ES2368293A1 - Long chain acyl coenzyme-a synthetases (acsls) as cyto-serological biomarkers in inflammatory or autoimmune diseases - Google Patents

Abstract

Acyl-Coenzyme-A long-chain synthetases (ACSLs) as cytoserological biomarkers of inflammatory or autoimmune diseases.Use of Acyl-Coenzyme-A long-chain synthetases (ACSLs) as cytoserological biomarkers of inflammatory or autoimmune diseases, and especially systemic lupus erythematosus, and method of obtaining useful data for the diagnosis and monitoring of said diseases.

Description

Acil Coenzyme-A synthetases of long chain (ACSLs) as biomarkers cyto-serological in inflammatory diseases or Autoimmune

The present invention is within the field of molecular biology and medicine, and refers to the use of Acyl-Coenzyme-A synthetases of long chain (ACSL) as biomarkers cyto-serological of inflammatory diseases or autoimmune, and especially systemic lupus erythematosus.

Prior art

Acyl-Coenzyme A long chain synthetases (ACSLs), are intracellular enzymes constituted by the isoenzymes ACSL1, ACSL2 (now called ACSL6) ACSL3, ACSL4 and ACSL5. Activate long-chain fatty acids (12 to 22 C) by ligation of Coenzyme A (CoA) as the first essential stage for subsequent use in lipid synthesis and degradation as well as in obtaining energy by robe-oxidation , regulation and signaling (Bu et al ., 2009. J Biol Chem . 2009 Sep 8) (Digel et al ., 2008. Mol Cell Biochem . 2009 Jun; 326 (1-2): 23-8) (Soupene & Kuypers 2008. Exp Biol Med (Maywood) May; 233 (5): 507-21) (Webster et al ., 2008. Endocrinology . Jul; 149 (7): 3679-87) (Watkins et al ., 2007. J Lipid Res . 2007 Dec; 48 (12): 2736-50) (Cano et al ., 2006. J Steroid Biochem Mol Biol . 2006 Jun; 99 (4-5): 197-202) (Yeh et al ., 2006 Cancer Lett . Feb 28; 233 (2): 297-308).

The metabolism of lipids and long-chain fatty acids and acyl-CoA synthetases are being linked to various diseases and important physiological processes in pathogenesis, including cancer (Mashima et al ., 2009. Cancer Sci . 100 (8) : 1556-62) (Celis et al , 2009. Mol Oncol . Feb 3) (Tomoda 1991. Arch Exp Med . Apr; 65 Suppl: 1-12), apoptosis (Gassler et al ., 2007. Gastroenterology . 2007 Aug; 133 (2): 587-98) (Heimli et al ., 2003. Lipids . Mar; 38 (3): 263-8), inflammation (Westerbacka 2007) (Ciapaite et al ., 2006. Biochim Biophys Acta . Feb; 1771 (2): 147-54), cardiovascular diseases, atherosclerosis (Brown et al ., 2007. Curr Atheroscler Rep . Dec; 9 (6): 494-500), obesity (Teng et al ., 2009. FASEB J. Jun; 23 (6): 1705-9) (Lobo et al ., 2009. J Biol Chem . 2009 Jul 3; 284 (27): 18347-56), diabetes (Wendel et al ., Diabetes. 2010 Mar 3. PubMed electronic pub in advance) (Gu et al ., 2009. PLoS One . Jun 2; 4 (6): e5767), diseases of the central nervous system (SNC) (McGuinness & Smith. 1999. Arch Immunol Ther Exp (Warsz). 1999; 47 (5): 281-7.) (Song et al ., 2007. J Neurosci Res . Dec; 85 (16): 3586-97) (Mirnics K 2006.) (Rapoport, 2008) (Lee et al ., 2007. Prostaglandins Leukot Essent Fatty Acids . Nov-Dec; 77 (5-6): 239-46. Epub 2007 Nov 26. Review), celiac disease (Gassler et al ., 2007. Gastroenterology . Aug; 133 (2): 587-98, Epub 2007 Jun 8) (Obermuller et al ., 2006. Int J Colorectal Dis . Mar; 21 (2): 130-4. Epub 2005 Apr 5), premature ovarian failure (Kang et al ., 2008. Epub 2008 Jun 13). ACSLs have also recently been implicated in arteriosclerosis (Askari et al , 2007. Diabetes . Apr; 56 (4): 1143-52. Epub 2007 Jan 26) and cardiovascular complications (Matsuda et al , 2008. J Antibiot ( Tokyo ). May; 61 (5): 318-21), which is interesting because it connects with SLE since these complications are highly frequent in SLE. Therefore, the proteins involved in the uptake of fatty acids and their metabolism can be important pharmaceutical targets.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that is mainly suffered by young women and has the potential to clinically affect all organ systems (D'Cruz et al 2007. Lancet 369 (9561): 587-96). These typically include a facial erythematous rash with a "butterfly" type distribution above the nose and cheeks (skin rashes). Arthritis and arthralgia that usually affect most phalangeal and carpal joints are seen in a majority of patients with SLE (joint pain). Likewise, a renal complication is observed in approximately 70% of SLE patients, and it is considered one of the main causes of death of SLE. Glomerulonephritis secondary to the deposition of the antigen-autoantibody complex in the kidney often leads to renal impairment, as observed by proteinuria, or ultimately to renal failure. Clinical presentations usually include asymptomatic hematuria or proteinuria, acute nephritic or nephrotic syndromes, rapid progressive glomerulonephritis and chronic renal failure. Clinical manifestations of SLE are also observed in the lymphatic, pulmonary, gastrointestinal, hemic, cardiovascular and central nervous (CNS) systems (Jain & Halusca 2009. J Clin Pathol . 2009 Jul; 62 (7): 584-92) (Guillevin 2009 Rheumatology (Oxford) 48 Suppl 3: iii54-7) (Greenberg 2009. Neurologist 15 (3): 115-21) (Buyon et al ., 2009. Nat Clin Pract Rheumatol . 5 (3): 139-48) (Seshan et al ., 2009. Arch Pathol Lab Med . 133 (2): 233-48) (Ryan 2009. Am J Physiol Regul Integr Comp Physiol . 296 (4): R1258-67).

The current treatment of SLE depends on the location and severity of the disease, the treatment method often being dictated by the affected organ system (Tuthill and Khamashta 2009. J Autoimmun . Sep; 33 (2): 92-8. Epub 2009 Jun 25. Review). Arthritis or arthralgia can often be controlled with aspirin or other non-steroidal anti-inflammatory drugs. The most severe manifestations of SLE, such as hemolytic anemia, thrombocytopenic purpura and severe lapoliserositis have been treated with prednisone. The currently recommended treatment for renal impairment uses combinations of prednisone with immunosuppressive agents such as azathioprine or cyclophosphamide. Very good results are being obtained with biological treatments directed against B cells (Levesque 2009. Clin Exp Immunol . Aug; 157 (2): 198-208. Review; D'Cruz et al ., 2007. Lancet . 2007 Feb 17; 369 (9561): 587-96. Review).

Current treatments have addressed nephritic lupus, although commonly used therapeutic regimens are potentially toxic and may be ineffective in some high-risk patients. Normally intensive immunosuppressive regimens are prescribed. For severe SLE, immunosuppressants such as chemotherapies and cyclosporine are used. Other treatments include treatment with corticosteroids and cytotoxic drugs. Alternative therapies include treatment with cyclophosphamide and prednisone. Side effects of long-term use of prednisone include the development of high blood pressure, diabetes and osteoporosis. Currently, several pharmaceutical companies are applying biological therapies such as anti-cytokine therapy, such as TNF-a blockers, IL1, IL6 and IL10 blockers; Also very important is targeted therapy against B lymphocytes as anti-CD20 antibodies (Rituximab, ocrelizumab) (D'Cruz et al ., 2007. Lancet 369 (9561): 587-96).

The prognosis of patients with SLE has improved in the last four decades, parallel to the most important advances in diagnosis and treatment, as well as our understanding of the pathophysiological process and its clinical expression. There is no unequivocal test for the diagnosis of lupus, which is based on the clinical and analytical findings (Yee et al ., 2009. Best Pract Res Clin Rheumatol . 23 (4): 457-67).

The presence of major visceral-organic compromise such as the CNS and more consistently kidney disease has been associated with poor prognosis. Additionally, there is an increased risk of progression to end-stage renal disease when vascular lesions are found in the pathology. Thrombocytopenia implies a poor prognosis in several studies, especially when it is an acute manifestation. Hypertension has been identified as a risk factor for mortality in SLE, although it may be part of late manifestations not related to disease activity. Metabolic syndrome and arteriosclerosis are especially increased in SLE (Sabio et al ., 2009. J Rheumatol 36 (10): 2204-11),

The criteria of the ACR (American College of Rheumatology) have a sensitivity of 96% and specificity of 96%. The global activity at the beginning of the disease as a predictor of mortality is a recently studied factor with the advent of the different indices (SLEDAI, LAI, SLAM, BILAG) and organic damage SLICC / ACR Damage Index (SDI) validated to measure in a way Reproducible and reliable degree of disease activity. Several studies have documented the degree of activity measured by SLEDAI ( Sistemic Lupus Erythematosous Diseases Activity Indexc ) as an activity index and poor prognosis factor for mortality when presenting with the onset of the disease in a Lupus clinic or at the time of renal biopsy initial.

Normally, SLE patients have elevated serum levels of autoantibodies against nuclear constituents. The elevation of the antinuclear antibody (ANA) to titers of 1:40 or higher is the most sensitive diagnostic criterion. More than 99% of patients with lupus have an ANA elevation. Although a significant proportion of patients may have negative ANA at the onset of the disease. The main antigens of antibodies produced in patients with SLE include protein-nucleic acid complexes, such as chromatin, the small nuclear ribonucleoprotein particles Sm and U1 (snRNP) and the Ro / SSA and La / SSB RNP complexes, i.e. antinuclear antibodies , particularly double-stranded DNA, and immune complex-mediated pathology (Yang 2009. Rheumatology ( Oxford ). 48 (9): 1083-7). Tested anti-nuclear antibodies and anti-ENA anti-nuclear antibodies form the mainstay of a serological study for lupus. Autoantibodies of phospholipids and cell surface molecules are also detected. Antiphospholipid antibodies may predispose to thrombosis. More specific is the anti-smith antibody. Nuclear autoantibody complexes with their respective antigens, which are subsequently deposited in small blood vessels, is a direct cause of many of the clinical manifestations of SLE. Other routine studies conducted on suspected SLE are complement system levels (low levels suggest consumption by the immune system, electrolytes and renal function (disturbed if the kidney is affected), liver enzymes and a complete blood count. They have found evidence to suggest that SLE may have an impact on lung cancer and testicular cancer.

However, very few biomarkers in lupus have been validated and used to make clinical decisions. The lack of reliable biomarkers, specific for lupus, worsens the adequate clinical management of SLE and prevents the development of new therapies for lupus, which makes the search for biomarkers that accurately reflect pathophysiology and clinical changes. So far several laboratory markers have shown promise for susceptibility to lupus, diagnosis and monitoring. These include certain SNP and copy variation (CNV) polymorphisms of the complement genes C4 and the receptor Fcgamma (disease susceptibility), C4d bound to erythrocytes (Yang 2009 Rheumatology (Oxford). 48 (9): 1083 -7) which is higher in SLE and specifically in lupus with hemolytic anemia. It does not correlate with SLEDAI or with other markers of disease activity such as anti-dsDNA, C3 and C4. Therefore it has limited value. CD27 (high) plasma cells (disease activity), interferon signature (disease activity) and anti-C1q and anti-NMDA (disease activity and organ involvement -SNC-). All these biomarkers have yet to be validated in rigorous and large-scale studies in different populations (Liu & Ahearn 2009. Best Pract Res Clin Rheumatol . 23 (4): 507-23).

A biopsy is currently required to diagnose lupus nephritis. However, it is an invasive technique not recommended with the daily clinic. A biomarker that is involved in the pathogenesis of LN would be needed. There are candidates that are not fully consolidated at the moment: CCL2 (chemokine ligand 2; also known as MCP-1); CCL5 ( chemokine ligand 5; also known as RANTES); and CX3CL1 ( chemokine ligand 1; also known as fractalkine ); IP-10 ( interferon-inducible protein 10; also known as chemokine ligand 10); neutrophil gelatinase associate lipocalin; hepcidin; adiponectin; transferrin; ceruloplasmin; lipocalin-like prostaglandin synthetase -D; and orosomucoid (Das L).

Several scales have been validated to measure the activity of SLE, such as the SLEDAI ( Systemic Lupus Erythematosus Activity Index ), BILAG ( British Isles Lupus Assessment Group ), SLAM ( Systemic Lupus Activity Measure), ECLAM (European Concensus Lupus Activity Measure ), LAI ( Lupus Activity Index ), which are showing a good correlation between them (Ward et al ., 2000. J. Rheumatol 27: 664-670). Together with the different indexes (SLEDAI, LAI, SLAM, BILAG) and organic damage (SLICC / ACR - Systemic Lupus International Collaborating Clinics / American College of Rheumatology Damage Index , Gladman et al ., 1996. Arthritis Rheum . 39 (3): 363-369). With the advent of different indices and organic damage, mortality predoctors are recently studied factors.

It is necessary, therefore, to find a method alternative diagnostic, less invasive, allowing the diagnosis of complex inflammatory or autoimmune diseases, that is, they are caused by numerous factors, and preferably, that allow the diagnosis of lupus erythematosus systemic (SLE), and subclassification of affected individuals for said disease based on its clinical manifestations and mortality predictors

Description of the invention

The present invention provides a method of Obtaining useful data for the diagnosis of diseases inflammatory or autoimmune, and among them lupus erythematosus systemic (SLE), also allowing early identification of patients with different clinical manifestations of lupus and the establishment of patient groups in combination with these clinical manifestations, as well as the evaluation of the response to Treatment of such diseases.

Therefore, a first aspect of the invention relates to the use of the genes of the Acyl-Coenzyme A long chain synthetases ( ACSLs ) family or their expression products, as markers for the diagnosis, prognosis, or monitoring of inflammatory or autoimmune diseases. In a preferred embodiment, the inflammatory or autoimmune disease is selected from the list comprising: Systemic lupus erythematosus (SLE), Rheumatoid arthritis (RA), Arteriosclerosis (AS), Hypertension (HT), Non-insulin-dependent type 2 diabetes (T2D) , Metabolic syndrome (includes several of those mentioned), Crohn's disease, or any combination thereof. In another more preferred embodiment, the inflammatory or autoimmune disease is systemic lupus erythematosus (SLE).

Another aspect of the invention relates to a method of obtaining useful data for diagnosis and follow-up of inflammatory or autoimmune diseases, from now on hereinafter method of the invention, comprising:

a) obtain an isolated biological sample from a individual, and

b) quantify the expression product of at least one of the genes of the long chain Acyl-Coenzyme A synthetases ( ACSLs ) family in the biological sample isolated from (a).

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In a preferred embodiment, the method of invention further comprises:

c) compare the amounts obtained in step (b) with a reference amount.

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The reference quantity is obtained from constitutive expression values of the gene, in a group of healthy patients Steps (b) and / or (c) of the methods described previously they can be totally or partially automated, by example, by means of a robotic sensor device for the detection of the amount in step (b) or the computerized comparison in step (C).

In another preferred embodiment of this aspect of the invention the isolated biological sample of an individual from step (a) comprises peripheral blood cells ( peripheral blood mononuclear cells PBMCs).

In this memory it is understood by cell peripheral blood mononuclear (PBMC) a blood cell characterized by having a single round core, such as lymphocytes, monocytes or macrophages. These blood cells they are a critical component in the immune system, specifically for fight infections. The lymphocyte population is formed by T cells (CD4 and CD8 positive approx75%).

These cells are often obtained from the blood using ficol, a hydrophilic polysaccharide that separates layers of blood, with monocytes and lymphocytes forming a buffy coat under the plasma layer. This buffy contains the PBMCs. There are other extraction methods known in the state of the art, such as, for example, extracting them from whole blood with a hypotonic lysis solution that preferably smooths the red blood cells. This method gives rise to neutrophils and other polymorphonuclear cells (PMN) important in innate immune defense.

In another preferred embodiment of this aspect of the invention the genes of the ACSL family are selected from the list comprising: ACSL1, ACSL6 (ACSL2), ACSL3, ACSL4, ACSL5 , or any combination thereof.

In another preferred embodiment, the disease Inflammatory or autoimmune is selected from the list comprising: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), arteriosclerosis (AS), hypertension (HT), non-insulin diabetes type 2 dependent (T2D), metabolic syndrome (includes several of the mentioned), Crohn's disease, or any of its combinations In another even more preferred embodiment, the disease inflammatory or autoimmune is systemic lupus erythematosus (THEM).

The term "diagnosis", as it is used in the present invention, to the ability to discriminate between individuals affected or not by an inflammatory disease or Autoimmune Preferably inflammatory or autoimmune disease is rheumatoid arthritis (RA), arteriosclerosis (AS, the hypertension (HT), non-insulin dependent diabetes type 2 (T2D), metabolic syndrome, Crohn's disease, or any of its combinations Even more preferably, it is lupus erythematosus systemic (SLE). It also refers, but not limited to, to the ability to discriminate between samples from patients who they have different states of systemic lupus erythematosus (SLE). TO in turn, according to the method of the present invention, they could establish other subclassifications within this principal, facilitating, therefore, the choice and establishment of adequate therapeutic regimens. This discrimination as it is understood by an expert in the field 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 an expert in the field by using different statistical tools, for example, but not limited, by determining intervals of confidence, determination of the significance value P, test of Student or Fisher discriminant functions, measures not parametric of Mann Whitney, Spearman correlation, regression logistics, 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, of 0.01, 0.005 or 0.0001. Preferably, the present invention allows to detect the disease correctly in a differential way at least 60%, at least 70%, at least 80%, or at least minus 90% of the subjects of a certain group or population analyzed.

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 isolated biological sample comprises peripheral blood mononuclear cells PBMCs.

The term "individual" as it is used in the description, refers to animals, preferably mammals, and more preferably, humans. The term "individual" is not intended to be limiting in any way, It can be this of any age, sex and physical condition.

ACSL1 ( acyl-CoA synthetase long-chain family member 1), is an isoenzyme of the family of Acyl-Coenzyme A long chain synthetases. Although they differ in their substrate specificity, subcellular location, and tissue distribution, all isoenzymes of this family convert long chain fatty acids into Acyl-CoA fatty acid esters, and therefore play a key role in biosynthesis. of membrane lipids and in the degradation of fatty acids by beta-oxidation. It is also called fatty-acid-Coenzyme A ligase, long-chain 1; fatty-acid-Coenzyme A ligase, long-chain 2; lignoceroyl-CoA synthase; long-chain acyl-CoA synthetase 1; long-chain acyl-CoA synthetase 2; long-chain fatty-acid-coenzyme A ligase 1; palmitoyl-CoA ligase 2; paltimoyl-CoA ligase 1. It is located on chromosome 4 (4q34-q35). Other abbreviations are: ACS1; LACS; FACL1; FACL2; LACS1; LACS2. Its amino acid sequence is found with access number in GenBank (NCBI) NP_001986.2; and / or in SEQ ID NO: 1.

In the context of the present invention, ACSL1 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 1, and which would comprise various variants from:

a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: one,

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

c) nucleic acid molecules whose sequence differs from (a) or (b) due to code degeneration genetic,

d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with a identity of at least 80%, 90%, 95%, 98% or 99% with the SEQ ID NO: 1, and in which the polypeptide encoded by said nucleic acids possesses the activity and characteristics Structural of the ACSL1 protein.

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ACSL6 (ACSL2) ( acyl-CoA synthetase long-chain family member 6 / (EC 6.2.1.3)), is an isoenzyme of the family of long-chain Acyl-Coenzyme A synthetases that is also called fatty-acid-Coenzyme A ligase, long-chain 6; long fatty acyl-CoA synthetase 2; long-chain acyl-CoA synthetase 6. It is located on chromosome 5 (5q31). Other abbreviations are: ACS2; FACL6; LACS2; LACS5; FLJ16173; KIAA0837 . Its amino acid sequence is found with access number in Gen Bank (NCBI) NP_056071.2; NP_001009185.1 and / or in SEQ ID NO: 2; SEQ ID NO: 3, isoform a / isoform b, among others with indicated homology.

In the context of the present invention, ACSL6 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 2 or SEQ ID NO: 3, and which would comprise various variants from from:

a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3,

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

c) nucleic acid molecules whose sequence differs from (a) or (b) due to code degeneration genetic,

d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with a identity of at least 80%, 90%, 95%, 98% or 99% with the SEQ ID NO: 2 or SEQ ID NO: 3, and in which the polypeptide encoded by said nucleic acids possesses the activity and Structural characteristics of the ACSL6 protein.

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ACSL3 ( acyl-CoA synthetase long-chain family member 3), is another isoenzyme of the family of Acyl-Coenzyme A long-chain synthetases that is also called fatty-acid-Coenzyme A ligase, long-chain 3; lignoceroyl-CoA synthase . It is located on chromosome 2 (2q34-q35). Other abbreviations are: ACS3, FACL3, PRO2194 . It is noticeably expressed in the brain and preferably uses myristate, arachidonate, and eicosapentaenoate as substrates. The amino acid sequence of this isoenzyme is 92% identical to that of its rat counterpart. Two transcripts have been found that encode this gene. Its amino acid sequence is found with access number in GenBank (NCBI) NP_004448.2 and / or in SEQ ID NO: 4.

In the context of the present invention, ACSL3 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 4, and which would comprise various variants from:

a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 4,

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

c) nucleic acid molecules whose sequence differs from (a) or (b) due to code degeneration genetic,

d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with a identity of at least 80%, 90%, 95%, 98% or 99% with the SEQ ID NO: 4, and in which the polypeptide encoded by said nucleic acids possesses the activity and characteristics Structural of the ACSL3 protein.

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ACSL4 ( acyl-CoA synthetase long-chain family member 4), is another isoenzyme of the family of Acyl-Coenzyme A long chain synthetases which is also called: acyl-CoA synthetase 4; fatty-acid-Coenzyme A ligase, long-chain 4; lignoceroyl-CoA synthase; long-chain fatty-acid-Coenzyme A ligase 4. It is located on the X chromosome (Xq22.3-q23). Other abbreviations are: ACS4, FACL4, LACS4, MRX63, MRX68 . This enzyme uses arachidonate as a substrate, preferably. The absence of this enzyme may contribute to mental retardation or Alport syndrome. The alternative splicing of this gene generates 2 transcripts. Its amino acid sequence is found with access number in GenBank (NCBI) NP_004449.1; NP_0765266.1 and / or in SEQ ID NO: 5 (isoform 1), SEQ ID NO: 6 (isoform 2).

In the context of the present invention, ACSL4 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 5 or SEQ ID NO: 6, and which would comprise various variants from from:

a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6,

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

c) nucleic acid molecules whose sequence differs from (a) or (b) due to code degeneration genetic,

d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with a identity of at least 80%, 90%, 95%, 98% or 99% with the SEQ ID NO: 5 or SEQ ID NO: 6, and in which the polypeptide encoded by said nucleic acids possesses the activity and Structural characteristics of the ACSL4 protein.

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ACSL5 ( acyl-CoA synthetase long-chain family member 5), is another isoenzyme of the family of Acyl-Coenzyme A long chain synthetases which is also called: FACL5 by fatty acid coenzyme A ligase 5; fatty acid coenzyme A ligase 5; fatty-acid-Coenzyme A ligase, long-chain 5; long-chain acyl-CoA synthetase 5; long-chain fatty acid coenzyme A ligase 5. It is located on chromosome 10 (10q25.1-q25.2). Other abbreviations are: RP11-32402.5, ACS2, ACS5, FACL5 . This isoenzyme is abundantly expressed in the uterus and spleen, and in trace amounts in the normal brain, but has elevated levels in malignant gliomas. This gene is involved in the cell growth of gliomas mediated by fatty acids. Three transcripts have been found encoding two isoforms for this gene. Its amino acid sequence is found with access number in GenBank (NCBI) NP_976313.1; NP_057318.2 and / or in SEQ ID NO: 7 (isoform b), SEQ ID NO: 8 (isoform a).

In the context of the present invention, ACSL5 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the protein collected in SEQ ID NO: 7, or SEQ ID NO: 8, and which would comprise various variants from:

a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 7, or SEQ ID NO: 8,

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

c) nucleic acid molecules whose sequence differs from (a) or (b) due to code degeneration genetic,

d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with a identity of at least 80%, 90%, 95%, 98% or 99% with the SEQ ID NO: 7, or SEQ ID NO: 8, and in which the polypeptide encoded by said nucleic acids possesses the activity and Structural characteristics of the ACSL5 protein.

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The detection of the amount of the expression product of the genes selected from the list comprising: ACSL1, ACSL2, ACSL3, ACSL4 , or ACSL5 , or any combination thereof, can be carried out by any means known in the state of the art. The authors of the present invention have shown that the detection of the quantity or concentration of these expression products semi-quantitatively or quantitatively allows differentiating between the different stages of SLE. In this way, a differential diagnosis can be established in individuals affected by SLE, which allows them to subclassify them.

The measure of quantity or concentration, preferably semi-quantitatively or quantitative, can be carried out directly or hint. The direct measure refers to the measure of the quantity or the concentration of gene expression product, based on a signal that is obtained directly from the transcripts of said genes, or proteins (ACSL enzymes), and that is correlated directly with the number of RNA molecules or proteins produced by genes. This signal - to which we can also refer to as intensity signal - can be obtained, for example, measuring an intensity value of a chemical or physical property of such products. The indirect measure includes the measure obtained from a secondary component or a biological measurement system (for example the measurement of cellular responses, ligands, "labels" or enzymatic reaction products).

The term "quantity" as it is used in the description, refers but is not limited to the absolute or relative quantity of the products of expression of the genes, as well as any other value or parameter related to the same or that may be derived from them. These values or parameters comprise signal strength values obtained at from any of the physical or chemical properties of said expression products obtained by direct measurement. Additionally, said values or parameters include all those obtained by indirect measure, for example, any of the measurement systems described elsewhere in this document.

The term "comparison", as used in the description, refers to, but is not limited to, the comparison of the amount of expression products of the ACSL1, ACSL2, ACSL3, ACSL4 , or ACSL5 genes of the biological sample to be analyzed, also called a biological problem sample, with an amount of the expression products of the ACSL1, ACSL2, ACSL3, ACSL4 , or ACSL5 genes of one or more desirable reference samples described elsewhere in this description. The reference sample can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample. The comparison described in section (c) of the method of the present invention can be performed manually or assisted by a computer.

The term "reference quantity", as used in the description, refers to the absolute or relative quantity (to the reference gene) of expression products of the ACSL1, ACSL2, ACSL3, ACSL4 , or ACSL5 genes that allows discriminate a certain stage of SLE from other stages, or from other diseases. Suitable reference amounts 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. Thus, for example but without limiting ourselves, the reference sample may be the negative controls, that is, the amounts detected by the method of the invention in samples of individuals who do not suffer from any inflammatory or autoimmune disease, and more preferably, who do not suffer THEM.

The reference amount will be, for example, in the case of differentiation between patients affected by the inflammatory disease of healthy patients, expression constitutive of the gene in a control group of healthy patients. Without However, in the case of subclassification of patients affected by lupus based on its manifestations, the group control will be formed by a group of patients with lupus that do not They had that clinical manifestation.

So, the sample or reference samples they can be, for example, obtained from a patient's serum with inflammatory or autoimmune disease, and more preferably with an SLE patient, in a certain clinical phase. In other preferred embodiment of this aspect of the present invention, the reference quantity is obtained from a sample of reference. The reference amount can also be obtained, by example, of the normal distribution limits of an amount found in samples obtained from a population of patients with inflammatory or autoimmune disease of interest in different phases, by well-known statistical techniques. Preferably, the inflammatory or autoimmune disease of interest It's LES.

In the sense used in this description, the term "variant" refers to a protein substantially homologous to any of the proteins ACSL1, ACSL2, ACSL3, ACSL4, or ACSL5. In general, a variant includes additions, deletions or amino acid substitutions The term "variant" includes also to the proteins resulting from modifications posttranslational, such as, but not limited to, glycosylation, methylation or acylation phosphorylation.

As used here, a protein is "substantially homologous" to any of the ACSL1 proteins, ACSL2, ACSL3, ACSL4, or ACSL5, when its amino acid sequence has a good alignment with the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively as has been previously described; that is, when its amino acid sequence It has a degree of identity regarding the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, at least 50%, typically of at least 80%, advantageously of at least one 85%, preferably at least 90%, more preferably, at less, 95%, and, even more preferably, at least 99%. The sequences homologous to any of the ACSL1, ACSL2 proteins, ACSL3, ACSL4, or ACSL5 can be easily identified by a subject matter expert, for example, with the help of a program appropriate computer to compare sequences.

The expression "functionally equivalent", as used herein, means that the proteins or the fragment / s of the protein / s in question maintains / n essentially the biological or immunological properties described in this document. This capacity can be determined by methods conventional.

In another preferred embodiment, the detection of the amount of any of the proteins ACSL1, ACSL2, ACSL3, ACSL4, or ACSL5 is performed by an immunoassay. The term "immunoassay," as used herein, refers to any analytical technique that is based on the reaction of conjugation of an antibody with an antigen. Examples of immunoassays known in the state of the art are, for example, but not limited to: immunoblot , enzyme-linked immunosorbent assay (ELISA), linear immunoassay (LIA), radioimmunoassay (RIA), immunofluorescence, x-map or protein chips .

In another preferred embodiment, the immunoassay is an enzyme-linked immunosorbent assay or ELISA ( Enzyme-Linked ImmunoSorbent Assay ). The ELISA is based on the premise that an immunoreactive (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. There are different types of ELISA: direct ELISA, indirect ELISA or sandwich ELISA.

The term "marker compound", as used in the present description, refers to a compound capable of giving rise to a chromogenic, fluorogenic, radioactive signal and / or chemiluminescent that allows the detection and quantification of the amount of antibodies against the ACSL1, ACSL2 antigens, ACSL3, ACSL4, and / or ACSL5. 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. Some examples of marker compounds that bind directly they are, but not limited to, enzymes such as alkaline phosphatase or peroxidase, radioactive isotopes such as 32 P or 35 S, fluorochromes such as fluorescein or metal particles, for direct detection by colorimetry, auto-radiography, fluorimetry, or metallography respectively.

Another aspect of the invention relates to a method of diagnosing systemic lupus erythematosus, hereafter referred to as the second method of the invention, comprising steps (a) - (c) according to the first method of the invention, and which further comprises assign the individual according to step (a) to the group of individuals with systemic lupus erythematosus disease when presenting an amount of expression product of the genes that are selected from the list comprising: ACSL1, ACSL3, ACSL5SI or any combination thereof, detected in step (b) minor and statistically significant compared to a reference amount.

In another preferred embodiment, the second method of the invention further comprises assigning the individual according to step (a) to the group of individuals with systemic lupus erythematosus disease when presenting an amount of expression product of the ACSL1A, ACSL1SI, ACSL2A, ACSL2SI genes , ACSL3SI, ACSL4SI, ACSL5 , or any combination thereof, detected in step (b) greater and statistically significant compared to a reference amount (obtained from the control group without lupus).

In another preferred embodiment of this aspect of the invention, the second method of the invention further comprises assigning the individual according to step (a) to the group of individuals with systemic lupus erythematosus disease with cutaneous manifestations when it presents a quantity of expression product of the genes that are selected from the list comprising: ACSL1A, ACSL1SI, ACSL3SI , or any combination thereof, detected in step (b) minor and statistically significant compared to a reference amount.

In another preferred embodiment of this aspect of the invention, the second method of the invention further comprises assigning the individual according to step (a) to the group of individuals with systemic lupus erythematosus disease with joint manifestations when presenting a quantity of product of expression of the ACSL4N gene detected in step (b) major, and (or simultaneously) a smaller and statistically significant amount of ACSL4SI expression product compared to a reference amount.

In another preferred embodiment of this aspect of the invention, the second method of the invention further comprises assigning the individual according to step (a) to the group of individuals with systemic lupus erythematosus with renal manifestations when presenting an amount of product of expression of the ACSL3SI gene detected in step (b) major and statistically significant compared to a reference amount.

In another preferred embodiment of this aspect of the invention, the second method of the invention further comprises assigning the individual according to step (a) to the group of individuals with systemic lupus erythematosus disease with hematological manifestations when presenting a quantity of product of expression of the ACSL4SI gene detected in step (b) major and statistically significant compared to a reference amount.

In another preferred embodiment of this aspect of the invention, the second or third method of the invention further comprises assigning the individual according to step (a) to the group of individuals with systemic lupus erythematosus disease with neurological manifestations when presenting a quantity of product ACSL4A gene expression detected in step (b) minor and statistically significant compared to a reference amount.

Several scales have been validated to measure the activity of SLE, such as the SLEDAI ( Systemic Lupus Erythematosus Activity Index ), BILAG ( British Isles Lupus Assessment Group ), SLAM ( Systemic Lupus Activity Measure ), ECLAM ( European Concensus Lupus Activity Measure ), LAI ( Lupus Activity Index ), which are showing a good correlation between them (Ward et al ., 2000. J. Rheumatol 27: 664-670). Together with the different indexes (SLEDAI, LAI, SLAM, BILAG) and organic damage (SLICC / ACR - Systemic Lupus International Collaborating Clinics / American College of Rheumatology Damage Index , Gladman et al ., 1996. Arthritis Rheum . 39 (3): 363-369).

So, at reference amount you can also obtain, for example, the distribution limits normal of an amount found in samples obtained from a population of patients with an inflammatory or autoimmune disease, and preferably with SLE, in different phases or with different clinical manifestations, using statistical techniques well known.

"Gene expression profile" means the gene profile obtained after quantification of mRNA and / or protein produced by the genes of interest or biomarkers, that is, by the genes ACSL1, ACSL2, ACSL3, ACSL4 , or ACSL5 , in An isolated biological sample. The expression profile of the genes is preferably performed by determining the level of mRNA derived from its transcription, after extracting the total RNA present in the isolated biological sample, which can be performed by protocols known in the state of the art. The level of mRNA derived from the transcription of the ACSL1, ACSL2, ACSL3, ACSL4 , or ACSL5 genes can be determined, for example, but not limited to, by polymerase chain reaction (PCR) amplification, back transcription in combination with polymerase chain reaction (RT-PCR), quantitative RT-PCR, back transcription in combination with ligase chain reaction (RT-LCR), or any other nucleic acid amplification method; serial analysis of gene expression (SAGE, SuperSAGE); DNA chips made with oligonucleotides deposited by any mechanism; DNA microarrays made with oligonucleotides synthesized in situ by photolithography or by any other mechanism; in situ hybridization using specific probes labeled with any method of marking; by electrophoresis gels; by membrane transfer and hybridization with a specific probe; by nuclear magnetic resonance or any other diagnostic imaging technique using paramagnetic nanoparticles or any other type of detectable nanoparticles functionalized with antibodies or by any other means. The gene expression profile could also be obtained by detecting and / or quantifying the proteins resulting from the translation of the mRNA derived from the transcription of the ACSL1, ACSL2, ACSL3, ACSL4 , or ACSL5 genes , for example, but not limited to, Western blot immunodetection.

Quantitative detection of the expression of the ACSL1, ACSL2, ACSL3, ACSL4 , or ACSL5 genes can be more preferably performed by real-time PCR (RT-PCR or RTqPCR). The real-time detection of the amplified products can be carried out by means of the use of fluorescent molecules that are intercalated in the double-stranded DNA or by hybridization with different types of probes. Thus, in another preferred embodiment, the expression of the ACSL1, ACSL2, ACSL3, ACSL4 , or ACSL5 genes is detected by RTqPCR using primers SEQ ID NO: 11-SEQ ID NO: 23, indicated in Table 1.

Another aspect of the present invention relates to a kit or device, hereafter kit of the invention, comprising the elements necessary to analyze the expression products of at least one, preferably two, more preferably three, more preferably four , and even more preferably the five ACSL1, ACSL2, ACSL3, ACSL4 , or ACSL5 genes in the sample obtained in step (a). More preferably it comprises the means necessary to compare the amount detected in step (b) with a reference amount.

Even more preferably, the kit of the present invention comprises the elements necessary to carry out any of the methods of the present invention.

Said kit may contain all those reagents necessary to analyze the quantity of the expression product of at least one of the genes selected from the list comprising: ACSL1, ACSL2, ACSL3, ACSL4 , or ACSL5 , in the isolated biological sample, by means of any of the methods described hereinbefore, for example, but not limited to, primers, probes and all those reagents necessary to determine the expression of the ACSL1, ACSL2, ACSL3, ACSL4 , or ACSL5 protein . More preferably, the kit comprises at least one pair of the primers listed in Table 1. (SEQ ID NO: 9 / SEQ ID NO: 10; SEQ ID NO: 11 / SEQ ID NO: 12, SEQ ID NO: 13 / SEQ ID NO: 14; SEQ ID NO: 15 / SEQ ID NO: 16; SEQ ID NO: 17 / SEQ ID NO: 18 or SEQ ID NO: 19 / SEQ ID NO: 20,). The kit can also include, without any limitation, the use of buffers, polymerases, cofactors to obtain optimum activity from these, agents to prevent contamination, etc.

In another preferred embodiment, the kit of the invention comprises antibodies specific to the ACSL1 protein, ACSL2, ACSL3, ACSL4, or ACSL5, secondary antibodies or controls positive and / or negative. The kit can also include, without any type of limitation, buffers, protein extraction solutions, agents to prevent contamination, inhibitors of protein degradation, etc. In the case of detection by RTqPCR can contain, but not limited to.

On the other hand, the kit can include all supports and containers necessary for commissioning and optimization Preferably, the kit further comprises the instructions for carrying out the methods of the invention.

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

Another aspect concerns the use of the kit invention, for the diagnosis, prognosis, or monitoring of inflammatory or autoimmune diseases. In one embodiment preferred, the kit comprises a pair of primers that are select from the list comprising: SEQ ID NO: 9 / SEQ ID NO: 10; SEQ ID NO: 11 / SEQ ID NO: 12, for SEQ ID NO: 13 / SEQ ID NO: 14; pair SEQ ID NO: 15 / SEQ ID NO: 16; SEQ ID NO: for SEQ ID NO: 17 / SEQ ID NO: 18 and SEQ ID NO: 19 / SEQ ID NO: 20, or any combination thereof. In another preferred embodiment, the inflammatory disease or Autoimmune is selected from the list comprising: lupus systemic erythematosus, rheumatoid arthritis, arteriosclerosis, hypertension, type 2 non-insulin dependent diabetes, syndrome metabolic, Crohn's disease, or any combination thereof. In an even more preferred embodiment, the inflammatory disease or Autoimmune is systemic lupus erythematosus.

The terms "amino acid sequence", "peptide", "oligopeptide", "polypeptide" and "protein" is used interchangeably here, and refers to a polymeric form of amino acids of any length, which they can be coding or non-coding, chemically or biochemically modified.

Throughout the description and the claims the word "comprises" and its variants not they intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be partly detached of the description and in part of the practice of the invention. The The following examples are provided by way of illustration, and are not It is intended to be limiting of the present invention.

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Examples

The invention will be illustrated below by means of tests carried out by the inventors.

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Expression levels of mRNAs encoding ACSLs in peripheral blood mononuclear cells (PBMC) and their association with THEM

The objective of this study was to investigate the level of transcription (relative to a reference gene) of the ACSL1, 2, 3, 4, 5 isoforms in PBMC of 45 samples of patients with SLE and 35 healthy controls, by quantitative real-time PCR, for comparison and association study.

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Subjects of study

To perform these measures, 45 patients with SLE have been used that met ≥ 4 criteria reviewed by the " American College of Rheumatology " (ACR) (Tan et al ., 1982. Arthritis Rheum 25: 1271) (see next section: Diagnosis in SLE) who were recruited from the Autoimmune Diseases Unit of the Virgen de las Nieves Hospital. All participants were white, Caucasian. Patients with SLE who were not monitored for at least 1 year by said unit were excluded, as well as patients with suspected active infection or another disease involving systemic inflammation, except SLE, at the time of inclusion. All participants gave Informed consent to participate in this study, which was approved by the local ethics committee. The healthy controls come from donors of the blood bank of Granada, specifically the blood material comes from the blood bags that are routinely removed, which pass the microbiological tests, but do not finish processing properly due to volume defects and constitute waste bags.

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SLE diagnosis

The diagnosis of systemic lupus erythematosus (SLE) is based on 11 criteria, of which 4 or more of these criteria, either sequentially or simultaneously, during any interval of observation. These criteria were published in 1982 by the diagnostic criteria committee and American College of Rheumatology (ACR), and They were revised in 1992. The criteria are as follows:

1. Malar rash: Fixed, flat or high erythema, about malar eminences, which does not usually affect the grooves nasogenians

2. Discoid eruption: High erythematous plaques, with adherent keratotic peeling and follicular plugs; may There are atrophic scars on older lesions.

3. Photosensitivity: Rash due to an unusual reaction to sunlight, referred by the patient or observed by the doctor.

4. Mouth ulcers: Nasopharyngeal ulceration, by The painless common, observed by a doctor.

5. Arthritis: Non-erosive arthritis that affects two or more peripheral joints, characterized by pain at palpation, swelling or spillage.

6. Serositis:

to.

Pleuritis: Clear history of pain pleuritic or rub, or signs of pleural effusion, or

b.

Pericarditis: proven by electrocardiogram or rub or signs of pericardial effusion.

7. Renal disorder:

to.

Persistent proteinuria greater than 0.5 g / day or greater than 3+ if not quantified, or

b.

Cellular cylinders: can be of erythrocytes, hemoglobin, granular, tubular or mixed.

8. Neurological disorder:

to.

Seizures: in the absence of known pharmacological treatments or metabolic disorders; eg Uremia, ketoacidosis, or electrolyte imbalance, or good

b.

Psychosis: in the absence of treatments known pharmacological or metabolic disorders; eg Uremia, ketoacidosis, or electrolyte imbalance.

9. Hematological disorder:

to.

Hemolytic anemia: with reticulocytosis, O well

b.

Leukopenia: less than 4,000 / mm3 in two or more times

C.

Lymphopenia: less than 1,500 / mm3 in two or more occasions, or

d.

Thrombocytopenia: less than 100,000 / mm3 in the absence of drugs that produce this disturbance.

10. Immune disorder:

to.

Cell preparation LE-positives (This item was removed from the diagnostic criteria in the review conducted in 1992), or good

b.

Anti-DNA: title abnormal antibody against native DNA, or

C.

Anti-Sm: Presence of antibodies against nuclear antigen Sm.

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d.

Positive Antibody Finding antiphospholipid (AFL) based on:

one.

level Abnormal serum of IgG or IgM anticardiolopin antibodies,

2.

Positive result for anticoagulant lupus using a standard method, or

3.

False positive in syphilis serological tests (VDRL), which persists for at least 6 months and is confirmed by Treponema pallidum tests or fluorescent treponemal antibody (FTA-Abs) absorption test.

11. Antinuclear antibody: An abnormal titer of ANA by immunofluorescence or equivalent analysis in any time and in the absence of medications related to the syndrome of lupus of pharmacological origin.

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Material for analysis

The material that is extracted from the patients is a volume of 3 ml of peripheral blood with anticoagulant. The blood is processed by density gradient centrifugation, specifically Histopaque 1077 from Sigma-Aldrich (commercial reference 10771) was used following its technical indications (Sigma-Aldrich Inc., 3050 Spruce Street, St. Louis Mo 63103, USA) to obtain peripheral blood mononuclear cells (PBMC) from periferal blood mononuclear cells ) without erythrocytes. PBMCs are processed to obtain RNA using the RNeasy Plus Mini kit (Qiagen, cat. No. 74134. Quiagen is a trademark of AMBION, Inc., Austin, Texas). Said material is processed to convert it into cDNA, using commercial RNA to cDNA conversion kits that use a reverse transcriptase enzyme, the material obtained is called complementary DNA (cDNA), which can already be used for amplification by real-time thermal cycler (RT-PCR) that will quantify the present amounts of cDNA corresponding to the different ACSL isoenzymes. The quantification of these molecules is made relative to that of the reference molecule of a very stable constitutive expression gene throughout the cell cycle (UbcH5B) (Hamalainem et al ., 2001. Anal Biochem . Dec 1; 299 (1): 63-70). The oligonucleotides for the amplification of each ACSL are indicated below:

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TABLE 1 Oligonucleotides used

one

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Statistics used for data analysis

Clinical data were collected on a basis. of data created in the statistical program SPSS for Windows, version 15.0.

The univariate analysis of the values that show the different ACSLs, in two independent groups (lupus sufferers and healthy controls) is indicated in Table 2 as median with interquartile range. Mann's test was used Whitney to determine the statistical significance of the Differences between patients and controls. For effect analyzes of the different ACSLs in lupus and determine what the ACSLs are essentials that best describe the probability of lupus, was performed bimodal logistic regression introducing as variables the different series, that is, the N, the A and the SI. It is indicated in the table the beta coefficient, the significance P and the effect as odds ratio (OR) with 95% confidence interval (95% CI). Likewise, the Logistic regression, was performed with the group of lupus sufferers to determine the effects of each ACSL on the different manifestations and diagnostic criteria of lupus. It was determined at area under the curve (AUC) to determine the predictive value of each ACSL from the probability data of the different conditional multivariate logistic regression models for select independent ACSL. For the correlation analysis was performed using the Pearson correlation coefficient (with two tails) (r) and Spearman. Multiple linear regression was performed to relate each ACSL with quantitative parameters of serum in lupus which constitute inflammation factors. The tables indicate the Beta regression coefficient, P significance, and coefficient of determination R2, to determine the% variation of that factor that explains a unit of variation of the ACSL considered.

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Results

These results indicate that ACSLs are clearly involved in the pathology of SLE. We have quantified the levels of mRNA encoding ACSLs of various conditions. The first one is in cells freshly extracted from the blood of patients with SLE or healthy controls. These values are indicative of the levels of ACSLs in PBMC (lymphocytes and monocytes) in the physiological conditions in which they are in patients or controls (they are called with number and letter N, not treated in vitro ). The ACSL3N has a median of 18 (13.0-29.7) in healthy controls versus 10 (8.1-15.3) in lupus sufferers.

As can be seen in Table 2, compared with healthy people, the PBMCs of SLE newly extracted from patients and without any additional activating treatment (normal cells, N) had significantly lower ACSL1N level (P = 0.008) and ACSL3N (P = 4.1 x 10 e-4) but was higher for ACSL5N (P = 3.4 x 10 e-4).

The multivariate logistic regression of this series indicates that ACSL3N and 5N constitute the best model predictive of lupus, with an odds ratio (OR) of 0.83 for 3N and of 1.14 for 5N (Table 3). The area under the curve for this model is 0.888 indicating a high diagnostic capacity of lupus (Table 4) versus healthy controls.

When these factors are analyzed by non-parametric association analysis or correlation Spearman's, only in the group of patients, in relation to different manifestations that occur in lupus, specifically, with the 11 criteria of clinical diagnosis of lupus (see Table 3 and Table 4), or with the activity indices, the ACSL2N correlates moderately and inversely with the index of accumulated organic damage chronic (SLICC / ACR Damage index) (r = -0.314, P = 0.048), with homocysteine levels, and IL-2. ACSL5N correlates with IL6 levels which in turn is an inflammatory factor important, and with the presence of clinical manifestations hematologic (hemogl. = hemolytic anemia), and inversely with the corticosteroid treatment In the same way, within the group of patients with SLE, ACSL5 levels correlate with the treatment of corticosteroid patients suggesting that Such levels may be affected by such treatment.

Another source of quantification of ACSLs is a from PBMCs that have been stimulated in culture for 24 hours with a potent cellular and mitogen activator (PMA + lonomycin) (it denominated with number and letter A, of activated). The values obtained in this case for the different ACSLs they are indicative, rather than the Immune system status of the people they come from the samples, of the maximum response potential of such cells. In This type of measure, only ACSL1A and 2A are significantly different. Thus the values that go from 47.8 to 71.5 are Lupus specific. For 2A, values ranging from 0.28 up to 0.6, which are the majority, are given only in lupus. It is appreciated in Table 2, that the relative expression was higher in SLE compared to the controls for ACSL1 (A) (P = 3.8x10 e-4) and ACSL2A (P = 1.9 x 10 e-5), leaving the others without change significantly with the activation of PBMCs. The Logistic regression indicates that 1A and 5A are variable independent that best explain the appearance of lupus (Table 3) and the area under the curve is 0.918, reflecting a large capacity predictive Within the group of patients with SLE and stratifying based on having or not having any of the clinical manifestations previously indicated (see Table 2 and Table 3), the ACSL1A is associated with the subgroup that suffers from skin manifestations (P = 0.019), ACSL2A correlates moderately and inversely with both the index of lupus activity SLEDAI (r = -0.375, P = 0.017) as with protein C reactive (PCR, r = -0400, P = 0.011) factor widely associated with inflammatory process in many diseases and processes infectious

A parameter that we have set for balance differences due to factors not visible between values obtained from normal and activated cells, with PMA + lo, thereof sample, and to make them more extrapolar and similar to Quantifications performed in other laboratories and even in others diseases or situations, is the stimulation index (stimulation index, SI). Stimulation index (SI), the quotient between normal and active PBMC ACSL levels were significantly higher in SLE for ACSL1SI (P = 4.9 x 10 e-7), ACSL2SI (P = 0.001), ACS3SI (P = 1.0 x 10 e-6), and ACSL4SI (P = 0.013), but it was lower for the ACSL5SI (P = 1.8 x 10 e-5) as shown in the Table 1. When these values are analyzed within the group with lupus, stratifying as before for having or not having a condition pathological determined or by correlation analysis (see Table 3 and Table 4), we found that ACSL1A, 1SI and 3SI is associated with skin manifestations (P = 0.021) and renal complications (P = 0.017); ACSL3SI is associated with skin manifestations (P = 0.019), renal complications (P = 0.002), and correlates inversely with PCR levels (r = -0.446, P = 0.004). ACSL4SI is associated with joint problems (P = 0.003), renal (P = 0.038), hematological (P = 0.0034), and correlates inversely with the PCR levels (r = - 0.316, P = 0.047). ACSL2SI and ACSL5SI showed no association or correlation with no pathological manifestation listed.

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Conclusions

Therefore, we suggest that the imbalances in the expression of the different ACSLs in mononuclear cells of peripheral blood (PBMCs) in physiological situation (healthy controls) or pathophysiological (lupus) may contribute to the pathogenesis of lupus, specifically the expression values of the genes of the ACSL1N, 3N, 4N, 1 A, 2A, 1 SI, 2SI, 3SI, 4SI and 5SI. Equally they can contribute according to their level of expression to the manifestations of facial erythema (ACSL1A, 5A), photosensitivity (ACSL1N), articular (ACSL4SI), oral ulcers (ACSL5N), renal (ACSL3SI), hematological (ACSL4SI) and neurological (ACSL4A) and 0presence of anti-DNA antibodies (ACSL1A) and that together as a preliminary suggestion, ACSLs may represent new targets for the treatment of lupus. The measures or quantifications of the different ACSLs can be useful as markers and in the prompt identification of patients with different manifestations lupus clinics and indicate response to treatment in patients With active pathology.

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TABLE 2 Relative expression levels (mRNA) of the different ACSLs in lupus and healthy controls. The quantification is performed on untreated PBMCs (N series), activated in vitro (A series) cells and the ratio between activated / not activated (A / N) called Stimulation index (SI). The relative levels of ACSLs are presented as the median (range) and statistical significance of the difference in the comparison between lupus and controls was performed using the Mann-Whitney test.

2

3

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TABLE 3 Logistic regression to delimit ACSL variables independent that better explain the probability of having lupus. Be adjust a model for each series (N, A and SI). The beta coefficient, significance P and OR with 95% confidence interval

4

to)

Variables included in the model: ACSL1N, 2N, 3N, 4N, 5N.

b)

Variables included in the model: ACSL1A, 2A, 3A, 4A, 5A.

C)

Variables included in the model: ACSL1SI, 2SI, 3SI, 4SI, 5SI.

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TABLE 4 Area under the curve (AUC) of the different models of Logistic regression

5

AUC, area under the curve; EE, standard error; P, probability .

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TABLE 5 Association analysis and quantity expression of different ACSL between the groups that have (yes) and that do not have (no) the lupus manifestation indicated with the number of samples in each group. It is also indicated on the bottom line of each ACSL the Significance P. Not significant is indicated by "n.s". Statistics: non-parametric contrast is used for 2 independent groups and ordinal dependent variable (affectations clinics 0 = no, 1 = yes) with the Mann test Whitney

6

TABLE 6 Logistic regression to delimit ACSL variables independent that better explain the probability of having the lupus manifestation indicated. One model fits for each series (N, A and YES). The beta coefficient, the significance P and the OR with 95% confidence interval

7

to)

Source : Tan EM, et al ., 1982. Arthritis Rheum 25: 1271, 1982.

N.S., no significant.

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TABLE 7 Spearman correlation analysis

8

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<212> PRT

           \ vskip0.400000 \ baselineskip
        

<213> Homo sapiens

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<221> ACSL 3

           \ vskip0.400000 \ baselineskip
        

<222> (1) .. (720)

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 4

           \ vskip1.000000 \ baselineskip
        

19

twenty

twenty-one

22

           \ vskip0.400000 \ baselineskip
        

<210> 5

           \ vskip0.400000 \ baselineskip
        

<211> 670

           \ vskip0.400000 \ baselineskip
        

<212> PRT

           \ vskip0.400000 \ baselineskip
        

<213> Homo sapiens

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<221> ACSL 4 isoform 1

           \ vskip0.400000 \ baselineskip
        

<222> (1) .. (670)

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 5

2. 3

24

25

           \ vskip0.400000 \ baselineskip
        

<210> 6

           \ vskip0.400000 \ baselineskip
        

<211> 711

           \ vskip0.400000 \ baselineskip
        

<212> PRT

           \ vskip0.400000 \ baselineskip
        

<213> Homo sapiens

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<221> ACSL 4 isoform 2

           \ vskip0.400000 \ baselineskip
        

<222> (1) .. (711)

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 6

           \ vskip1.000000 \ baselineskip
        

26

27

28

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 7

           \ vskip0.400000 \ baselineskip
        

<211> 683

           \ vskip0.400000 \ baselineskip
        

<212> PRT

           \ vskip0.400000 \ baselineskip
        

<213> Homo sapiens

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<221> ACSL 5 isoform b

           \ vskip0.400000 \ baselineskip
        

<222> (1) .. (683)

           \ newpage
        

           \ vskip0.400000 \ baselineskip
        

<400> 7

29

31

32

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 8

           \ vskip0.400000 \ baselineskip
        

<211> 739

           \ vskip0.400000 \ baselineskip
        

<212> PRT

           \ vskip0.400000 \ baselineskip
        

<213> Homo sapiens

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<221> ACSL 5 isoform a

           \ vskip0.400000 \ baselineskip
        

<222> (1) .. (739)

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 8

           \ vskip1.000000 \ baselineskip
        

33

3. 4

35

36

           \ vskip0.400000 \ baselineskip
        

<210> 9

           \ vskip0.400000 \ baselineskip
        

<211> 20

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Artificial Sequence

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<223> direct primer ACSL1

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 9

           \ vskip1.000000 \ baselineskip
        

           \ hskip1cm
        

37

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 10

           \ vskip0.400000 \ baselineskip
        

<211> 20

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Artificial Sequence

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<223> ACSLl1 reverse primer

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 10

           \ vskip1.000000 \ baselineskip
        

           \ hskip1cm
        

38

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 11

           \ vskip0.400000 \ baselineskip
        

<211> 20

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Artificial Sequence

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<223> ACSL3 direct primer

           \ newpage
        

           \ vskip0.400000 \ baselineskip
        

<400> 11

           \ vskip1.000000 \ baselineskip
        

           \ hskip1cm
        

39

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 12

           \ vskip0.400000 \ baselineskip
        

<211> 20

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Artificial Sequence

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<223> ACSL3 reverse primer

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 12

           \ vskip1.000000 \ baselineskip
        

           \ hskip1cm
        

40

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 13

           \ vskip0.400000 \ baselineskip
        

<211> 20

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Artificial Sequence

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<223> ACSL4 direct primer

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 13

           \ vskip1.000000 \ baselineskip
        

           \ hskip1cm
        

41

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 14

           \ vskip0.400000 \ baselineskip
        

<211> 20

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Artificial Sequence

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<223> ACSL4 reverse primer

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 14

           \ vskip1.000000 \ baselineskip
        

           \ hskip1cm
        

42

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 15

           \ vskip0.400000 \ baselineskip
        

<211> 20

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Artificial Sequence

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<223> ACSL5 direct primer

           \ newpage
        

           \ vskip0.400000 \ baselineskip
        

<400> 15

           \ vskip1.000000 \ baselineskip
        

           \ hskip1cm
        

43

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 16

           \ vskip0.400000 \ baselineskip
        

<211> 20

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Artificial Sequence

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<223> ACSL5 reverse primer

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 16

           \ vskip1.000000 \ baselineskip
        

           \ hskip1cm
        

44

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 17

           \ vskip0.400000 \ baselineskip
        

<211> 20

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Artificial Sequence

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<223> direct primer ACSL6 (2)

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 17

           \ vskip1.000000 \ baselineskip
        

           \ hskip1cm
        

Four. Five

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 18

           \ vskip0.400000 \ baselineskip
        

<211> 20

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Artificial Sequence

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<223> ACLS6 reverse primer (2)

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 18

           \ vskip1.000000 \ baselineskip
        

           \ hskip1cm
        

46

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 19

           \ vskip0.400000 \ baselineskip
        

<211> 22

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Artificial Sequence

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<223> direct primer UbcH5B

           \ newpage
        

           \ vskip0.400000 \ baselineskip
        

<400> 19

           \ vskip1.000000 \ baselineskip
        

           \ hskip1cm
        

47

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<210> 20

           \ vskip0.400000 \ baselineskip
        

<211> 24

           \ vskip0.400000 \ baselineskip
        

<212> DNA

           \ vskip0.400000 \ baselineskip
        

<213> Artificial Sequence

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<220>

           \ vskip0.400000 \ baselineskip
        

<223> UbcH5B reverse primer

           \ vskip1.000000 \ baselineskip
        

           \ vskip0.400000 \ baselineskip
        

<400> 20

           \ vskip1.000000 \ baselineskip
        

           \ hskip1cm
        

48

Claims (16)

1. Use of the genes of the family of Acyl-Coenzyme A long chain synthetases ( ACSLs ) or their expression products, as markers for the diagnosis, prognosis, or monitoring of inflammatory or autoimmune diseases. 2. Use of the genes of the family of Acyl-Coenzyme A long chain synthetases ( ACSLs ) or their expression products according to the preceding claim, wherein the inflammatory or autoimmune disease is selected from the list comprising: systemic lupus erythematosus , rheumatoid arthritis, arteriosclerosis, hypertension, non-insulin dependent diabetes type 2, metabolic syndrome, Crohn's disease, or any combination thereof. 3. Use of the genes of the Acyl-Coenzyme A long chain synthetases ( ACSLs ) family or their expression products according to any of claims 1-2, wherein the inflammatory or autoimmune disease is systemic lupus erythematosus (SLE) ). 4. Method of obtaining useful data for diagnosis and monitoring of inflammatory diseases or autoimmune, comprising:

to.

obtain an isolated biological sample of an individual, and

b.

Detect the amount of the expression product of at least one of the genes of the family of Acyl-Coenzyme A long chain synthetases ( ACSLs ) in the sample isolated from (a).

           \ vskip1.000000 \ baselineskip
        

5. Method according to the preceding claim, which It also includes:

C.

compare the amounts obtained in the step (b) with a reference amount.

           \ vskip1.000000 \ baselineskip
        

6. A method according to any of claims 4-5, wherein the isolated biological sample of an individual from step (a) comprises peripheral blood mononuclear cells (PBMCs). 7. Method according to any of claims 4-6, wherein the genes of the ACSL family are selected from the list comprising: ACSL1, ACSL2 (ACSL6), ACSL3, ACSL4, ACSL5 , or any combination thereof. 8. Method according to any of the claims 4-7, wherein the disease Inflammatory or autoimmune is selected from the list comprising: systemic lupus erythematosus, rheumatoid arthritis, arteriosclerosis, hypertension, type 2 non-insulin dependent diabetes, syndrome metabolic, Crohn's disease, or any of its combinations 9. Method according to any of the claims 4-8, wherein the disease Inflammatory or autoimmune is systemic lupus erythematosus. 10. Method according to any of claims 4-9, further comprising assigning the individual of step (a) to the group of individuals with systemic lupus erythematosus disease when presenting an amount of expression product of the genes that are selected from the list which comprises: ACSL1, ACSL3, ACSL5SI , or any combination thereof, detected in step (b) minor and statistically significant compared to a reference amount. 11. Method according to any of claims 4-10, and further comprising assigning the individual of step (a) to the group of individuals with systemic lupus erythematosus disease when presenting an amount of product of expression of the genes that are selected from the list comprising: ACSL1A, ACSL1SI, ACSL2A, ACSL2SI, ACSL3SI, ACSL4SI, ACSL5 , or any combination thereof, detected in step (b) greater and statistically significant compared to a reference amount. 12. Method according to any of claims 4-11, further comprising assigning the individual of step (a) to the group of individuals with systemic lupus erythematosus disease with renal manifestations when presenting an amount of ACSL3SI gene expression product detected in the step (b) greater and statistically significant compared to a reference amount. 13. A method according to any of claims 4-12, further comprising assigning the individual according to step (a) to the group of individuals with systemic lupus erythematosus disease with hematological manifestations when presenting an amount of ACSL4SI gene expression product detected in the step (b) greater and statistically significant compared to a reference amount. 14. A method according to any of claims 4-13, further comprising assigning the individual according to step (a) to the group of individuals with systemic lupus erythematosus disease with neurological manifestations when there is an amount of ACSL4A gene expression product detected in the step (b) minor and statistically significant compared to a reference amount. 15. Diagnostic kit comprising a couple of primers selected from the list comprising: SEQ ID NO: 9 / SEQ ID NO: 10; SEQ ID NO: 11 / SEQ ID NO: 12, for SEQ ID NO: 13 / SEQ ID NO: 14; for SEQ ID NO: 15 / SEQ ID NO: 16; for SEQ ID NO: 17 / SEQ ID NO: 18 and SEQ ID NO: 19 / SEQ ID NO: 20, or any of its combinations 16. Use of diagnostic kit according to previous claim, for diagnosis, prognosis, or follow-up of inflammatory or autoimmune diseases.