individual NOD mice have been selected at 5 weeks of age and high quality T-RNAs have been prepared. The criteria for selecting these mice have been as follows: animals have been checked for IAA at 3, 4 and 5 weeks of age. Mice, at least twice E- IAA positive between 3 and 5 weeks of age, were used representing the autoimmunity positive samples (3 mice). Animals negative for E-IAA at the same ages constituted the negative controls 5 (6 mice).
  • the transcriptome analysis of the pre-diabetes phenotypes in the 20 NOD mouse revealed 125 genes showing statistically significant differences of gene expression between the 2 groups: the IAApos and the lAAneg. It is however to be noted that, in analyzing the present data, one mouse that although has been E-IAA negative between 3 and 5 weeks of age, is placed by the clustering program together with the E-IAA positive samples Interestingly 40 of these 125 genes showed differences between the two groups of samples over 25 10 fold and reaching up to 122 fold. The inventor has assessed for the presence of genes coding for secreted proteins within this set of genes. Indeed 32 in total code for potential secreted proteins (Table II). From this set of genes, 25 belong to the 40 highly regulated genes and 7 are less regulated showing 3 to 8 fold differences between the two sets of samples (Table II).
  • Secreted proteins are found in the extracellular space and represent the major class of molecules implicated in intercellular communication in multicellular organisms. In the mouse this class of proteins is referred to as the mouse "secretome” ⁇ Greenbaum, 2001 ⁇ .
  • Spp1 codes for the secreted phosphoprotein named also osteopontin (opn), or Eta1 gene, for Early T lymphocyte activation protein.
  • Spp1 has pleiotropic functions (Figure 2) and it has been proposed to play a role in various inflammatory diseases such as rheumatoid arthritis ⁇ Yamamoto, 2003 ⁇ , bone tumors ⁇ Natasha, 2006 ⁇ , multiple sclerosis ⁇ Chabas, 2001 ⁇ , bacterial infections ⁇ Patarca, 1993 ⁇ , as well as it may act as a cytokine and promote T cell polarization and macrophage migration ⁇ Ashkar, 2000 ⁇ . Therefore Spp1 is part of all groups of secreted proteins (Table III & IV).
  • Tff2 has been found to be expressed in the gastrointestinal tract and it is up regulated in epithelial damage ⁇ Tomasetto, 1990; Thim, 2005; Baus-Loncar, 2005; Dhar, 2005 ⁇ , with a potential role in tissue regeneration. Clps have been recently found to be implicated in type 2 diabetes ⁇ Lindner, 2005 ⁇ .
  • CeI carboxyl ester lipase
  • Tissue kallikreins are serine proteases with a high degree of tissue specificity, thought to be involved in the generation of bioactive peptides, the kinines, in many organs, such as the kidney, salivary glands, pancreas and blood vessels. They constitute a large multigene family highly similar between humans and rodents ⁇ Yousef, 2000 ⁇ .
  • KIkG has a region showing homology with the protease serine 1 (PRSS1) (OMIM 276000).
  • PRSS9 mRNA was expressed in several primary tumors and cell lines from mammary, prostate, and ovarian cancers, but was not detected in any metastases of these cancers ⁇ Stamey, 1987 ⁇ .
  • the mucin (Muc 1) gene or episialin has been found to be highly expressed in gastric carcinoma and proposed to be involved in gastric carcinogenesis ⁇ Silva, 2001 ⁇ .
  • the genes coding for Cholecystkinin (Cckar), gamma glutamyl hydrolase (Ggh) and angiogenin (Ang) have been found implicated in the formation of various tumors ⁇ Takata, 1997; Cheng, 2005; Esaki, 1998; He, 2004; Bond, 1990 ⁇ .
  • Angiogenin in particular is involved in the Notch signaling pathway related to neurovascular progression of pancreatic cancer ⁇ Buchler, 2005 ⁇ and metastasis, as well as in multiple myeloma ⁇ Politou, 2005 ⁇ .
  • nucleobindin 2 (Nucbl) is implicated in B cell lymphomas ⁇ de Vos, 2003 ⁇ . It is a DNA binding protein called also NEFA and its sequence contains a signal peptide suggesting that it is a secreted or transmembrane protein.
  • Pancreatic lipase related protein 2 (Pnliprp2) is expressed in cytotoxic T cells and it is an IL-4 inducible gene, playing a role in cellular defense response ⁇ Grusby, 1990 ⁇ .
  • Phospholipase A2 group 1 B [Pla2g1b) is an anti-inflammatory molecule implicated in oxidative stress and cell proliferation ⁇ Snyder, 1999 ⁇ .
  • Pancreatic lipase related protein 1 (Pnliprp ⁇ ) has been found in acute pancreatitis with an infectious pathogens etiology ⁇ Reimund, 2005 ⁇ .
  • Elastase 1 (E/a1) is an enzyme secreted by macrophages or lymphocytes ⁇ Yamasaki, 1987 ⁇ . It is a pro inflammatory protein and has been found to be related in adjuvant induced arthritis ⁇ Escandell, 2006 ⁇ , asthma, lung disease and cystic fibrosis ⁇ Bines, 2005 ⁇ .
  • the integral membrane-associated protein 1 (Itmap) contains a CUB domain and plays an essential role in trypsinogen activation and both impaired and augmented trypsinogen activation can be associated with protection of increased severity of pancreatitis ⁇ Imamura, 2002 ⁇ .
  • the CUB domain is an extracellular domain of approximately 110 residues which is found in functionally diverse, mostly developmentally regulated proteins.
  • CUB domains contain four conserved cysteines which probably form two disulfide bridges (C1-C2, C3-C4).
  • the structure of the CUB domain has been predicted to be a ⁇ -barrel, similar to that of immunoglobulins. It is interesting to note that two of the genes identified in these microarray experiments contain the CUB domain or a zinc metalloprotease domain: the Itmap and the complement subcomponent C1q. The biological significance of the CUB domain in these proteins remains to be elucidated. Finally two genes showed a lower level of differential gene expression in this experimental design: the Vtn and C1qb ( Figure 6).
  • vitronectin (Vtn) coding gene is a cell adhesion molecule involved in the pathway of inflammatory response and expressed in hepatocytes ⁇ Seiffert, 1991 ⁇ . Vitronectin together with metalloproteases have been found to increase in the serum of patients with hepatocellular carcinoma ⁇ Paradis, 2005 ⁇ .
  • Complement component 1 , q subcomponent (C1qb) gene is a beta-defensin homologue and its isoforms have been found to cooperatively contribute to innate immunity in the urogenital system ⁇ Yamaguchi, 2002 ⁇ .
  • C1qb The Complement component 1 , q subcomponent (C1qb) gene is a beta-defensin homologue and its isoforms have been found to cooperatively contribute to innate immunity in the urogenital system ⁇ Yamaguchi, 2002 ⁇ .
  • one unknown gene (Genbank accession number: AE000663) codes for a protein possessing a signal peptide and is located within the T cell receptor beta (Tcrb) locus. It is an unknown protein and due to similarity with the Tcrb segments, it has been suggested to play a role in the immunologic synapse.
  • calcineurin acts by interaction with their cognate intracellular receptors, cyclophilin and FK506 Binding Protein, respectively.
  • FK506 immunosuppressive drugs
  • Jurkat cell line has derived from human T-cell leukemia and used to determine the mechanism of differential susceptibility to anti-cancer drugs and radiation.
  • Calcineurin has been identified as a key enzyme in the T-cell signal transduction cascade and biological evidence has been provided to support the notion that the interaction of drug-isomerase complexes with calcineurin underlies the molecular basis of CsA/FK506-mediated immunosuppression.
  • amylase Amy 1
  • Sycn Syncollin
  • the nature of the immune response in various pathophysiological conditions is determined by the dynamic interaction of cells of the immune system with other cells, antigens and secreted factors. Understanding the complexity of such responses under physiological or pathological conditions might contribute to the comprehension of the origin of several common complex disorders in which the immune system plays the leading role.
  • Transcriptome offers the possibility to assess simultaneously several thousands of genes.
  • Tissue architecture might be in the core of the initial steps of autoimmune destruction as well as of other immune responses as it is observed in the case of tumorigenesis.
  • the transformed cell escapes recognition by the immune system and by proliferating allows transforming tissue architecture.
  • tissue integrity is also attained by the immune destructive lymphocytes. Therefore proteins involved in tissue regeneration and integrity might play a key role in these diseases and they represent potential diagnostic tools and therapeutic targets.
  • the genes found in this transcriptome data to have a somehow collective immune function role might have a universal task in the initiation of various conditions including cancer, infection and autoimmunity. After initiating the disease, other genetic factors might take over the disease specific steps, characteristic of the particular clinical signs.
  • tumorigenesis is the reverse process of autoimmunity as far as the involvement of the immune system is concerned.
  • the immune system In tumor formation the immune system is inactive or inoperative since incapable to provide proper protection of the organ against the tumor cells. In contrast during the autoimmune process the immune system is over activated and attacks the self tissue. While in both situations the reverse actions should take place if a physiological response has to be considered.
  • the respective orthologs in different mammalian species can be found with the UNIGENE database or with the Eukaryotic Gene Orthologs (EGO) database: http : //www. tig r. orq/td b/tq i/eq o/. This is a database for orthologous genes in eukaryotes.
  • the following antibodies are directed against human marker proteins of the invention. These antibodies can be used in different detection and measurement methods according to the invention.
  • Example 4 determination of plasma level of a marker protein.
  • the marker protein chosen is Osteopontin (OPN); the same protocol may however easily be adapted in order to determine the levels of another marker protein of the invention in the peripheral blood.
  • Opn levels in plasma can be measured using a solid-phase sandwich enzyme-linked immunosorbant assay kit (Immuno-Biological Laboratory, Gumma, Japan). In a published report ⁇ Schorge 2004 ⁇ , the methodology suggested is as follows:
  • Microplates were first pre coated with antihuman OPN rabbit IgG [100 ⁇ l of 20 ⁇ g/ml in 0.1 M carbonate buffer (pH, 9.5)] and blocked with 1% BSA and 0.05% Tween 20. Plasma and standard OPN samples were diluted with 1% BSA and 0.05% Tween 20 in PBS and incubated for 1 h at 37 0 C. After seven washes with 0.05% Tween 20 in phosphate buffer, horseradish peroxidase- labeled conjugated antihuman OPN (10A16) mouse monoclonal antibody (100 ⁇ l of 2 ng/ml) was added and incubated for 30 min at 4°C.
  • antihuman OPN rabbit IgG 100 ⁇ l of 20 ⁇ g/ml in 0.1 M carbonate buffer (pH, 9.5)
  • Plasma and standard OPN samples were diluted with 1% BSA and 0.05% Tween 20 in PBS and incubated for 1 h at 37 0 C. After seven washe
  • Example 5 detection of autoantibodies directed against a marker protein.
  • the marker protein chosen is Osteopontin (OPN); the same protocol may however easily be adapted to other marker proteins of the invention.
  • OPN Osteopontin
  • Example 6 ostepontin gene as early marker for type 1 diabetes
  • This example concerns the experimental validation of the osteopontin gene as potential early diagnostic marker for type 1 diabetes.
  • the inventor has evaluated the OPN concentration in the serum of NOD (Non Obese Diabetic) mice at an early age between 4 and 6 weeks corresponding to the pre inflammatory stages of type 1 diabetes.
  • NOD mice were also tested for the presence of OPN in the serum at 16 weeks of age corresponding to insulitis lesions in the pancreatic islets and at 30 weeks of age of a diabetic mouse.
  • the corresponding evaluations of OPN concentrations in human are advantageously carried out at the same stages, i.e. at prediabetic and diabetic stages, and in control subjects.
  • the inventor reports data of OPN concentrations in the mouse serum. These data are in agreement with the proposition that osteopontin levels vary with time and mark with high levels the early pre inflammatory stages corresponding to an increase of OPN concentrations in the serum of 5 weeks old mice when compared with 4, 6, 12 and 30 weeks old.
  • mice NOD mice were purchased from the Jackson laboratory (Bar Harbor, USA). Mice were housed in specific pathogen-free barrier facilities at the lnstitut Pasteur. Experimental protocols were under conditions approved by the Institutional Animal Care and Use Committee. Animals were bled at 4 weeks, 5, 6, 16 and 30 weeks of age by retro orbital vein. For the identification of the presence of osteopontin protein 3 females and 5 males have been used for each group with a total of 28 NOD and one C57BI/6.Spp1 Knock Out mouse and its corresponding wild type control have been used.
  • Osteopontin protein has been found in the NOD mouse to range between 213 and 317 ng/ml. Higher OPN concentrations are observed prior to inflammation (317 ng/ml) while the lower concentrations are observed after 16 weeks of age (Table V). Data are shown in detail on Table Vl.
  • Table V OPN concentrations vary with age in the serum of Non Obese Diabetic mouse. For comparison C57BI/6 serum OPN content is: 287, 53 ng/ml and C57BI/6.Spp1 KO is: 0 as expected. The data are reported in detail in Table Vl.
  • NOD-1 F 0,659 1098,41993 329525,98 329,53
  • NOD-2 F 0,533 888,419933 266525,98 266,53
  • NOD-1 F 0,625 1041,75327 312525,98 312,53
  • NOD-2 F 0,7 1166,75327 350025,98 350,03
  • NOD-1 F 0,584 973,419933 292025,98 292,03
  • NOD-3 F 0,493 821 ,753267 246525,98 246,53
  • the inventor has also aimed to test for the simultaneous presence in the serum or lymphocytes, of at least few of the other genes of the invention identified as part of the secretome. Detection of the proteins coded by these genes simultaneously will strengthen the validity of these markers in early diagnosis of type 1 diabetes. The correlation with the presence of Insulin Auto Antibodies will give additional strength to the use of these genes and their products in diagnostic tests.
  • the inventor has initially studied simultaneously the genes which are in bold on Table VII. These networks have been proposed according to gene function. Following the hypothesis implying a prior to inflammation tissue remodelling that might influence the autoimmune process; it was proposed to include in the studies a set of five genes found to be highly differentially expressed in the data set reported here ("tissue integrity" group on table).
  • Elastase 1 (E/a1) is an enzyme secreted by macrophages or lymphocytes ⁇ Yamasaki, 1987 ⁇ . It is a pro inflammatory protein and has been found to be related in adjuvant induced arthritis ⁇ Escandell, 2006 ⁇ , asthma, lung disease and cystic fibrosis ⁇ Bines, 2005 ⁇ . EIaI has been a marker for exocrine pancreas function and correlates with both residual ⁇ cell secretion and metabolic control of type 1 diabetes. Neutrophil elastase (Ela2) plays a role in the destruction of tissues at sites of inflammation.
  • Mucins in which belongs the gene Mud present also in the data set have been found to confer gut dysfunction in the BB rat diabetes model (Malaisse et al, 2004).
  • mucin 1 has been found to be also an autoantigen in cases of tissue malignancies.
  • the present inventor investigated a projection method classification that allows decomposing the dataset into components that have a desired property as described in the literature [Su-In Lee and Bartzoglou, 2003 ⁇ .
  • PCA Principal Component Analysis
  • This statistical method allows to a better understanding of data in a complex measurement environment ⁇ Su-ln Lee and Bartzoglou S, 2003 ⁇ .
  • the technique has the potential to significantly increase the quality of the resulting data and improve the biological activity of subsequent analysis.
  • Example 7 ostepontin in human samples
  • Osteopontin concentrations have been measured by ELISA in Human Control, Recent onset Diabetic patients and long term Diabetics. The values correspond to corrected Optical Density values corresponding to OPN in the serum (OD-Blanc).
  • the kits used are Human osteopontin (OPN) quantikine ELISA kit DOSTOO and Human osteopontin (OPN) quantikine SixPak (6 plates) SOSTOO; they were used according to the manufacturer's recommendations.
  • -Diabetics-1 corresponds to patients with disease onset prior to 2 years from the collection of the serum.
  • -Diabetics-2 corresponds to patients with diagnosed disease. However the onset of disease of these patients relative to the time of collection of serum was not determined. This group has been placed in a separate group in this analysis due to consistently higher OPN concentrations.
  • -Diabetics-3 group comprises only one individual. The onset of disease of this patient relative to the time of collection of serum was not determined. Data for this group was separated from the two other groups because of its very high OPN concentration. As seen in table IX and Figure 11 , 3 groups of diagnosed diabetics can be distinguished on the basis of OPN protein concentrations in the serum. In group-1 (diabetics-1), values do not differ from the control whilst group-2 (diabetics-2) OPN values are higher than the control values. Group-3 (diabetics-3) OPN concentration is very high, however only one patient is included in this group.
  • Osteopontin concentrations in Human Serum from Control and Diabetic patients correspond to Experiment 1 as described above.
  • Data values are osteopontin concentrations presented as Optical Density measures.
  • Osteopontin concentrations in Human serum from control and recent onset diabetic patients are osteopontin concentrations presented as Optical Density measures.
  • Eta-1 an early component of type-1 (cell-mediated) immunity. Science, 287, 860-864.
  • HIP/PAP-I Hepatocarcinoma-lntestine-Pancreas/Pancreatitis-associated Protein I
  • Dihydrocucurbitacin B isolated from Cayaponia tayuya, reduces damage in adjuvant-induced arthritis.
  • Eur J Pharmacol. Eto, M. et al Clinical significance of elevated osteopontin levels in head and neck cancer patients. Auris Nasus Larynx. [Epub ahead of print]
  • Circulating levels of angiogenic cytokines can predict tumour progression and prognosis in 20 neuroendocrine carcinomas.
  • Clin Endocrinol (Oxf) 62, 434-443.
  • Osteopontin is an adhesive factor for myeloma cells and is found in increased levels in plasma from patients with multiple myeloma.
  • Yamaguchi, Y., et al. (2002) Identification of multiple novel epididymis-specific beta-defensin isoforms in humans and mice. J Immunol, 169, 2516-2523. Yamamoto, N., et al. (2003) Essential role of the cryptic epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis. J Clin Invest, 112, 181-188.
  • pancreatitis-associated protein and REG1A expression in hepatocellular carcinoma Opposite roles of human pancreatitis-associated protein and REG1A expression in hepatocellular carcinoma: association of pancreatitis-associated protein expression with low-stage hepatocellular carcinoma, beta-catenin mutation, and favorable prognosis. Clin Cancer Res, 11, 2568-2575.

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Abstract

La présente invention concerne une méthode de diagnostic précoce d'une maladie présentant une phase pré-inflammatoire, ou de diagnostic d'une prédisposition à ladite maladie, chez un mammifère, avant tout signe clinique, comprenant la mesure du niveau d'au moins une protéine marqueur choisie parmi les protéines murines suivantes : Pap, Reg3a, Reg2, Cel, Reg1, Tff2, Clps, Spp1, Klk9, Klk6, Rib1, Klk5, Muc1, Cckar, Ggh, Ang, Nucb2, Pnliprp2, Pla2g1b, Ela1, Ela2, 2210010C04Rik, Pnliprpi, ltmap1, Vtn, C1qb, Sycn, Amy1, Ctrb1, 1110002O23Rik, 1810014L12Rik et leurs orthologues mammaliens respectifs, dans un fluide corporel ou dans un échantillon de tissu prélevé sur ledit mammifère; la comparaison du niveau mesuré à un niveau de référence pour ladite protéine marqueur et le diagnostic de l'apparition ultérieure de ladite maladie si le niveau mesuré est significativement supérieur au niveau de référence. L'invention concerne également des procédés associés et des utilisations de ces marqueurs précoces de maladies présentant une phase pré-inflammatoire. Parmi les mammifères, la présente invention s'applique de préférence à l'homme et les maladies sont de préférence des maladies auto-immunes, notamment le diabète de type 1. Les protéines sont de préférence la spp1 murine ou son orthologue humain l'ostéopontine, ou un autre orthologue mammalien de spp1.
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CN113092757A (zh) * 2021-02-23 2021-07-09 承德医学院 一种肺癌肝转移早期诊断试剂盒及制备使用方法
CN113092757B (zh) * 2021-02-23 2024-02-06 承德医学院 一种肺癌肝转移早期诊断试剂盒及制备使用方法

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