Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/475—Assays involving growth factors
  • G01N2333/50—Fibroblast growth factors [FGF]
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/285—Demyelinating diseases; Multipel sclerosis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/50—Determining the risk of developing a disease

Definitions

• the present invention falls within the field of biomedicine. Specifically, it refers to the use of the FGF-2 and Anosmin-1 proteins, to predict the histopathology of the lesions of a subject with a demyelinating disease of the Central Nervous System (CNS) by detecting the amount of said proteins in a sample of isolated biological fluid. Furthermore, the present invention relates to a method for knowing the histopathological characteristics of the lesions of a subject with a CNS demyelinating disease to a kit for carrying out said method.
• the CNS demyelinating disease is multiple sclerosis and the biological fluid is cerebrospinal fluid (CSF).
• Demyelinating diseases are a serious clinical and social problem. It is a set of neurological diseases in which, for various reasons, the myelin sheath that covers the axons of neurons is damaged, which acts by protecting and facilitating the conduction of the nerve impulse. The most important of these diseases is multiple sclerosis. In many cases, multiple sclerosis begins as a disease that occurs with outbreaks (or acute phases) in which the neurological abnormalities typical of this disease are experienced. Each outbreak is followed by a remission phase after which the patient may or may not be left with sequelae that produce different degrees of involvement.
• oligodendrocytes myelin-producing cells in the CNS, die in the areas of injury, also known as demyelination plaques. Oligodendrocytes originate during pre- and postnatal development from oligodendroglial precursors, cells that also exist in the adult brain of all mammals.
• oligodendrocyte precursors move towards the area of the injured brain, although, depending on the stage of evolution of the lesion, they can enter the lesion and repair the myelin sheaths or not (cf. Wolswijk, J Neurosci. 1998, vol. 18, pp. 601-609; Chang et al., J. Neurosci. 2000, vol. 20, pp. 6404-6412; Chang et co., N. Engl. J. Med. 2002 , vol. 346, pp. 165-173; Reynolds et al., J. Neurocytol. 2002, vol. 31, pp. 523-536; Chandran et al., Philos. Trans. R. Soc. Lond. B. Biol Sci. 2008, vol. 363, pp. 171-183).
• the demyelinating lesions observed in the CNS of a multiple sclerosis patient can be classified histopathologically in three types based on the distribution of myelin-destroying cells (lymphocytes, macrophages and microglia) and with the degree of myelin destruction ( cf. Trapp et al., J. Neuroimmunol., 1999, vol. 98, pp. 49-56; Benito et al., J. Neurosci., 2007, vol. 27, pp. 2396-2402):
• Knowing the histopathology of demyelinating lesions of a subject is extremely useful. By the uses and methods described in the present invention, said lesions can be detected before the subject exhibits symptoms and is diagnosed.
• the authors of this invention provide a new tool that allows to know through the levels of FGF-2 and / or Anosmin-1 in the patient's cerebrospinal fluid (CSF), the degree to which their lesions are brain, the environment that favors or inhibits the migration of oligodendrocyte precursors, myelin sheath repair cells, the prognosis of the possible evolution of the disease towards neuronal damage in the gray matter, as well as the assignment of a treatment or another for them (including the design of clinical trials).
• the present invention therefore provides a non-invasive tool for histopathological cataloging of the lesions present in the substance. target of multiple sclerosis patients only by analyzing the level of FGF-2 and / or Anosmin-1 in the CSF.
• the CSF levels of the FGF-2 and / or Anosmin-1 molecule reflect the type or types of lesions of the Central Nervous System (CNS) of a multiple sclerosis patient, without Need for more aggressive tests or diagnostic errors, with the same level of certainty as if a biopsy were performed, the only reliable test of the degree of involvement in the CNS of any neurological patient, including patients with multiple sclerosis.
• the present invention demonstrates that the CSF levels of FGF-2 and / or Anosmin-1 reflect the state of the molecular environment that favors or inhibits the arrival of myelin sheath repair cells to the demyelinating lesion within of the white substance.
• ACTIVE lesions are associated with the presence of the motogenic and chemoattractant molecule of oligodendrocyte precursors, FGF- 2, expressed only by some microglia / macrophage cells inside the lesion, but FGF-2 or its receptor, FGFR1 is never detected in the vicinity of the lesion. demyelination plate. Within the active lesions, the presence of molecules that, such as Anosmin-1, prevent or hinder the migration of oligodendrocyte precursors is not detected, clearly indicating that in such lesions the appropriate circumstances for possible remyelination occur and, therefore, the repair of them.
• the measurement of the level of FGF-2 and / or of Anosmin-1 in the CSF of multiple sclerosis patients reflects alterations in the gray matter, both at the cellular level and the state of the blood-brain barrier of the gray substance, so it serves as a prognosis for the appearance of damage to neurons.
• a bioindicator also called a biomarker, is a substance that can be measured and whose values can be objectively associated with a biological state, such as suffering from a pathology or not. Bioindicators or biomarkers are also used in the study of the response to a treatment.
• a first aspect of the invention relates to the use of the FGF-2, Anosmin-1 or both proteins, to predict the histopathology of the lesions of a subject with a possible demyelinating disease, preferably of the CNS, by detecting the amount of said proteins in a sample of isolated biological fluid.
• the FGF-2 and Anosmin-1 proteins are used to predict the histopathology of the lesions of a subject with a possible demyelinating disease, preferably of the CNS, by detecting the amount of said proteins in an isolated biological fluid sample.
• the FGF-2 and Anosmin-1 proteins are used as remyelination biomarkers in a hypothetical cell therapy in a patient with demyelinating pathology. These proteins can therefore serve as biomarkers to follow the evolution of a patient diagnosed with a demyelinating disease and to follow the evolution during and after a treatment, since the levels of these proteins are indicative of the state of myelination that a patient presents.
• a patient can be treated, for example, but not limited to, with a cell therapy aimed at the regeneration of myelin.
• the FGF-2, Anosmin-1, or both proteins are used to predict the presence of at least one CHRONIC or INACTIVE demyelinating lesion.
• the protein FGF-2 protein is used.
• the protein Anosmin-1 protein is used.
• the combination of both is used.
• the Anosmin-1 protein is used as a biomarker for the detection of a demyelinating disease, preferably of the CNS.
• the Anosmin-1 protein is used to detect a demyelinating disease in an isolated biological sample, preferably in an isolated biological fluid.
• isolated biological fluid sample refers to a biological fluid of a subject, obtained by any method known to a person skilled in the art that serves for that end.
• the biological fluid may include excreted or secreted fluids from the body, as well as fluids that are not normally.
• a biological fluid may include, amniotic fluid that surrounds the fetus, aqueous humor, interstitial fluid, lymph, breast milk, mucus (including nasal drainage and phlegm), saliva, sebum (skin fat), sweat, urine, sperm and pericardial fluid.
• the isolated biological sample can be, for example, but not limited, fresh, frozen or fixed.
• the isolated biological fluid sample is any body fluid and is preferably selected from the list comprising: CSF, blood, blood serum, blood plasma and tears.
• the isolated biological fluid sample is CSF.
• subject refers to animals, preferably mammals, and more preferably, humans. Preferably, this term refers to a subject who has been diagnosed with a CNS demyelinating disease. A subject can therefore be a patient who can suffer from a demyelinating disease or can be a healthy individual.
• CNS demyelinating disease refers to a disease characterized by the loss or dysfunction of myelin in the CNS.
• the demyelinating disease affects the CNS and is selected from the list comprising: multiple sclerosis, Devic optic neuromyelitis, acute disseminated encephalitis, acute transverse myelitis, acute or subacute haemorrhagic leukoencephalitis, acute disseminated demyelination, diffuse sclerosis, demyelination central corpus callosum, pontic central myelinolysis, necrotizing myelitis subacute, concentric sclerosis, adrenoleukodystrophy, Alexander's disease, Canavan's disease, Krabbe's disease and Zellweger's syndrome.
• Multiple sclerosis is the most common CNS demyelinating disease.
• Pathological findings include multiple well-defined areas of demyelination of all white CNS substance.
• Clinical manifestations include loss of vision, abnormal extraocular movements, paraesthesia, loss of sensation, weakness, dysarthria, spasticity, ataxia and bladder dysfunction.
• the CNS demyelinating disease is multiple sclerosis, in any of its clinical variants.
• the most common clinical variants of the disease are: Recurrent-Remitting Multiple Sclerosis, Progressive Secondary Multiple Sclerosis, Progressive Primary Multiple Sclerosis, Recurrent Progressive Multiple Sclerosis and Isolated Clinical Syndrome (CIS).
• Recurrent-Remitting Multiple Sclerosis is the most common clinical variant of Multiple Sclerosis, characterized by acute and recurrent outbreaks or exacerbations of neurological dysfunction followed by partial or complete recoveries. Common clinical manifestations include loss of visual, motor, sensory or bladder function. Acute episodes of demyelination can occur at any CNS site, which commonly affect the optic nerves, spinal cord, brain, brainstem and cerebellum.
• Progressive Secondary Multiple Sclerosis is the usual clinical evolution of Recurrent-Remitting Multiple Sclerosis; It involves decreasing the number of outbreaks and the progression of disability. Progressive Primary Multiple Sclerosis is characterized by a gradual progression of disability from the beginning; patients do not experience relapses, but a constant functional deterioration since the disease begins.
• Recurrent Progressive Multiple Sclerosis begins with a progressive primary course, on which outbreaks overlap.
• the CNS demyelinating disease is multiple sclerosis in its progressive primary or secondary progressive clinical variant.
• the demyelinating disease affects the Peripheral Nervous System (SNP) and is preferably Guillain-Barré syndrome.
• SNP Peripheral Nervous System
• both proteins are used as biomarkers for the detection of a demyelinating disease, preferably of the CNS.
• the levels of both proteins are detected in an isolated biological sample, preferably an isolated biological fluid.
• a second aspect of the invention relates to a method for obtaining useful data for the detection of a demyelinating disease or for predicting the histopathology of the lesions of a subject or a patient with a CNS demyelinating disease (hereinafter, method of the invention) comprising the following steps: a) obtaining an isolated biological sample from a subject, preferably an isolated biological fluid; b) detect the amount of FGF-2 protein and / or Anosmin-1 in the biological sample of step (a); Y
• step (c) compare the amount detected in step (b) with a reference amount.
• the method of the invention comprises the following step: d) predicting the presence of at least one chronic or inactive lesion in the white substance and / or the existence of alteration in the blood-brain barrier of the gray substance in the subject.
• steps (b) and / or (c) of the method of the invention can be totally or partially automated, for example, by means of a robotic sensor device for the detection of the amount in step (b) or the computerized comparison in step (c).
• predict refers to the ability to classify the lesions of a subject with a CNS demyelinating disease according to histopathological characteristics such as, for example, the type of lesions present in the white substance or the existence of alterations in the blood-brain barrier of the gray substance, when a sample classification method is applied based on the analysis of the amount of the FGF-2 and / or Anosmin-1 protein and the comparison of said quantity detected with respect to a reference quantity.
• This discrimination 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 correctly classified.
• the amount that is significantly statistical can be established by one skilled in the art by using different statistical tools, for example, but not limited, by determining confidence intervals, determining the p-value, Student's test or discriminant functions of Fisher
• the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%.
• the value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001.
• the present invention allows the disease to be correctly detected in at least 60%, in at least 70%, in at least 80%, or in at least 90% of the subjects of a certain group or population analyzed.
• white substance refers to the part of the CNS and, more preferably, the brain, composed mainly of myelinated nerve fibers.
• white substance refers to the part of the CNS and, more preferably, the brain, composed mainly of dendrites and neuronal bodies, which do not possess myelin.
• FGF-2 protein fibroblast growth factor-2
• bFGF basic fibroblast growth factor
• FGF-2 protein refers to any of the proteins resulting from the expression of the human FGF-2 gene (GenID: 2247).
• the sequence of Prototypic amino acids of the FGF-2 protein is SEQ ID NO: 1 (NP 001997.5). Therefore, the term “FGF-2 protein” also refers to the protein whose amino acid sequence is SEQ ID NO: 1, a variant thereof or a fragment thereof, as long as said variant or said fragment is functionally equivalent.
• extracellular matrix protein Anosmin-1 also known as "Kallmann syndrome protein” and "Adhesion molecule-like X-linked" is encoded in the human KAL1 gene and has been implicated in various biological processes in both the development of the CNS and in pathological conditions (Kallmann syndrome, cancer).
• Anosmin-1 protein refers to any of the proteins resulting from the expression of the human KAL1 gene (GenID: 3730).
• the prototypic amino acid sequence of the Anosmin-1 protein is SEQ ID NO: 2 (NP 000207.2). Therefore, the term “Anosmin-1 protein” also refers to the protein whose amino acid sequence is SEQ ID NO: 2, a variant thereof or a fragment thereof, provided that said variant or said fragment is functionally equivalent.
• variant refers to a protein substantially homologous to the FGF-2 and / or Anosmin-1 proteins. In general, a variant includes additions, deletions or substitutions of amino acids. The term “variant” also includes proteins resulting from posttranslational modifications such as, but not limited to, glycosylation, phosphorylation or methylation.
• a protein is "substantially homologous to the FGF-2 or Anosmin-1 protein" when its amino acid sequence exhibits a good alignment with the SEQ amino acid sequences.
• ID NO: 1 and / or SEQ ID NO: 2 that is, when its amino acid sequence has a degree of identity with respect to the amino acid sequence SEQ ID NO: 1, of at least 50%, typically of, at least 80%, advantageously at least 85%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% .
• identity refers to the proportion of identical amino acids between two amino acid sequences that are compared. The percentage of identity existing between two sequences can be easily identified by a person skilled in the art, for example, with the help of an appropriate computer program to compare sequences.
• fragment refers to a portion of the FGF-2 and Anosmin-1 protein or one of its variants.
• the expression "detection of the amount of FGF-2 and / or Anosmin-1 protein" in a sample of biological fluid isolated from a subject refers to the measure of the amount or the concentration, preferably semi-quantitatively or quantitatively.
• the measure can be carried out directly or indirectly.
• the direct measure refers to the measure of the quantity or FGF-2 and / or Anosmin-1 protein concentration, based on a signal that is obtained directly from the protein, and that is directly correlated with the number of protein molecules present in the sample.
• Said signal - which we can also refer to as an intensity signal - can be obtained, for example, by measuring an intensity value of a chemical or physical property of the FGF-2 and / or Anosmin-1 protein.
• the indirect measurement includes the measurement obtained from a secondary component (for example, a component other than the product of gene expression) or a biological measurement system (for example the measurement of cellular responses, ligands, "tags” or enzymatic reaction products ).
• Quantity refers to, but is not limited to, the absolute or relative amount of the protein, as well as any other value or parameter related to or derived from them.
• Said values or parameters comprise values of signal intensity obtained from any of the physical or chemical properties of the protein, obtained by direct measurement, for example, intensity values in an immunoassay, mass spectroscopy or nuclear magnetic resonance. Additionally, said values or parameters include all those obtained by indirect measurement, for example, any of the measurement systems described elsewhere in this document.
• comparison refers to, but is not limited to, the comparison of the amount of the FGF-2 and / or Anosmin-1 protein of the biological sample obtained in step ( a) of the method of the invention with an amount of the FGF-2 protein and / or reference Anosmin-1.
• the comparison described in section (b) of the method of the present invention can be performed manually or assisted by a computer.
• reference amount refers to the amount of the expression product that allows classifying the lesions of a subject with a CNS demyelinating disease according to the presence or absence of at least a chronic or inactive lesion in the white substance and / or according to the existence or not of alteration in the blood-brain barrier of the gray substance.
• Said reference quantity can be calculated by means of statistical methods known in the state of the art from a population of subjects for whom the amount of expression of FGF-2 and / or of Anosmin-1 present in their CSF is known and Histopathological characteristics of the lesions present in its white substance and / or gray substance.
• the detection of the amount of FGF-2 and / or Anosmin-1 expression present in the CSF and the analysis of the histopathological characteristics of the lesions present in its white substance and / or gray matter of this population of subjects can be performed by Any procedure described in the state of the art, such as, but not limited to, those described in the examples of the present description.
• said reference quantity defines a threshold quantity.
• Suitable reference amounts or thresholds can be determined by the method of the present invention from a reference sample that can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample. More preferably, the threshold amount may be derived from the normal distribution limits of a physiological amount found in a population of control subjects.
• step (c) is indicative of at least one CHRONIC or INACTIVE lesion in the white matter and / or the existence of alteration in the blood-brain barrier of the gray matter.
• the amount of FGF-2 and the amount of Anosmin-1 in the control individuals is, on average, at least twice less than the average amount detected in patients with at least one chronic or inactive demyelinating lesion.
• the biological sample isolated in step (a) is a body biological fluid, and is preferably selected from the list comprising: CSF, blood, blood serum, blood plasma and tears. More preferably, the sample is CSF.
• the method of the invention preferably serves to detect or predict the histopathology associated with the demyelinating diseases described above.
• the detection of the amount of FGF-2 and / or Anosmin-1 protein is performed by incubation with a specific antibody in an immunoassay.
• immunoassay refers to any analytical technique that is based on the reaction of conjugation of an antibody with the sample obtained. Examples of immunoassays known in the state of the art are, for example, but not limited to: immunoblot, enzyme-linked immunosorbent assay (ELISA) or protein micmarrays.
• antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain a site. of antigen binding that specifically binds (immunoreacts) with the FGF-2 or Anosmin-1 protein.
• portions of immunologically active immunoglobulin molecules include F (ab) and F (ab ') 2 fragments that can be generated by treating the antibody with an enzyme such as pepsin.
• the antibodies can be polyclonal (typically include different antibodies directed against different determinants or epitopes) or monoclonal (directed against a single determinant in the antigen).
• the monoclonal antibody may be biochemically altered, by genetic manipulation, or it may be synthetic, possibly lacking the antibody in whole or in part, from portions that are not necessary for recognition of the FGF-2 and / or Anosmin protein -1 and being replaced by others that communicate additional advantageous properties to the antibody.
• the antibody can also be recombinant, chimeric, humanized, synthetic or a combination of any of the foregoing.
• a "recombinant antibody or polypeptide” (rAC) is an antibody that has been produced in a host cell that has been transformed or transfected with the nucleic acid encoding the polypeptide, or produces the polypeptide as a result of homologous recombination.
• Antibodies that recognize the FGF-2 protein or Anosmin-1 are known in the state of the art, such as those described, but not limited to, in the examples of the present description.
• the immunoassay is an immunoblot or Western blot.
• a protein extract is obtained from a sample of biological fluid isolated from a subject and the proteins are separated in a support medium capable of retaining them by electrophoresis. Once separated, the proteins are transferred to a different support where they can be detected through the use of specific antibodies that recognize the FGF-2 protein or Anosmin-1.
• Electrophoresis is an analytical technique of separation of kinetic foundation based on the movement or migration of macromolecules dissolved in a certain medium (electrophoresis buffer solution), through a matrix or cross-linked support as a result of the action of an electric field.
• the behavior of the molecule is given by its electrophoretic mobility and this by the charge, size and shape thereof. The higher the charge / size ratio, the faster an ion migrates within the electric field.
• This technique depending on the equipment used, support and physical-chemical conditions in which the separation will be carried out. Some variations of this technique are, for example, but not limited to, capillary electrophoresis, paper electrophoresis, agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), isoelectric focusing or two-dimensional electrophoresis (2D-PAGE). Electrophoresis can be carried out under denaturing or non-denaturing conditions.
• the proteins are transferred to a support or to a membrane, for example, but not limited to, PDVF, nitrocellulose or cellulose acetate.
• a membrane for example, but not limited to, PDVF, nitrocellulose or cellulose acetate.
• This membrane is hybridized with a specific antibody (also called primary antibody) that recognizes the FGF-2 protein or Anosmin-1.
• the membrane is then hybridized with an antibody (also called secondary antibody) capable of specifically recognizing the primary antibody and which is conjugated or bound with a marker compound.
• an antibody also called secondary antibody
• it is the antibody that recognizes FGF-2 or Anosmin-1 proteins that is conjugated or bound to a marker compound, and the use of a secondary antibody is not necessary.
• the immunoassay is an enzyme-linked immunosorbent assay or ELISA (Enzyme-Linked ImmunoSorbent Assay).
• ELISA Enzyme-Linked ImmunoSorbent Assay
• the ELISA is based on the premise that an immunoreactive (biological sample antigen or antibody) can be immobilized on a solid support, then bringing that system into contact with a fluid phase containing the complementary reagent that can bind to a marker compound .
• ELISA There are different types of ELISA.
• the solid support In the direct ELISA or simple two-layer ELISA assay, the solid support is coated with the biological sample and incubated with an antibody that recognizes the FGF-2 protein or Anosmin-1, conjugated or bound to a marker compound.
• the solid support In the indirect ELISA, the solid support is coated with the biological sample and incubated with a primary antibody, which recognizes FGF-2 or Anosmin-1 proteins and then a secondary antibody, which recognizes the primary antibody, conjugate or attached to a marker compound.
• the well is coated with a first antibody that recognizes and binds to the FGF-2 protein or to Anosmin-1, the problem biological sample is applied, so that the FGF-2 or Anosmin-1 protein will be retained in the well when recognized by the first antibody, and then a second antibody that recognizes FGF-2 or Anosmin-1 proteins, conjugated or bound to a marker compound is applied.
• the immunoassay is a protein microarray consisting of a collection of proteins immobilized on a solid support in a regular and preset arrangement.
• marker compound refers to a compound capable of giving rise to a chromogenic, fluorogenic, radioactive and / or chemiluminescent signal that allows the detection and quantification of the amount of the protein. FGF-2 and / or Anosmin-1.
• the marker compound is selected from the list comprising radioisotopes, enzymes, fluorophores or any molecule capable of being conjugated with another molecule or detected and / or quantified directly.
• This marker compound can bind to the antibody directly, or through another compound.
• directly binding marker compounds are, but are not limited to, enzymes such as alkaline phosphatase or peroxidase, radioactive isotopes such as 33P or 35S, fluorochromes such as fluorescein or metal particles, for direct detection by colorimetry, autoradiography, fluorimetry , or metallography respectively.
• the levels of the FGF-2 and Anosmin-1 proteins are detected in biological samples isolated from the same patient or subject, and said levels are compared with the respective reference levels.
• the biological sample where FGF-2 levels are detected may be the same or different from the biological sample where Anosmin-1 levels are detected.
• the present invention also relates to a kit comprising the elements necessary to carry out the method of the invention. Therefore, a third aspect of the present invention relates to a kit for carrying out any of the embodiments of the method of the invention.
• Said kit may contain all those reagents necessary to detect the amount of the FGF-2 and / or Anosmin-1 protein by means of any of the methods described hereinbefore, such as, but not limited to, specific antibodies against FGF-2 and / or Anosmin-1 proteins, secondary antibodies or positive and / or negative controls.
• the kit can also include, without any limitation, buffers, protein extraction solutions, agents to prevent contamination, inhibitors of protein degradation, etc.
• the kit can include all the supports and containers necessary for its start-up and optimization.
• the kit further comprises instructions for carrying out the method of the invention.
• the methods and kits described in the present invention allow classifying the lesions of a subject with a CNS demyelinating disease according to the presence or absence of at least one chronic or inactive lesion in the white matter.
• the methods and kits described in the present invention are therefore useful for selecting the type of treatment that can be performed in a patient with this disease (including clinical trials), since elevated levels of this molecule in the CSF indicate a greater degree of affectation and, therefore, the impossibility of remyelination, including through the use of reparative cell therapies based on the use of transplants with oligodendrocyte precursors (or other cell types), regardless of their origin (autotransplantation, donors, etc. ).
• the methods and kits described in the present invention allow classifying the lesions of a subject with a CNS demyelinating disease according to the existence or not of alterations in the blood-brain barrier of the gray matter.
• the methods and kits described in the present invention are, therefore, useful for predicting the evolution of a patient with a demyelinating disease towards possible damage to neurons, which would occur in the case of finding the level of FGF- in their CSF. 2 and / or of Anosmin-1 significantly higher than that found in conditions of non-demyelination.
• this measurement at the patient's CSF level serves as the basis for the use of other therapies (including new clinical trial designs) that lead to neuroprotection, not just neuroreparation, that is, oriented towards the protection of neurons more than towards the repair of the myelin sheaths that wrap their axons.
• a particular preferred embodiment of the aspects of the present invention relates to the use of the FGF-2 protein.
• a particular preferred embodiment of the aspects of the present invention relates to the use of the Anosmin-1 protein.
• a particular preferred embodiment of the aspects of the present invention relates to the use of the combination of the FGF-2 protein and the Anosmin-1 protein.
• Figure 1 illustrates how the comparison of the level of FGF-2 in the CSF of a multiple sclerosis patient with respect to the level of the control patients reflects at histopathological level the type of demyelination plaques that it presents in its white matter.
• A Average levels of FGF-2 in the CSF of control patients with multiple sclerosis. " ** " is P ⁇ 0.01 (Student's t-test or Mann-Whithney test for non-parametric samples).
• B Correlation between the level of FGF-2 and the type of lesions present in the white matter of patients with multiple sclerosis. Correlation shown according to Pearson's correlation test.
• CD Double immunohistochemical screening for HLD-DR (MHC-II) and myoin chromium cyanine staining of a control patient (C), with an active lesion (D) and with an inactive lesion (E). Below, the level of FGF-2 that each of these patients presented in their CSF.
• Figure 2 illustrates how only with the measurement of the level of FGF-2 in the CSF of multiple sclerosis patients can we distinguish between those who present only ACTIVE lesions and those who present at least one CHRONIC and / or INACTIVE lesion in their CNS .
• A Western blot of protein extracts from tissue sections.
• B The quantitative blot analysis of section A shows that both multiple sclerosis patients who only have active lesions and those who have at least one chronic and / or inactive lesion, have levels significantly higher than FGF-2 than control patients. However, the analysis of the level of FGF-2 from these extracts does not show differences between both types of patients.
• C The analysis of the level of FGF-2 in the CSF of the same patients represented in AB does show significant differences between both groups of multiple sclerosis patients.
• Figure 3 illustrates how the comparison of the level of FGF-2 in the CSF of a multiple sclerosis patient with respect to the level of control patients reflects the molecular environment related to the migration of oligodendrocyte precursors.
• A-B Double immunohistochemical screening for HLA-DR (A) or Anosmin-1 (B) and staining of myelin eriochrome cyanine (A and B) showing an active lesion that does not have immunostaining for Anosmin-1.
• C-D Adjacent sections of a patient with a chronic lesion where it is seen that the area of lesion stained with eriochrome cyanine and HLA-DR (C) has an immunolabel on the outside for FGF-2 (D).
• Figure 4 shows the relationship between the comparison of the level of FGF-2 in the CSF of multiple sclerosis patients with respect to that of control patients and the presence of alterations in the gray matter, both at the level of FGF expression -2 and of Anosmin-1 as in the integrity of the blood brain barrier.
• AF Immunohistochemistry for FGF-2 (AC) and Anosmin-1 (DF) in the cerebral cortex of a control patient (A, D) and one with multiple sclerosis (BC, EF) where there is a strong increase in the marking for FGF- 2 and Anosmin-1 in the patient injured, corresponding to perivascular cells.
• Gl Example of double fluorescent immunohistochemistry of said perivascular cells in this case for FGF-2 (G) and GFAP (H), as well as the mixture of both with a counterstain for nuclei (I) where their astrocytic nature is demonstrated .
• JO Double fluorescent immunohistochemistry showing the tracing for the blood brain barrier protein ZO-1 (J, M), together with FGF-2 (K, N) as well as the mixture of both together with a nucleus counterstain (L, O) in the gray matter of a control patient (JL) and in that of one with chronic and inactive lesions in its white substance (MO). It can be seen how the alteration of the blood brain barrier (M) is associated with the appearance of astrocytes positive for FGF-2 (N). In the lower part of the figure, the level of FGF-2 in the CSF that each of the patients referred to in the images is shown.
• Figure 5 shows that the active lesions presented exclusively immunostained cells for FGF-2 inside (Figure 5A-C) with characteristics of microglia / macrophages and inflammatory cells since they appeared doubly labeled for CD68 (Figure 5D-F) and HLA-DR ( Figure 5G-I), respectively.
• Figure 5D-F characteristics of microglia / macrophages and inflammatory cells since they appeared doubly labeled for CD68
• Figure 5G-I Figure 5G-I
• CHRONIC and INACTIVE lesions did not show cells marked for FGF-2 in their interior but mostly in the periplaca ( Figure 5J-L).
• double immunostaining characterized these cells as inflammatory cells immunostained for HLA-DR ( Figure 5M-0) with microglia / macrophage characteristics (Figure 5P-R).
• CHRONIC and INACTIVE lesions presented precursors of oligodendrocytes immunostained with PDGFRa in the periplach ( Figure 5S), which presented immunomarking for FGFR1 ( Figure 5T), but not for GFAP ( Figure 5U-V).
• the inventors show how the measurement of the level of FGF-2 in the CSF of multiple sclerosis patients correlates with the type of lesions found histopathologically in the cerebral cortex of said patients.
• CO control
• M woman
• MS multiple sclerosis
• PP progressive primary
• SP progressive secondary
• TP post-mortem time
• V male. 1
• HLA-DR HLA-DR
• eriochrome cyanine eriochrome cyanine for myelin. 50 pm thick histological sections were obtained using a sliding microtome coupled to a cold source and collected and stored until use.
• the sections were immersed in a medium containing citrate buffer (pH 6.0) at 90 ° C, for 10 minutes. Subsequently, the endogenous peroxidase was inactivated by a 20-minute pretreatment in phosphate buffer (0.1 M, pH 7.4), with 10% methanol and 3% H2O2. They were then pre-incubated for 1 hour in a medium containing phosphate buffered saline (pH 7.4), with 5% (v / v) normal horse serum and 0.2% Triton X-100 ( v / v), and subsequently incubated in this same pre-incubation medium with the anti-HLA-DR antibody (1: 200) overnight (at 4 ° C).
• phosphate buffer 0.1 M, pH 7.4
• Triton X-100 v / v
• the sections were stained for one hour with cyanoine eriochrome and subsequently washed in water, after which the differentiation process was carried out: i) 5% iron alum (w / v) for 5 minutes, ii) washing in running water and iii) 1% (w / v) potassium borax-ferricyanide and 1.25% (w / v), respectively, for 10 minutes, controlling the color under a microscope.
• the CSFs of multiple sclerosis patients were centrifuged at 14,000 rpm for 5 minutes at 4 ° C and analyzed using a specific ELISA kit for human FGF-2, with a sensitivity limit of 2 pg / ml.
• A Active injuries.
• C Chronic injuries.
• I Inactive injuries. indicates the presence of injury, while - indicates absence.
• Ast Astrocytes.
• Per Periplaca. + indicates that the tide was positive and - that it was negative.
• the inventors show how the measurement of the level of FGF-2 in the CSF manages to delimit the presence of two groups of patients while it cannot be differentiated if FGF-2 is measured directly from the tissue (WB).
• Proteins were extracted from the histological sections of the cerebral cortex using a protein extraction kit from fixed tissue, and the presence of FGF-2 or ⁇ -tubulin (as a load control) was immunodetected using the Western blot technique ( Figure 2A) hybridizing with a polyclonal primary antibody made in goat (1: 200) or monoclonal antibody made in mouse (1: 30,000) followed by a biotinylated anti-goat secondary antibody made in horse (1: 5,000) or polyclonal anti-mouse made in rabbit conjugated with HRP, respectively.
• the inventors show how the measurement of the level of FGF-2 and / or Anosmin-1 in the CSF of patients with multiple sclerosis is a good bioindicator of the molecular environment found in white matter demyelination lesions. .
• the antibody used was polyclonal made in rabbit (1: 500), to which a biotinylated goat (1: 200) polyclonal antibody was subsequently attached.
• the immunomarking process was similar and was revealed with DAB and H2O2.
• the active type lesions found in the tissue of some of the patients with a high FGF-2 level in the CSF showed no difference compared to those observed in multiple sclerosis patients with FGF-2 levels in their CSF. similar to those of control individuals, which indicates that the presence of FGF-2, FGFR1 and Anosmin-1 is specific to the type of chronic or inactive lesion and not the age of the subject, the type of multiple sclerosis they presented as well as of the postmortem time that passed from death to the collection of the samples.
• the inventors show the identity of the FGF-2 and FGFR1 producing cells in the ACTIVE and CHRONIC and INACTIVE lesions on the other.
• a double histological immunostaining was performed using an antibody for rabbit polyclonal FGF-2 (1: 200 v / v) together with the HLA-DR inflammatory cell marker (see previous Examples) and the specific microglia / macrophage CD68 (antibody mouse monoclonal; 1: 200).
• FGFR1 horse-made polyclonal antibody
• PDGFRa rabbit-made polyclonal antibody
• GFAP mouse-made monoclonal antibody
• immunofluorescence was intensified by the use of a biotinylated goat anti-rabbit secondary antibody, the ABC kit as well as with the Tiramida intensification kit (TSA) and Streptavidin Texas Red (1: 500 v / v) .
• GFAP visualization used a polyclonal donkey-made mouse antibody linked to Alexa 633 and for the PDGFRa a polyclonal donkey-made rabbit antibody linked to Alexa 488.
• the nuclei were contracted with Hoechst.
• EXAMPLE 5 the inventors show that the measurement of the level of FGF-2 in the CSF correlates with alterations in the substance gray of multiple sclerosis patients, regardless of the presence or not of demyelinating lesions in the white matter.
• FGFR1 and Anosmin-1 In addition to the immunostaining for FGF-2, FGFR1 and Anosmin-1 and the staining of myochrome cyanine myelin, similar to that described in examples 1 and 3, a double immunohistochemistry against FGF-2 or Anosmin- 1 and the protein present in occluding zonules that are part of the blood brain barrier (ZO-1) or glial fibrillary acidic protein (GFAP), a typical astrocyte marker.
• ZO-1 blood brain barrier
• GFAP glial fibrillary acidic protein
• the detection of GFAP was carried out with a polyclonal antibody made in rabbit to which a secondary polyclonal antibody against rabbit was made, made in donkey, and which had a fluorescent molecule attached.
• the measurement of the level of FGF-2 in the CSF is a good bioindicator of the state of involvement of the blood-brain barrier of the gray matter, even in regions where demyelination has not occurred, which can serve as a prognostic test to prevent future neurodegenerations.

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