WO2013178858A1 - Use of vip as a prognostic marker of autoimmune diseases - Google Patents

Use of vip as a prognostic marker of autoimmune diseases Download PDF

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Publication number
WO2013178858A1
WO2013178858A1 PCT/ES2013/070347 ES2013070347W WO2013178858A1 WO 2013178858 A1 WO2013178858 A1 WO 2013178858A1 ES 2013070347 W ES2013070347 W ES 2013070347W WO 2013178858 A1 WO2013178858 A1 WO 2013178858A1
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Prior art keywords
vip
arthritis
disease
patients
autoimmune
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PCT/ES2013/070347
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Spanish (es)
French (fr)
Inventor
Rosa PÉREZ GOMARIZ
Mª Carmen MARTÍNEZ MORA
Yasmina JUARRANZ MORATILLA
Javier LECETA MARTÍNEZ
Isidoro GONZÁLEZ ÁLVARO
Ana María ORTIZ GARCÍA
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Fundación De Investigación Biomédica Del Hospital Universitario La Princesa
Universidad Complutense De Madrid
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Publication of WO2013178858A1 publication Critical patent/WO2013178858A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2278—Vasoactive intestinal peptide [VIP]; Related peptides (e.g. Exendin)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints

Definitions

  • the present invention relates to the use of vasoactive intestinal peptide (VIP) as a prognostic marker of the progression of an autoimmune disease, preferably spopoarthritis or arthritis of autoimmune origin, especially of newly initiated arthritis or rheumatoid arthritis. Therefore, the invention could be framed in the field of biomedicine.
  • VIP vasoactive intestinal peptide
  • RA Rheumatoid arthritis
  • RA Rheumatoid arthritis
  • other autoimmune diseases such as psoriasis, inflammatory bowel disease, spondyloarthritis or systemic lupus erythematoses, a series of etiopathogenic mechanisms that have caused them to be globally referred to as EIMMI (inflammatory diseases mediated by immune mechanisms) and also share their response to similar or equal immunomodulatory or immunosuppressive treatments.
  • EIMMI inflammatory diseases mediated by immune mechanisms
  • the treatment is established following a trial-error scheme in which methotrexate is usually started because it has a good cost / benefit / adverse effect ratio, although in a non-negligible number of patients this leads to a delay in the initiation of an effective treatment since about 40% of patients are refractory to methotrexate or respond partially.
  • the present invention relates to the use of at least one VIP expression product as a prognostic marker of autoimmune diseases, preferably arthritis, as well as a method for the prognosis of autoimmune diseases, preferably arthritis, and the use of a kit comprising at least an element for the quantification of at least one expression product of the VIP gene for the prognosis of autoimmune diseases, preferably arthritis.
  • the authors of the present invention demonstrate that at higher levels of VIP patients with autoimmune diseases, and more specifically Autoimmune arthritis, present a better evolution of the disease.
  • statistical data are also shown by which the association of VIP levels with the level of autoimmune arthritis activity is demonstrated throughout its follow-up in a cohort of patients with autoimmune arthritis. It is also shown that patients with autoimmune arthritis who initially have higher levels of VIP, show a better evolution of the disease compared to those patients who have low levels of VIP, demonstrating the usefulness of this element as a prognostic marker of the disease . Since VIP is encoded by the VIP gene, which results in transcripts that code for VIP, these transcripts have the same utility as VIP as a prognostic marker.
  • VIP as demonstrated in the present invention allows to predict the development of autoimmune arthritis, and that is involved in the modulation of autoimmune elements, this element is also useful as a prognostic factor in other autoimmune diseases with which it shares etiopathogenic mechanisms.
  • These diseases would be inflammatory diseases mediated by immune mechanisms or EIMMI and that includes for example in addition to autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus.
  • a simple and precise method of prognosis of different autoimmune diseases is also described.
  • Said method requires the determination of the amount of at least one expression product of the VIP gene, that is, the amount of mRNA or VIP peptide (vasoactive intestinal peptide) in a biological sample.
  • the amount of at least one VIP gene expression product can be determined by various technical procedures known in the state of the art.
  • the prognosis should be carried out, preferably, but not limited, in the first year of the onset of autoimmune disease symptoms, even before the definitive diagnosis of the disease is definitively established.
  • the method can also be applied at any time during the development of the disease.
  • the method of the invention is applied in patients without treatment, since some drugs can modify the levels of the VIP gene expression products, so it should ideally be used before starting treatment with immunomodulatory drugs.
  • the determination of the level of expression of at least one expression product of the VIP gene makes it possible to have useful information for making therapeutic decisions, allowing patients to be selected with the greatest probability of needing intensive treatment. This results in a reduction in the direct and indirect costs of the disease and an improvement in the patient's quality of life.
  • a first aspect of the invention relates to the use of at least one VIP gene expression product as a prognostic marker of an autoimmune disease in a subject previously diagnosed with said disease.
  • the autoimmune disease is selected from the list comprising autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus.
  • the autoimmune disease is autoimmune arthritis.
  • the autoimmune arthritis is newly started arthritis or rheumatoid arthritis.
  • Another preferred embodiment of this aspect of the invention relates to the use of a VIP gene expression product as a prognostic marker of an autoimmune disease, preferably autoimmune arthritis, disease.
  • an autoimmune disease preferably autoimmune arthritis, disease.
  • Inflammatory bowel, psoriasis, spondyloarthritis or systemic lupus erythematosus more preferably autoimmune arthritis, and even more preferably recent onset arthritis or rheumatoid arthritis in a subject previously diagnosed with the disease where the expression product is mRNA.
  • Another preferred embodiment of this aspect of the invention relates to the use of a VIP gene expression product as a prognostic marker of an autoimmune disease, preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably recently started arthritis or rheumatoid arthritis in a subject previously diagnosed with the disease, where the expression product is VIP.
  • an autoimmune disease preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably recently started arthritis or rheumatoid arthritis in a subject previously diagnosed with the disease, where the expression product is VIP.
  • VIP in the present invention is understood as that gene that has the ability to code for VIP.
  • the gene can be, for example, but not limited to, the VIP gene with access number to the NCBI GenBank ⁇ National Center for Biotechnology Information) AH003027.
  • VIP gene product mRNA is understood in the present invention, for example, but not limited to, the mRNA with access number to the NCBI GenBank NM_003381, and whose translation results in the vasoactive intestinal peptide or VIP.
  • vasoactive intestinal peptide or "VIP” in the present invention refers to the peptide with GenBank accession number of NCBI AAB22264.1 of amino acid sequence SEQ ID NO: 1 (hsdavftdny trlrkqmavk kylnsiln).
  • a second aspect of the invention relates to an in vitro method of prognosis of an autoimmune disease, preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably arthritis. of recent onset or rheumatoid arthritis characterized in that it comprises the quantification of at least the VIP gene expression product in a biological sample isolated from a subject suffering from autoimmune disease.
  • in vitro refers to the method of the invention being performed outside the subject's body.
  • “Autoimmune disease” is understood in the present invention as a disease in which one or several cell types of an individual are attacked by the individual's own immune system since it recognizes said cell types as foreign elements. This causes the deterioration and even the destruction of one or several tissues of the individual.
  • the term "inflammatory bowel disease” or “EN” in the present invention refers to chronic inflammation of the intestine in an individual, where said inflammation is due to the individual's own immune system. The two most frequent forms correspond to ulcerative colitis and Crohn's disease.
  • "psoriasis” is understood to be that skin disease which is characterized by a malfunction of the immune system, which causes excess production of skin cells. This disease leads to the formation of reddish bulges covered with scaling. In addition, excess cell production also causes the infiltration of white blood cells into the skin. Injuries are generally locate in regions with greater friction, such as, but not limited to, elbows, knees or English.
  • Spondyloarthritis in the present invention is understood as those autoimmune diseases with axial and / or peripheral involvement that meet the classification criteria of the Assessment of SpondyloArthritis International Society (ASAS) (Rudwaleit et al. Ann Rheum Dis 2009; 68: 777-783; Rudwaleit et al. Ann Rheum Dis 201 1; 70: 25-31).
  • ASAS SpondyloArthritis International Society
  • Systemic lupus erythematosus is understood in the present invention as a systemic autoimmune disease defined by the criteria of the American College of Rheumatology (Tan et al. Arthritis Rheum- 1982; 25: 1271-1277)
  • autoimmune arthritis would encompass both the terms Rheumatoid Arthritis and Undifferentiated Arthritis, whether it is of recent onset or if it is well established.
  • Undifferentiated or Al Arthritis This last case is called “undifferentiated or Al Arthritis” which, in many cases, left to its free evolution, ends up leading to an RA (van der Helm-van Mil et al. Arthritis Rheum. 2007; 56: 433 -440).
  • RA van der Helm-van Mil et al. Arthritis Rheum. 2007; 56: 433 -440.
  • ARC, AR or Al refer to a chronic and progressive autoimmune systemic disease, which causes chronic inflammation mainly of the joints, and that given its progressive nature, produces the destruction of them, with their consequent deformation and loss of functional capacity.
  • this disease can cause extraarticular alterations in various organs (Carmona, González-Alvaro et al. Ann Rheum Dis 2003; 62: 897-900).
  • the disease activity can be determined by compound indices that provide a number that brings together the information of different clinical and analytical variables such as DAS (van der Heide et al. J Rheumatol 1993; 20: 579-581), DAS28 (Prevoo et al. Arthritis Rheum 1995; 38: 44-48), SDAI, CDAI (Smolen et al. Rheumatology (Oxford) 2003; 42: 244-257) and others.
  • prognosis is understood as the ability to determine in patients of an autoimmune disease, how the disease will evolve in terms of its severity.
  • prognosis also refers to the ability to detect subjects already diagnosed with an autoimmune disease with a high probability of suffering a worsening of the disease. This bad evolution can be defined concretely as a lower possibility of achieving disease remission, taking into account that there are multiple ways to define this ideal state of remission in each disease.
  • isolated biological sample in the present invention refers to any sample that makes it possible to determine the VIP levels of the individual from whom said sample was obtained, and includes, but is not limited to, biological fluids of an individual, obtained by any method known by an expert in the field that serves this purpose.
  • the biological sample could be, for example, but not limited to, a sample of fluid, such as blood, plasma, serum, synovial fluid or lymph.
  • serum or plasma are serum or plasma since they provide more accurate data on VIP levels.
  • Other samples of interest would be, for example, peripheral blood mononuclear cells, preferably T lymphocytes, since they have the highest expression and therefore allow a simpler determination of the levels.
  • the blood is also extracted routinely in tests that can be performed periodically to patients.
  • the sample is blood, plasma or serum.
  • tissue samples obtained that can be analyzed for example by immunohistochemistry, or that can be homogenized to obtain the transcription mRNA of the VIP or VIP gene in order to qualify them.
  • An example is the synovial membrane, which comes from the specific tissue affected by the disease, and is therefore interesting for the determination of an expression product of the VIP gene.
  • the isolated biological sample is blood, serum, plasma or synovial membrane.
  • the biological sample is serum.
  • the biological sample can be, for example, but not limited, fresh, frozen, fixed or fixed and embedded in paraffin.
  • the combination of the quantification of at least one VIP gene expression product in an isolated biological sample with other clinical variables, or markers helps or complements the usefulness of the quantification of at least one product of VIP gene expression in the prognosis of the disease.
  • These variables or markers can be, for example, but not limited to the patient's sex, the activity status at the onset of the disease, as well as others known to the person skilled in the art.
  • An example of these useful clinical variables is, for example, but not limited to, citrullinated antiseptic antibodies (ACPA) if you are studying recently started arthritis or rheumatoid arthritis. These antibodies have been previously described and have some utility in the prognosis of arthritis.
  • ACPA citrullinated antiseptic antibodies
  • the method may further comprise determining the amount of VIP gene expression product in a biological sample isolated from a subject suffering from an autoimmune disease, preferably autoimmune arthritis , inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably recent onset arthritis or rheumatoid arthritis in a subject previously diagnosed with autoimmune disease, the determination of other clinical variables, or markers.
  • the clinical variables or Markers are the patient's sex, the activity status at the onset of the disease.
  • Another preferred embodiment of the second aspect of the invention relates to an in vitro method of prognosis of autoimmune arthritis, preferably recently started arthritis or rheumatoid arthritis characterized in that it comprises the quantification of at least VIP gene expression product in an isolated biological sample. of a subject suffering from autoimmune arthritis and who also includes the determination of the sex of the subject, the presence of citrullinated antiseptic antibodies (ACPA) and / or the state of activity at the onset of the disease.
  • ACPA citrullinated antiseptic antibodies
  • a third aspect of the present invention relates to an in vitro method for the prognosis of an autoimmune disease, preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably recent arthritis.
  • autoimmune arthritis preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably recent arthritis.
  • onset or rheumatoid arthritis in a previously diagnosed subject of autoimmune disease comprising the following stages:
  • autoimmune disease preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably arthritis of recent onset or rheumatoid arthritis, b. compare the value obtained in step (a) with a reference amount, and
  • step (a) Associate the individual from step (a) to the group of patients with poor prognosis of autoimmune disease when the quantity quantified in step (a) is less than the 50th percentile of the reference amount of step (b).
  • the comparison with the 50th percentile of reference amount is useful when classifying patients. It is also shown that as this percentile is reduced, the value is more restrictive and therefore allows a better classification, and greater security in their classification. Examples of these more restrictive and useful percentiles would be, for example, but not limited to, the 25th percentile or the 10th percentile.
  • step (c) it is associated with individual from step (a) to the group of patients with poor prognosis of autoimmune disease when the quantity quantified in step (a) is less than the 25th percentile of the reference amount of step (b).
  • step (c) the individual of step (a) is associated with the group of patients with poor prognosis of autoimmune disease when the amount quantified in step (a) is less than the 10th percentile of the reference amount of step (b).
  • the term "reference quantity" refers to any value or range of values derived from the quantification of a VIP gene expression product in a collection of biological samples from individuals healthy and that are representative of the population in which the test will be applied. This sample can also be a mixture of biological samples obtained from different healthy subjects. The reference amount must be measured in the same way, and be obtained in the same type of isolated biological sample as the amount of interest in the patients. Therefore, in a preferred embodiment of this aspect of the invention, the reference amount in step (b) is the amount of VIP in a collection of biological samples isolated from individuals who do not have autoimmune diseases.
  • “healthy""healthyindividual" or “healthy subject” is understood in the present invention that subject or individual who does not suffer from any autoimmune disease.
  • “Healthy population” is understood in the present invention, a set of individuals who do not have autoimmune diseases.
  • Healthy individuals representative of the population in which the test is to be applied means those people who do not suffer from autoimmune diseases at the time of extraction and who as a group have a similar pattern in terms of race, age, distribution by gender than the population of patients to whom the test is to be applied.
  • the quantification of the amount of an expression product of the VIP gene in an isolated biological sample refers to the quantification of the mRNA, of the complementary DNA (cDNA) to this mRNA, and / or of the VIP protein in an isolated biological sample.
  • the quantification can therefore be performed by determining the level of mRNA derived from its transcription, after extracting the total RNA present in the isolated biological sample, which can be performed by protocols known in the state of the art.
  • the isolated biological sample can be physically or mechanically treated to break up the cellular tissue or structures and release the intracellular components to an aqueous or organic solution to prepare the nucleic acids for further analysis. Nucleic acids are extracted from the sample by procedures known to those skilled in the art and commercially available.
  • the level of mRNA derived from VIP transcription can be determined, for example, but not limited to, by amplification by polymerase chain reaction (PCR), back transcription in combination with polymerase chain reaction (RT-PCR ), Quantitative RT-PCR, retrotranscription in combination with ligase chain reaction (RT-LCR), or any other nucleic acid amplification method; serial analysis of gene expression (SAGE, SuperSAGE); DNA chips made with oligonucleotides deposited by any mechanism; DNA microarrays made with oligonucleotides synthesized in situ by photolithography or by any other mechanism; in situ hybridization using specific probes labeled with any method of marking; by electrophoresis gels; by membrane transfer and hybridization with a specific probe; by nuclear magnetic resonance or any other diagnostic imaging technique using paramagnetic nanoparticles or any other type of detectable nanoparticles functionalized with antibodies or by any other means. Therefore, in a preferred embodiment of this aspect of the invention, the expression product quantified in step (a) is
  • Quantification on the other hand can also be performed by determining the level of VIP protein derived from the translation of the mRNA transcribed from the gene.
  • This protein quantification can be carried out by any method known to a person skilled in the art that serves this purpose, such as, but not limited to, immunodetection methods (such as western blot, ELISA, immunohistochemistry), methods based on isobaric labeling (such as ⁇ TRAQ - isobaric Tag for Relative and Absolute Quantitation-, or ICAT -Isotope Coded Affinity Tag-) or in isotopic (like SILAC -Stable Isotopes Labeling by Amino Acids in Cell Culture-) or based on fluorescent (like 2D-) DIGE -Difference in Gel Electrophoresis-), as well as methods based on mass spectrometry (MRM, -Multiple Reaction Monitoring-) .For all this in another preferred embodiment of this aspect of the invention the expression product quantified in step
  • Subject in the present invention is understood as that individual susceptible to suffering from an autoimmune disease.
  • the subject is a human.
  • the biological sample is selected from blood, plasma, serum, or synovial membrane.
  • the biological sample is serum.
  • Another preferred embodiment of the third aspect of the invention relates to the method where the subject is a human.
  • any of the steps of the methods of the invention can be totally or partially automated, for example, but not limited to, by means of a robotic sensor device for obtaining the quantities of the VIP gene expression products in step (a ), or the computerized comparison in step (b) of the third aspect of the invention.
  • the methods described herein may comprise additional steps, for example, related to the pretreatment of the sample or the extraction of the protein and / or genetic material necessary for subsequent analysis.
  • the third aspect of the invention may further comprise the determination of other clinical variables, or markers such as, but not limited to, sex, the state of activity at the onset of the disease that helps or complements the prognosis of the disease.
  • other clinical variables or markers
  • markers such as, but not limited to, sex, the state of activity at the onset of the disease that helps or complements the prognosis of the disease.
  • the method further comprises determining the sex of the subject, and / or the state of activity at the onset of the disease.
  • the method further comprises determining the sex of the subject, the presence of citrullinated anti-peptide antibodies and / or the state of activity at the onset of the disease.
  • All methods in the present invention may additionally include a step of treating the patient based on the prognosis obtained. This treatment step will be determined based on the severity of the prognosis of the disease analyzed.
  • the treatment to be followed can be determined, such as, for example, glucocorticoids, biological therapies such as TNF blocking agents, tocilizumab, abatacept or rituximab, or disease modifying drugs (DMARDs) which include antimalarials, methotrexate, leflunomide, sulfasalacin , aurothiomalate, and / or cyclosporine A.
  • biological therapies such as TNF blocking agents, tocilizumab, abatacept or rituximab
  • DMARDs disease modifying drugs
  • a fourth aspect of the invention relates to the use of a kit comprising the elements necessary to determine the amount of VIP for obtaining useful data for the prognosis of an autoimmune disease, preferably recent arthritis.
  • rheumatoid arthritis inflammatory bowel disease, psoriasis or systemic lupus erythematosus, and more preferably recent onset arthritis or rheumatoid arthritis previously diagnosed with autoimmune disease.
  • kits may also include, without any limitation, the use of buffers, enzymes, polymerase enzymes, cofactors to obtain optimum activity thereof, agents to prevent contamination, etc.
  • the kits may include all the supports and containers necessary for their implementation and optimization.
  • the kits may also contain other molecules, genes, proteins or probes of interest, which serve as positive and negative controls.
  • the kits further comprise the instructions for carrying out the methods of the invention.
  • the kit comprises probes that allow the determination of the amount of mRNA transcribed from the VIP gene for the prognosis of an autoimmune disease, preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or Systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably newly started arthritis or rheumatoid arthritis previously diagnosed with autoimmune disease.
  • an autoimmune disease preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or Systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably newly started arthritis or rheumatoid arthritis previously diagnosed with autoimmune disease.
  • the kit comprises antibodies that allow the determination of the amount of VIP for the prognosis of an autoimmune disease, preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus, more preferably Autoimmune arthritis, and even more preferably newly started arthritis or rheumatoid arthritis previously diagnosed with autoimmune disease.
  • the subject is a human.
  • the kit can also include, without any limitation, buffers, enzymes, agents to prevent contamination, etc.
  • the kit can include all the supports and containers necessary for commissioning and optimization.
  • the kit may also contain other molecules, antibody antibodies, or proteins, which serve as positive and negative controls or for the normalization of the values obtained.
  • the kit further comprises instructions for carrying out the methods of the invention.
  • FIG. 1 Variability of VIP levels in healthy controls and patients with rheumatoid arthritis or undifferentiated arthritis. Data are shown as the median (horizontal line inside the boxes), the 25th and 75th percentiles (bottom and top lines of the boxes respectively) and the 10th and 90th percentiles (small horizontal lines at the end of the vertical lines below and above the boxes respectively). Points represent out of range values.
  • the dashed horizontal line refers to the 25th percentile of the healthy population.
  • FIG. 2 Relationship of VIP levels with gender (A), age (B) and disease activity (DAS28) (C). Data are shown as the median (horizontal line inside the boxes), the 25th and 75th percentiles (bottom and top lines of the boxes respectively) and the 10th and 90th percentiles (small horizontal lines at the end of the vertical lines below and above the boxes respectively) in the case of figure A.
  • the different points are represented with the estimated regression line (continuous black line), as well as the 95% confidence interval (gray zone around the black line).
  • FIG. 3. VIP levels adjusted for initial visits (first column), 6 months (second column), 12 months (third column) and 24 months (fourth column).
  • FIG. 4. VIP levels at the initial visit according to the level of activity of the disease (according to DAS28) on visits at 2 (A) and 5 (B) years of follow-up.
  • Panel C shows the degree of arthritis activity (DAS28) at the initial visit and after two and five years of follow-up in patients who have high VIP (white boxes) or low (gray boxes).
  • FIG. 5 Intensity of treatment in patients studied as they present high or low VIP levels at the first follow-up visit.
  • the intensity of the treatment was quantified according to the number of days with treatment with all DMARDs (disease modifying drugs) that each patient has received during the first two years of their follow-up.
  • DMARDs disease modifying drugs
  • FIG. 6 Effect of the combination of gender (A), age (B) and activity level at the beginning of the disease (C) with VIP levels to predict the disease activity status at two years of follow-up .
  • Data are shown as the median (horizontal line inside the boxes), the 25th and 75th percentiles (bottom and top lines of the boxes respectively) and the 10th and 90th percentiles (small horizontal lines at the end of the vertical lines below and above the boxes respectively) of the DAS28 at two years of follow-up in patients who have high VIP (white boxes) or low (gray boxes).
  • FIG. 7 Effect of positivity of citrullinated antiseptic antibodies (ACPA) on the predictive capacity of VIP levels.
  • FIG. 8 VIP levels before (white boxes) and after (gray boxes) to start the different treatments. It should be noted that patients treated with anti-TNF were very scarce and that may be the cause of statistical significance not being achieved, the effect observed being greater.
  • FIG. 9 Serum VIP levels in patients of the initial Spondyloarthritis office, diagnosed or not of Spondyloarthritis. Data are shown as the median of the VIP levels (line inside the box), the 25th and 75th percentiles (bottom and top edges of the box, respectively) and the 10th and 90th percentiles (bottom and top lines outside of the box, respectively). The dots represent cases outside these ranges.
  • FIG. 10 A) Serum VIP levels according to the patient's age at the first visit of the study. The data are shown as the average value for each age point adjusted by sex, visit and measuring plate of the VIP levels (solid round), together with the 95% confidence interval (lines above and below the round) . The estimation of these values was carried out by means of the "margins" command of Stata 12, after performing a multivariable linear regression by means of generalized linear models with the "glm” command of Stata.
  • FIG. 11 Relationship of serum VIP levels with different clinical variables of patients.
  • the data is shown as the median of the VIP levels (line inside the box), the 25th and 75th percentiles (bottom and top edges of the box, respectively) and the 10th and 90th percentiles (lower and upper lines outside the box, respectively). The dots represent cases outside these ranges.
  • the data is shown as the average of the VIP value (normalized by logarithmic transformation) for each BASFI value shown in the figure adjusted for the rest of the variables that were included in the multivariable analysis (solid round).
  • the 95% confidence interval is shown (lines above and below the round). The estimation of these values was carried out by means of the "margins” command of Stata 12, after performing a multivariable analysis for longitudinal data nested by visit and patient by means of the "xtgee" command of Stata.
  • FIG. 12 Relationship of VIP levels with radiological and analytical variables.
  • B Relationship between VIP levels and hemoglobin levels. The data are shown as the average of the VIP value (normalized by logarithmic transformation) for each Hemoglobin (Hb) value shown in the figure, adjusted for the rest of the variables that were included in the multivariable analysis (solid round).
  • the recently started registry of arthritis patients of the La Princesa University Hospital Health Research Institute includes patients derived from Primary Care who have one or more inflamed joints for a minimum of four weeks and a maximum of one year of evolution.
  • the only exclusion criterion is that throughout the follow-up patients will be diagnosed with microcrystalline arthritis, septic or viral arthritis, spondyloarthropathies or connective tissue diseases.
  • the study protocol establishes four visits in a five-year follow-up period: a baseline visit, at six months, at one year, at two and at five years. At each visit demographic data (gender, level of studies and marital status), clinical (date of onset of the disease, count of 28 painful joints [NAD28] and swollen [NAT28], the assessment are collected).
  • FAME includes: antimalarials, methotrexate, leflunomide, sulfasalacin, aurothiomalate, cyclosporin A and biological therapies that in turn comprise TNF blocking agents, tocilizumab, abatacept and rituximab. Of all these drugs, not only the dose is collected, but also the start date and, if appropriate, suspension.
  • Treatment Intensity is the result of adding the number of days the patient has received each of the drugs in the first two years of treatment, weighted by multiplying by a correction factor (related with its theoretical efficacy): 1x for antimalarials, 1'5x for methotrexate, leflunomide, sulfasalacin, aurothiomalate and cyclosporine A and 2x for biological therapies.
  • correction factor related with its theoretical efficacy
  • VIP levels have been studied in 93 patients of this consultation, in a total of 376 visits. Likewise, VIP levels have been determined in 100 healthy blood donors. The determination of VIP levels is done through a competitive ELISA from Phoenix Pharmaceuticals, INC. In order to carry out this test, it is necessary to lyophilize the samples of about 250 microliters of serum which are subsequently diluted in 120 microliters of a test buffer of the same commercial house and added to a plate whose wells are coated with a secondary antibody . A biotinylated primary and VIP antibody is loaded together with the samples. After two hours of incubation, unbound material is removed by washing.
  • the distribution of VIP values is quite variable in the 3 populations studied: healthy controls, patients with RA and Al.
  • the 50, 25 and 10 percentiles of the healthy population have been considered as the limit to define low VIP because this is a common procedure in statistics and epidemiology that divides the population into two distinct groups.
  • some analyzes have been performed with the continuous variable that provides greater sensitivity.
  • VIP levels were analyzed at the baseline study visit according to the activity of the patients at two years of follow-up. As can be seen in Figure 4, the lowest VIP levels at the initial visit accumulate among patients with high activity, despite treatment, at two ( Figure 4A) and five ( Figure 4B) years of follow-up.
  • FIG. 4C shows that in the initial visit patients have similar activity levels have high or low VIP, while at two or five years of follow-up patients with low VIP tend to have higher values of DAS28. Therefore, patients with low levels of VIP tend to have a worse evolutionary course throughout the disease, although as shown in Figure 5 they receive a more intense treatment with DMARD than patients with high VIP.
  • the predictive effect of low VIP levels can be modulated or enhanced by other confounding variables. As shown in Figure 6A, women with low VIP had activity levels at two years of follow-up greater than those of women with normal or high VIP, while these differences were less marked in men.
  • Figure 6B age did not interfere with the predictive capacity of VIP (Figure 6B).
  • activity levels during the initial visit can also modulate the predictive capacity of VIP levels.
  • Figure 6C shows that there were no patients in remission or low activity at the beginning of follow-up among patients with low VIP. These patients were among those who had moderate or high activity. But among the latter, those with low VIP levels were the worst at the end of the follow-up.
  • Example 2 Study of VIP levels as a Prognostic factor in Patients with Spondyloarthritis.
  • the study protocol establishes an annual visit in which data on disease activity (BASDAI), functional capacity (BASFI), conventional analytics and determination of HLA-B27, prescribed treatment and collection of serum samples are collected. and peripheral blood cells for RNA and DNA extraction. All patients undergo a MR of sacroiliac and lumbar spine to determine the presence of inflammatory signs and conventional radiographic study of lumbar spine and sacroiliac to objectify structural changes typical of the disease. The presence of comorbidities related to spondyloarthritis (uveitis, psoriasis, inflammatory bowel disease, enthesitis, etc.) is systematically collected.

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Abstract

The invention relates to the use of an expression product of the VIP gene as a prognostic marker of an autoimmune disease, preferably autoimmune spondyloarthritis, in patients that have previously been diagnosed with said autoimmune disease. The invention also relates to a method for the prognosis of an autoimmune disease, preferably autoimmune spondyloarthritis, said method being characterised by the determination of the quantity of an expression product of the VIP gene.

Description

USO DE VIP COMO MARCADOR PRONÓSTICO DE ENFERMEDADES  USE OF VIP AS A FORECAST MARKER OF DISEASES
AUTOINMUNES  AUTOIMMUNES
DESCRIPCIÓN DESCRIPTION
La presente invención se refiere al uso del péptido intestinal vasoactivo (VIP) como marcador pronóstico de la evolución de una enfermedad autoinmune, preferiblemente espodiloartritis o artritis de origen autoinmune, especialmente de la artritis de reciente comienzo o la artritis reumatoide. Por tanto, la invención se podría encuadrar en el campo de la biomedicina. The present invention relates to the use of vasoactive intestinal peptide (VIP) as a prognostic marker of the progression of an autoimmune disease, preferably spopoarthritis or arthritis of autoimmune origin, especially of newly initiated arthritis or rheumatoid arthritis. Therefore, the invention could be framed in the field of biomedicine.
ESTADO DE LA TÉCNICA STATE OF THE TECHNIQUE
La artritis reumatoide (AR) es la enfermedad reumática autoinmune más común. Comparte con otras enfermedades autoinmunes, como la psoriasis, la enfermedad inflamatoria intestinal, espondiloartritis o el lupus eritematosos sistémico, una serie de mecanismos etiopatogénicos que ha hecho que se las denomine globalmente como EIMMI (enfermedades inflamatorias mediadas por mecanismos inmunes) y que también comparten su respuesta a tratamientos inmunomoduladores o inmunosupresores similares o ¡guales. Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease. It shares with other autoimmune diseases, such as psoriasis, inflammatory bowel disease, spondyloarthritis or systemic lupus erythematoses, a series of etiopathogenic mechanisms that have caused them to be globally referred to as EIMMI (inflammatory diseases mediated by immune mechanisms) and also share their response to similar or equal immunomodulatory or immunosuppressive treatments.
Aunque la variabilidad en el curso clínico de la AR es marcada, dejada a su evolución natural esta enfermedad provoca inflamación crónica de la membrana sinovial de las articulaciones diartrodiales, lo cual causa su progresivo deterioro. Como consecuencia provoca una discapacidad y deterioro de la calidad de vida del paciente. Además, con cierta frecuencia se acompaña de manifestaciones sistémicas y de un aumento en el número de comorbilidades, e incluso de la mortalidad en comparación con la población general (Carmona et al. Ann Rheum Dis 2003;62:897-900). Su etiología no ha sido completamente dilucidada, aunque se acepta que es la consecuencia de la influencia de factores ambientales en un huésped genéticamente predispuesto. Su prevalencia oscila entre un 0,3 y un 1 %, con un claro gradiente norte-sur de mayor a menor prevalencia (Alamanos and Drosos Autoimmun Rev 2005;4: 130-136). En España la prevalencia es de un 0,5% (Carmona et al. Ann Rheum Dis 2001 ;60: 1040-1045), lo que supone unos 200.000 pacientes con AR en España. Dada su capacidad para producir incapacidad funcional, la AR es una de las principales causas de incapacidad laboral definitiva en España y a finales de los 90, se estimó que los costes anuales directos de la enfermedad alcanzaban los 14.500 dólares por paciente y año (Lajas et al. Arthritis Rheum 2003;49:64-70). Por tanto, se trata de una enfermedad con una gran relevancia económica y social puesto que la edad más frecuente de comienzo de la enfermedad es entre los 35 y los 55 años, momento del mayor aporte en el sector laboral. Pero en pacientes mayores de 65 años también representa un problema económico importante por aumentar la dependencia y los costes indirectos derivados de ésta. Although the variability in the clinical course of RA is marked, left to its natural evolution this disease causes chronic inflammation of the synovial membrane of the diartrodial joints, which causes its progressive deterioration. As a result it causes a disability and deterioration of the patient's quality of life. In addition, with some frequency it is accompanied by systemic manifestations and an increase in the number of comorbidities, and even mortality compared to the general population (Carmona et al. Ann Rheum Dis 2003; 62: 897-900). Its etiology has not been fully elucidated, although it is accepted that it is the consequence of influence of environmental factors in a genetically predisposed host. Its prevalence ranges between 0.3 and 1%, with a clear north-south gradient from highest to lowest prevalence (Alamanos and Drosas Autoimmun Rev 2005; 4: 130-136). In Spain the prevalence is 0.5% (Carmona et al. Ann Rheum Dis 2001; 60: 1040-1045), which is about 200,000 patients with RA in Spain. Given its ability to produce functional disability, RA is one of the main causes of permanent work disability in Spain and in the late 1990s, it was estimated that the direct annual costs of the disease reached $ 14,500 per patient per year (Lajas et al Arthritis Rheum 2003; 49: 64-70). Therefore, it is a disease with great economic and social relevance since the most frequent age of onset of the disease is between 35 and 55 years, the time of the greatest contribution in the labor sector. But in patients over 65 years of age it also represents an important economic problem due to the increase in dependence and indirect costs derived from it.
Durante los últimos años se ha puesto de manifiesto que el inicio de tratamiento intensivo y precoz con fármacos modificadores de la enfermedad (FAME) puede mejorar este sombrío panorama (Anderson et al. Arthritis Rheum 2000; 43:22-29). El establecimiento de consultas específicas de artritis de reciente comienzo ha supuesto un hito en la evolución de la enfermedad, al permitir una derivación más rápida al especialista y el inicio precoz de FAME. De hecho, se ha podido establecer que la implementación de programas para establecer consultas de artritis de reciente comienzo ha mejorado el curso evolutivo de la enfermedad (Descalzo et al. Arthritis Care Res (Hoboken) 2012;64:321 -330). Aún así, muchos pacientes no alcanzan el estado ideal de remisión, probablemente porque no se establece un tratamiento lo suficientemente intenso desde el inicio, por no poderse determinar la gravedad con la que va a cursar la enfermedad. Como consecuencia de la generalización de las consultas de artritis de reciente comienzo, se han publicado recientemente unos criterios que permiten clasificar como AR de forma más precoz a los pacientes (Aletaha et al. Ann Rheum Dis 2010; 69: 1580-1588). Aún así, aproximadamente un 40% de los pacientes de estas consultas no cumplen criterios de AR al inicio del seguimiento, a pesar de lo cual, pueden acabar precisando un tratamiento intenso para el control de su enfermedad. Por ello, es importante el desarrollo de marcadores pronósticos de un curso evolutivo de la enfermedad más agresivo, actualmente poco desarrollados, así como de marcadores que predigan la respuesta a determinados fármacos, en la actualidad casi inexistentes. Como consecuencia, el tratamiento se establece siguiendo un esquema de ensayo-error en el que se suele empezar por metotrexato por tener este una buena relación coste/beneficio/efectos adversos, aunque en un número no desdeñable de pacientes esto conduce a una demora en el inicio de un tratamiento eficaz ya que cerca del 40% de los pacientes son refractarios al metotrexato o responden parcialmente. In recent years it has been shown that the initiation of intensive and early treatment with disease-modifying drugs (DMARDs) can improve this bleak picture (Anderson et al. Arthritis Rheum 2000; 43: 22-29). The establishment of specific consultations of arthritis of recent beginning has been a milestone in the evolution of the disease, allowing a faster referral to the specialist and the early onset of DMARD. In fact, it has been established that the implementation of programs to establish newly started arthritis consultations has improved the evolutionary course of the disease (Descalzo et al. Arthritis Care Res (Hoboken) 2012; 64: 321-330). Even so, many patients do not reach the ideal state of remission, probably because a sufficiently intense treatment is not established from the beginning, because the severity with which the disease is going to be determined cannot be determined. As a consequence of the generalization of the newly started arthritis consultations, criteria have been published recently that allow patients to be classified as RA earlier (Aletaha et al. Ann Rheum Dis 2010; 69: 1580-1588). Even so, approximately 40% of patients in these consultations do not meet RA criteria at the beginning of the follow-up, despite which, they may end up requiring an intense treatment to control their disease. Therefore, it is important to develop prognostic markers of a more aggressive course of the disease, currently underdeveloped, as well as markers that predict the response to certain drugs, which are now almost non-existent. As a consequence, the treatment is established following a trial-error scheme in which methotrexate is usually started because it has a good cost / benefit / adverse effect ratio, although in a non-negligible number of patients this leads to a delay in the initiation of an effective treatment since about 40% of patients are refractory to methotrexate or respond partially.
Paradójicamente, frente a esta situación se ha propuesto que el uso precoz de terapias biológicas (anti-TNF, anti-IL6 etc.) podría aumentar el porcentaje de pacientes en los que la enfermedad entra en remisión (Allaart et al. J Rheumatol Suppl 2007;80:25-33). Sin embargo, el uso generalizado de este tipo de fármacos resultaría insostenible para cualquier sistema sanitario y expondría, de forma innecesaria, a algunos pacientes a los efectos adversos que las terapias biológicas pueden producir. Paradoxically, in the face of this situation it has been proposed that the early use of biological therapies (anti-TNF, anti-IL6 etc.) could increase the percentage of patients in whom the disease goes into remission (Allaart et al. J Rheumatol Suppl 2007 ; 80: 25-33). However, the widespread use of this type of drugs would be unsustainable for any healthcare system and would unnecessarily expose some patients to the adverse effects that biological therapies can produce.
Por todo ello, se hace necesaria la búsqueda de elementos que permitan determinar cuál va a ser el curso evolutivo de la enfermedad y que, por tanto, permitan determinar precozmente la intensidad del tratamiento adecuada para cada paciente, asumiendo los riesgos de los tratamientos más agresivos para pacientes que vayan a tener un peor pronóstico y evitando estos tratamientos agresivos en los pacientes que vayan a tener una forma leve de la enfermedad. En este sentido ya se han realizado algunos estudios como por ejemplo el uso de anticuerpos anti-proteínas citrulinadas (Puszczewicz M et al. Arch Med Sci 201 1 ; 7(2): 189-194). Sin embargo, este marcador solo aparece en alrededor de un 40 % de pacientes con artritis de reciente comienzo y no es capaz de detectar a todos los pacientes con curso evolutivo desfavorable. Así pues, se hace necesaria la búsqueda de otros marcadores que permitan una mejor determinación del pronóstico de la enfermedad, o que puedan suponer alternativas o complementen los biomarcadores ya disponibles. Therefore, it is necessary to search for elements that allow to determine what will be the evolutionary course of the disease and, therefore, allow early determination of the intensity of the appropriate treatment for each patient, assuming the risks of the most aggressive treatments for patients who are going to have a worse prognosis and avoiding these treatments aggressive in patients who are going to have a mild form of the disease. In this sense, some studies have already been carried out, such as the use of anti-citrullinated protein antibodies (Puszczewicz M et al. Arch Med Sci 201 1; 7 (2): 189-194). However, this marker only appears in about 40% of patients with arthritis of recent onset and is not able to detect all patients with an unfavorable evolutionary course. Thus, it is necessary to search for other markers that allow a better determination of the prognosis of the disease, or that may involve alternatives or complement the biomarkers already available.
Por lo tanto, el desarrollo de test pronósticos que se basen en biomarcadores fácilmente detectables que puedan identificar a los pacientes con peor pronóstico es una necesidad evidente en la reumatología actual. La posibilidad de que algunos de estos biomarcadores puedan detectar a pacientes que respondan mejor o peor a un determinado tratamiento permitiría ahorrar tiempo y costes en el manejo de la AR. Therefore, the development of prognostic tests that are based on easily detectable biomarkers that can identify patients with a worse prognosis is an obvious need in current rheumatology. The possibility that some of these biomarkers can detect patients who respond better or worse to a certain treatment would save time and costs in the management of RA.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
La presente invención se refiere al uso de al menos un producto de expresión de VIP como marcador pronóstico de enfermedades autoinmunes, preferiblemente artritis, así como un método para el pronóstico de enfermedades autoinmunes, preferiblemente artritis, y al uso de un kit que comprende al menos un elemento para la cuantificación de al menos un producto de expresión del gen VIP para el pronóstico de enfermedades autoinmunes, preferiblemente artritis. The present invention relates to the use of at least one VIP expression product as a prognostic marker of autoimmune diseases, preferably arthritis, as well as a method for the prognosis of autoimmune diseases, preferably arthritis, and the use of a kit comprising at least an element for the quantification of at least one expression product of the VIP gene for the prognosis of autoimmune diseases, preferably arthritis.
Los autores de la presente invención demuestran que a mayores niveles de VIP los pacientes de enfermedades autoinmunes, y de forma más concreta artritis autoinmune, presentan una mejor evolución de la enfermedad. En la presente invención además se muestran datos estadísticos mediante los cuales se demuestra la asociación de los niveles de VIP con el nivel de actividad de la artritis autoinmune a lo largo de su seguimiento en una cohorte de pacientes con artritis autoinmune. También se demuestra que los pacientes con artritis autoinmune que de inicio presentan unos mayores niveles de VIP, muestran una mejor evolución de la enfermedad con respecto a aquellos pacientes que presentan niveles bajos de VIP, demostrando la utilidad de este elemento como marcador pronóstico de la enfermedad. Dado que VIP se encuentra codificado por el gen VIP, el cual da lugar a transcritos que codifican para VIP, estos transcritos presentan la misma utilidad que VIP como marcador pronóstico. The authors of the present invention demonstrate that at higher levels of VIP patients with autoimmune diseases, and more specifically Autoimmune arthritis, present a better evolution of the disease. In the present invention, statistical data are also shown by which the association of VIP levels with the level of autoimmune arthritis activity is demonstrated throughout its follow-up in a cohort of patients with autoimmune arthritis. It is also shown that patients with autoimmune arthritis who initially have higher levels of VIP, show a better evolution of the disease compared to those patients who have low levels of VIP, demonstrating the usefulness of this element as a prognostic marker of the disease . Since VIP is encoded by the VIP gene, which results in transcripts that code for VIP, these transcripts have the same utility as VIP as a prognostic marker.
Dado que VIP tal y como se demuestra en la presente invención permite pronosticar el desarrollo de la artritis autoinmune, y que se encuentra implicado en la modulación de elementos autoinmunes, dicho elemento también es de utilidad como factor pronóstico en otras enfermedades autoinmunes con las que comparte mecanismos etiopatogénicos. Estas enfermedades serían las enfermedades inflamatorias mediadas por mecanismos inmunes o EIMMI y que incluye por ejemplo además de la artritis autoinmune, enfermedad inflamatoria intestinal, psoriasis, espondiloartritis o el lupus eritematoso sistémico. Since VIP as demonstrated in the present invention allows to predict the development of autoimmune arthritis, and that is involved in the modulation of autoimmune elements, this element is also useful as a prognostic factor in other autoimmune diseases with which it shares etiopathogenic mechanisms. These diseases would be inflammatory diseases mediated by immune mechanisms or EIMMI and that includes for example in addition to autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus.
En la presente invención también se describe un método sencillo y preciso de pronóstico de diferentes enfermedades autoinmunes. Dicho método requiere la determinación de la cantidad de al menos un producto de expresión del gen VIP, es decir, la cantidad de mRNA o del péptido VIP (péptido intestinal vasoactivo) en una muestra biológica. La cantidad de, al menos, un producto de expresión del gen VIP puede determinarse mediante diversos procedimientos técnicos conocidos en el estado de la técnica. El pronóstico debe ser realizado, de forma preferible, aunque sin limitarse, en el primer año del inicio de los síntomas de la enfermedad autoinmune, incluso antes de que se establezca definitivamente el diagnóstico definitivo de la enfermedad. De cualquier forma, el método también puede ser aplicado a lo largo de cualquier momento del desarrollo de la enfermedad. También de forma preferible el método de la invención se aplica en pacientes sin tratamiento, ya que algunos fármacos pueden modificar los niveles de los productos de expresión del gen VIP, por lo que idealmente debería ser usado antes de iniciar tratamiento con fármacos inmunomoduladores. In the present invention a simple and precise method of prognosis of different autoimmune diseases is also described. Said method requires the determination of the amount of at least one expression product of the VIP gene, that is, the amount of mRNA or VIP peptide (vasoactive intestinal peptide) in a biological sample. The amount of at least one VIP gene expression product can be determined by various technical procedures known in the state of the art. The prognosis should be carried out, preferably, but not limited, in the first year of the onset of autoimmune disease symptoms, even before the definitive diagnosis of the disease is definitively established. However, the method can also be applied at any time during the development of the disease. Also preferably the method of the invention is applied in patients without treatment, since some drugs can modify the levels of the VIP gene expression products, so it should ideally be used before starting treatment with immunomodulatory drugs.
La determinación del nivel de expresión de al menos un producto de expresión del gen VIP permite disponer de información útil para la toma de decisiones terapéuticas, permitiendo seleccionar a los pacientes con mayor probabilidad de necesitar un tratamiento intenso. Esto deriva en un abaratamiento de los costes directos e indirectos de la enfermedad y una mejora de la calidad de vida del paciente. The determination of the level of expression of at least one expression product of the VIP gene makes it possible to have useful information for making therapeutic decisions, allowing patients to be selected with the greatest probability of needing intensive treatment. This results in a reduction in the direct and indirect costs of the disease and an improvement in the patient's quality of life.
Por todos estos motivos, un primer aspecto de la invención se refiere al uso de, al menos, un producto de expresión del gen VIP como marcador pronóstico de una enfermedad autoinmune en un sujeto previamente diagnosticado de dicha enfermedad. En una realización preferida la enfermedad autoinmune se selecciona de la lista que comprende artritis autoinmune, enfermedad inflamatoria intestinal, psoriasis, espondiloartritis o el lupus eritematoso sistémico. En una realización más preferida la enfermedad autoinmune es artritis autoinmune. En una realización aun más preferida la artritis autoinmune es artritis de reciente comienzo o artritis reumatoide. For all these reasons, a first aspect of the invention relates to the use of at least one VIP gene expression product as a prognostic marker of an autoimmune disease in a subject previously diagnosed with said disease. In a preferred embodiment the autoimmune disease is selected from the list comprising autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus. In a more preferred embodiment the autoimmune disease is autoimmune arthritis. In an even more preferred embodiment, the autoimmune arthritis is newly started arthritis or rheumatoid arthritis.
Otra realización preferida de este aspecto de la invención se refiere al uso de un producto de expresión del gen VIP como marcador pronóstico de una enfermedad autoinmune, preferiblemente artritis autoinmune, enfermedad inflamatoria intestinal, psoriasis, espondiloartritis o lupus eritematoso sistémico, más preferiblemente artritis autoinmune, y aun más preferiblemente artritis de reciente comienzo o artritis reumatoide en un sujeto previamente diagnosticado de la enfermedad donde el producto de expresión es mRNA. Otra realización preferida de este aspecto de la invención se refiere al uso de un producto de expresión del gen VIP como marcador pronóstico de una enfermedad autoinmune, preferiblemente artritis autoinmune, enfermedad inflamatoria intestinal, psoriasis, espondiloartritis o lupus eritematoso sistémico, más preferiblemente artritis autoinmune, y aun más preferiblemente artritis de reciente comienzo o artritis reumatoide en un sujeto previamente diagnosticado de la enfermedad, donde el producto de expresión es VIP. Another preferred embodiment of this aspect of the invention relates to the use of a VIP gene expression product as a prognostic marker of an autoimmune disease, preferably autoimmune arthritis, disease. Inflammatory bowel, psoriasis, spondyloarthritis or systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably recent onset arthritis or rheumatoid arthritis in a subject previously diagnosed with the disease where the expression product is mRNA. Another preferred embodiment of this aspect of the invention relates to the use of a VIP gene expression product as a prognostic marker of an autoimmune disease, preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably recently started arthritis or rheumatoid arthritis in a subject previously diagnosed with the disease, where the expression product is VIP.
Se entiende por " VIP' en la presente invención aquel gen que presente capacidad de codificar para VIP. De cualquier forma el gen puede ser, por ejemplo, aunque sin limitarse, el gen VIP con número de acceso al GenBank del NCBI {National Center for Biotechnology Information) AH003027. "VIP" in the present invention is understood as that gene that has the ability to code for VIP. In any case, the gene can be, for example, but not limited to, the VIP gene with access number to the NCBI GenBank {National Center for Biotechnology Information) AH003027.
Se entiende por "mRNA producto del gen VIP' en la presente invención, por ejemplo, aunque sin limitarse, el mRNA con número de acceso al GenBank del NCBI NM_003381 , y cuya traducción da lugar al péptido intestinal vasoactivo o VIP. "VIP gene product mRNA" is understood in the present invention, for example, but not limited to, the mRNA with access number to the NCBI GenBank NM_003381, and whose translation results in the vasoactive intestinal peptide or VIP.
El término "péptido intestinal vasoactivo" o "VIP" en la presente invención, se refiere al péptido con número de acceso al GenBank del NCBI AAB22264.1 de secuencia aminoacídica SEQ ID NO: 1 (hsdavftdny trlrkqmavk kylnsiln). The term "vasoactive intestinal peptide" or "VIP" in the present invention refers to the peptide with GenBank accession number of NCBI AAB22264.1 of amino acid sequence SEQ ID NO: 1 (hsdavftdny trlrkqmavk kylnsiln).
Los términos "secuencia de aminoácidos", "péptido" o "proteína" se usan aquí de manera intercambiable, y se refieren a una forma polimérica de aminoácidos de cualquier longitud, que pueden estar, o no, química o bioquímicamente modificados. El término "residuo" corresponde a un aminoácido. Por otro lado, un segundo aspecto de la invención se refiere a un método in vitro de pronóstico de una enfermedad autoinmune, preferiblemente artritis autoinmune, enfermedad inflamatoria intestinal, psoriasis, espondiloartritis o lupus eritematoso sistémico, más preferiblemente artritis autoinmune, y aun más preferiblemente artritis de reciente comienzo o artritis reumatoide caracterizado porque comprende la cuantificación de al menos producto de expresión del gen VIP en una muestra biológica aislada de un sujeto que padece la enfermedad autoinmune. The terms "amino acid sequence", "peptide" or "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which may or may not be chemically or biochemically modified. The term "residue" corresponds to an amino acid. On the other hand, a second aspect of the invention relates to an in vitro method of prognosis of an autoimmune disease, preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably arthritis. of recent onset or rheumatoid arthritis characterized in that it comprises the quantification of at least the VIP gene expression product in a biological sample isolated from a subject suffering from autoimmune disease.
El término "in vitro" tal y como se utiliza en la presente invención, se refiere a que el método de la invención se realiza fuera del cuerpo del sujeto. The term "in vitro" as used in the present invention, refers to the method of the invention being performed outside the subject's body.
Se entiende por "enfermedad autoinmune" en la presente invención aquella enfermedad en la cual uno o varios tipos celulares de un individuo son atacados por el sistema inmune del propio individuo ya que reconoce dichos tipos celulares como elementos extraños. Esto provoca el deterioro e incluso la destrucción de uno o varios tejidos del individuo. El término "enfermedad inflamatoria intestinal" o "EN" en la presente invención se refiere a la inflamación crónica del intestino en un individuo, donde dicha inflamación es debida al sistema inmune del propio individuo. Las dos formas más frecuentes corresponden a la colitis ulcerosa y la enfermedad de Crohn. En la presente invención se entiende por "psoriasis" aquella enfermedad cutánea la cual está caracterizada por un mal funcionamiento del sistema inmune, lo que provoca un exceso de producción de células cutáneas. Esta enfermedad da lugar a la formación de abultamientos rojizos cubiertas de descamaciones. Además, el exceso de producción de células también produce la infiltración de glóbulos blancos en la piel. Las lesiones de forma general se localizan en regiones con un mayor rozamiento, como por ejemplo, aunque sin limitarse, los codos, las rodillas o ingles. "Autoimmune disease" is understood in the present invention as a disease in which one or several cell types of an individual are attacked by the individual's own immune system since it recognizes said cell types as foreign elements. This causes the deterioration and even the destruction of one or several tissues of the individual. The term "inflammatory bowel disease" or "EN" in the present invention refers to chronic inflammation of the intestine in an individual, where said inflammation is due to the individual's own immune system. The two most frequent forms correspond to ulcerative colitis and Crohn's disease. In the present invention "psoriasis" is understood to be that skin disease which is characterized by a malfunction of the immune system, which causes excess production of skin cells. This disease leads to the formation of reddish bulges covered with scaling. In addition, excess cell production also causes the infiltration of white blood cells into the skin. Injuries are generally locate in regions with greater friction, such as, but not limited to, elbows, knees or English.
Se entiende por "espondiloartritis" en la presente invención como aquellas enfermedades autoinmunes con afectación axial y/o periférica que cumplen los criterios de clasificación de la Assessment of SpondyloArthritis International Society (ASAS o Sociedad Internacional de Evaluación de Espodiloartritis) (Rudwaleit et al. Ann Rheum Dis 2009;68:777-783; Rudwaleit et al. Ann Rheum Dis 201 1 ;70:25-31 ). "Spondyloarthritis" in the present invention is understood as those autoimmune diseases with axial and / or peripheral involvement that meet the classification criteria of the Assessment of SpondyloArthritis International Society (ASAS) (Rudwaleit et al. Ann Rheum Dis 2009; 68: 777-783; Rudwaleit et al. Ann Rheum Dis 201 1; 70: 25-31).
Se entiende por "lupus eritematoso sistémico" en la presente invención aquella enfermedad autoinmune sistémica definida por los criterios del Colegio Americano de Reumatología (Tan et al. Arthritis Rheum- 1982;25:1271 -1277) En la presente invención el término "artritis autoinmune" englobaría tanto los términos Artritis Reumatoide como Artritis Indiferenciada tanto si es de reciente comienzo como si está bien establecida. "Systemic lupus erythematosus" is understood in the present invention as a systemic autoimmune disease defined by the criteria of the American College of Rheumatology (Tan et al. Arthritis Rheum- 1982; 25: 1271-1277) In the present invention the term "autoimmune arthritis "would encompass both the terms Rheumatoid Arthritis and Undifferentiated Arthritis, whether it is of recent onset or if it is well established.
Se entiende por "Artritis de Reciente Comienzo o ARC" en la presente invención, aquella enfermedad consistente en inflamación de al menos una articulación, de menos de un año de evolución que cumple los criterios preestablecidos por el Colegio Americano de Reumatología (American College of Rheumatology o ACR) y la Liga Europea Contra el Reumatismo (European League Against Rheumatism o EULAR) de "Artritis reumatoide o AR" (Aletaha, Neogi et al. Ann Rheum Dis 2010;69: 1580-1588) o, que sin cumplir dichos criterios, no cumple criterios de otras enfermedades autoinmunes, degenerativas o metabólicas que puedan explicar los síntomas. Este último caso, viene siendo denominado "Artritis Indiferenciada o Al" que, en muchos de los casos, dejados a su libre evolución, acaban desembocando en una AR (van der Helm-van Mil et al. Arthritis Rheum. 2007;56:433-440). La definición de Artritis Indiferenciada está ganando progresivamente aceptación, ya que se entiende que cuanto más precozmente se trate a los pacientes más opciones de inducir remisión tenemos y en la actualidad se acepta establecer tratamiento inmunomodulador en pacientes que no cumplen criterios de AR. En esta invención los términos ARC, AR o Al hacen referencia a una enfermedad sistémica autoinmune crónica y progresiva, la cual provoca la inflamación crónica fundamentalmente de las articulaciones, y que dada su naturaleza progresiva, produce la destrucción de las mismas, con su consecuente deformación y pérdida de capacidad funcional. Además, esta enfermedad puede provocar alteraciones extraarticu lares en diversos órganos (Carmona, González-Alvaro et al. Ann Rheum Dis 2003;62:897-900). La actividad de la enfermedad se puede determinar mediante índices compuestos que proporcionan un numero que aglutina la información de diferentes variables clínicas y analíticas como el DAS (van der Heide et al. J Rheumatol 1993;20:579-581 ), DAS28 (Prevoo et al. Arthritis Rheum 1995;38:44-48), SDAI, CDAI (Smolen et al. Rheumatology (Oxford) 2003;42:244-257) y otros. It is understood by "Recent Arthritis or ARC" in the present invention, that disease consisting of inflammation of at least one joint, less than one year old that meets the criteria pre-established by the American College of Rheumatology (American College of Rheumatology or ACR) and the European League Against Rheumatism (European League Against Rheumatism or EULAR) of "Rheumatoid Arthritis or RA" (Aletaha, Neogi et al. Ann Rheum Dis 2010; 69: 1580-1588) or, that without meeting those criteria , does not meet criteria for other autoimmune, degenerative or metabolic diseases that may explain the symptoms. This last case is called "undifferentiated or Al Arthritis" which, in many cases, left to its free evolution, ends up leading to an RA (van der Helm-van Mil et al. Arthritis Rheum. 2007; 56: 433 -440). The definition of Undifferentiated arthritis is progressively gaining acceptance, since it is understood that the earlier patients are treated, the more options for inducing remission we have and it is currently accepted to establish immunomodulatory treatment in patients who do not meet RA criteria. In this invention the terms ARC, AR or Al refer to a chronic and progressive autoimmune systemic disease, which causes chronic inflammation mainly of the joints, and that given its progressive nature, produces the destruction of them, with their consequent deformation and loss of functional capacity. In addition, this disease can cause extraarticular alterations in various organs (Carmona, González-Alvaro et al. Ann Rheum Dis 2003; 62: 897-900). The disease activity can be determined by compound indices that provide a number that brings together the information of different clinical and analytical variables such as DAS (van der Heide et al. J Rheumatol 1993; 20: 579-581), DAS28 (Prevoo et al. Arthritis Rheum 1995; 38: 44-48), SDAI, CDAI (Smolen et al. Rheumatology (Oxford) 2003; 42: 244-257) and others.
En la presente invención se entiende por "pronóstico" la capacidad de determinar en pacientes de una enfermedad autoinmune, cómo va a evolucionar dicha enfermedad en cuanto a su gravedad. El término "pronóstico" también se refiere a la capacidad de detectar sujetos ya diagnosticados de una enfermedad autoinmune con alta probabilidad de sufrir un empeoramiento de la enfermedad. Esta mala evolución puede definirse de forma concreta como una menor posibilidad de alcanzar la remisión de la enfermedad, teniendo en cuenta que existen múltiples formas de definir este estado ideal de remisión en cada enfermedad. In the present invention, "prognosis" is understood as the ability to determine in patients of an autoimmune disease, how the disease will evolve in terms of its severity. The term "prognosis" also refers to the ability to detect subjects already diagnosed with an autoimmune disease with a high probability of suffering a worsening of the disease. This bad evolution can be defined concretely as a lower possibility of achieving disease remission, taking into account that there are multiple ways to define this ideal state of remission in each disease.
Por ejemplo en la artritis existen diferentes formas de definir el estado ideal de remisión, como por ejemplo aunque sin limitarse los descritos en Felson et al. Arthritis Rheum 201 1 ;63:573-586. Otras situaciones que también pueden considerarse mala evolución son no alcanzar un estado de mínima actividad de la enfermedad (Wells et al. J Rheumatol 2005;32:2016-2024) o como la posibilidad de tener peor respuesta terapéutica bien según criterios ACR20, 50 o 70 (Felson et al. Arthritis Rheum 1995;38:727-735; Felson J Rheumatol 2004;31 :835-837) o según criterios EULAR (van Gestel et al. Arthritis Rheum 1996;39:34-40). For example, in arthritis there are different ways of defining the ideal state of remission, such as, but not limited to those described in Felson et al. Arthritis Rheum 201 1; 63: 573-586. Other situations that may also considered bad evolution are not to reach a state of minimal disease activity (Wells et al. J Rheumatol 2005; 32: 2016-2024) or as the possibility of having a worse therapeutic response either according to ACR20, 50 or 70 criteria (Felson et al Arthritis Rheum 1995; 38: 727-735; Felson J Rheumatol 2004; 31: 835-837) or according to EULAR criteria (van Gestel et al. Arthritis Rheum 1996; 39: 34-40).
El término "muestra biológica aislada" en la presente invención se refiere a cualquier muestra que permita determinar los niveles de VIP del individuo del que se ha obtenido dicha muestra, e incluye, pero sin limitarnos, fluidos biológicos de un individuo, obtenidos mediante cualquier método conocido por un experto en la materia que sirva para tal fin. La muestra biológica podría ser, por ejemplo, pero sin limitarse, una muestra de fluido, como sangre, plasma, suero, líquido sinovial o linfa. Dentro de las muestras biológicas unas de las que presentan mayor utilidad son el suero o el plasma ya que proporcionan datos más precisos de los niveles de VIP. Otras muestras de interés serían por ejemplo células mononucleares de sangre periférica, preferentemente linfocitos T, ya que son las que presentan mayor expresión y por tanto permiten una determinación más sencilla de los niveles. La sangre además se extrae de forma rutinaria en análisis que se pueden realizar periódicamente a los pacientes. Por ello, preferentemente la muestra es sangre, plasma o suero. También resultan útiles muestras de tejido obtenidas que se puedan analizar por ejemplo mediante ¡nmunohistoquímica, o que se puedan homogeneizar para obtener el mRNA de la transcripción del gen VIP o VIP para poder cualificarlos. Un ejemplo es la membrana sinovial, la cual procede del tejido concreto afectado por la enfermedad, y por tanto resulta interesante para la determinación de un producto de expresión del gen VIP. Por todo ello, en una realización preferida del segundo aspecto de la invención, la muestra biológica aislada es sangre, suero, plasma o membrana sinovial. En una realización más preferida, la muestra biológica es suero. Asimismo, la muestra biológica puede ser, por ejemplo, pero sin limitarse, fresca, congelada, fijada o fijada y embebida en parafina. The term "isolated biological sample" in the present invention refers to any sample that makes it possible to determine the VIP levels of the individual from whom said sample was obtained, and includes, but is not limited to, biological fluids of an individual, obtained by any method known by an expert in the field that serves this purpose. The biological sample could be, for example, but not limited to, a sample of fluid, such as blood, plasma, serum, synovial fluid or lymph. Among the biological samples, some of those that are most useful are serum or plasma since they provide more accurate data on VIP levels. Other samples of interest would be, for example, peripheral blood mononuclear cells, preferably T lymphocytes, since they have the highest expression and therefore allow a simpler determination of the levels. The blood is also extracted routinely in tests that can be performed periodically to patients. Therefore, preferably the sample is blood, plasma or serum. Also obtained are tissue samples obtained that can be analyzed for example by immunohistochemistry, or that can be homogenized to obtain the transcription mRNA of the VIP or VIP gene in order to qualify them. An example is the synovial membrane, which comes from the specific tissue affected by the disease, and is therefore interesting for the determination of an expression product of the VIP gene. Therefore, in a preferred embodiment of the second aspect of the invention, the isolated biological sample is blood, serum, plasma or synovial membrane. In a more preferred embodiment, the biological sample is serum. Likewise, the biological sample can be, for example, but not limited, fresh, frozen, fixed or fixed and embedded in paraffin.
En la presente invención se muestra también, que la combinación de la cuantificación de al menos un producto de expresión del gen VIP en una muestra biológica aislada con otras variables clínicas, o marcadores ayudan o complementan la utilidad de la cuantificación de al menos un producto de expresión del gen VIP en el pronóstico de la enfermedad. Estas variables o marcadores, pueden ser por ejemplo, aunque sin limitarse el sexo del paciente, el estado de actividad al inicio de la enfermedad, así como otras conocidas por el experto en la materia. Un ejemplo de estas variables clínicas útiles es, por ejemplo, aunque sin limitarse, los anticuerpos antipéptidos citrulinados (ACPA) en caso de estarse estudiando la artritis de reciente comienzo o artritis reumatoide. Estos anticuerpos han sido previamente descritos y tienen cierta utilidad en el pronóstico de la artritis. In the present invention it is also shown that the combination of the quantification of at least one VIP gene expression product in an isolated biological sample with other clinical variables, or markers helps or complements the usefulness of the quantification of at least one product of VIP gene expression in the prognosis of the disease. These variables or markers can be, for example, but not limited to the patient's sex, the activity status at the onset of the disease, as well as others known to the person skilled in the art. An example of these useful clinical variables is, for example, but not limited to, citrullinated antiseptic antibodies (ACPA) if you are studying recently started arthritis or rheumatoid arthritis. These antibodies have been previously described and have some utility in the prognosis of arthritis.
Por todo ello, en otra realización preferida del segundo aspecto de la invención, el método puede comprender además de la determinación de la cantidad de producto de expresión del gen VIP en una muestra biológica aislada de un sujeto que padece una enfermedad autoinmune, preferiblemente artritis autoinmune, enfermedad inflamatoria intestinal, psoriasis, espondiloartritis o lupus eritematoso sistémico, más preferiblemente artritis autoinmune, y aun más preferiblemente artritis de reciente comienzo o artritis reumatoide en un sujeto previamente diagnosticado de la enfermedad autoinmune, la determinación de otras variables clínicas, o marcadores. En una realización más preferida del segundo aspecto de la invención, las variables clínicas o marcadores son el sexo del paciente, el estado de actividad al inicio de la enfermedad. Otra realización preferida del segundo aspecto de la invención, se refiere a un método in vitro de pronóstico de artritis autoinmune, preferiblemente artritis de reciente comienzo o artritis reumatoide caracterizado porque comprende la cuantificación de al menos producto de expresión del gen VIP en una muestra biológica aislada de un sujeto que padece la artritis autoinmune y que además comprende la determinación del sexo del sujeto, la presencia de anticuerpos antipéptidos citrulinados (ACPA) y/o el estado de actividad al inicio de la enfermedad. Therefore, in another preferred embodiment of the second aspect of the invention, the method may further comprise determining the amount of VIP gene expression product in a biological sample isolated from a subject suffering from an autoimmune disease, preferably autoimmune arthritis , inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably recent onset arthritis or rheumatoid arthritis in a subject previously diagnosed with autoimmune disease, the determination of other clinical variables, or markers. In a more preferred embodiment of the second aspect of the invention, the clinical variables or Markers are the patient's sex, the activity status at the onset of the disease. Another preferred embodiment of the second aspect of the invention relates to an in vitro method of prognosis of autoimmune arthritis, preferably recently started arthritis or rheumatoid arthritis characterized in that it comprises the quantification of at least VIP gene expression product in an isolated biological sample. of a subject suffering from autoimmune arthritis and who also includes the determination of the sex of the subject, the presence of citrullinated antiseptic antibodies (ACPA) and / or the state of activity at the onset of the disease.
Un tercer aspecto de la presente invención se refiere a un método in vitro para el pronóstico de una enfermedad autoinmune, preferiblemente artritis autoinmune, enfermedad inflamatoria intestinal, psoriasis, espondiloartritis o lupus eritematoso sistémico, más preferiblemente artritis autoinmune, y aun más preferiblemente artritis de reciente comienzo o artritis reumatoide en un sujeto previamente diagnosticado de la enfermedad autoinmune que comprende las siguientes etapas: A third aspect of the present invention relates to an in vitro method for the prognosis of an autoimmune disease, preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably recent arthritis. onset or rheumatoid arthritis in a previously diagnosed subject of autoimmune disease comprising the following stages:
a. cuantificar al menos un producto de expresión del gen VIP en una muestra biológica aislada de un sujeto que padece una enfermedad autoinmune, preferiblemente artritis autoinmune, enfermedad inflamatoria intestinal, psoriasis, espondiloartritis o lupus eritematoso sistémico, más preferiblemente artritis autoinmune, y aun más preferiblemente artritis de reciente comienzo o artritis reumatoide, b. comparar el valor obtenido en el paso (a) con una cantidad de referencia, y  to. quantify at least one VIP gene expression product in an isolated biological sample of a subject suffering from an autoimmune disease, preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably arthritis of recent onset or rheumatoid arthritis, b. compare the value obtained in step (a) with a reference amount, and
c. asociar al individuo del paso (a) al grupo de pacientes con mal pronóstico de la enfermedad autoinmune cuando la cantidad cuantificada en el paso (a) es menor que el percentil 50 de la cantidad de referencia del paso (b). En los ejemplos de la presente invención se muestra que la comparación con el percentil 50 de cantidad de referencia, presenta utilidad a la hora de clasificar los pacientes. Además se demuestra que según se va reduciendo este percentil, el valor es más restrictivo y por tanto permite una mejor clasificación, y una mayor seguridad en la clasificación de los mismos. Ejemplos de estos percentiles más restrictivos y que presentan utilidad serían por ejemplo, aunque sin limitarse, el percentil 25 o el percentil 10. Por todo esto, en una realización preferida del tercer aspecto de la invención, en el paso (c) se asocia al individuo del paso (a) al grupo de pacientes con mal pronóstico de la enfermedad autoinmune cuando la cantidad cuantificada en el paso (a) es menor que el percentil 25 de la cantidad de referencia del paso (b). En una realización más preferida del tercer aspecto de la invención, en el paso (c) se asocia al individuo del paso (a) al grupo de pacientes con mal pronóstico de la enfermedad autoinmune cuando la cantidad cuantificada en el paso (a) es menor que el percentil 10 de la cantidad de referencia del paso (b). C. Associate the individual from step (a) to the group of patients with poor prognosis of autoimmune disease when the quantity quantified in step (a) is less than the 50th percentile of the reference amount of step (b). In the examples of the present invention it is shown that the comparison with the 50th percentile of reference amount is useful when classifying patients. It is also shown that as this percentile is reduced, the value is more restrictive and therefore allows a better classification, and greater security in their classification. Examples of these more restrictive and useful percentiles would be, for example, but not limited to, the 25th percentile or the 10th percentile. For all this, in a preferred embodiment of the third aspect of the invention, in step (c) it is associated with individual from step (a) to the group of patients with poor prognosis of autoimmune disease when the quantity quantified in step (a) is less than the 25th percentile of the reference amount of step (b). In a more preferred embodiment of the third aspect of the invention, in step (c) the individual of step (a) is associated with the group of patients with poor prognosis of autoimmune disease when the amount quantified in step (a) is less than the 10th percentile of the reference amount of step (b).
El término "cantidad de referencia", tal y como se utiliza en el paso (b), se refiere a cualquier valor o rango de valores derivado de la cuantificación de un producto de expresión del gen VIP en una colección de muestras biológicas procedente de individuos sanos y que son representativos de la población en la que se va a aplicar el test. Esta muestra también puede ser una mezcla de muestras biológicas obtenidas de diferentes sujetos sanos. La cantidad de referencia se ha de medir de la misma forma, y ser obtenida en el mismo tipo de muestra biológica aislada que la cantidad de interés en los pacientes. Por ello en una realización preferida de este aspecto de la invención la cantidad de referencia del paso (b) es la cantidad de VIP en una colección de muestras biológicas aislada de individuos que no presentan enfermedades autoinmunes. Se entiende por "sano" "individuo sano" o "sujeto sano" en la presente invención aquel sujeto o individuo que no padece ninguna enfermedad autoinmune. Se entiende por "población sana" en la presente invención, un conjunto de individuos que no presentan enfermedades autoinmunes. The term "reference quantity", as used in step (b), refers to any value or range of values derived from the quantification of a VIP gene expression product in a collection of biological samples from individuals healthy and that are representative of the population in which the test will be applied. This sample can also be a mixture of biological samples obtained from different healthy subjects. The reference amount must be measured in the same way, and be obtained in the same type of isolated biological sample as the amount of interest in the patients. Therefore, in a preferred embodiment of this aspect of the invention, the reference amount in step (b) is the amount of VIP in a collection of biological samples isolated from individuals who do not have autoimmune diseases. By "healthy""healthyindividual" or "healthy subject" is understood in the present invention that subject or individual who does not suffer from any autoimmune disease. "Healthy population" is understood in the present invention, a set of individuals who do not have autoimmune diseases.
Se entiende por "individuos sanos representativos de la población en la que se va a aplicar el test" a aquellas personas que no padecen enfermedades autoinmunes en el momento de la extracción y que como grupo tienen un patrón similar en cuanto a raza, edad, distribución por género que la población de pacientes a los que se va a aplicar el test. "Healthy individuals representative of the population in which the test is to be applied" means those people who do not suffer from autoimmune diseases at the time of extraction and who as a group have a similar pattern in terms of race, age, distribution by gender than the population of patients to whom the test is to be applied.
La cuantificación de la cantidad de un producto de expresión del gen VIP en una muestra biológica aislada se refiere a la cuantificación del mRNA, del ADN complementario (ADNc) a este ARNm, y/o de la proteína VIP en una muestra biológica aislada. La cuantificación por tanto se puede realizar determinando el nivel de mRNA derivado de su transcripción, previa extracción del ARN total presente en la muestra biológica aislada, lo cual puede realizarse mediante protocolos conocidos en el estado de la técnica. Para ello la muestra biológica aislada puede tratarse física o mecánicamente para romper el tejido o las estructuras celulares y liberar los componentes intracelulares a una solución acuosa u orgánica para preparar los ácidos nucleicos para un posterior análisis. Los ácidos nucleicos se extraen de la muestra por procedimientos conocidos por el experto en la materia y comercialmente disponibles. La determinación del nivel de mRNA derivado de la transcripción de VIP puede realizarse, por ejemplo, aunque sin limitarnos, mediante amplificación por reacción en cadena de la polimerasa (PCR), retrotranscripción en combinación con la reacción en cadena de la polimerasa (RT-PCR), RT-PCR cuantitativa, retrotranscripción en combinación con la reacción en cadena de la ligasa (RT-LCR), o cualquier otro método de amplificación de ácidos nucleicos; análisis en serie de la expresión génica (SAGE, SuperSAGE); chips de ADN elaborados con oligonucleótidos depositados por cualquier mecanismo; microarrays de ADN elaborados con oligonucleótidos sintetizados in situ mediante fotolitografía o por cualquier otro mecanismo; hibridación in situ utilizando sondas específicas marcadas con cualquier método de mareaje; mediante geles de electroforesis; mediante transferencia a membrana e hibridación con una sonda específica; mediante resonancia magnética nuclear o cualquier otra técnica de diagnóstico por imagen utilizando nanopartículas paramagnéticas o cualquier otro tipo de nanopartículas detectables funcionalizadas con anticuerpos o por cualquier otro medio. Por todo ello en una realización preferida de este aspecto de la invención el producto de expresión cuantificado en el paso (a) es mRNA. The quantification of the amount of an expression product of the VIP gene in an isolated biological sample refers to the quantification of the mRNA, of the complementary DNA (cDNA) to this mRNA, and / or of the VIP protein in an isolated biological sample. The quantification can therefore be performed by determining the level of mRNA derived from its transcription, after extracting the total RNA present in the isolated biological sample, which can be performed by protocols known in the state of the art. For this, the isolated biological sample can be physically or mechanically treated to break up the cellular tissue or structures and release the intracellular components to an aqueous or organic solution to prepare the nucleic acids for further analysis. Nucleic acids are extracted from the sample by procedures known to those skilled in the art and commercially available. The level of mRNA derived from VIP transcription can be determined, for example, but not limited to, by amplification by polymerase chain reaction (PCR), back transcription in combination with polymerase chain reaction (RT-PCR ), Quantitative RT-PCR, retrotranscription in combination with ligase chain reaction (RT-LCR), or any other nucleic acid amplification method; serial analysis of gene expression (SAGE, SuperSAGE); DNA chips made with oligonucleotides deposited by any mechanism; DNA microarrays made with oligonucleotides synthesized in situ by photolithography or by any other mechanism; in situ hybridization using specific probes labeled with any method of marking; by electrophoresis gels; by membrane transfer and hybridization with a specific probe; by nuclear magnetic resonance or any other diagnostic imaging technique using paramagnetic nanoparticles or any other type of detectable nanoparticles functionalized with antibodies or by any other means. Therefore, in a preferred embodiment of this aspect of the invention, the expression product quantified in step (a) is mRNA.
La cuantificación por otro lado también se puede realizar determinando el nivel de proteína VIP derivado de la traducción de los mRNA transcritos a partir del gen. Esta cuantificación proteica se puede realizar mediante cualquier método conocido por un experto en la materia que sirva para tal fin, como por ejemplo, pero sin limitarnos, métodos de inmunodetección (como western blot, ELISA, ¡nmunohistoquímica), métodos basados en mareajes isobáricos (como ¡TRAQ - isobaric Tag for Relative and Absolute Quantitation-, o ICAT -Isotope Coded Affinity Tag-) o en mareajes isotópicos (como SILAC -Stable Isotopes Labeling by Amino Acids in Cell Culture-) o basados en mareajes fluorescentes (como 2D-DIGE -Difference in Gel Electrophoresis-), así como métodos basados en espectrometría de masas (MRM, -Múltiple Reaction Monitoring-) .Por todo ello en otra realización preferida de este aspecto de la invención el producto de expresión cuantificado en el paso (a) es VIP. Quantification on the other hand can also be performed by determining the level of VIP protein derived from the translation of the mRNA transcribed from the gene. This protein quantification can be carried out by any method known to a person skilled in the art that serves this purpose, such as, but not limited to, immunodetection methods (such as western blot, ELISA, immunohistochemistry), methods based on isobaric labeling ( such as ¡TRAQ - isobaric Tag for Relative and Absolute Quantitation-, or ICAT -Isotope Coded Affinity Tag-) or in isotopic (like SILAC -Stable Isotopes Labeling by Amino Acids in Cell Culture-) or based on fluorescent (like 2D-) DIGE -Difference in Gel Electrophoresis-), as well as methods based on mass spectrometry (MRM, -Multiple Reaction Monitoring-) .For all this in another preferred embodiment of this aspect of the invention the expression product quantified in step (a ) is VIP.
Se entiende por "sujeto" en la presente invención aquel individuo susceptible de padecer una enfermedad autoinmune. Preferiblemente el sujeto es un humano. En otra realización preferida del tercer aspecto de la invención la muestra biológica se selecciona de entre sangre, plasma, suero, o membrana sinovial. En una realización más preferida, la muestra biológica es suero. Otra realización preferida del tercer aspecto de la invención se refiere al método donde el sujeto es un humano. "Subject" in the present invention is understood as that individual susceptible to suffering from an autoimmune disease. Preferably the subject is a human. In another preferred embodiment of the third aspect of the invention the biological sample is selected from blood, plasma, serum, or synovial membrane. In a more preferred embodiment, the biological sample is serum. Another preferred embodiment of the third aspect of the invention relates to the method where the subject is a human.
Cualquiera de los pasos de los métodos de la invención pueden ser total o parcialmente automatizados, por ejemplo, pero sin limitarnos, por medio de un equipo robótico sensor para la obtención de las cantidades de los productos de expresión del gen VIP en el paso (a), o la comparación computerizada en el paso (b) del tercer aspecto de la invención. Además de los pasos especificados anteriormente, los métodos descritos en la presente memoria pueden comprender otros pasos adicionales, por ejemplo, relacionados con el pre- tratamiento de la muestra o con la extracción del material proteico y/o genético necesario para su posterior análisis. Any of the steps of the methods of the invention can be totally or partially automated, for example, but not limited to, by means of a robotic sensor device for obtaining the quantities of the VIP gene expression products in step (a ), or the computerized comparison in step (b) of the third aspect of the invention. In addition to the steps specified above, the methods described herein may comprise additional steps, for example, related to the pretreatment of the sample or the extraction of the protein and / or genetic material necessary for subsequent analysis.
El tercer aspecto de la invención puede comprender además la determinación de otras variables clínicas, o marcadores como por ejemplo, aunque sin limitarse, sexo, el estado de actividad al inicio de la enfermedad que ayuden o complementen el pronóstico de la enfermedad. Además, en el caso de tratarse de artritis autoinmune, se puede utilizar como variable clínica además la presencia de anticuerpos antipéptidos citrulinados. Por todo ello, en una realización preferida del tercer aspecto de la invención, el método comprende además la determinación del sexo del sujeto, y/o el estado de actividad al inicio de la enfermedad. En otra realización preferida del tercer aspecto de la invención, en el caso de tratarse de artritis autoinmune, el método comprende además la determinación del sexo del sujeto, la presencia de anticuerpos antipéptidos citrulinados y/o el estado de actividad al inicio de la enfermedad. Todos los métodos en la presente invención pueden incluir adicionalmente un paso de tratamiento del paciente en función del pronóstico obtenido. Este paso de tratamiento será determinado en función de la gravedad del pronóstico de la enfermedad analizada. De esta forma se puede determinar el tratamiento a seguir como pueden ser por ejemplo glucocorticoides, terapias biológicas como agentes bloqueantes del TNF, tocilizumab, abatacept o rituximab, o fármacos modificadores de la enfermedad (FAME) los cuales incluyen antimaláricos, metotrexato, leflunomida, sulfasalacina, aurotiomalato, y/o ciclosporina A. Un cuarto aspecto de la invención se refiere al uso de un kit que comprende los elementos necesarios para determinar la cantidad de VIP para la obtención de datos útiles para el pronóstico de una enfermedad autoinmune preferiblemente artritis de reciente comienzo, artritis reumatoide, enfermedad inflamatoria intestinal, psoriasis o lupus eritematoso sistémico, y más preferiblemente artritis de reciente comienzo o artritis reumatoide previamente diagnosticado de la enfermedad autoinmune. The third aspect of the invention may further comprise the determination of other clinical variables, or markers such as, but not limited to, sex, the state of activity at the onset of the disease that helps or complements the prognosis of the disease. In addition, in the case of autoimmune arthritis, the presence of citrullinated antiseptic antibodies can also be used as a clinical variable. Therefore, in a preferred embodiment of the third aspect of the invention, the method further comprises determining the sex of the subject, and / or the state of activity at the onset of the disease. In another preferred embodiment of the third aspect of the invention, in the case of autoimmune arthritis, the method further comprises determining the sex of the subject, the presence of citrullinated anti-peptide antibodies and / or the state of activity at the onset of the disease. All methods in the present invention may additionally include a step of treating the patient based on the prognosis obtained. This treatment step will be determined based on the severity of the prognosis of the disease analyzed. In this way, the treatment to be followed can be determined, such as, for example, glucocorticoids, biological therapies such as TNF blocking agents, tocilizumab, abatacept or rituximab, or disease modifying drugs (DMARDs) which include antimalarials, methotrexate, leflunomide, sulfasalacin , aurothiomalate, and / or cyclosporine A. A fourth aspect of the invention relates to the use of a kit comprising the elements necessary to determine the amount of VIP for obtaining useful data for the prognosis of an autoimmune disease, preferably recent arthritis. onset, rheumatoid arthritis, inflammatory bowel disease, psoriasis or systemic lupus erythematosus, and more preferably recent onset arthritis or rheumatoid arthritis previously diagnosed with autoimmune disease.
En la presente descripción se entiende por "elementos adecuados para cuantificar un producto de expresión del gen VIP' los cebadores, sondas, anticuerpos poli o monoclonales, específicos del gen VIP o de sus mRNA, o de VIP, y todos aquellos reactivos necesarios para llevar a cabo cualquiera de los métodos de la invención descritos anteriormente en este documento. Los kits además pueden incluir, sin ningún tipo de limitación, el uso de tampones, enzimas, enzimas polimerasas, cofactores para obtener una actividad óptima de éstas, agentes para prevenir la contaminación, etc. Por otro lado los kits pueden incluir todos los soportes y recipientes necesarios para su puesta en marcha y optimización. Los kits pueden contener además otras moléculas, genes, proteínas o sondas de interés, que sirvan como controles positivos y negativos. Preferiblemente, los kits comprenden además las instrucciones para llevar a cabo los métodos de la invención. En una realización preferida del cuarto aspecto de la invención, el kit comprende sondas que permiten la determinación de la cantidad de mRNA transcritos a partir del gen VIP para el pronóstico de una enfermedad autoinmune, preferiblemente artritis autoinmune, enfermedad inflamatoria intestinal, psoriasis, espondiloartritis o lupus eritematoso sistémico, más preferiblemente artritis autoinmune, y aun más preferiblemente artritis de reciente comienzo o artritis reumatoide previamente diagnosticado de la enfermedad autoinmune. In the present description, "suitable elements for quantifying an expression product of the VIP gene 'are understood to be primers, probes, poly or monoclonal antibodies, specific to the VIP gene or its mRNA, or VIP, and all those reagents necessary to carry any of the methods of the invention described above herein, the kits may also include, without any limitation, the use of buffers, enzymes, polymerase enzymes, cofactors to obtain optimum activity thereof, agents to prevent contamination, etc. On the other hand, the kits may include all the supports and containers necessary for their implementation and optimization.The kits may also contain other molecules, genes, proteins or probes of interest, which serve as positive and negative controls. , the kits further comprise the instructions for carrying out the methods of the invention. In a preferred embodiment of the fourth aspect of the invention, the kit comprises probes that allow the determination of the amount of mRNA transcribed from the VIP gene for the prognosis of an autoimmune disease, preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or Systemic lupus erythematosus, more preferably autoimmune arthritis, and even more preferably newly started arthritis or rheumatoid arthritis previously diagnosed with autoimmune disease.
En otra realización preferida del cuarto aspecto de la invención, el kit comprende anticuerpos que permiten la determinación de la cantidad de VIP para el pronóstico de una enfermedad autoinmune, preferiblemente artritis autoinmune, enfermedad inflamatoria intestinal, psoriasis, espondiloartritis o lupus eritematoso sistémico, más preferiblemente artritis autoinmune, y aun más preferiblemente artritis de reciente comienzo o artritis reumatoide previamente diagnosticado de la enfermedad autoinmune. Preferiblemente, el sujeto es un humano. El kit además puede incluir, sin ningún tipo de limitación, tampones, enzimas, agentes para prevenir la contaminación, etc. Por otro lado el kit puede incluir todos los soportes y recipientes necesarios para su puesta en marcha y optimización. El kit puede contener además otras moléculas, anticuerpos anticuerpos, o proteínas, que sirvan como controles positivos y negativos o para la normalización de los valores obtenidos. Preferiblemente, el kit comprende además las instrucciones para llevar a cabo los métodos de la invención. In another preferred embodiment of the fourth aspect of the invention, the kit comprises antibodies that allow the determination of the amount of VIP for the prognosis of an autoimmune disease, preferably autoimmune arthritis, inflammatory bowel disease, psoriasis, spondyloarthritis or systemic lupus erythematosus, more preferably Autoimmune arthritis, and even more preferably newly started arthritis or rheumatoid arthritis previously diagnosed with autoimmune disease. Preferably, the subject is a human. The kit can also include, without any limitation, buffers, enzymes, agents to prevent contamination, etc. On the other hand, the kit can include all the supports and containers necessary for commissioning and optimization. The kit may also contain other molecules, antibody antibodies, or proteins, which serve as positive and negative controls or for the normalization of the values obtained. Preferably, the kit further comprises instructions for carrying out the methods of the invention.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y figuras se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
BREVE DESCRIPCION DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Variabilidad de los niveles de VIP en controles sanos y pacientes con artritis reumatoide o artritis indiferenciada. Los datos se muestran como la mediana (línea horizontal dentro de las cajas), los percentiles 25 y 75 (líneas inferior y superior de las cajas respectivamente) y los percentiles 10 y 90 (pequeñas líneas horizontales al final de las líneas verticales por debajo y encima de las cajas respectivamente). Los puntos representan valores fuera de rango. La línea horizontal en trazo discontinuo se refiere al percentil 25 de la población sana. FIG. 1. Variability of VIP levels in healthy controls and patients with rheumatoid arthritis or undifferentiated arthritis. Data are shown as the median (horizontal line inside the boxes), the 25th and 75th percentiles (bottom and top lines of the boxes respectively) and the 10th and 90th percentiles (small horizontal lines at the end of the vertical lines below and above the boxes respectively). Points represent out of range values. The dashed horizontal line refers to the 25th percentile of the healthy population.
FIG. 2. Relación de los niveles de VIP con el género (A), la edad (B) y la actividad de la enfermedad (DAS28) (C). Los datos se muestran como la mediana (línea horizontal dentro de las cajas), los percentiles 25 y 75 (líneas inferior y superior de las cajas respectivamente) y los percentiles 10 y 90 (pequeñas líneas horizontales al final de las líneas verticales por debajo y encima de las cajas respectivamente) en el caso de la figura A. En las figuras B y C, se representan los diferentes puntos con la recta de regresión estimada (línea negra continua), así como el intervalo de confianza al 95% (zona gris alrededor de la línea negra). FIG. 2. Relationship of VIP levels with gender (A), age (B) and disease activity (DAS28) (C). Data are shown as the median (horizontal line inside the boxes), the 25th and 75th percentiles (bottom and top lines of the boxes respectively) and the 10th and 90th percentiles (small horizontal lines at the end of the vertical lines below and above the boxes respectively) in the case of figure A. In figures B and C, the different points are represented with the estimated regression line (continuous black line), as well as the 95% confidence interval (gray zone around the black line).
FIG. 3. Niveles de VIP ajustado en las visitas inicial (primera columna), a los 6 meses (segunda columna), a los 12 meses (tercera columna) y a los 24 meses (cuarta columna). FIG. 4. Niveles de VIP en la visita inicial según el nivel de actividad de la enfermedad (según DAS28) en las visitas a los 2 (A) y 5 (B) años de seguimiento. En el panel C se muestra el grado de actividad de la artritis (DAS28) en la visita inicial y tras dos y cinco años de seguimiento en los pacientes que tienen VIP alto (cajas blancas) o bajo (cajas grises). FIG. 3. VIP levels adjusted for initial visits (first column), 6 months (second column), 12 months (third column) and 24 months (fourth column). FIG. 4. VIP levels at the initial visit according to the level of activity of the disease (according to DAS28) on visits at 2 (A) and 5 (B) years of follow-up. Panel C shows the degree of arthritis activity (DAS28) at the initial visit and after two and five years of follow-up in patients who have high VIP (white boxes) or low (gray boxes).
FIG. 5. Intensidad de tratamiento en los pacientes estudiados según presenten niveles de VIP alto o bajo en la primera visita del seguimiento. La intensidad del tratamiento se cuantifico según el número de días con tratamiento con todos los FAME (fármacos modificadores de la enfermedad) que cada paciente ha recibido a lo largo de los dos primeros años de su seguimiento. FIG. 5. Intensity of treatment in patients studied as they present high or low VIP levels at the first follow-up visit. The intensity of the treatment was quantified according to the number of days with treatment with all DMARDs (disease modifying drugs) that each patient has received during the first two years of their follow-up.
FIG. 6. Efecto de la combinación del genero (A), la edad (B) y el nivel de actividad al inicio de la enfermedad (C) con los niveles de VIP para predecir el estado de actividad de la enfermedad a los dos años de seguimiento. Los datos se muestran como la mediana (línea horizontal dentro de las cajas), los percentiles 25 y 75 (líneas inferior y superior de las cajas respectivamente) y los percentiles 10 y 90 (pequeñas líneas horizontales al final de las líneas verticales por debajo y encima de las cajas respectivamente) del DAS28 a los dos años de seguimiento en los pacientes que tienen VIP alto (cajas blancas) o bajo (cajas grises). FIG. 6. Effect of the combination of gender (A), age (B) and activity level at the beginning of the disease (C) with VIP levels to predict the disease activity status at two years of follow-up . Data are shown as the median (horizontal line inside the boxes), the 25th and 75th percentiles (bottom and top lines of the boxes respectively) and the 10th and 90th percentiles (small horizontal lines at the end of the vertical lines below and above the boxes respectively) of the DAS28 at two years of follow-up in patients who have high VIP (white boxes) or low (gray boxes).
FIG. 7. Efecto de la positividad de los anticuerpos antipéptidos citrulinados (ACPA) en la capacidad predictiva de los niveles de VIP. A) Actividad de la enfermedad a los dos años de seguimiento. B) Intensidad de tratamiento. Los datos se muestran como la mediana (línea horizontal dentro de las cajas), los percentiles 25 y 75 (líneas inferior y superior de las cajas respectivamente) y los percentiles 10 y 90 (pequeñas líneas horizontales al final de las líneas verticales por debajo y encima de las cajas respectivamente) del DAS28 a los dos años de seguimiento en los pacientes que tienen VIP alto (cajas blancas) o bajo (cajas grises). FIG. 7. Effect of positivity of citrullinated antiseptic antibodies (ACPA) on the predictive capacity of VIP levels. A) Disease activity after two years of follow-up. B) Intensity of treatment. Data are shown as the median (horizontal line inside the boxes), the 25th and 75th percentiles (bottom and top lines of the boxes respectively) and the 10th and 90th percentiles (small horizontal lines at the end of the vertical lines below and above the boxes respectively) of DAS28 at Two years of follow-up in patients who have high VIP (white boxes) or low (gray boxes).
FIG. 8. Niveles de VIP antes (cajas blancas) y después (cajas grises) de iniciar los diferentes tratamientos. Hay que reseñar que los pacientes en tratamiento con anti-TNF fueron muy escasos y esa puede ser la causa de que no se alcance significación estadística siendo mayor el efecto observado. FIG. 8. VIP levels before (white boxes) and after (gray boxes) to start the different treatments. It should be noted that patients treated with anti-TNF were very scarce and that may be the cause of statistical significance not being achieved, the effect observed being greater.
FIG. 9. Niveles séricos de VIP en pacientes de la consulta de Espondiloartritis de inicio, diagnosticados o no de Espondiloartritis. Los datos se muestran como la mediana de los niveles de VIP (línea en el interior de la caja), los percentiles 25 y 75 (bordes inferior y superior de la caja, respectivamente) y los percentiles 10 y 90 (líneas inferior y superior fuera de la caja, respectivamente). Los puntos representan casos fuera de estos rangos. FIG. 9. Serum VIP levels in patients of the initial Spondyloarthritis office, diagnosed or not of Spondyloarthritis. Data are shown as the median of the VIP levels (line inside the box), the 25th and 75th percentiles (bottom and top edges of the box, respectively) and the 10th and 90th percentiles (bottom and top lines outside of the box, respectively). The dots represent cases outside these ranges.
FIG. 10. A) Niveles séricos de VIP según la edad del paciente en la primera visita del estudio. Los datos se muestran como la medía del valor para cada punto de edad ajustado por sexo, visita y placa de medición de los niveles de VIP (redondel sólido), junto al intervalo de confianza al 95% (líneas por encima y debajo del redondel). La estimación de estos valores ser realizó mediante el comando "margins" de Stata 12, tras realizar una regresión lineal multivariable mediante modelos lineales generalizados con el comando "glm" de Stata. B) Niveles séricos de VIP según el género del paciente. Los datos se muestran como la mediana de los niveles de VIP (línea en el interior de la caja), los percentiles 25 y 75 (bordes inferior y superior de la caja, respectivamente) y los percentiles 10 y 90 (líneas inferior y superior fuera de la caja, respectivamente). Los puntos representan casos fuera de estos rangos. FIG. 10. A) Serum VIP levels according to the patient's age at the first visit of the study. The data are shown as the average value for each age point adjusted by sex, visit and measuring plate of the VIP levels (solid round), together with the 95% confidence interval (lines above and below the round) . The estimation of these values was carried out by means of the "margins" command of Stata 12, after performing a multivariable linear regression by means of generalized linear models with the "glm" command of Stata. B) Serum VIP levels according to the patient's gender. Data are shown as the median of the VIP levels (line inside the box), the 25th and 75th percentiles (bottom and top edges of the box, respectively) and the 10th and 90th percentiles (bottom and top lines outside of the box, respectively). The dots represent cases outside these ranges.
FIG. 11. Relación de los niveles séricos de VIP con diferentes variables clínicas de los pacientes. A) Distribución de los niveles séricos de VIP según el paciente presentase entesitis o no. B) Distribución de los niveles séricos de VIP según el paciente presentase psoriasis o no. En los paneles A y B los datos se muestran como la mediana de los niveles de VIP (línea en el interior de la caja), los percentiles 25 y 75 (bordes inferior y superior de la caja, respectivamente) y los percentiles 10 y 90 (líneas inferior y superior fuera de la caja, respectivamente). Los puntos representan casos fuera de estos rangos. C) Relación entre los niveles de VIP a lo largo del seguimiento y la puntuación del índice de capacidad funcional BASFI. Los datos se muestran como la medía del valor de VIP (normalizado mediante transformación logarítmica) para cada valor de BASFI que se muestra en la figura ajustado por el resto de variables que se incluyeron en el análisis multivariable (redondel sólido). Se muestra, además, el intervalo de confianza al 95% (líneas por encima y debajo del redondel). La estimación de estos valores ser realizó mediante el comando "margins" de Stata 12, tras realizar un análisis multivariable para datos longitudinales anidado por visita y paciente mediante el comando "xtgee" de Stata. FIG. 11. Relationship of serum VIP levels with different clinical variables of patients. A) Distribution of serum VIP levels according to patient presenting enthesitis or not. B) Distribution of serum VIP levels according to the patient presenting with psoriasis or not. In panels A and B the data is shown as the median of the VIP levels (line inside the box), the 25th and 75th percentiles (bottom and top edges of the box, respectively) and the 10th and 90th percentiles (lower and upper lines outside the box, respectively). The dots represent cases outside these ranges. C) Relationship between VIP levels throughout the follow-up and the BASFI functional capacity index score. The data is shown as the average of the VIP value (normalized by logarithmic transformation) for each BASFI value shown in the figure adjusted for the rest of the variables that were included in the multivariable analysis (solid round). In addition, the 95% confidence interval is shown (lines above and below the round). The estimation of these values was carried out by means of the "margins" command of Stata 12, after performing a multivariable analysis for longitudinal data nested by visit and patient by means of the "xtgee" command of Stata.
FIG. 12. Relación de los niveles de VIP con variables radiológicas y analíticas. A) Distribución de los niveles de VIP según los pacientes presentasen lesiones asociadas a Espondiloartritis en la resonancia magnética (RM) o no. Los datos se muestran como la mediana de los niveles de VIP (línea en el interior de la caja), los percentiles 25 y 75 (bordes inferior y superior de la caja, respectivamente) y los percentiles 10 y 90 (líneas inferior y superior fuera de la caja, respectivamente). Los puntos representan casos fuera de estos rangos. B) Relación entre los niveles de VIP y los de hemoglobina. Los datos se muestran como la medía del valor de VIP (normalizado mediante transformación logarítmica) para cada valor de Hemoglobina (Hb) que se muestra en la figura, ajustado por el resto de variables que se incluyeron en el análisis multivariable (redondel sólido). Se muestra, además, el intervalo de confianza al 95% (líneas por encima y debajo del redondel). La estimación de estos valores ser realizó mediante el comando "margins" de Stata 12, tras realizar un análisis multivahable para datos longitudinales anidado por visita y paciente mediante el comando "xfgee" de Stata. FIG. 12. Relationship of VIP levels with radiological and analytical variables. A) Distribution of the VIP levels according to the patients presenting lesions associated with Spondyloarthritis on magnetic resonance imaging (MRI) or not. Data are shown as the median of the VIP levels (line inside the box), the 25th and 75th percentiles (bottom and top edges of the box, respectively) and the 10th and 90th percentiles (bottom and top lines outside of the box, respectively). The dots represent cases outside these ranges. B) Relationship between VIP levels and hemoglobin levels. The data are shown as the average of the VIP value (normalized by logarithmic transformation) for each Hemoglobin (Hb) value shown in the figure, adjusted for the rest of the variables that were included in the multivariable analysis (solid round). In addition, the 95% confidence interval is shown (lines above and below the round). The estimate of These values were performed using the "margins" command of Stata 12, after performing a multivahable analysis for longitudinal data nested by visit and patient using the "xfgee" Stata command.
EJEMPLOS EXAMPLES
Los siguientes ejemplos específicos que se proporcionan en este documento de patente sirven para ¡lustrar la naturaleza de la presente invención. Estos ejemplos se incluyen solamente con fines ilustrativos y no han de ser interpretados como limitaciones a la invención que aquí se reivindica. Por tanto, los ejemplos descritos más adelante ¡lustran la invención sin limitar el campo de aplicación de la misma. A continuación se ¡lustrará la invención mediante unos ensayos realizados por los inventores que pone de manifiesto la utilidad de VIP como marcador pronóstico de la evolución de enfermedades autoinmunes. The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope. The invention will now be illustrated by tests carried out by the inventors that show the usefulness of VIP as a prognostic marker of the evolution of autoimmune diseases.
Ejemplo 1 Example 1
El registro de pacientes con artritis de reciente comienzo del Instituto de Investigación Sanitaria Hospital Universitario de La Princesa incluye pacientes derivados desde Atención Primaria que presentan una o más articulaciones inflamadas durante un mínimo de cuatro semanas y un máximo de un año de evolución. El único criterio de exclusión es que a lo largo del seguimiento los pacientes sean diagnosticados de artritis microcristalina, artritis sépticas o víricas, espondiloartropatías o enfermedades del tejido conectivo. El protocolo de estudio establece la realización de cuatro visitas en un período de seguimiento de cinco años: una visita basal, a los seis meses, al año, a los dos y a los cinco años. En cada visita se recogen datos demográficos (género, nivel de estudios y estado civil), clínicos (fecha de inicio de la enfermedad, recuento de 28 articulaciones dolorosas[NAD28] y tumefactas [NAT28], la valoración global de la enfermedad por el médico [VGEM] y por el paciente [VGEP] y evaluación del dolor en una escala visual analógica de 0 a 100 mm) y analíticos (velocidad de sedimentación globular [VSG; medida por el método de Westergren], proteína C reactiva [PCR; medida por nefelometría], factor reumatoide [FR; nefelometría niveles de anticuerpos antipéptido cíclico citrulinado [ACPA; determinados por ELISA: immunoscan CCPIus®. Euro- Diagnostica. Arnhem, Holanda] y hemograma y bioquímica básica. La capacidad funcional se estima mediante el cuestionario HAQ en su versión validada para población española (Esteve-Vives et al. J Rheumatol 1993;20:21 16-2122) y en cada visita se calcula el DAS28 con VSG como ha sido descrito previamente: 0,56*V(NAD28) + 0,28*V(NAT28) + 0,70*ln(VSG) + 0,014*(VGEP) (Prevoo, van't Hof et al. Arthritis Rheum 1995;38:44-48). También se realiza una recogida exhaustiva del tipo de fármacos que recibe el paciente para el tratamiento de su artritis, tanto glucocorticoides, como de fármacos modificadores de la enfermedad (FAME). El término FAME incluye a: antimaláricos, metotrexato, leflunomida, sulfasalacina, aurotiomalato, ciclosporina A y terapias biológicas que a su vez comprenden los agentes bloqueantes del TNF, tocilizumab, abatacept y rituximab. De todos estos fármacos se recoge, no solo la dosis, sino también la fecha de inicio y, si procede, de suspensión. Ello nos ha permitido generar una variable, denominada Intensidad de Tratamiento, que es el resultado de sumar el número de días que el paciente ha recibido cada uno de los fármacos en los dos primeros años de tratamiento, ponderados multiplicando por un factor de corrección (relacionado con su eficacia teórica): 1x para antimaláricos, 1 '5x para metotrexato, leflunomida, sulfasalacina, aurotiomalato y ciclosporina A y 2x para las terapias biológicas. Esta variable es de gran importancia ya que, en estudios observacionales como el que se describe, la cuantificación de la intensidad del tratamiento se acepta como una variable subrogada de gravedad. El motivo es que, a diferencia de los ensayos clínicos doble ciego en los que hay un protocolo preestablecido que no se puede modificar, en los estudios observacionales se modifica el tratamiento en cada visita según el estado en el que se encuentra el paciente. Además, se recogen muestras de suero, RNA de células mononucleares de sangre periférica y DNA genómico que se procesan adecuadamente para su conservación hasta la realización de diferentes determinaciones. El protocolo del Registro ha sido evaluado y aprobado por el Comité Ético de Investigación Clínica del Hospital Universitario La Princesa y todos los pacientes son informados del estudio y firman un consentimiento informado antes de su inclusión en el Registro. The recently started registry of arthritis patients of the La Princesa University Hospital Health Research Institute includes patients derived from Primary Care who have one or more inflamed joints for a minimum of four weeks and a maximum of one year of evolution. The only exclusion criterion is that throughout the follow-up patients will be diagnosed with microcrystalline arthritis, septic or viral arthritis, spondyloarthropathies or connective tissue diseases. The study protocol establishes four visits in a five-year follow-up period: a baseline visit, at six months, at one year, at two and at five years. At each visit demographic data (gender, level of studies and marital status), clinical (date of onset of the disease, count of 28 painful joints [NAD28] and swollen [NAT28], the assessment are collected). global disease by the doctor [VGEM] and by the patient [VGEP] and pain assessment on an analog visual scale from 0 to 100 mm) and analytical (globular sedimentation rate [VSG; measured by the Westergren method], C-reactive protein [PCR; measured by nephelometry], rheumatoid factor [FR; nephelometry levels of citrullinated cyclic antiseptic antibodies [ACPA; determined by ELISA: immunoscan CCPIus®. Euro-Diagnostics. Arnhem, Netherlands] and hemogram and basic biochemistry. Functional is estimated using the HAQ questionnaire in its version validated for the Spanish population (Esteve-Vives et al. J Rheumatol 1993; 20: 21 16-2122) and at each visit the DAS28 with ESR is calculated as previously described: 0, 56 * V (NAD28) + 0.28 * V (NAT28) + 0.70 * ln (VSG) + 0.014 * (VGEP) (Prevoo, van't Hof et al. Arthritis Rheum 1995; 38: 44-48) An exhaustive collection of the type of drugs that the patient receives for the treatment of his arthritis is also performed, both glucocorticoids and disease modifying drugs (FAME). The term FAME includes: antimalarials, methotrexate, leflunomide, sulfasalacin, aurothiomalate, cyclosporin A and biological therapies that in turn comprise TNF blocking agents, tocilizumab, abatacept and rituximab. Of all these drugs, not only the dose is collected, but also the start date and, if appropriate, suspension. This has allowed us to generate a variable, called Treatment Intensity, which is the result of adding the number of days the patient has received each of the drugs in the first two years of treatment, weighted by multiplying by a correction factor (related with its theoretical efficacy): 1x for antimalarials, 1'5x for methotrexate, leflunomide, sulfasalacin, aurothiomalate and cyclosporine A and 2x for biological therapies. This variable is of great importance since, in observational studies such as the one described, the quantification of the intensity of the treatment is accepted as a subrogated variable of severity. The reason is that, unlike double-blind clinical trials in which there is a pre-established protocol that cannot be modified, in the Observational studies modify the treatment at each visit according to the state in which the patient is. In addition, serum samples, peripheral blood mononuclear cell RNA and genomic DNA are collected and processed properly for preservation until different determinations are made. The Registry protocol has been evaluated and approved by the Clinical Research Ethics Committee of La Princesa University Hospital and all patients are informed of the study and sign an informed consent before being included in the Registry.
Se han estudiado los niveles de VIP en 93 pacientes de esta consulta, en un total de 376 visitas. Así mismo, se han determinado los niveles de VIP en 100 donantes de sangre sanos. La determinación de los niveles de VIP se realiza por medio de un ELISA competitivo de la casa Phoenix Pharmaceuticals, INC. Para llevar a cabo este ensayo, previamente hay que liofilizar las muestras de unos 250 microlitros de suero que posteriormente se diluyen en 120 microlitros de un tampón de ensayo de la misma casa comercial y se añaden a una placa cuyos pocilios están recubiertos con un anticuerpo secundario. Junto con las muestras se carga un anticuerpo primario y VIP biotinilado. Tras dos horas de incubación, el material no unido es retirado por lavados. A continuación se añade estreptavidina conjugada con peroxidasa y en el último paso se agrega el sustrato de la peroxidasa, tetrametilbencidina (TMB). Las muestras desarrollan color azul, que vira a amarillo cuando la reacción se detiene con ácido clorhídrico. Por último, se mide la absorbancia a 450nm. Utilizando una curva estándar, se calcula la concentración de péptido en las muestras, aplicando el correspondiente factor de dilución. VIP levels have been studied in 93 patients of this consultation, in a total of 376 visits. Likewise, VIP levels have been determined in 100 healthy blood donors. The determination of VIP levels is done through a competitive ELISA from Phoenix Pharmaceuticals, INC. In order to carry out this test, it is necessary to lyophilize the samples of about 250 microliters of serum which are subsequently diluted in 120 microliters of a test buffer of the same commercial house and added to a plate whose wells are coated with a secondary antibody . A biotinylated primary and VIP antibody is loaded together with the samples. After two hours of incubation, unbound material is removed by washing. Then, peroxidase-conjugated streptavidin is added and in the last step the peroxidase substrate, tetramethylbenzidine (TMB) is added. The samples develop blue, which turns yellow when the reaction stops with hydrochloric acid. Finally, absorbance at 450nm is measured. Using a standard curve, the concentration of peptide in the samples is calculated, applying the corresponding dilution factor.
Como inconveniente de este procesado de las muestras hemos obtenido una cierta variabilidad interensayo. Además algunos valores de pacientes concretos tenían una importante dispersión por encima de 1000 pg/ml. Para el análisis de los datos que se presentan a continuación, ambos problemas se han solventado favorablemente mediante herramientas estadísticas. En el primer caso, el alto número de muestras estudiadas ha permitido incluir como variable independiente la placa en la que se determinaba cada grupo de muestras, ajusfando así la variabilidad interensayo. En el segundo caso, se ha utilizado una variable censurada en el valor 1000 pg/ml de la curva patrón del ELISA. Utilizando modelos lineales generalizados ello nos ha permitido determinar asociaciones significativas, aunque los coeficientes no dan una información perfecta del grado de asociación de los niveles de VIP con las diferentes variables. As a disadvantage of this sample processing, we have obtained a certain interstability variability. In addition, some specific patient values had a significant dispersion above 1000 pg / ml. For the analysis of the data presented below, both problems have been favorably solved by statistical tools. In the first case, the high number of samples studied has allowed to include as an independent variable the plate in which each group of samples was determined, thus adjusting the inter-assay variability. In the second case, a censored variable in the value 1000 pg / ml of the ELISA standard curve has been used. Using generalized linear models, this has allowed us to determine significant associations, although the coefficients do not give perfect information on the degree of association of the VIP levels with the different variables.
Resultados Results
Como se puede apreciar en la figura 1 , la distribución de los valores de VIP es bastante variable en las 3 poblaciones estudiadas: controles sanos, pacientes con AR y con Al. Se han considerado los percentiles 50, 25 y 10 de la población sana como el límite para definir VIP bajo porque este es un proceder común en estadística y epidemiología que divide a la población en dos grupos bien diferenciados. No obstante, algunos análisis se han realizado con la variable continua que proporciona una mayor sensibilidad. As can be seen in Figure 1, the distribution of VIP values is quite variable in the 3 populations studied: healthy controls, patients with RA and Al. The 50, 25 and 10 percentiles of the healthy population have been considered as the limit to define low VIP because this is a common procedure in statistics and epidemiology that divides the population into two distinct groups. However, some analyzes have been performed with the continuous variable that provides greater sensitivity.
El análisis multivariable de los resultados permite determinar que el género no influye en los niveles de VIP (Figura 2A). Por otra parte, se ha observado que existe una pequeña tendencia a aumentar los niveles de VIP con la edad (Figura 2B), aunque esta variación atribuible a la edad es mucho menor que la variabilidad individual de los diferentes pacientes. Por último, hay una correlación inversa entre los niveles de actividad de la artritis y la concentración de VIP en suero. Así pacientes con niveles de VIP más bajos tienden a tener mayores niveles de actividad de la enfermedad (Figura 2C). En relación con la actividad de la enfermedad, es interesante saber si los niveles de VIP varían al hacerlo la actividad de la enfermedad o si se mantienen más o menos estables para cada paciente. Así, se puede observar que no existe una variación significativa de los niveles de VIP a lo largo del seguimiento (figura 3), a diferencia de lo que ocurre con la actividad de la enfermedad que mejora en las visitas sucesivas respecto a la basal como efecto del tratamiento administrado. The multivariable analysis of the results makes it possible to determine that gender does not influence VIP levels (Figure 2A). On the other hand, it has been observed that there is a small tendency to increase VIP levels with age (Figure 2B), although this variation attributable to age is much smaller than the individual variability of different patients. Finally, there is an inverse correlation between arthritis activity levels and serum VIP concentration. Thus patients with lower VIP levels tend to have higher levels of disease activity (Figure 2C). In relation to the activity of the disease, it is interesting to know if the VIP levels vary when doing the activity of the disease or if they remain more or less stable for each patient. Thus, it can be observed that there is no significant variation in VIP levels throughout the follow-up (Figure 3), unlike what happens with the disease activity that improves in successive visits with respect to the baseline as an effect of the treatment administered.
Por lo tanto, la asociación vista entre niveles de VIP y la actividad de la enfermedad se refiere a que los pacientes con VIP bajo tienden a tener mayor actividad al no ser capaces de incrementar el VIP endógeno (cuya función es la inmunoregulación negativa) en situaciones de inflamación intensa. Por ello se analizaron los niveles de VIP en la visita basal del estudio según la actividad de los enfermos a los dos años de seguimiento. Como se aprecia en la figura 4, los niveles de VIP más bajos en la visita inicial se acumulan entre los pacientes con actividad alta, a pesar del tratamiento, a los dos (Figura 4A) y cinco (figura 4B) años de seguimiento. Therefore, the association seen between VIP levels and disease activity refers to patients with low VIP tend to be more active because they are not able to increase endogenous VIP (whose function is negative immunoregulation) in situations of intense inflammation Therefore, VIP levels were analyzed at the baseline study visit according to the activity of the patients at two years of follow-up. As can be seen in Figure 4, the lowest VIP levels at the initial visit accumulate among patients with high activity, despite treatment, at two (Figure 4A) and five (Figure 4B) years of follow-up.
Por el contrario, los niveles de VIP más elevados se acumularon en los grupos de pacientes con enfermedad en remisión o con actividad baja a los dos ó cinco años de seguimiento. Vista de la forma inversa, en la figura 4C se observa que en la visita inicial los pacientes tiene similares niveles de actividad tengan VIP alto o bajo, mientras que a los dos ó cinco años de seguimiento los pacientes con VIP bajo tienden a tener mayores valores de DAS28. Por lo tanto, los pacientes con niveles bajos de VIP tienden a tener un peor curso evolutivo a lo largo de la enfermedad, a pesar de que como se muestra en la figura 5 reciben un tratamiento con FAME más intenso que los pacientes con VIP alto. El efecto predictivo de los niveles bajos de VIP pueden verse modulados o potenciados por otras variables de confusión. Como se observa en la figura 6A, las mujeres con VIP bajo tuvieron niveles de actividad a los dos años de seguimiento mayores que los de las mujeres con VIP normal o alto, mientras que estas diferencias fueron menos marcadas en los varones. Por su parte, la edad no interfirió con la capacidad predictiva de VIP (figura 6B). Además, los niveles de actividad en la visita inicial, también pueden modular la capacidad predictiva de los niveles de VIP. La figura 6C pone de manifiesto que no había pacientes en remisión o actividad baja al inicio del seguimiento entre los pacientes con VIP bajo. Estos pacientes se encontraban entre los que tenían actividad moderada o alta. Pero entre estos últimos, aquellos con niveles de VIP bajos eran los que peor se encontraban al final del seguimiento. On the contrary, higher levels of VIP accumulated in the groups of patients with remission disease or with low activity at two or five years of follow-up. Seen in reverse, Figure 4C shows that in the initial visit patients have similar activity levels have high or low VIP, while at two or five years of follow-up patients with low VIP tend to have higher values of DAS28. Therefore, patients with low levels of VIP tend to have a worse evolutionary course throughout the disease, although as shown in Figure 5 they receive a more intense treatment with DMARD than patients with high VIP. The predictive effect of low VIP levels can be modulated or enhanced by other confounding variables. As shown in Figure 6A, women with low VIP had activity levels at two years of follow-up greater than those of women with normal or high VIP, while these differences were less marked in men. On the other hand, age did not interfere with the predictive capacity of VIP (Figure 6B). In addition, activity levels during the initial visit can also modulate the predictive capacity of VIP levels. Figure 6C shows that there were no patients in remission or low activity at the beginning of follow-up among patients with low VIP. These patients were among those who had moderate or high activity. But among the latter, those with low VIP levels were the worst at the end of the follow-up.
Más compleja es la interacción con la presencia de ACPA +, ya que este es un reconocido marcador de mal pronóstico y ante su presencia los reumatólogos tienden a ser más agresivos en las pautas de tratamiento. Así, podemos apreciar en la figura 7A que los pacientes con ACPA negativo y VIP bajo tenían mayor nivel de actividad a los dos años de seguimiento que los pacientes ACPA negativo y con VIP normal o alto. Esta diferencia no es tan clara en los pacientes ACPA +. Muy probablemente ello es debido a que todos los pacientes con ACPA positivo recibieron tratamiento más intenso (figura 7B). Posiblemente, los que además tenían VIP bajo, precisaron un tratamiento un poco más intenso. Los pacientes con ACPA negativo y VIP normal o alto precisaron tratamiento menos intenso, mientras que en los pacientes con ACPA negativo y VIP bajo el tratamiento tendió a ser más alto aunque de forma muy heterogénea por no tener marcador de mal pronóstico conocido. More complex is the interaction with the presence of ACPA +, since this is a recognized marker of poor prognosis and in the presence of rheumatologists tend to be more aggressive in treatment patterns. Thus, we can see in Figure 7A that patients with negative ACPA and low VIP had a higher level of activity at two years of follow-up than patients with ACPA negative and with normal or high VIP. This difference is not so clear in ACPA + patients. This is most likely due to the fact that all patients with positive ACPA received more intense treatment (Figure 7B). Possibly, those who also had low VIP, required a slightly more intense treatment. Patients with ACPA negative and normal or high VIP required less intense treatment, while in patients with negative ACPA and VIP under treatment, it tended to be higher although in a very heterogeneous way because they had no known poor prognosis marker.
Por último, también se estudio si los diferentes fármacos utilizados en el tratamiento de la AR afectan a los niveles de VIP. Como se observa en la figura 8, la prescripción de leflunomida y posiblemente de anti-TNF, podrían ejercer un efecto sobre los niveles de VIP incrementándolos. Sin embargo, este dato precisa un mayor número de pacientes a estudiar pues leflunomida y terapias biológicas suelen utilizarse en segunda línea con pacientes que han fracasado a antimaláricos y/o metotrexato. Finally, it was also studied if the different drugs used in the treatment of RA affect VIP levels. As can be seen in figure 8, the prescription of leflunomide and possibly anti-TNF could exert an effect on VIP levels by increasing them. However, this data requires a larger number of patients to study because leflunomide and biological therapies are often used in the second line with patients who have failed antimalarials and / or methotrexate.
En conclusión, los pacientes con niveles bajos de VIP tienen un peor curso evolutivo y la determinación de este biomarcador en fases precoces podría ser de utilidad para seleccionar a los pacientes candidatos a pautas terapéuticas más intensa contra la enfermedad. In conclusion, patients with low levels of VIP have a worse evolutionary course and the determination of this biomarker in early stages could be useful to select the candidate patients for more intense therapeutic guidelines against the disease.
Ejemplo 2: Estudio de los niveles de VIP como factor Pronóstico en Pacientes con Espondiloartritis. Example 2: Study of VIP levels as a Prognostic factor in Patients with Spondyloarthritis.
Se trata de pacientes que asisten a una consulta de Espondiloartritis de inicio, cuyos criterios de inclusión son dolor lumbar inflamatorio de menos de 2 años de duración y edad menor de 45 años al inicio de los síntomas. These are patients who attend an initial Spondyloarthritis consultation, whose inclusion criteria are inflammatory low back pain of less than 2 years duration and age less than 45 years at the onset of symptoms.
El protocolo de estudio establece la realización de una visita anual en la que se recogen datos de actividad de la enfermedad (BASDAI), de capacidad funcional (BASFI), analítica convencional y determinación de HLA-B27, tratamiento prescrito y recogida de muestras de suero y células de sangre periférica para extracción de RNA y DNA. A todos los pacientes se les realiza una RM de sacroiliacas y columna lumbar para determinar la presencia de signos inflamatorios y estudio radiográfico convencional de columna lumbar y sacroiliacas para objetivar cambios estructurales típicos de la enfermedad. Se recoge de forma sistemática la presencia de comorbilidades relacionadas con espondiloartritis (uveítis, psoriasis, enfermedad inflamatoria intestinal, entesitis, etc). Se han estudiado 54 pacientes (32 [59%] mujeres) de los cuales alcanzaron la 2a y 3a visitas 31 y 17 pacientes respectivamente. La mediana de la edad al inicio de la enfermedad fue de 35 años (rango intercuartílico 31 - 43). De los 54 pacientes, 36 cumplieron criterios de Espondiloartritis y 12 cumplieron criterios de Nueva York de Espondilitis Anquilosante. Dieciocho pacientes no cumplieron criterios de Espondiloartritis y, por tanto, fueron diagnosticados de lumbalgia crónica inespecífica. The study protocol establishes an annual visit in which data on disease activity (BASDAI), functional capacity (BASFI), conventional analytics and determination of HLA-B27, prescribed treatment and collection of serum samples are collected. and peripheral blood cells for RNA and DNA extraction. All patients undergo a MR of sacroiliac and lumbar spine to determine the presence of inflammatory signs and conventional radiographic study of lumbar spine and sacroiliac to objectify structural changes typical of the disease. The presence of comorbidities related to spondyloarthritis (uveitis, psoriasis, inflammatory bowel disease, enthesitis, etc.) is systematically collected. We studied 54 patients (32 [59%] females) of which reached the 2 nd and 3 visits 31 and 17 patients respectively. The median age at the onset of the disease was 35 years (interquartile range 31 - 43). Of the 54 patients, 36 met Spondyloarthritis criteria and 12 met New York criteria for Ankylosing Spondylitis. Eighteen patients did not meet Spondyloarthritis criteria and, therefore, were diagnosed with nonspecific chronic low back pain.
En quince pacientes existían antecedentes familiares de EA, 14 de ellos cumplían criterios de Espondiloartritis. Un 35% de la población presentaba entesitis desde el punto de vista clínico, un 22% tenían artritis periférica, 7% tenían uveítis, 9% psoriasis, 5'5% antecedentes de infección compatible con artritis reactiva y 4% enfermedad inflamatoria intestinal. En todos los pacientes se determinaron los niveles séricos de VIP en las diferentes visitas por medio de un ELISA competitivo de la casa Phoenix Pharmaceuticals, INC, según el mismo procedimiento descrito para los pacientes con artritis de reciente comienzo. De los 54 pacientes en 23 presentaron hallazgos compatibles con espondiloartritis en la RM de sacroiliacas y/o columna lumbar. De ellos 21 cumplían criterios de espondiloartritis de inicio y solo 6 pacientes cumplían criterios de Espondilitis Anquilosante según criterios de Nueva York. Como ocurrió en el caso de los pacientes con Artritis Reumatoide, no se observaron diferencias significativas entre los pacientes que cumplieron criterios de Espondiloartritis y los pacientes con lumbalgia crónica que no cumplieron criterios de esta enfermedad (Figura 9). Aunque no se observaron diferencias significativas (p=0'36), la mediana de los niveles de VIP fue menor en los pacientes con Espondiloartritis. Se consideraron niveles bajos de VIP aquellos por debajo del percentil 25 de la población con lumbalgia crónica inespecífica. In fifteen patients there was a family history of AD, 14 of them met Spondyloarthritis criteria. 35% of the population had clinical enthesitis, 22% had peripheral arthritis, 7% had uveitis, 9% psoriasis, 5.5% history of infection compatible with reactive arthritis and 4% inflammatory bowel disease. In all patients, serum VIP levels were determined at different visits by means of a competitive ELISA from Phoenix Pharmaceuticals, INC, according to the same procedure described for patients with recent onset arthritis. Of the 54 patients in 23 presented findings compatible with spondyloarthritis in the MR of sacroiliac and / or lumbar spine. Of these, 21 met criteria for onset spondyloarthritis and only 6 patients met criteria for Ankylosing Spondylitis according to New York criteria. As in the case of patients with Rheumatoid Arthritis, no significant differences were observed between patients who met Spondyloarthritis criteria and patients with chronic low back pain who did not meet criteria for this disease (Figure 9). Although no significant differences were observed (p = 0.36), the median VIP levels were lower in patients with Spondyloarthritis. Low VIP levels were considered those below the 25th percentile of the population with nonspecific chronic low back pain.
En relación con los datos sociodemográficos, observamos una tendencia no significativa a que los niveles de VIP aumentasen con la edad (Figura 10A), de igual manera que ocurría en pacientes con artritis de inicio. Es muy probable que esta tendencia no sea significativa por el menor número de pacientes estudiados en el grupo de espondiloartritis, que por protocolo solo se incluyen si su enfermedad comienza antes de los 45 años. Por el contrario, en la población de artritis de inicio el rango de edad de los pacientes era mucho más amplio (desde 17 a más de 80 años). Tampoco observamos diferencias significativas por género. Regarding sociodemographic data, we observed a non-significant tendency for VIP levels to increase with age (Figure 10A), in the same way as in patients with onset arthritis. It is very likely that this trend is not significant due to the lower number of patients studied in the spondyloarthritis group, which by protocol are only included if their disease begins before the age of 45. On the contrary, in the population of onset arthritis the age range of the patients was much wider (from 17 to more than 80 years). Nor do we observe significant differences by gender.
Por otra parte, diferentes variables clínicas, analíticas y radiológicas recogidas a lo largo del seguimiento mostraron asociación con los niveles de VIP en el análisis bivanable. Para poder determinar mejor su relación con los niveles de VIP se decidió realizar un análisis multivahable en el que se incluyeron también las variables placa del ensayo en la que se realizó la medición de VIP, edad y sexo ya que previamente, en los pacientes con artritis de inicio, determinamos que se comportaban como variables de confusión. Respecto a los datos clínicos, los pacientes con entesitis (proceso inflamatorio de las inserciones de los tendones que puede evolucionar de forma independiente de la afectación de las articulaciones de la columna vertebral en las espondiloartritis y puede considerarse como una complicación; Figura 1 1A y Tabla 1 ), aquellos que tenían psoriasis cutánea (Figura 1 1 B y Tabla 1 ) y los que a lo largo del seguimiento tuvieron peor evolución de la capacidad funcional, medida por el BASFI (Figura 1 1 B y Tabla 1 ), tenían menores niveles de VIP. Respecto a variables analíticas y radiológicas, también presentaron niveles más bajos de VIP los pacientes con datos de afectación inflamatoria en la RM de columna lumbar o sacroiliacas (Figura 12A y Tabla 1 )y aquellos pacientes con niveles bajos de VIP también tenían mayor tendencia a la anemia o a presentar niveles más bajos de hemoglobina (Figura 12B y Tabla 1 ). On the other hand, different clinical, analytical and radiological variables collected throughout the follow-up showed association with VIP levels in the bivanable analysis. In order to better determine their relationship with VIP levels, it was decided to carry out a multivahable analysis in which the plaque variables of the trial in which the measurement of VIP, age and sex were also included since previously, in patients with arthritis Initially, we determined that they behaved as confounding variables. Regarding clinical data, patients with enthesitis (inflammatory process of tendon insertions that can evolve independently of the involvement of the spinal joints in spondyloarthritis and can be considered as a complication; Figure 1 1A and Table 1), those who had cutaneous psoriasis (Figure 1 1 B and Table 1) and those who throughout the follow-up had worse evolution of functional capacity, measured by BASFI (Figure 1 1 B and Table 1), had lower levels VIP Regarding analytical and radiological variables, patients with inflammatory involvement data in lumbar spine MRI or sacroiliac MR (Figure 12A and Table 1) also presented lower levels of VIP. Low VIPs also had a greater tendency to anemia or to have lower levels of hemoglobin (Figure 12B and Table 1).
Por el contrario, en las visitas en las que los pacientes recibieron tratamiento con anti-TNF se observó una tendencia, no significativa, a que aumentasen los niveles de VIP. On the contrary, in the visits in which the patients received treatment with anti-TNF, there was a tendency, not significant, to increase VIP levels.
Tabla 1 Table 1
Ln_vip_v Coef. Std. Err. z P>|z| [95% Conf. Interval] Ln_vip_v Coef. Std. Err. z P> | z | [95% Interval Conf.]
Placa_vip_v Vip_v_plate
0 referencia  0 reference
1 -.3216598 .0760749 -4.23 0.000 -.4707638 -.1725558 1 -.3216598 .0760749 -4.23 0.000 -.4707638 -.1725558
2 -.6308785 -.0834766 -7.56 0.000 -.7944896 -.4672674 2 -.6308785 -.0834766 -7.56 0.000 -.7944896 -.4672674
Hombre referencia Reference man
Mujer .0390876 .0729475 0.54 0.592 -.1038869 .182062 edad .0005991 .0030796 0.19 0.846 -.0054367 .006635  Female .0390876 .0729475 0.54 0.592 -.1038869 .182062 age .0005991 .0030796 0.19 0.846 -.0054367 .006635
Visita Visit
0 referencia  0 reference
2 -.1020435 .04963 -2.06 0.040 -.1993164 -.0047705 2 -.1020435 .04963 -2.06 0.040 -.1993164 -.0047705
3 -.094458 .0622457 -1 .52 0.129 -.2164573 .0275413 3 -.094458 .0622457 -1 .52 0.129 -.2164573 .0275413
Entesistis -.1087062 .0590265 -1 .84 0.066 -.2243959 .0069836Entesistis -.1087062 .0590265 -1 .84 0.066 -.2243959 .0069836
Uveítis .3960885 .1073499 3.69 0.000 .1856866 .6064904 psoriasis -.2184889 .0998546 -2.19 0.029 -.4142004 -.0227775Uveitis .3960885 .1073499 3.69 0.000 .1856866 .6064904 psoriasis -.2184889 .0998546 -2.19 0.029 -.4142004 -.0227775
RM positiva -.1578492 .057184 -2.76 0.006 -.2699278 -.0457706 Anti-TNF .1817787 .1177944 1.54 0.123 -.0490942 .4126515Positive RM -.1578492 .057184 -2.76 0.006 -.2699278 -.0457706 Anti-TNF .1817787 .1177944 1.54 0.123 -.0490942 .4126515
Hamoglobina .0347743 .0149374 2.33 0.020 .0054974 .0640511Hamoglobin .0347743 .0149374 2.33 0.020 .0054974 .0640511
BASFI -.0032958 .0010917 -3.02 0.003 -.0054356 -.001156 BASFI -.0032958 .0010917 -3.02 0.003 -.0054356 -.001156

Claims

REIVINDICACIONES
1 . - Uso de al menos un producto de expresión del gen VIP como marcador pronóstico de una enfermedad autoinmune en un sujeto que padece dicha enfermedad autoinmune. one . - Use of at least one VIP gene expression product as a prognostic marker of an autoimmune disease in a subject suffering from said autoimmune disease.
2. - Uso según la reivindicación 1 donde el producto de expresión es VIP. 2. - Use according to claim 1 wherein the expression product is VIP.
3. - Uso según cualquiera de las reivindicaciones 1 o 2 donde la enfermedad autoinmune es artritis autoinmune o espondiloartritis. 3. - Use according to any of claims 1 or 2 wherein the autoimmune disease is autoimmune arthritis or spondyloarthritis.
4. - Uso según la reivindicación 3 donde la artritis autoinmune es artritis de reciente comienzo o artritis reumatoide. 4. - Use according to claim 3 wherein the autoimmune arthritis is newly started arthritis or rheumatoid arthritis.
5.- Método in vitro para el pronóstico de una enfermedad autoinmune caracterizado porque comprende la cuantificación de al menos un producto de expresión del gen VIP en una muestra biológica aislada de un sujeto que padece dicha enfermedad autoinmune. 5.- In vitro method for the prognosis of an autoimmune disease characterized in that it comprises the quantification of at least one VIP gene expression product in a biological sample isolated from a subject suffering from said autoimmune disease.
6.- Método in vitro para el pronóstico de una enfermedad autoinmunede un sujeto que padece dicha enfermedad autoinmune que comprende: 6.- In vitro method for the prognosis of an autoimmune disease of a subject suffering from said autoimmune disease comprising:
a. cuantificar al menos un producto de expresión del gen VIP en una muestra biológica aislada de un sujeto que padece dicha enfermedad autoinmune,  to. quantify at least one VIP gene expression product in an isolated biological sample of a subject suffering from said autoimmune disease,
b. comparar el valor obtenido en el paso (a) con una cantidad de referencia, y  b. compare the value obtained in step (a) with a reference amount, and
c. asociar al individuo del paso (a) al grupo de pacientes con mal pronóstico de enfermedad autoinmune cuando la cantidad cuantificada en el paso (a) es menor que el percentil 50 de la cantidad de referencia del paso (b). C. Associate the individual from step (a) to the group of patients with poor prognosis of autoimmune disease when the quantity quantified in step (a) is less than the 50th percentile of the reference amount of step (b).
7. - Método según la reivindicación 6 donde en el paso (c) se asocia al individuo del paso (a) al grupo de pacientes con mal pronóstico de enfermedad autoinmune cuando la cantidad cuantificada en el paso (a) es menor que el percentil 25 de la cantidad de referencia del paso (b). 7. - Method according to claim 6 wherein in step (c) the individual from step (a) is associated with the group of patients with poor prognosis of autoimmune disease when the amount quantified in step (a) is less than the 25th percentile of the reference amount of step (b).
8. - Método según cualquiera de las reivindicaciones 6 ó 7 donde en el paso (c) se asocia al individuo del paso (a) al grupo de pacientes con mal pronóstico de enfermedad autoinmune cuando la cantidad cuantificada en el paso (a) es menor que el percentil 10 de la cantidad de referencia del paso (b). 8. - Method according to any of claims 6 or 7 wherein in step (c) the individual of step (a) is associated with the group of patients with poor prognosis of autoimmune disease when the amount quantified in step (a) is less than the 10th percentile of the reference amount of step (b).
9. - Método según cualquiera de las reivindicaciones 5 a 8 donde la muestra biológica aislada es suero, plasma o membrana sinovial. 9. - Method according to any of claims 5 to 8 wherein the isolated biological sample is serum, plasma or synovial membrane.
10.- Método según cualquiera de las reivindicaciones 5 a 9 que además comprende la determinación del sexo del sujeto, la presencia de anticuerpos antipéptidos citrulinados y/o el estado de actividad al inicio de la enfermedad. 10. Method according to any of claims 5 to 9 which further comprises determining the sex of the subject, the presence of citrullinated antiseptic antibodies and / or the state of activity at the onset of the disease.
1 1 . - Método según cualquiera de las reivindicaciones 5 a 10 donde el producto de expresión del gen VIP es VIP. eleven . - Method according to any of claims 5 to 10 wherein the VIP gene expression product is VIP.
12. - Método según cualquiera de las reivindicaciones 5 a 1 1 donde el sujeto es humano. 12. - Method according to any of claims 5 to 1 1 wherein the subject is human.
13.- Método según cualquiera de las reivindicaciones 5 a 12 donde la enfermedad autoinmune es artritis autoinmune o espondiloartritis 13. Method according to any of claims 5 to 12 wherein the autoimmune disease is autoimmune arthritis or spondyloarthritis
14.- Método según la reivindicación 13 donde la artritis autoinmune es artritis de reciente comienzo o artritis reumatoide. 14. Method according to claim 13 wherein the autoimmune arthritis is newly started arthritis or rheumatoid arthritis.
15.- Uso de un kit que comprende anticuerpos específicos para VIP para la realización de un método según cualquiera de las reivindicaciones 5 a 14. 15. Use of a kit comprising antibodies specific for VIP for the performance of a method according to any of claims 5 to 14.
16.- Uso de un kit que comprende sondas que permiten la determinación de la cantidad de mRNA transcrito a partir del gen VIP para la realización de un método según cualquiera de las reivindicaciones 5 a 14. 16. Use of a kit comprising probes that allow the determination of the amount of mRNA transcribed from the VIP gene to perform a method according to any of claims 5 to 14.
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