ES2648694B1 - Proinflammatory cytokines as a diagnostic marker in episodic vestibular syndrome. - Google Patents

Abstract

Proinflammatory cytokines as a diagnostic marker in episodic vestibular syndrome. # Use of proinflammatory cytokines IL1 {be}, IL6, TNF {al} to obtain useful data for the diagnosis and classification of a disease that develops with episodic vestibular syndrome, method to obtain useful data for the diagnosis and classification of said disease, kit or device and uses.

Description

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DESCRIPTION

Proinflammatory cytokines as a diagnostic marker in vestibular syndrome

episodic.

The present invention is within the field of biomedicine and biotechnology, and refers to the use of proinflammatory cytokines as a diagnostic marker in episodic vestibular syndrome, and especially refers to a method for the classification of patients presenting with episodic vestibular syndrome with migraine and / or hearing loss, and specifically the differential diagnosis of vestibular migraine and Meniere's disease.

STATE OF THE PREVIOUS TECHNIQUE

The classification of episodic vestibular syndrome (EVS) is based on clinical symptoms and there are no biological markers for diagnosis. Currently, consensus diagnoses have been developed for the most frequent causes of spontaneous vertigo that are: vestibular migraine (MV) and Meniere's disease (MS). However, the diagnostic criteria of these entities are not exclusive and the same patient can meet diagnostic criteria for both diseases. In addition, there are patients with an incomplete phenotype that do not meet the clinical criteria and that could be partial forms of the disease. MS is a chronic disorder that affects the inner ear, characterized by recurrent episodes of vertigo, progressive instability, hearing pressure, tinnitus and hearing loss at low and medium frequencies. There are several epidemiological evidences that indicate that MS could have a genetic basis, as well as the role of the immune response and allergy. In this sense, patients with MS have a high prevalence of autoimmune diseases as well as allergic symptoms. However, there is no biological marker to identify which patients with MS have an impaired immune response. Thus, nonspecific autoantibodies such as antinuclear antibodies or circulating immunocomplexes are only elevated in a very low percentage of patients and have no practical diagnostic profitability.

Autoimmune inner ear disease (EAOI) is a chronic disorder of the inner ear that is caused by an immune attack on the inner ear, mediated by antibodies or immunologically active cells. It is characterized by bilateral sensorineural hearing loss, rapidly progressive and often fluctuating, which occurs over a period of weeks to years. Vestibular symptoms, such as instability

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Generalized, ataxia, positional vertigo and episodic vertigo, may be present in almost 50% of patients. Occasionally, only one ear is initially affected, but bilateral hearing loss occurs in most patients, with symmetric or asymmetric audiometric thresholds. Almost 25% to 50% of patients also have tinnitus and a sensation of otic fullness, which can be fluctuating.

A wide variety of treatments, both medical and surgical, have been proposed for hearing loss, and specifically for MS. All of them with different results regarding the remission of symptoms and side effects.

Among the medical treatments are the hyposodic diet, antihistamines, diuretics, subcutaneous histamine, antivertiginous drugs, benzodiazepines and transtympanic therapy, especially in that group of patients who do not respond to the usual therapies. However, these treatments have very limited efficacy, due to the clinical heterogeneity of patients with MS.

In some centers, intratympanic injection of ototoxic drugs, such as gentamicin, into the middle ear cavity has been practiced for some time. The objective of this procedure would be to produce a medical labytectomy, which would cause the decrease or disappearance of symptoms. The risk of this procedure is the deterioration of the patient's hearing capacity, especially described in those patients undergoing treatment regimens in short time periods. The most commonly used agents have been, initially, streptomycin and, currently, gentamicin.

In very severe cases, surgery has been resorted to, but this resource worsens the patient's quality of life because it eliminates not only the symptoms, but also the sense of balance.

Therefore, the treatments currently available for hearing loss can be excessively aggressive, such as ablative surgery (vestibular neurectomy or labyrinthyctomy) or the application of intratympanic gentamicin, and can put the patient's hearing at risk. The identification of an autoimmune variant through a high proinflammatory cytokine profile would allow immunosuppressive treatment in this subgroup of patients. In addition, a correct differential diagnosis between MV and MS would allow the administration of appropriate therapies, avoiding the aggressive treatments described above in misdiagnosed patients.

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BRIEF DESCRIPTION OF THE INVENTION

The examples of the invention show that ILip, IL6 and TNFa are useful markers to differentiate a disease that occurs with episodic vestibular syndrome (attacks of vertigo) with migrainous symptoms (headache and / or sensory aura) and / or sensorineural hearing loss, preferably MS and MV, and even more preferably, autoimmune MS.

Therefore, a first aspect of the invention relates to the use of the proinflammatory cytokines ILip, IL6, TNFa or any combination thereof to obtain useful data for the diagnosis and classification of a disease that occurs with episodic vestibular syndrome. In a preferred embodiment, the ILin, IL6 and TNFa proinflammatory cytokines are used simultaneously. In another preferred embodiment, the determination of proinflammatory cytokines is performed in vitro in human mononuclear cells. In another even more preferred embodiment, the classification of the disease with vestibular syndrome allows obtaining useful data for the differential diagnosis between MS and MV, and more specifically, for the diagnosis of autoimmune MS.

MS may present with migrainous symptoms, including headache and MV may have sensorineural hearing loss, being indistinguishable in the early stages of the disease.

A second aspect of the invention relates to a method of obtaining useful data for the diagnosis and classification of a disease that develops with episodic vestibular syndrome, hereafter referred to as the first method of the invention, comprising:

a) detect the levels of ILin, IL6 and TNFa proinflammatory cytokines in an isolated biological sample from an individual.

b) compare the levels of proinflammatory cytokines detected in step (a), with baseline levels in a healthy individual.

In a preferred embodiment of this aspect the invention, the sample isolated in step (a) is peripheral blood, and even more preferably they are mononuclear cells obtained from peripheral blood.

In another even more preferred embodiment, the classification of the disease with vestibular syndrome allows obtaining useful data for the differential diagnosis between MS and MV, and more specifically, for the diagnosis of autoimmune MS.

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A third aspect of the invention relates to a method for the diagnosis and classification of a disease that develops with episodic vestibular syndrome, hereafter referred to as the second method of the invention, comprising steps (a) and (b) of the first method. of the invention, and also comprises:

c) include the individual in the group of individuals with MS of autoimmune origin.

In a preferred embodiment of this aspect the invention, the sample isolated in step (a) is peripheral blood, and even more preferably they are mononuclear cells obtained from peripheral blood.

Steps (a), (b), and of the methods described above can be totally or partially automated, for example, by means of a robotic sensor device for the quantification of step (a) or the computerized calculation of any of the indices of steps (b), (c) and / or (d), or the computerized classification in the steps

A fourth aspect of the invention relates to a kit or device for the diagnosis and classification of a disease that develops with episodic vestibular syndrome, hereafter referred to as the kit or device of the invention, which comprises the elements necessary to detect the levels of Proinflammatory cytokines ILip, IL6 and TNFa in an isolated biological sample of an individual. In a preferred embodiment of this aspect the invention, the isolated sample is peripheral blood, and even more preferably they are mononuclear cells obtained from peripheral blood. In another preferred embodiment of this aspect, the kit or device of the invention comprises primers, probes and / or antibodies capable of detecting and quantifying the proinflammatory cytokines ILip, IL6 and TNFa in an isolated biological sample. Even more preferably, the detection of proinflammatory cytokine levels is performed by immunological techniques, with antibodies, and even more preferably by ELISA. In another preferred embodiment of this aspect of the invention, the antibodies are modified. In another preferred embodiment of this aspect of the invention, the antibody is human, humanized or synthetic. In another preferred embodiment, the antibody is monoclonal and / or is labeled with a fluorochrome. Preferably, the fluorochrome is selected from the list comprising Fluorescein (FITC), Tetramethylrodamine and derivatives, Phycoerythrin (PE), PerCP, Cy5, Texas, allophycocyanin, or any combination thereof.

In another preferred embodiment of this aspect of the invention, the kit or device of the invention is a kit of parts, comprising a component A, formed by a device

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for the collection of the sample from step a), and a component B, formed by the elements necessary to carry out the quantitative analysis in the sample from step a) or any of the methods of the invention.

A fifth aspect of the invention relates to the use of the kit of the invention, for the diagnosis and classification of a disease that occurs with episodic vestibular syndrome. In a preferred embodiment of this aspect of the invention, the disease that occurs with vestibular syndrome is MS or MV, being used for the differential diagnosis of MS and MV, and more specifically, for the diagnosis of autoimmune MS.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1. Levels of interleukin ip in patients with Meniere's disease and controls obtained after the in vitro mononuclear cell assay

Fig. 2. Interleukin 6 levels in patients with Meniere's disease and controls obtained after the in vitro mononuclear cell assay

Fig. 3. Levels of TNFa in patients with Meniere's disease and controls obtained after the in vitro mononuclear cell assay.

Fig. 4. Analysis of hierarchical clusters that allows patients to be classified into 2 groups according to their cytokine levels.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of determining the concentration of proinflammatory cytokines for the differential diagnosis of vestibular migraine and Meniere's disease. The clinical picture is characterized by episodic vestibular symptoms associated with migrainous symptoms, including migraine headache, sensory aura, sensorineural hearing loss, tinnitus and vertigo crisis. The authors of the present invention are the first to demonstrate a relationship between proinflammatory cytokine levels in an in vitro assay using peripheral blood mononuclear cells, which allows the vestibular migraine to be differentiated from Meniere's disease.

Therefore, a first aspect of the invention relates to the use of the proinflammatory cytokines ILip, IL6, TNFa or any combination thereof to obtain useful data for the diagnosis and classification of a disease that occurs with episodic vestibular syndrome. In a preferred embodiment, the proinflammatory cytokines ILip, IL6 and

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TNFa are used simultaneously. In another preferred embodiment, the determination of proinflammatory cytokines is performed in vitro in human mononuclear cells. In another even more preferred embodiment, the classification of the disease with vestibular syndrome allows obtaining useful data for the differential diagnosis between MS and MV, and more specifically, for the diagnosis of autoimmune MS.

MS may present with migrainous symptoms, including headache and MV may have sensorineural hearing loss, being indistinguishable in the early stages of the disease.

A second aspect of the invention relates to a method of obtaining useful data for the diagnosis and classification of a disease that develops with episodic vestibular syndrome, hereafter referred to as the first method of the invention, comprising:

a) detect the levels of ILin, IL6, TNFa proinflammatory cytokines, or any combination thereof, in an isolated biological sample of an individual.

b) compare the levels of proinflammatory cytokines detected in step (a), with baseline levels in a healthy individual.

In another preferred embodiment the sample isolated in step (a) is peripheral blood, and even more preferably they are mononuclear cells obtained from peripheral blood.

In another even more preferred embodiment, the classification of the disease with vestibular syndrome allows obtaining useful data for the differential diagnosis between MS and MV, and more specifically, for the diagnosis of autoimmune MS.

That is, step (b) consists in detecting the concentrations of ILip, IL6 or TNFa cytokines or any of their combinations. Preferably, all cytokines of the diagnostic panel of the invention are detected. Simultaneously it means that all are detected to carry out the method of the invention, although they can be detected in any order, without time limit.

An "isolated biological sample" includes, but is not limited to, cells, tissues and / or biological fluids of an organism, obtained by any method known to a person skilled in the art. Preferably, the biological sample isolated from step (a) is a peripheral blood sample.

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The isolated biological sample is peripheral blood, and / or comprises peripheral blood cells (PBCs), more preferably mononuclear cells. These cells are isolated, for example but not limited to, by density gradient separation using Ficoll, which is a density gradient medium based on the principle of differential migration of the blood cells through the media during the centrifugation stage of the process. Once isolated, these cells are incubated under controlled conditions, which make subsequent determinations reliable.

Basal concentrations of the cytokines ILip, IL6 and TNFa are obtained after maintaining the mononuclear cells for 16 hours in RPMI1640 medium supplemented at conditions of 37 ° C and 7% CO2.

IL-ip - Interleukin-ip is a member of the cytokine family 1. This is produced through macrophages activated as pro-protein, which by proteolytic processes is processed to its active form thanks to caspase 1 (CASP1). This protein is a very important mediator in the inflammatory response, and is involved in a wide variety of cellular activities, including cell proliferation, differentiation and apoptosis. The increase in the production of this cytokine is observed in different diseases such as autoimmune disease of the inner ear, Muckle Wells syndrome or systemic inflammatory disease of neonatal onset.

IL-6 - Interleukin-6 is a glycoprotein secreted by macrophages, T cells and endothelial cells to stimulate the immune response. It also helps in the maturation of B cells and is an antagonist of regulatory T cells. It is a protein with both inflammatory and anti-inflammatory capacity. This cytokine is first produced at sites of acute or chronic inflammation, where it is secreted to the plasma and induces an inflammatory response. It is an important mediator of fever and acute response. IL-6 stimulates inflammatory and autoimmune processes in various diseases, such as diabetes, lupus or rheumatoid arthritis.

TNFa - Tumor necrosis factor alpha is a proinflammatory cytokine that belongs to the tumor necrosis factor superfamily. It is mostly secreted by macrophages and is involved in a broad spectrum of biological processes that include lipid metabolism, coagulation, proliferation, differentiation and cell apoptosis. This cytokine is involved in a wide variety of diseases such as cancer, insulin resistance and

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autoimmune diseases (rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, psoriasis).

In the context of the present invention, IL-ip, IL-6 and TNFa are also defined, but not limited to, by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the sequences collected respectively in the SEQ IDs collected in the Table 3, and that would include various variants from:

a) nucleic acid molecules encoding said cytokines comprising the nucleotide sequence of SEQ ID listed in table 1,

b) nucleic acid molecules whose complementary hybrid chain with the polynucleotide sequence of a),

c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code,

d) nucleic acid molecules encoding said cytokines comprising the nucleotide sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with the SEQ IDs listed in Table 1, respectively, and in which the cytokines encoded by said nucleic acids possess the activity and structural characteristics of the cytokines IL-ip, IL-6 and TNFa. Among these nucleic acid molecules are those collected in the SEQ IDs indicated in the table.

Sequences of the invention:

SEQ ID NO: 1

GTGTCTGAAGCAGCCATGGCAGAAGTACCTGAGCTCGCCAGTGAAATGATGGCTTATTA

CAGTGGCAATGAGGATGACTTGTTCTTTGAAGCTGATGGCCCTAAACAGATGAAGTGCT

CCTTCCAGGACCTGGACCTCTGCCCTCTGGATGGCGGCATCCAGCTACGAATCTCCGA

CCACCACTACAGCAAGGGCTTCAGGCAGGCCGCGTCAGTTGTTGTGGCCATGGACAAG

CTGAGGAAGATGCTGGTTCCCTGCCCACAGACCTTCCAGGAGAATGACCTGAGCACCT

TCTTTCCCTTCATCTTTGAAGAAGGTAGTTAGCCAAGAGCAGGCAGTAGATCTCCACTTG

TGTCCTCTTGGAAGTCATCAAGCCCCAGCCAACTCAATTCCCCCAGAGCCAAAGCCCTT

TAAAGGTAGAAGGCCCAGCGGGGAGACAAAACAAAGAAGGCTGGAAACCAAAGCAATC

ATCTCTTTAGTGGAAACTATTCTTAAAGAAGATCTTGATGGCTACTGACATTTGCAACTC

CCTCACTCTTTCTCAGGGGCCTTTCACTTACATTGTCACCAGAGAACCTATCTTCTTCGA

CACATGGGATAACGAGGCTTATGTGCACGATGCACCTGTACGATCACTGAACTGCACGC

TCCGGGACTCACAGCAAAAAAGCTTGGTGATGTCTGGTCCATATGAACTGAAAGCTCTC

CACCTCCAGGGACAGGATATGGAGCAACAAGTGGTGTTCTCCATGTCCTTTGTACAAGG

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AGAAGAAAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACC

TGTCCTGCGTGTTGAAAGATGATAAGCCCACTCTACAGCTGGAGAGTGTAGATCCCAAA

AATTACCCAAAGAAGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGAAATCAATAAC

AAGCTGGAATTTGAGTCTGCCCAGTTCCCCAACTGGTACATCAGCACCTCTCAAGCAGA

AAACATGCCCGTCTTCCTGGGAGGGACCAAAGGCGGCCAGGATATAACTGACTTCACC

ATGCAATTTGTGTCTTCCTAAAGAGAGCTGTACCCAGAGAGTCCTGTGCTGAATGTGGA

CTCAATCCCTAGGGCTGGCAGAAAGGGAACAGAAAGGTTTTTGAGTACGGCTATAGCCT

GGACTTTCCTGTTGTCTACACCAATGCCCAACTGCCTGCCTTAGGGTAGTGCTAAGAGG

ATCTCCTGTCCATCAGCCAGGACAGTCAGCTCTCTCCTTTCAGGGCCAATCCCCAGCCC

TTTTGTTGAGCCAGGCCTCTCTCACCTCTCCTACTCACTTAAAGCCCGCCTGACAGAAA

CCACGGCCACATTTGGTTCTAAGAAACCCTCTGTCATTCGCTCCCACATTCTGATGAGC

AACCGCTTCCCTATTTATTTATTTATTTGTTTGTTTGTTTTATTCATTGGTCTAATTTATTCA

AAGGGGGCAAGAAGTAGCAGTGTCTGTAAAAGAGCCTAGTTTTTAATAGCTATGGAATC

AATTCAATTTGGACTGGTGTGCTCTCTTTAAATCAAGTCCTTTAATTAAGACTGAAAATAT

ATAAGCTCAGATTATTTAAATGGGAATATTTATAAATGAGCAAATATCATACTGTTCAATG

GTTCTGAAATAAACTTCACTGAAGAAAAAAAAA

SEQ ID NO: 2

MATDICNSLTLSQGPFTYIVTREPIFFDTWDNEAYVHDAPVRSLNCTLRDSQQKSLVMSGPY

ELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESV

DPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFT

MQFVSS

SEQ ID NO: 3

ACCAAACCTCTTCGAGGCACAAGGCACAACAGGCTGCTCTGGGATTCTCTTCAGCCAAT

CTTCATTGCTCAAGTGTCTGAAGCAGCCATGGCAGAAGTACCTGAGCTCGCCAGTGAAA

TGATGGCTTATTACAGTGGCAATGAGGATGACTTGTTCTTTGAAGCTGATGGCCCTAAA

CAGATGAAGTGCTCCTTCCAGGACCTGGACCTCTGCCCTCTGGATGGCGGCATCCAGC

TACGAATCTCCGACCACCACTACAGCAAGGGCTTCAGGCAGGCCGCGTCAGTTGTTGT

GGCCATGGACAAGCTGAGGAAGATGCTGGTTCCCTGCCCACAGACCTTCCAGGAGAAT

GACCTGAGCACCTTCTTTCCCTTCATCTTTGAAGAAGAACCTATCTTCTTCGACACATGG

GATAACGAGGCTTATGTGCACGATGCACCTGTACGATCACTGAACTGCACGCTCCGGG

ACTCACAGCAAAAAAGCTTGGTGATGTCTGGTCCATATGAACTGAAAGCTCTCCACCTC

CAGGGACAGGATATGGAGCAACAAGTGGTGTTCTCCATGTCCTTTGTACAAGGAGAAGA

AAGTAATGACAAAATACCTGTGGCCTTGGGCCTCAAGGAAAAGAATCTGTACCTGTCCT

GCGTGTTGAAAGATGATAAGCCCACTCTACAGCTGGAGAGTGTAGATCCCAAAAATTAC

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CCAAAGAAGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGAAATCAATAACAAGCT

GGAATTTGAGTCTGCCCAGTTCCCCAACTGGTACATCAGCACCTCTCAAGCAGAAAACA

TGCCCGTCTTCCTGGGAGGGACCAAAGGCGGCCAGGATATAACTGACTTCACCATGCA

ATTTGTGTCTTCCTAAAGAGAGCTGTACCCAGAGAGTCCTGTGCTGAATGTGGACTCAA

TCCCTAGGGCTGGCAGAAAGGGAACAGAAAGGTTTTTGAGTACGGCTATAGCCTGGAC

TTTCCTGTTGTCTACACCAATGCCCAACTGCCTGCCTTAGGGTAGTGCTAAGAGGATCT

CCTGTCCATCAGCCAGGACAGTCAGCTCTCTCCTTTCAGGGCCAATCCCCAGCCCTTTT

GTTGAGCCAGGCCTCTCTCACCTCTCCTACTCACTTAAAGCCCGCCTGACAGAAACCAC

GGCCACATTTGGTTCTAAGAAACCCTCTGTCATTCGCTCCCACATTCTGATGAGCAACC

GCTTCCCTATTTATTTATTTATTTGTTTGTTTGTTTTATTCATTGGTCTAATTTATTCAAAG

GGGGCAAGAAGTAGCAGTGTCTGTAAAAGAGCCTAGTTTTTAATAGCTATGGAATCAATT

CAATTTGGACTGGTGTGCTCTCTTTAAATCAAGTCCTTTAATTAAGACTGAAAATATATAA

GCTCAGATTATTTAAATGGGAATATTTATAAATGAGCAAATATCATACTGTTCAATGGTTC

TGAAATAAACTTCACTGAAG

SEQ ID NO: 4

MAEVPELASEMMAYYSGNEDDLFFEADGPKQMKCSFQDLDLCPLDGGIQLRISDHHYSKG

FRQAASVVVAMDKLRKMLVPCPQTFQENDLSTFFPFIFEEEPIFFDTWDNEAYVHDAPVRSL

NCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKN

LYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAE

NMPVFLGGTKGGQDITDFTMQFVSS

SEQ ID NO: 5

GTATTGTGTCACTCAGTTCAAGTACTTGAAATTTATTGAATTGTATTTTCTAAAAAATAGAT

AGTTGAGTAAAAGCAAGCTCACATTACATAGACGGATCACAGTGCACGGCTGCGGAGCT

GGGAGCAGTGGCTTCGTTTCATGCAGGAAAGAGAACTTGGTTCAGGAGTGTCTACGTT

GCTTAAGACAGGAGAGCACTAAAAATGAAACCATCCAGCCATCCTCCCCCATTTTCATTT

TCACACCAAAGAATCCCACCGCGGCAGAGGACCACCGTCTCTGTTTAGACAATCGGTG

AAGAATGGATGACCTCACTTTCCCCAACAGGCGGGTCCTGAAATGTTATGCACGAAACA

AAACTTGAGTAAATGCCCAACAGAGGTCACTGTTTTATCGATCTTGAAGAGATCTCTTCT

TAGCAAAGCAAAGAAACCGATTGTGAAGGTAACACCATGTTTGGTAAATAAGTGTTTTGG

TGTTGTGCAAGGGTCTGGTTTCAGCCTGAAGCCATCTCAGAGCTGTCTGGGTCTCTGGA

GACTGGAGGGACAACCTAGTCTAGAGCCCATTTGCATGAGACCAAGGATCCTCCTGCA

AGAGACACCATCCTGAGGGAAGAGGGCTTCTGAACCAGCTTGACCCAATAAGAAATTCT

TGGGTGCCGACGCGGAAGCAGATTCAGAGCCTAGAGCCGTGCCTGCGTCCGTAGTTTC

CTTCTAGCTTCTTTTGATTTCAAATCAAGACTTACAGGGAGAGGGAGCGATAAACACAAA

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CTCTGCAAGATGCCACAAGGTCCTCCTTTGACATCCCCAACAAAGAGGACTGGAGATGT

CTGAGGCTCATTCTGCCCTCGAGCCCACCGGGAACGAAAGAGAAGCTCTATCTCCCCT

CCAGGAGCCCAGCTATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCC

CTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGTACCCCCAGGAGAAG

ATTCCAAAGATGTAGCCGCCCCACACAGACAGCCACTCACCTCTTCAGAACGAATTGAC

AAACAAATTCGGTACATCCTCGACGGCATCTCAGCCCTGAGAAAGGAGACATGTAACAA

GAGTAACATGTGTGAAAGCAGCAAAGAGGCACTGGCAGAAAACAACCTGAACCTTCCAA

AGATGGCTGAAAAAGATGGATGCTTCCAATCTGGATTCAATGAGGAGACTTGCCTGGTG

AAAATCATCACTGGTCTTTTGGAGTTTGAGGTATACCTAGAGTACCTCCAGAACAGATTT

GAGAGTAGTGAGGAACAAGCCAGAGCTGTGCAGATGAGTACAAAAGTCCTGATCCAGT

TCCTGCAGAAAAAGGCAAAGAATCTAGATGCAATAACCACCCCTGACCCAACCACAAAT

GCCAGCCTGCTGACGAAGCTGCAGGCACAGAACCAGTGGCTGCAGGACATGACAACTC

ATCTCATTCTGCGCAGCTTTAAGGAGTTCCTGCAGTCCAGCCTGAGGGCTCTTCGGCAA

ATGTAGCATGGGCACCTCAGATTGTTGTTGTTAATGGGCATTCCTTCTTCTGGTCAGAAA

CCTGTCCACTGGGCACAGAACTTATGTTGTTCTCTATGGAGAACTAAAAGTATGAGCGTT

AGGACACTATTTTAATTATTTTTAATTTATTAATATTTAAATATGTGAAGCTGAGTTAATTT

ATGTAAGTCATATTTATATTTTTAAGAAGTACCACTTGAAACATTTTATGTATTAGTTTTGA

AATAATAATGGAAAGTGGCTATGCAGTTTGAATATCCTTTGTTTCAGAGCCAGATCATTT

CTTGGAAAGTGTAGGCTTACCTCAAATAAATGGCTAACTTATACATATTTTTAAAGAAATA

TTTATATTGTATTTATATAATGTATAAATGGTTTTTATACCAATAAATGGCATTTTAAAAAAT

TCAGCAA

SEQ ID NO: 6

MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDG

ISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYL

EYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQD

MTTHLILRSFKEFLQSSLRALRQM

SEQ ID NO: 7

CCCGCTCTGGCCCCACCCTCACCCTCCAACAAAGATTTATCAAATGTGGGATTTTCCCA

TGAGTCTCAATATTAGAGTCTCAACCCCCAATAAATATAGGACTGGAGATGTCTGAGGCT

CATTCTGCCCTCGAGCCCACCGGGAACGAAAGAGAAGCTCTATCTCCCCTCCAGGAGC

CCAGCTATGAACTCCTTCTCCACAAGTAAGTGCAGGAAATCCTTAGCCCTGGAACTGCC

AGCGGCGGTCGAGCCCTGTGTGAGGGAGGGGTGTGTGGCCCAGGGAGGGCTGGCGG

GCGGCCAGCAGCAGAGGCAGGCTCCCAGCTGTGCTGTCAGCTCACCCCTGCGCTCGC

TCCCCTCCGGCACAGGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGGCTGCTCCTGGT

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GTTGCCTGCTGCCTTCCCTGCCCCAGTACCCCCAGGAGAAGATTCCAAAGATGTAGCC

GCCCCACACAGACAGCCACTCACCTCTTCAGAACGAATTGACAAACAAATTCGGTACAT

CCTCGACGGCATCTCAGCCCTGAGAAAGGAGACATGTAACAAGAGTAACATGTGTGAAA

GCAGCAAAGAGGCACTGGCAGAAAACAACCTGAACCTTCCAAAGATGGCTGAAAAAGA

TGGATGCTTCCAATCTGGATTCAATGAGGAGACTTGCCTGGTGAAAATCATCACTGGTC

TTTTGGAGTTTGAGGTATACCTAGAGTACCTCCAGAACAGATTTGAGAGTAGTGAGGAA

CAAGCCAGAGCTGTGCAGATGAGTACAAAAGTCCTGATCCAGTTCCTGCAGAAAAAGGT

GGGTGTGTCCTCATTCCCTCAACTTGGTGTGGGGGAAGACAGGCTCAAAGACAGTGTC

CTGGACAACTCAGGGATGCAATGCCACTTCCAAAAGAGAAGGCTACACGTAAACAAAAG

AGTCTGAGAAATAGTTTCTGATTGTTATTGTTAAATCTTTTTTTGTTTGTTTGGTTGGTTG

GCTCTCTTCTGCAAAGGACATCAATAACTGTATTTTAAACTATATATTAACTGAGGTGGAT

TTTAACATCAATTTTTAATAGTGCAAGAGATTTAAAACCAAAGGCGGGGGGGCGGGCAG

AAAAAAGTGCATCCAACTCCAGCCAGTGATCCACAGAAACAAAGACCAAGGAGCACAAA

ATGATTTTAAGATTTTAGTCATTGCCAAGTGACATTCTTCTCACTGTGGTTGTTTCAATTC

TTTTTCCTACCTTTTACCAGAGAGTTAGTTCAGAGAAATGGTCAGAGACTCAAGGGTGGA

AAGAGGTACCAAAGGCTTTGGCCACCAGTAGCTGGCTATTCAGACAGCAGGGAGTAGA

CTTGCTGGCTAGCATGTGGAGGAGCCAAAGCTCAATAAGAAGGGGCCTAGAATGAAAC

CCTTGGTGCTGATCCTGCCTCTGCCATTTCTACTTAAGCCAGGGTTTCTCATATGTTAAC

ATGCATGGGAATTCCCTGGGCATCTTCTTGTGGTGTGGAGTCTGACTTAGCAAGCCTCG

GGTGGGTTTGAGGGTCAAATTTCTACCAGGCTTATATCCCTGGTGATGCTGCAGAATTC

CAGGACCACACTTGGAGGTTTAAGGCCTTCCACAAGTTACTTATCCCATATGGTGGGTC

TATGGAAAGGTGTTTCCCAGTCCTCTTTACACCACCGGATCAGTGGTCTTTCAACAGATC

CTAAAGGGATGGTGAGAGGGAAACTGGAGAAAAGTATCAGATTTAGAGGCCACTGAAG

AACCCATATTAAAATGCCTTTAAGTATGGGCTCTTCATTCATATACTAAATATGAACTATG

TGCCAGGCATTATTTCATATGACAGAATACAAACAAATAAGATAGTGATGCTGGTCAGGC

TTGGTGGCTCATGCCTGTATTCCCTAAACTTTGGGAGCCTAAGGTGAGAACTCCTTGAA

CTCCTAAGGCCAGGAGTTCAAGACCAGCCTGGATAACATAGCAAGACCCCATCTCTACA

AAAAACCAAAACCAAACAAACAAAAATGATAGTGGTGCTTCCCTCAGGATGCTTGTGGT

CTAATGGGAGACAGAACAGCAAAGGGATGATTAGAAGTTGGTTGCTGTGAGCCAGGCA

CAGTGCTGATATAATCCCAGCGCTATGGGAGGCTGAGGTGGGTGGATCATTTGAGGCC

AGGAGTTTAAGACCAGCCTGGTCAACATGGTAAAACCCCATCTCTACTTAAAAATACAAA

AAAGTTAGCCAGGCATGGTGGCATACACCTGTAACCCAGCTACTCAGGAGGCTGAGGC

ACATGAATCACTTGAACCCAGGAGGCAGAGGTTGCTGTGCACCACTGCACTCCAGCCT

GGGTGACAGAACGAGACCTTGACTCAAAAAAAAAAAAAAGAAGTTTGTTGCTATGGAAG

GGTCCTACTCAGAGCAGGCACCCCAGTTAATCTCATTCACCCCACATTTCACATTTGAAC

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AAGCTGAGGGGCAGCTCCAGTGGCTGAACCGCCGGGCCAATGCCCTCCTGGCCAATG

GCGTGGAGCTGAGAGATAACCAGCTGGTGGTGCCATCAGAGGGCCTGTACCTCATCTA

CTCCCAGGTCCTCTTCAAGGGCCAAGGCTGCCCCTCCACCCATGTGCTCCTCACCCAC

ACCATCAGCCGCATCGCCGTCTCCTACCAGACCAAGGTCAACCTCCTCTCTGCCATCAA

GAGCCCCTGCCAGAGGGAGACCCCAGAGGGGGCTGAGGCCAAGCCCTGGTATGAGCC

CATCTATCTGGGAGGGGTCTTCCAGCTGGAGAAGGGTGACCGACTCAGCGCTGAGATC

AATCGGCCCGACTATCTCGACTTTGCCGAGTCTGGGCAGGTCTACTTTGGGATCATTGC

CCTGTGAGGAGGACGAACATCCAACCTTCCCAAACGCCTCCCCTGCCCCAATCCCTTTA

TTACCCCCTCCTTCAGACACCCTCAACCTCTTCTGGCTCAAAAAGAGAATTGGGGGCTT

AGGGTCGGAACCCAAGCTTAGAACTTTAAGCAACAAGACCACACACTACTGAGAACCTGGGA

TTCAGGAATGTGTGGCCTGCACAGTGAAGTGCTGGCAACCACTAAGAATTCAAACTGGG

GCCTCCAGAACTCACTGGGGCCTACAGCTTTGATCCCTGACATCTGGAATCTGGAGACC

AGGGAGCCTTTGGTTCTGGCCAGAATGCTGCAGGACTTGAGAAGACCTCACCTAGAAAT

TGACACAAGTGGACCTTAGGCCTTCCTCTCTCCAGATGTTTCCAGACTTCCTTGAGACA

CGGAGCCCAGCCCTCCCCATGGAGCCAGCTCCCTCTATTTATGTTTGCACTTGTGATTA

TTTATTATTTATTTATTATTTATTTATTTACAGATGAATGTATTTATTTGGGAGACCGGGGT

ATCCTGGGGGACCCAATGTAGGAGCTGCCTTGGCTCAGACATGTTTTCCGTGAAAACG

GAGCTGAACAATAGGCTGTTCCCATGTAGCCCCCTGGCCTCTGTGCCTTCTTTTGATTA

TGTTTTTTAAAATATTTATCTGATTAAGTTGTCTAAACAATGCTGATTTGGTGACCAACTG

TCACTCATTGCTGAGCCTCTGCTCCCCAGGGGAGTTGTGTCTGTAATCGCCCTACTATT

CAGTGGCGAGAAATAAAGTTTGCTTAGAAAAGAAAAAAAAAAAAA

SEQ ID NO: 14

MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEF

PRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQL

VVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEA

KPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

Table 3. Sequences of the invention

 Gene Name

 Description Gen ID Nucleotide sequences Amino acid sequences

 IL1B

 Interleukin 1 beta 3553 SEQ ID NO: 1 or SEQ ID NO: 3 SEQ ID NO: 2 or SEQ ID NO: 4

 IL6

 Interleukin 6 3569 SEQ ID NO: SEQ ID NO: 6,

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 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12

 TNF alpha

 Tumor necrosis factor 7124 SEQ ID NO: 13 SEQ ID NO: 14

In another preferred embodiment of this aspect of the invention, the disease that occurs with the course of vestibular syndrome is Meniere's disease (MS) or autoimmune disease of the inner ear.

In this report it is understood as a disease that presents with episodic vestibular syndrome that in which the patient presents recurrent signs and symptoms that indicate an alteration of the vestibular function, such as vertigo, ataxia, imbalance, nystagmus or oscilopsia. These symptoms may be associated with auditory symptoms or headache of various origin.

In this report, Méniere's disease is understood as the syndrome characterized by uni or bilateral neurosensory hearing loss that affects low and medium frequencies (<2000Hz) and associated with episodes of vertigo and auditory symptoms, such as tinnitus, or otic fullness.

In this report it is understood as autoimmune disease of the inner ear or autoimmune MS to the syndrome characterized by uni or bilateral sensorineural hearing loss greater than 30dB that affects one or more frequencies and that progresses in a period between 3-90 days. The progression of hearing loss should be greater than 15dB for a frequency or 10dB for 2 frequencies and demonstrated by audiometry. Autoimmune disease of the inner ear is associated with vestibular symptoms in 50% of cases. The onset of sudden hearing loss (<3 days) with an autoimmune risk profile should make autoimmune disease of the inner ear suspect.

As shown in the examples of the present invention, proinflammatory cytokine levels are elevated in a subset of patients with MS (Table 1).

Table 1. Proinflammatory cytokine levels used for the diagnosis of autoimmune Meniere's disease.

 CONTROLS CASES <5 CASES> 5 Cases <5 vs Controls Cases> 5 vs Controls

 N Media DS N Media DS N Media DS P-value Reason P-value Reason

 IL1BCN

 50 2.30 1.92 42 1.31 1.04 18 26.38 14.06 6.45E-03 0.57 6.71E-10 11.49

 IL1BIII

 44 1286.38 846.28 41 1780.66 1898.96 19 1961.27 1896.66 6.04E-01 1.38 2.03E-01 1.52

 IL1BIV

 44 1193.97 895.35 41 1940.82 2677.93 19 1562.28 2365.90 6.74E-02 1.63 7.31 E-01 1.31

 IL6CN

 50 4.82 6.10 42 4.66 5.11 19 130.72 118.10 7.96E-01 0.97 3.69E-09 11.79

 IL6III

 44 3400.20 1917.44 41 5673.73 3701.98 19 6629.20 2604.09 9.97E-03 1.67 1.61E-05 1.26

 IL6IV

 44 3318.11 1888.07 41 5685.69 3592.94 19 6098.16 2284.40 5.16E-03 1.71 7.23E-05 1.47

 TNFaCN

 50 5.32 6.35 42 7.11 5.92 19 47.69 24.54 2.16E-02 1.34 5.91E-10 27.14

 TNFalll

 44 817.55 393.75 41 2241.68 2541.29 19 3536.68 3208.61 7.91 E-06 2.74 3.57E-05 1.95

 TNFalV

 44 947.15 693.52 41 2583.56 2887.26 19 2534.08 2722.64 1 24E-05 2.73 1.10E-03 1.84

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Table 2. Cytokine levels in the in vitro assay with mononuclear cells of patients with vestibular migraine and controls

CASE CONTROLS

 N Media DS N Media DS P-value Reason

 IL1B CN

 50 2,297 1,919 28 1,372 2,226 7.09E-05 0.597

 IL1B III

 44 1286.383 846.279 28 1582.245 2011.046 5.64E-01 1.230

 IL1B IV

 44 1193.972 895.353 28 2157.995 5246.685 8.90E-01 1.807

 IL6 CN

 50 4,816 6,096 28 4,847 5,641 9.42E-01 1,006

 IL6 III

 44 3400.200 1917.436 28 7949.572 8101.327 6.87E-03 2.388

 IL6 IV

 44 3318.110 1888.066 28 6603.886 4726.272 1.32E-03 1.990

 TNFa CN

 50 5,322 6,349 28 5,874 3,911 5,6E-02 1,104

 TNFa III

 44 817.553 393.755 28 1315.375 1310.003 1.73E-01 1.609

 TNFa IV

 44 947.145 693.524 28 1163.634 713.176 5.37E-02 1.229

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Therefore, a third aspect of the invention relates to a method for the diagnosis and classification of a disease that develops with episodic vestibular syndrome, hereafter referred to as the second method of the invention, comprising steps (a) and (b) of the first method of the invention, and further comprises:

15 c) include the individual in the group of individuals with MS of autoimmune origin.

In a preferred embodiment of this aspect the invention, the sample isolated in step (a) is peripheral blood, and even more preferably they are mononuclear cells obtained from peripheral blood.

The term "individual" or "subject", as used in the description, refers to an animal, preferably a mammal, and more preferably a human being. The term "individual" herein is synonymous with "patient", and is not intended to be limiting in

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no aspect, this being of any age, sex and physical condition. The individual can be anyone, an individual predisposed to a disease (for example, lung cancer) or an individual suffering from said disease.

The cytokine determination of the invention can be done by any method known in the state of the art, for example, but not limited to, by techniques based on the antigen-antibody interaction such as immunoassays (radioimmunoassay, enzyme immunoassay, fluoroimmunoassay, immunochemiluminescent assay), western blot, immunoprecipitation, immunohistochemistry or immunofluorescence, or by means of quantification techniques of genetic material (RNA) such as PCR, real-time PCR, or expression assays.

The term "diagnosis", as used in the present invention, at the greater or lesser risk of suffering from Meniere's disease or at the greater or lesser risk of suffering from the autoimmune disease of the inner ear.

In turn, according to the method of the present invention, other subclassifications could be established within this principal, thus facilitating the choice and establishment of appropriate therapeutic regimens. This discrimination, as understood by one skilled in the art, is not intended to be correct in 100% of the samples analyzed. However, it requires that a statistically significant amount of the analyzed samples be classified correctly. The amount that is statistically significant can be established by a person skilled in the art by using different statistical tools, for example, but not limited, by determining confidence intervals, determining the significance value P, Student test or discriminant functions. Fisher, non-parametric measurements of Mann Whitney, Spearman correlation, logistic regression, linear regression, area under the ROC curve (AUC). Preferably, the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. Preferably, the value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001. Preferably, the present invention makes it possible to correctly detect the predisposition to the disease or disease differentially by at least 60%, more preferably at least 70%, much more preferably at least 80%, or even much more preferably in at least 90% of the subjects of a certain group or population analyzed.

Steps (a), (b), and of the methods described above may be wholly or partially

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automated, for example, by means of a robotic sensor device for the quantification of step (a) or computerized calculation of any of the indices of steps (b), (c) and / or (d), or computerized classification in the steps

MEDICAL USES

A fourth aspect of the invention relates to the use of an immunosuppressant for the treatment of an individual from the group of individuals with MS of autoimmune origin identified according to the second method of the invention. In a preferred embodiment of this aspect the immunosuppressants are selected from the list consisting of: cytostatic agents, glucocorticoids, calcineurin enzyme inhibitors, mTOR inhibitors, antibodies, or any combination thereof.

Even more preferably, cytostatic agents are selected from among Azathioprine, nucleoside synthesis inhibitors such as, but not limited to, mycophenolate mofetil and mycophenolic acid, cyclophosphamide, methotrexate, or any combination thereof.

In another preferred embodiment, the glucocorticoids are selected from prednisone, fludrocortisone, triamcinolone, betamethasone, or any combination thereof.

In another preferred embodiment, the calcineurin enzyme inhibitors are selected from cyclosporine, tacrolimus, or their combination.

In another preferred embodiment, mTOR inhibitors are selected from Sirolimus, Everolimus, or their combination.

In another preferred embodiment the antibodies are selected from: anti-CD20, Rituximab; anti-IL-2R, Daclizumab, anti-TNF, Remicade, anti-SR-TNF, Enbrel, Anti-chemokines, or any combination thereof.

The different treatments of the diseases that occur with episodic vestibular syndrome are described below.

Preventive treatment of vestibular migraine attacks

1. Anticonvulsants (Valproate, topiramate)

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2. Antidepressants (amitriptyline, nortriptyline, venlafaxine)

3. Beta-blockers (propranolol, metoprolol, atenolol)

4. Calcium antagonists (flunarizine)

5. Carbonic anhydrase inhibitor: acetazolamide

The treatment is personalized based on other associated pathologies.

Preventive treatment of Meniere's disease crises

1. Diet without salt and water overload (> 2000ml water / day)

2. Betahistine

3. Diuretics

4. Inttimimpanic Gentamicin

5. Intrathymic corticosteroids

6. Surgery (vestibular neurectomy, labytectomy according to auditory function)

Treatment of autoimmune Meniere's disease and / or autoimmune inner ear disease

1. Glucocorticoids (prednisone, methyl-prednisolone)

These are all potential treatments, which could be applied in CD:

2. Immunosuppressants such as cytostatics (cyclophosphamide), folic acid analogs (methotrexate), purine analogs (azathioprine), immunophilin regulatory drugs (cyclosporine, tacrolimus)

3. Monoclonal antibodies against interleukins or their receptors such as

3.1. AntiTNFalfa: infliximab, etanercept, adalimumab.

3.2. Anti IL-1beta receptor: Anakinra (this drug is already patented for the treatment of autoimmune disease of the inner ear, but not for the Autoimmune Meniere).

3.3. Anti IL-6: Tolizizumab, siltuximab, sarilumab, olokizumab.

3.4. Anti TWEAK / FN14: BIIB023 (BIOGEN)

KIT OR DEVICE OF THE INVENTION

A fifth aspect of the invention relates to a kit or device for the diagnosis and classification of a disease that occurs with episodic vestibular syndrome, hereafter referred to as the kit or device of the invention, comprising the elements necessary to detect the levels of Proinflammatory cytokines ILip, IL6 and TNFa in an isolated biological sample of an individual. In a preferred embodiment of this aspect the invention, the isolated sample is peripheral blood, and even more preferably they are cells

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mononuclear obtained from peripheral blood. In another preferred embodiment of this aspect, the kit or device of the invention comprises primers, probes and / or antibodies capable of detecting and quantifying the proinflammatory cytokines ILip, IL6 and TNFa in an isolated biological sample. Even more preferably, the detection of proinflammatory cytokine levels is performed by immunological techniques, with antibodies, and even more preferably by ELISA. In another preferred embodiment of this aspect of the invention, the antibodies are modified. In another preferred embodiment of this aspect of the invention, the antibody is human, humanized or synthetic. In another preferred embodiment, the antibody is monoclonal and / or is labeled with a fluorochrome. Preferably, the fluorochrome is selected from the list comprising Fluorescein (FITC), Tetramethylrodamine and derivatives, Phycoerythrin (PE), PerCP, Cy5, Texas, allophycocyanin, or any combination thereof.

Therefore, preferably, the antibodies of the kit of the invention are conjugated to molecules that emit detectable signals such as a radioactive isotope, an enzyme, a fluorescent particle or a chemiluminescent substance or assays that are measured by light scattering or direct visualization such as they are precipitation, agglutination or radial diffusion.

Said kit or device may contain all those reagents necessary to determine the cytokines of the invention by means of any of the methods existing in the state of the art and / or described herein. The kit can also include, without any limitation, buffers, agents to prevent contamination, inhibitors of protein degradation, etc. On the other hand, the kit can include all the supports and containers necessary for commissioning and optimization. Preferably, the kit further comprises instructions for carrying out any of the methods of the invention.

It is also possible that the antibody (s) are immobilized in spots on a (preferably solid) surface. In one of its embodiments, the kit comprises a microarray, or microarray of the invention. An antibody microarray is a matrix on a solid substrate (usually a glass holder or a thin silicon film cell). Detection is performed colorimetrically by the interaction of a chromogenic substrate and an enzyme that has been coupled to a detector antibody. Each stain contains the antibody (s). Although the number of spots is not limited in any way, there is a preferred embodiment in which the matrix is customized for

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methods of the invention. In one embodiment, said custom matrix comprises fifty spots or less, such as thirty spots or less, including twenty spots or less. Therefore, another aspect of the invention relates to a matrix comprising the antibody (s) of the invention.

Synthesis in situ on a solid support (for example, glass) could be done using ink-jet technology, which requires longer probes. The supports could be, but not limited to, filters or membranes of NC or nylon (charged), silicon, or glass slides for microscopes covered with aminosilanes, polylysine, aldehydes or epoxy.

In another preferred embodiment of this aspect of the invention, the kit or device of the invention is a kit of parts, comprising a component A, formed by a device for collecting the sample from step a), and a component B, formed by the elements necessary to carry out the quantitative analysis in the sample from step a) or any of the methods of the invention.

A sixth aspect of the invention relates to the use of the kit of the invention, for the diagnosis and classification of a disease that occurs with episodic vestibular syndrome. In a preferred embodiment of this aspect of the invention, the disease that occurs with vestibular syndrome is MS or MV, being used for the differential diagnosis of MS and MV, and more specifically, for the diagnosis of autoimmune MS.

The invention also extends to computer programs adapted so that any processing means can implement the methods of the invention. Such programs may have the form of source code, object code, an intermediate source of code and object code, for example, as in partially compiled form, or in any other form suitable for use in the implementation of the processes according to the invention . Computer programs also cover cloud applications based on that procedure.

Therefore, a seventh aspect of the invention relates to a computer program comprising program instructions to make a computer carry out the method according to any of the methods of the invention.

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In particular, the invention encompasses computer programs arranged on or within a carrier. The carrier can be any entity or device capable of supporting the program. When the program is incorporated into a signal that can be directly transported by a cable or other device or medium, the carrier may be constituted by said cable or other device or means. As a variant, the carrier could be an integrated circuit in which the program is included, the integrated circuit being adapted to execute, or to be used in the execution of, the corresponding processes.

For example, the programs could be incorporated into a storage medium, such as a ROM, a CD ROM or a semiconductor ROM, a USB memory, or a magnetic recording medium, for example, a floppy disk or a disk Lasted. Alternatively, the programs could be supported on a transmissible carrier signal. For example, it could be an electrical or optical signal that could be transported through an electrical or optical cable, by radio or by any other means.

An eighth aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of having a computer perform the steps of any of the methods of the invention.

A ninth aspect of the invention relates to a transmissible signal comprising program instructions capable of having a computer perform the steps of any of the methods of the invention.

The terms "polynucleotide" and "nucleic acid" are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) and deoxyribonucleotides (DNA or DNA).

The terms "amino acid sequence", "peptide", "oligopeptide", "polypeptide" and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which may be coding or non-coding, Chemically or biochemically modified.

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 characteristics of the invention are

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they will come partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLES OF THE INVENTION

The invention will now be illustrated by tests carried out by the inventors.

Materials and Methods Subjects and samples

The present study included a total of 64 patients with Meniere's disease and 29 patients with vestibular migraine. Patients were included in 6 centers: Granada University Hospital, Poniente de El Ejido Hospital, Almería, San Agustín de Linares Hospital, Getafe University Hospital, Santiago de Compostela Clinical Hospital, Salamanca University Hospital, Lugo Hospital, between November from 2014 and March 2016. Peripheral blood samples were obtained for the separation of mononuclear cells and the determination of cytokines after the in vitro assay. These samples were obtained in patients who had not had vertigo crisis at present, nor had they presented it at least 30 days before the date of obtaining the sample. Patients were diagnosed using the diagnostic scale of the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) (Committee on Hearing and Equilibrium guidelines for the diagnosis and evaluation of therapy in Meniere's disease. American Academy of Otolaryngology-Head and Neck Foundation, Inc. Otolaryngol Head Neck Surg. 1995. 113, 181-185).

A total of 54 samples of Spanish volunteers from the cities of Almería, Granada, Salamanca, Jaén, Lugo and Santiago de Compostela were included as healthy controls. Quality controls were used on the samples of the individuals to exclude those that did not exceed the quality filters (elimination of duplicate samples, extreme values).

A basic neurotological examination including pure tone audiometry, nystagmus in the primary position, nystagmus evoked by the gaze, nystagmus of cephalic agitation and standard caloric test was performed in all patients. The auditory stage for each patient

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With definitive MS, it is defined as the average of the four-tone average of 0.5, 1, 2 and 3 kHz according to the AAO-HNS criteria: stage 1, <25 dB; stage 2, 26-40dB, stage 3, 41-70dB, and stage 4,> 70 dB. The diagnosis of migraine was made using the criteria of the International Headache Society and the diagnosis of vestibular migraine using the criteria of the Barany Society (2012).

The study was carried out in accordance with the principles of the Helsinki Declaration for research with human beings. The hospital research committee approved the research protocol. Peripheral blood mononuclear cells were isolated by Ficoll gradient. All patients and healthy individuals gave their informed consent for the study.

In vitro test of mononuclear cells and measurement of cytokines in supernatant using Multiplex plates.

Peripheral blood mononuclear cells were isolated by Ficoll density gradients and cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, pyruvate and essential amino acids, plated at 1x106 cells / ml in 12-well plates and incubated 16h to 37 ° C at 7% CO2. At the end of the incubation the cells were centrifuged, the supernatant was stored and the RNA was extracted from the cells.

Multiplex analysis

The levels of the cytokines IL-1p, IL-6, and TNFa were quantified simultaneously by an immunoassay with magnetic beads (EMD Millipore, Billerica, MA, USA) using a Luminex 2000 reader (Luminex Corp., Austin, TX, USA) and analyzed with Luminex x PONENT 3.1 software (Luminex Corp., Austin, TX, USA).

ELISAS

The levels of IL-1P and Il-6 were determined in supernatant by a sandwich ELISA (R&D Systems, Minneapolis, MN, USA) as described in the manufacturer's protocol. All samples were made in duplicate to ensure reproducibility.

The results obtained by Multiplex and ELISA were compared for validation.

Statistic analysis.

A descriptive statistical analysis was done using the SPSS v.22 program (SPSS Inc, Chicago, IL, USA). The variables were compared using the T-student test.

5 A cluster analysis was carried out using the "gplots" package with the Heatmap.2 function of the R studio program.

Results

10 Two subgroups of patients were observed according to baseline levels of proinflammatory cytokines. Forty-two patients (68%) had low cytokine levels (IL-1p = 1.55 ± 1.53; IL-6 = 4.62 ± 4.99 and TNFa = 7.53 ± 6.17) while 20 patients (32%) exhibited elevated baseline levels of cytokines (IL-1p = 28.86 ± 12.84; IL-6 = 153.26 ± 115.26 and TNFa = 47.22 ± 16.01).

15 Individuals with vestibular migraine ((IL-1 p = 1.4 ± 2.2; IL-6 = 4.8 ± 5.6 and TNFa = 5.9 ± 3.9) had cytokine levels very similar to that of healthy controls ((IL-1p = 2.3 ± 1.92; IL-6 = 4.8 ± 6.1 and TNFa = 5.32 ± 6.35).

The diagnostic profitability of IL-1p was assessed to discriminate between MV and MS, obtaining a sensitivity of 35%, a specificity of 89%, a positive predictive value of 88% and a negative predictive value of 65%.

Claims (10)

5 10 fifteen twenty 25 30 35 1. A method of obtaining useful data for the diagnosis and classification of a disease that occurs with episodic vestibular syndrome, comprising: a) detect the levels of IL1p, IL6 and TNFa proinflammatory cytokines in an isolated biological sample from an individual, and b) compare the levels of proinflammatory cytokines detected in step (a), with baseline levels in a healthy individual. 2. A method for the diagnosis and classification of a disease that develops with episodic vestibular syndrome comprises steps (a) and (b) according to the preceding claim, and further comprises: c) include the individual in the group of individuals with MS of autoimmune origin. 3. The method according to any of claims 1-2, wherein the sample isolated in step (a) is peripheral blood. 4. The method according to any of claims 1-3, wherein the sample isolated in step (a) are mononuclear cells obtained from peripheral blood. 5. The method according to any of claims 1-4, wherein the cytokine determination of the invention is performed by techniques based on the antigen-antibody interaction. 6. The method according to any of claims 1-5, wherein the cytokine determination of the invention is performed by immunoassay, western blotting, immunoprecipitation, immunohistochemistry or immunofluorescence. 7. The use of an immunosuppressant in the preparation of a medicament for the treatment of an individual from the group of individuals with MS of autoimmune origin identified by the diagnostic method according to any of claims 1-6. 8. A kit or device for the diagnosis and classification of a disease that develops with episodic vestibular syndrome comprises antibodies capable of detecting and quantify the proinflammatory cytokines IL1p, IL6 and TNFa in an isolated biological sample, preferably peripheral blood, and even more preferably, peripheral blood mononuclear cells. 9. The use of a kit or device according to the previous reinvindication, for the diagnosis and classification of a disease that occurs with episodic vestibular syndrome. 10. The use of a kit or device according to reinvindication 9, where the disease that occurs with episodic vestibular syndrome is MS. The use of a kit or device according to any of claims 9-10, wherein the disease that occurs with episodic vestibular syndrome is autoimmune MS. fifteen